WO2021222597A2 - Rapid diagnostic immunoassay for presence of antibodies - Google Patents

Rapid diagnostic immunoassay for presence of antibodies Download PDF

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Publication number
WO2021222597A2
WO2021222597A2 PCT/US2021/029936 US2021029936W WO2021222597A2 WO 2021222597 A2 WO2021222597 A2 WO 2021222597A2 US 2021029936 W US2021029936 W US 2021029936W WO 2021222597 A2 WO2021222597 A2 WO 2021222597A2
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coronavirus
sars
bat
batcov
cov
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PCT/US2021/029936
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French (fr)
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WO2021222597A3 (en
Inventor
Bobby Brooke HERRERA
Irene Bosch
Adam GOMEZ
Nol SALCEDO
Ankita REDDY
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E25Bio, Inc.
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Publication of WO2021222597A2 publication Critical patent/WO2021222597A2/en
Publication of WO2021222597A3 publication Critical patent/WO2021222597A3/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • a viral infection is the proliferation of a harmful virus inside a subject’s body. Viruses depend on host cells for their reproduction. Coronavirus (CoV) disease COVID-19 is a fast spreading worldwide pandemic caused by a coronavirus strain. Symptoms can appear 2-14 days after exposure and include fever, cough, and shortness of breath. Flavivirus strains cause widespread morbidity and mortality throughout the world. They are transmitted by ticks and are responsible of encephalitis and hemorrhagic diseases.
  • the invention provides an immunoassay for detecting a first and a second antigen in a biological sample from a subject, the immunoassay comprising: a first and a second capture reagent, wherein the first and the second capture reagents are immobilized on a solid support; a first and a second detection reagent; and an at least one visualizing reagent.
  • the first capture reagent comprises an anti-human IgG antibody. In some embodiments, the second capture reagent comprises an anti-human IgM antibody. In some embodiments, the first detection reagent is at least a portion of a first virus protein. In some embodiments, the first virus protein is coronavirus Spike protein.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the second detection reagent is at least a portion of a second virus protein.
  • the second virus protein is coronavirus Nucleocapsid protein.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63,
  • HKU1 Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rml/
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
  • the first virus protein is flavivirus non-structural protein 1 (NS1).
  • the second virus protein is flavivirus precursor membrane (PrM) protein.
  • the first and second virus protein is from a flavivirus non-structural protein (NS1) strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4),
  • Zika virus ZIKV
  • Yellow Fever YFV
  • Ilheus Virus IHV
  • POWA Powassan
  • JE Japanese encephalitis
  • WNV West Nile virus
  • DTV Deer tick
  • TEV tick-borne encephalitis virus
  • USTV Usuto
  • SLEV Saint Louis Encephalitis
  • Omsk hemorrhagic fever virus OHFV
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
  • PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID
  • the first antigen is a human IgG antibody capable of specifically recognizing at least one virus protein.
  • the at least one virus protein is coronavirus Spike protein.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the at least one virus protein is coronavirus Nucleocapsid protein.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhin
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
  • the at least one virus protein is a flavivirus non- structural protein 1 (NS1).
  • the at least one virus protein is a flavivirus precursor membrane (PrM) protein.
  • NS1 flavivirus non- structural protein 1
  • PrM flavivirus precursor membrane
  • (1) IgG antibodies recognizing only NS1 are present in the sample, indicating a primary viral infection of the subject; or (2) IgG antibodies to PrM only or IgG antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
  • the flavivirus non- structural protein 1 and flavivirus precursor membrane (PrM) protein is from a flavivirus strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
  • DV1 Dengue virus serotype 1
  • DV2 Dengue virus serotype 2
  • DV3 Dengue virus serotype 3
  • Dengue virus serotype 4 DV4
  • ZIKV Zika virus
  • YFV Yellow Fever
  • IHV Ilhe
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
  • PrM 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%,
  • the second antigen is a human IgM antibody capable of specifically recognizing at least one virus protein.
  • the at least one virus protein is coronavirus Spike protein.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the at least one virus protein is coronavirus Nucleocapsid protein.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhin
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
  • the at least one virus protein is a flavivirus non- structural protein 1 (NS1).
  • the at least one virus protein is a flavivirus precursor membrane (PrM) protein.
  • (1) IgM antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgM antibodies to PrM only or IgM antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
  • the flavivirus non- structural protein 1 and flavivirus precursor membrane (PrM) protein is from a flavivirus strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2),
  • Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
  • ZIKV Zika virus
  • YFV Yellow Fever
  • IHV Ilheus Virus
  • POWA Powassan
  • JE Japanese encephalitis
  • WNV West Nile virus
  • DTV Deer tick
  • TEV tick-borne encephalitis virus
  • USTV tick-borne encephalitis virus
  • SLEV Saint Louis Encephalitis
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
  • PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%,
  • the first detection reagent comprises an anti-human IgG antibody. In some embodiments, the second detection reagent comprises an anti-human IgM antibody. In some embodiments, the first capture reagent is at least a portion of a first virus protein.
  • the first virus protein is coronavirus Spike protein.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the second capture reagent is at least a portion of a second virus protein.
  • the second virus protein is coronavirus Nucleocapsid protein.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related human coronavirus Urbani (SARS Co
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
  • the first virus protein is a flavivirus non-structural protein 1 (NS1).
  • the second virus protein is a flavivirus precursor membrane (PrM) protein.
  • the flavivirus non-structural protein 1 and flavivirus precursor membrane (PrM) protein is from a flavivirus strain is selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
  • DV1 Dengue virus serotype 1
  • DV2 Dengue virus serotype 2
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
  • PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%,
  • the at least one visualizing reagent is at least one nanoparticle with a structure and an emission spectrum. In some embodiments, the at least one nanoparticle is conjugated to the first and second detection reagents. In some embodiments, the visualizing reagent comprises: (1) at least one first nanoparticle with a first structure and a first emission spectrum; and (2) at least one second nanoparticle with a second structure and a second emission spectrum; wherein the first and second emission spectra are visually distinct. In some embodiments, the first nanoparticle is conjugated to the first detection reagent and the second nanoparticle is conjugated to the second detection reagent. [0026] In some embodiments, the subject is suspected of having or is diagnosed with a viral infection.
  • the viral infection is a coronavirus (CoV) infection.
  • the coronavirus (CoV) is selected from the coronavirus (CoV) strains consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus
  • the viral infection is a flavivirus infection.
  • the flavivirus strain is selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
  • DV1 Dengue virus serotype 1
  • DV2 Dengue virus serotype 2
  • DV3 Dengue virus serotype 3
  • Dengue virus serotype 4 DV4
  • Zika virus ZIKV
  • Yellow Fever YFV
  • IHV Ilheus Virus
  • the subject is suspected of having or is diagnosed with a coronavirus (CoV) disease.
  • the coronavirus disease is COVID-19.
  • the subject is suspected of having or is diagnosed with asymptomatic COVID-19.
  • the subject is suspected of having or is diagnosed with symptomatic COVID-19.
  • the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof.
  • the at least one visualizing reagent comprises a colorimetric readout.
  • the at least one visualizing reagent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
  • the invention provides a lateral flow immunoassay device for detecting a first and a second antigen in a biological sample from a subject, the device comprising: an elongated holder having at least one aperture for sample application and at least one aperture for readout viewing; a first and a second immunoassay test strips secured within the holder, wherein the first and second test strips are parallel to each other along a long edge; a first capture reagent embedded in a first test area, wherein the first test area comprises at least a portion of the first test strip; a second capture reagent embedded in a second test area, wherein the second area comprises at least a portion of the second test strip; a first detection reagent embedded in at least a portion of a first conjugate pad, wherein the first conjugate pad comprises at least a portion of the first test strip and at least a portion of the second test strip; a second detection reagent embedded in at least a portion of a second conjugate pad, wherein the second conjug
  • the first capture reagent comprises an anti-human IgG antibody. In some embodiments, the second capture reagent comprises an anti-human IgM antibody. In some embodiments, the first detection reagent is at least a portion of a coronavirus Spike protein.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the second detection reagent is at least a portion of a coronavirus Nucleocapsid protein.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
  • the first antigen is a human IgG antibody capable of specifically recognizing at least one virus protein.
  • the at least one virus protein is coronavirus Spike protein.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the at least one virus protein is coronavirus Nucleocapsid protein.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhin
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
  • the second antigen is a human IgM antibody capable of specifically recognizing at least one virus protein.
  • the at least one virus protein is a coronavirus Spike protein.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the at least one virus protein is coronavirus Nucleocapsid protein.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhin
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
  • the first detection reagent comprises an anti-human IgG antibody. In some embodiments, the second detection reagent comprises an anti-human IgM antibody. In some embodiments, the first capture reagent is at least a portion of a coronavirus Spike protein.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the second capture reagent is at least a portion of coronavirus Nucleocapsid protein.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
  • the at least one visualizing reagent is at least one nanoparticle with a structure and an emission spectrum. In some embodiments, the at least nanoparticle is conjugated to the first and second detection reagents. In some embodiments, the visualizing reagent comprises (1) an at least one first nanoparticle with a first structure and a first emission spectrum; and (2) an at least one second nanoparticle with a second structure and a second emission spectrum; wherein the first and second emission spectra are visually distinct. In some embodiments, the at least one first nanoparticle is conjugated to the first detection reagent and the at least one second nanoparticle is conjugated to the second detection reagent.
  • the first control reagent comprises an anti-coronavirus Spike protein antibody.
  • the second control reagent comprises an anti- coronavirus Nucleocapsid protein antibody.
  • the subject is suspected of having or is diagnosed with a viral infection.
  • the viral infection is a coronavirus (CoV) infection.
  • the coronavirus (CoV) is selected from the coronavirus (CoV) strains consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rh
  • the subject is suspected of having or is diagnosed with a coronavirus (CoV) disease.
  • the coronavirus disease is COVID-19.
  • the subject is suspected of having or is diagnosed with asymptomatic COVID-19.
  • the subject is suspected of having or is diagnosed with symptomatic COVID-19.
  • the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof.
  • the at least one visualizing reagent comprises a colorimetric readout.
  • the at least one visualizing agent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
  • the invention provides a lateral flow immunoassay device for detecting a first and a second antigen in a biological sample from a subject, the device comprising: an elongated holder having at least one aperture for sample application and at least one aperture for readout viewing; a first and a second immunoassay test strip secured within the holder, wherein the first and second test strips are parallel to each other along a long edge; a first capture reagent embedded in a first test area, wherein the first area comprises at least a portion of the first test strip; a second capture reagent embedded in a second test area, wherein the second area comprises at least a portion of the second test strip; a first detection reagent embedded in at least a portion of a first conjugate pad, wherein the first conjugate pad comprises at least a portion of the first test strip and at least a portion of the second test strip; a second detection reagent embedded in at least a portion of a second conjugate pad, wherein the second conjugate
  • the first capture reagent comprises an anti-human IgG antibody.
  • the second capture reagent comprises an anti-human IgM antibody.
  • the first detection reagent is at least a portion of a flavivirus non- structural protein 1 (NS1).
  • the second virus protein is at least a portion of a flavivirus precursor membrane (PrM) protein.
  • the flavivirus non- structural protein 1 and the flavivirus precursor membrane (PrM) protein is from a flavivirus strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
  • DV1 Dengue virus serotype 1
  • DV2 Dengue virus serotype 2
  • DV3 Dengue virus serotype 3
  • Dengue virus serotype 4 DV4
  • ZIKV Zika virus
  • YFV Yellow Fever
  • IHV Il
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
  • PrM comprises is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%,
  • the first antigen is a human IgG antibody capable of specifically recognizing at least one virus protein.
  • the at least one virus protein is a flavivirus non- structural protein 1 (NS1).
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3,
  • the at least one virus protein is a flavivirus precursor membrane (PrM) protein.
  • PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID
  • IgG antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgG antibodies to PrM only or IgG antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
  • the second antigen is a human IgM antibody capable of specifically recognizing at least one virus protein.
  • the at least one virus protein is a flavivirus non- structural protein 1 (NS1).
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3,
  • the at least one virus protein is a flavivirus precursor membrane (PrM) protein.
  • PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or is 50%, 55%, 60%,
  • the first detection reagent comprises an anti-human IgG antibody.
  • the second detection reagent comprises an anti-human IgM antibody.
  • the first capture reagent is at least a portion of a flavivirus non- structural protein 1 (NS1).
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%,
  • the second capture reagent is at least a portion of is a flavivirus precursor membrane (PrM) protein.
  • PrM flavivirus precursor membrane
  • PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID
  • the at least one visualizing reagent is at least one nanoparticle with a structure and an emission spectrum. In some embodiments, the at least one nanoparticle is conjugated to the first and second detection reagents. In some embodiments, the visualizing reagent comprises (1) at least one first nanoparticle with a first structure and a first emission spectrum; and (2) at least one second nanoparticle with a second structure and a second emission spectrum; wherein the first and second emission spectra are visually distinct. In some embodiments, the first nanoparticle is conjugated to the first detection reagent and the second nanoparticle is conjugated to the second detection reagent.
  • the first control reagent comprises an anti -human immunoglobulin or an anti-human IgG and IgM.
  • the second control reagent comprises an anti-PrM antibody or an NS1 antibody.
  • the subject is suspected of having or is diagnosed with a viral infection.
  • the viral infection is a flavivirus infection.
  • the flavivirus infection is selected from the strains consisting of Dengue virus serotype 1 (DVl), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
  • DVl Dengue virus serotype 1
  • DV2 Dengue virus serotype 2
  • DV3 Dengue virus serotype 3
  • Dengue virus serotype 4 DV4
  • Zika virus ZIKV
  • the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof.
  • the at least one visualizing reagent comprises a colorimetric readout.
  • the at least one visualizing agent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
  • the invention provides a lateral flow immunoassay device for detecting IgG and IgM antibodies in a biological sample from a subject, the device comprising: an elongated holder having at least one aperture for sample application and at least one aperture for readout viewing; a first and a second immunoassay test strips secured within the holder, wherein the first and second test strips are parallel to each other along a long edge; a first capture reagent embedded in a first test area, wherein the first test area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the first capture reagent comprises anti-IgG antibodies; a second capture reagent embedded in a second test area, wherein the second area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the second capture reagent comprises anti-IgM antibodies; a first detection reagent embedded in at least a portion of a first conjugate pad, wherein the first and a second immunoas
  • the first capture reagent comprises an anti-human IgG antibody.
  • the second capture reagent comprises an anti-human IgM antibody.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat cor
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
  • the IgG is a human IgG antibody capable of specifically recognizing at least one coronavirus protein.
  • the at least one coronavirus protein is coronavirus Spike protein.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS- CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani (SARS CoV Urbani (SARS CoV Urban
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the at least one coronavirus protein is coronavirus Nucleocapsid protein.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RFl, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
  • the IgM antibody is a human IgM antibody capable of specifically recognizing at least one virus protein.
  • the at least one virus protein is a coronavirus Spike protein.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related human coronavirus Urbani (SARS Co
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the at least one virus protein is coronavirus a Nucleocapsid protein.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
  • the visualization reagent comprises a species of nanoparticles with a structure and an emission spectrum. In some embodiments, the visualization reagent is conjugated to the first and second detection reagents. In some embodiments, the species of nanoparticles comprises 80 nm Gold NanoUrchins nanoparticles. In some embodiments, wherein the species of nanoparticles comprises 40 nm InnovaCoat® Gold nanoparticles. In some embodiments, the control reagent is an anti-human Fc antibody.
  • the subject is suspected of having or is diagnosed with a coronavirus (CoV) infection.
  • the coronavirus (CoV) is selected from the coronavirus (CoV) strains consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related human coronavirus, SARS
  • the subject is suspected of having or is diagnosed with a coronavirus (CoV) disease.
  • the coronavirus disease is COVID-19.
  • the subject is suspected of having or is diagnosed with asymptomatic COVID-19.
  • the subject is suspected of having or is diagnosed with symptomatic COVID-19.
  • the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof.
  • the visualization reagent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
  • the invention provides a lateral flow immunoassay device for detecting IgG and IgM antibodies in a biological sample from a subject, the device comprising: an elongated holder having at least one aperture for sample application and at least one aperture for readout viewing; a first and a second immunoassay test strip secured within the holder, wherein the first and second test strips are parallel to each other along a long edge; a first capture reagent embedded in a first test area, wherein the first area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the first capture reagent comprises anti-IgG antibodies; a second capture reagent embedded in a second test area, wherein the second area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the second capture reagent comprises anti-IgM antibodies; a first detection reagent embedded in at least a portion of a first conjugate pad, wherein the first conjug
  • the first capture reagent comprises an anti-human IgG antibody.
  • the second capture reagent comprises an anti-human IgM antibody.
  • the flavivirus non- structural protein 1 and the flavivirus precursor membrane (PrM) protein is from a flavivirus strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
  • DV1 Dengue virus serotype 1
  • DV2 Dengue virus serotype 2
  • DV3 Dengue virus serotype 4
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8.
  • PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%
  • the IgG antibody is a human IgG antibody capable of specifically recognizing at least one virus protein.
  • the at least one virus protein is a flavivirus non- structural protein 1 (NS1).
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%,
  • the at least one vims protein is a flavivirus precursor membrane (PrM) protein.
  • PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
  • IgG antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgG antibodies to PrM only or IgG antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
  • the IgM antibody is a human IgM antibody capable of specifically recognizing at least one vims protein.
  • the at least one vims protein is a flavivirus non-stmctural protein 1 (NS1).
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
  • the at least one vims protein is a flavivirus precursor membrane (PrM) protein.
  • PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%,
  • the visualization reagent comprises a species of nanoparticles with a structure and an emission spectrum. In some embodiments, the visualization reagent is conjugated to the first and second detection reagents. In some embodiments, the species of nanoparticles comprises 80 nm Gold NanoUrchins nanoparticles. In some embodiments, the species of nanoparticles comprises 40 nm InnovaCoat® Gold nanoparticles. In some embodiments, the control reagent comprises an anti-human Fc antibody.
  • the subject is suspected of having or is diagnosed with a flavivirus infection.
  • the flavivirus infection is selected from the strains consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
  • DV1 Dengue virus serotype 1
  • DV2 Dengue virus serotype 2
  • DV3 Dengue virus serotype 3
  • Dengue virus serotype 4 DV4
  • Zika virus ZIKV
  • Yellow Fever YFV
  • IHV
  • the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof.
  • the at least one visualizing agent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
  • FIG. 1 shows two-color technology to determine coronavirus convalescence (IgG) infection based on detection of Spike (S) and Nucleocapsid (N) in a coronavirus-infected subject.
  • FIG. 2 shows two-color technology to determine coronavirus acute (IgM) infection based on detection of Spike (S) and Nucleocapsid (N) in a coronavirus-infected subject.
  • FIG. 3 shows two-color technology to determine coronavirus convalescence (IgG) infection based on detection of Spike (S) and Nucleocapsid (N) in a coronavirus-non-infected subject.
  • FIG. 4 shows two-color technology to determine coronavirus acute (IgM) infection based on detection of Spike (S) and Nucleocapsid (N) in a coronavirus-non-infected subject.
  • FIG. 5 shows single-color technology to determine coronavirus acute (IgM) and convalescence (IgG) infection based on detection of antibodies against Spike (S) protein in a coronavirus-infected subject.
  • FIG. 6 shows single-color technology to determine coronavirus acute (IgM) and convalescence (IgG) infection based on detection of antibodies against Nucleocapsid (N) in a coronavirus-infected subject.
  • FIG. 7 shows two-color technology to determine primary flavivirus infection based on IgG and IgM antibodies against NS1 and PrM proteins.
  • FIG. 8 shows two-color technology to determine secondary flavivirus infection based on IgG and IgM antibodies against NS1 and PrM proteins.
  • FIG. 9 shows single-color technology to determine primary flavivirus infection based on IgG and IgM antibodies against NS1 and PrM proteins.
  • FIG. 10 shows single-color technology to determine secondary flavivirus infection based on IgG and IgM antibodies against NS1 and PrM proteins.
  • the term “subject” refers to a vertebrate animal.
  • the subject is a mammal or a mammalian species.
  • the subject is a human.
  • the subject is a healthy human adult.
  • the subject is a non-human vertebrate animal, including, without limitation, non-human primates, laboratory animals, livestock, racehorses, domesticated animals, and non-domesticated animals.
  • the term “human subjects” means a population of healthy human adults.
  • the term “patient” refers to a human or animal.
  • mammal includes, but is not limited to, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, such as a monkey, chimpanzee, baboon or rhesus. In one embodiment, the mammal is a human.
  • the invention provides an immunoassay for detecting a first and a second antigen in a biological sample from a subject, the immunoassay comprising: a first and a second capture reagent, wherein the first and the second capture reagents are immobilized on a solid support; a first and a second detection reagent; and an at least one visualizing reagent.
  • the first capture reagent comprises an anti-human IgG antibody. In some embodiments, the second capture reagent comprises an anti-human IgM antibody. In some embodiments, the first detection reagent is at least a portion of a first virus protein. In some embodiments, the first virus protein is coronavirus Spike protein.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the second detection reagent is at least a portion of a second virus protein.
  • the second virus protein is coronavirus Nucleocapsid protein.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani),
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
  • the first virus protein is flavivirus non-structural protein 1 (NS1).
  • the second virus protein is flavivirus precursor membrane (PrM) protein.
  • the first and second virus protein is from a flavivirus non-structural protein (NS1) strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4),
  • Zika virus ZIKV
  • Yellow Fever YFV
  • Ilheus Virus IHV
  • POWA Powassan
  • JE Japanese encephalitis
  • WNV West Nile virus
  • DTV Deer tick
  • TEV tick-borne encephalitis virus
  • USTV Usuto
  • SLEV Saint Louis Encephalitis
  • Omsk hemorrhagic fever virus OHFV
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
  • PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID
  • the first antigen is a human IgG antibody capable of specifically recognizing at least one virus protein.
  • the at least one virus protein is coronavirus Spike protein.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the at least one virus protein is coronavirus Nucleocapsid protein.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhin
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
  • the at least one virus protein is a flavivirus non- structural protein 1 (NS1).
  • the at least one virus protein is a flavivirus precursor membrane (PrM) protein.
  • NS1 flavivirus non- structural protein 1
  • PrM flavivirus precursor membrane
  • (1) IgG antibodies recognizing only NS1 are present in the sample, indicating a primary viral infection of the subject; or (2) IgG antibodies to PrM only or IgG antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
  • the flavivirus non- structural protein 1 and flavivirus precursor membrane (PrM) protein is from a flavivirus strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
  • DV1 Dengue virus serotype 1
  • DV2 Dengue virus serotype 2
  • DV3 Dengue virus serotype 3
  • Dengue virus serotype 4 DV4
  • ZIKV Zika virus
  • YFV Yellow Fever
  • IHV Ilhe
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8.
  • PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
  • the second antigen is a human IgM antibody capable of specifically recognizing at least one virus protein.
  • the at least one virus protein is coronavirus Spike protein.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the at least one virus protein is coronavirus Nucleocapsid protein.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhin
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
  • the at least one virus protein is a flavivirus non- structural protein 1 (NS1).
  • the at least one virus protein is a flavivirus precursor membrane (PrM) protein.
  • (1) IgM antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgM antibodies to PrM only or IgM antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
  • the flavivirus non- structural protein 1 and flavivirus precursor membrane (PrM) protein is from a flavivirus strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2),
  • Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
  • ZIKV Zika virus
  • YFV Yellow Fever
  • IHV Ilheus Virus
  • POWA Powassan
  • JE Japanese encephalitis
  • WNV West Nile virus
  • DTV Deer tick
  • TEV tick-borne encephalitis virus
  • USTV tick-borne encephalitis virus
  • SLEV Saint Louis Encephalitis
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8.
  • PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
  • the first detection reagent comprises an anti-human IgG antibody. In some embodiments, the second detection reagent comprises an anti-human IgM antibody. In some embodiments, the first capture reagent is at least a portion of a first virus protein. [0109] In some embodiments, the first virus protein is coronavirus Spike protein.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the second capture reagent is at least a portion of a second virus protein.
  • the second virus protein is coronavirus Nucleocapsid protein.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related human coronavirus Urbani (SARS Co
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
  • the first virus protein is a flavivirus non-structural protein 1 (NS1).
  • the second virus protein is a flavivirus precursor membrane (PrM) protein.
  • the flavivirus non-structural protein 1 and flavivirus precursor membrane (PrM) protein is from a flavivirus strain is selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
  • DV1 Dengue virus serotype 1
  • DV2 Dengue virus serotype 2
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
  • PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or is 50%, 55%, 60%, 65%, 70%, 75%,
  • the at least one visualizing reagent is at least one nanoparticle with a structure and an emission spectrum. In some embodiments, the at least one nanoparticle is conjugated to the first and second detection reagents. In some embodiments, the visualizing reagent comprises: (1) at least one first nanoparticle with a first structure and a first emission spectrum; and (2) at least one second nanoparticle with a second structure and a second emission spectrum; wherein the first and second emission spectra are visually distinct. In some embodiments, the first nanoparticle is conjugated to the first detection reagent and the second nanoparticle is conjugated to the second detection reagent. [0114] In some embodiments, the subject is suspected of having or is diagnosed with a viral infection.
  • the viral infection is a coronavirus (CoV) infection.
  • the coronavirus (CoV) is selected from the coronavirus (CoV) strains consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus
  • the viral infection is a flavivirus infection.
  • the flavivirus strain is selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
  • the subject is suspected of having or is diagnosed with a coronavirus (CoV) disease.
  • the coronavirus disease is COVID-19.
  • the subject is suspected of having or is diagnosed with asymptomatic COVID-19.
  • the subject is suspected of having or is diagnosed with symptomatic COVID-19.
  • the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof.
  • the at least one visualizing reagent comprises a colorimetric readout.
  • the at least one visualizing reagent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
  • the invention provides a lateral flow immunoassay device for detecting a first and a second antigen in a biological sample from a subject, the device comprising: an elongated holder having at least one aperture for sample application and at least one aperture for readout viewing; a first and a second immunoassay test strips secured within the holder, wherein the first and second test strips are parallel to each other along a long edge; a first capture reagent embedded in a first test area, wherein the first test area comprises at least a portion of the first test strip; a second capture reagent embedded in a second test area, wherein the second area comprises at least a portion of the second test strip; a first detection reagent embedded in at least a portion of a first conjugate pad, wherein the first conjugate pad comprises at least a portion of the first test strip and at least a portion of the second test strip; a second detection reagent embedded in at least a portion of a second conjugate pad, wherein the second conjug
  • the first capture reagent comprises an anti-human IgG antibody.
  • the second capture reagent comprises an anti-human IgM antibody.
  • the first detection reagent is at least a portion of a coronavirus Spike protein.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the second detection reagent is at least a portion of a coronavirus Nucleocapsid protein.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
  • the first antigen is a human IgG antibody capable of specifically recognizing at least one virus protein.
  • the at least one virus protein is coronavirus Spike protein.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the at least one virus protein is coronavirus Nucleocapsid protein.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RFl, SARS-related Rh
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
  • the second antigen is a human IgM antibody capable of specifically recognizing at least one virus protein.
  • the at least one virus protein is a coronavirus Spike protein.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the at least one virus protein is coronavirus Nucleocapsid protein.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhin
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
  • the first detection reagent comprises an anti-human IgG antibody.
  • the second detection reagent comprises an anti-human IgM antibody.
  • the first capture reagent is at least a portion of a coronavirus Spike protein.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RFl, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the second capture reagent is at least a portion of coronavirus Nucleocapsid protein.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF
  • the at least one visualizing reagent is at least one nanoparticle with a structure and an emission spectrum. In some embodiments, the at least nanoparticle is conjugated to the first and second detection reagents. In some embodiments, the visualizing reagent comprises (1) an at least one first nanoparticle with a first structure and a first emission spectrum; and (2) an at least one second nanoparticle with a second structure and a second emission spectrum; wherein the first and second emission spectra are visually distinct. In some embodiments, the at least one first nanoparticle is conjugated to the first detection reagent and the at least one second nanoparticle is conjugated to the second detection reagent.
  • the first control reagent comprises an anti-coronavirus Spike protein antibody.
  • the second control reagent comprises an anti- coronavirus Nucleocapsid protein antibody.
  • the subject is suspected of having or is diagnosed with a viral infection.
  • the viral infection is a coronavirus (CoV) infection.
  • the coronavirus (CoV) is selected from the coronavirus (CoV) strains consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rh
  • the subject is suspected of having or is diagnosed with a coronavirus (CoV) disease.
  • the coronavirus disease is COVID-19.
  • the subject is suspected of having or is diagnosed with asymptomatic COVID-19.
  • the subject is suspected of having or is diagnosed with symptomatic COVID-19.
  • the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof.
  • the at least one visualizing reagent comprises a colorimetric readout.
  • the at least one visualizing agent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
  • the invention provides a lateral flow immunoassay device for detecting a first and a second antigen in a biological sample from a subject, the device comprising: an elongated holder having at least one aperture for sample application and at least one aperture for readout viewing; a first and a second immunoassay test strip secured within the holder, wherein the first and second test strips are parallel to each other along a long edge; a first capture reagent embedded in a first test area, wherein the first area comprises at least a portion of the first test strip; a second capture reagent embedded in a second test area, wherein the second area comprises at least a portion of the second test strip; a first detection reagent embedded in at least a portion of a first conjugate pad, wherein the first conjugate pad comprises at least a portion of the first test strip and at least a portion of the second test strip; a second detection reagent embedded in at least a portion of a second conjugate pad, wherein the second conjugate
  • the first capture reagent comprises an anti-human IgG antibody.
  • the second capture reagent comprises an anti-human IgM antibody.
  • the first detection reagent is at least a portion of a flavivirus non- structural protein 1 (NS1).
  • the second virus protein is at least a portion of a flavivirus precursor membrane (PrM) protein.
  • the flavivirus non- structural protein 1 and the flavivirus precursor membrane (PrM) protein is from a flavivirus strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
  • DV1 Dengue virus serotype 1
  • DV2 Dengue virus serotype 2
  • DV3 Dengue virus serotype 3
  • Dengue virus serotype 4 DV4
  • ZIKV Zika virus
  • YFV Yellow Fever
  • IHV Il
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8.
  • PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
  • the first antigen is a human IgG antibody capable of specifically recognizing at least one virus protein.
  • the at least one virus protein is a flavivirus non- structural protein 1 (NS1).
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the at least one virus protein is a flavivirus precursor membrane (PrM) protein.
  • PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%,
  • IgG antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgG antibodies to PrM only or IgG antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
  • the second antigen is a human IgM antibody capable of specifically recognizing at least one virus protein.
  • the at least one virus protein is a flavivirus non- structural protein 1 (NS1).
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the at least one virus protein is a flavivirus precursor membrane (PrM) protein.
  • PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%,
  • IgM antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgM antibodies to PrM only or IgM antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
  • the first detection reagent comprises an anti-human IgG antibody.
  • the second detection reagent comprises an anti-human IgM antibody.
  • the first capture reagent is at least a portion of a flavivirus non- structural protein 1 (NS1).
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8.
  • the second capture reagent is at least a portion of is a flavivirus precursor membrane (PrM) protein.
  • PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 5
  • the at least one visualizing reagent is at least one nanoparticle with a structure and an emission spectrum. In some embodiments, the at least one nanoparticle is conjugated to the first and second detection reagents. In some embodiments, the visualizing reagent comprises (1) at least one first nanoparticle with a first structure and a first emission spectrum; and (2) at least one second nanoparticle with a second structure and a second emission spectrum; wherein the first and second emission spectra are visually distinct. In some embodiments, the first nanoparticle is conjugated to the first detection reagent and the second nanoparticle is conjugated to the second detection reagent.
  • the first control reagent comprises an anti -human immunoglobulin or an anti-human IgG and IgM.
  • the second control reagent comprises an anti-PrM antibody or an NS1 antibody.
  • the subject is suspected of having or is diagnosed with a viral infection.
  • the viral infection is a flavivirus infection.
  • the flavivirus infection is selected from the strains consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
  • DV1 Dengue virus serotype 1
  • DV2 Dengue virus serotype 2
  • DV3 Dengue virus serotype 3
  • Dengue virus serotype 4 DV4
  • Zika virus ZIKV
  • the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof.
  • the at least one visualizing reagent comprises a colorimetric readout.
  • the at least one visualizing agent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
  • the invention provides a lateral flow immunoassay device for detecting IgG and IgM antibodies in a biological sample from a subject, the device comprising: an elongated holder having at least one aperture for sample application and at least one aperture for readout viewing; a first and a second immunoassay test strips secured within the holder, wherein the first and second test strips are parallel to each other along a long edge; a first capture reagent embedded in a first test area, wherein the first test area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the first capture reagent comprises anti-IgG antibodies; a second capture reagent embedded in a second test area, wherein the second area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the second capture reagent comprises anti-IgM antibodies; a first detection reagent embedded in at least a portion of a first conjugate pad, wherein the first and a second immunoas
  • the first capture reagent comprises an anti-human IgG antibody.
  • the second capture reagent comprises an anti-human IgM antibody.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat cor
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
  • the IgG is a human IgG antibody capable of specifically recognizing at least one coronavirus protein.
  • the at least one coronavirus protein is coronavirus Spike protein.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS- CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani (SARS CoV Urbani (SARS CoV Urban
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the at least one coronavirus protein is coronavirus Nucleocapsid protein.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
  • the IgM antibody is a human IgM antibody capable of specifically recognizing at least one virus protein.
  • the at least one virus protein is a coronavirus Spike protein.
  • the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related human coronavirus Urbani (SARS Co
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
  • the at least one virus protein is coronavirus a Nucleocapsid protein.
  • the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RFl, SARS-
  • the coronavirus strain is SARS-CoV-2.
  • the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
  • the visualization reagent comprises a species of nanoparticles with a structure and an emission spectrum. In some embodiments, the visualization reagent is conjugated to the first and second detection reagents. In some embodiments, the species of nanoparticles comprises 80 nm Gold NanoUrchins nanoparticles. In some embodiments, wherein the species of nanoparticles comprises 40 nm InnovaCoat® Gold nanoparticles. In some embodiments, the control reagent is an anti-human Fc antibody.
  • the subject is suspected of having or is diagnosed with a coronavirus (CoV) infection.
  • the coronavirus (CoV) is selected from the coronavirus (CoV) strains consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related human coronavirus, SARS
  • the subject is suspected of having or is diagnosed with a coronavirus (CoV) disease.
  • the coronavirus disease is COVID-19.
  • the subject is suspected of having or is diagnosed with asymptomatic COVID-19.
  • the subject is suspected of having or is diagnosed with symptomatic COVID-19.
  • the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof.
  • the visualization reagent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
  • the invention provides a lateral flow immunoassay device for detecting IgG and IgM antibodies in a biological sample from a subject, the device comprising: an elongated holder having at least one aperture for sample application and at least one aperture for readout viewing; a first and a second immunoassay test strip secured within the holder, wherein the first and second test strips are parallel to each other along a long edge; a first capture reagent embedded in a first test area, wherein the first area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the first capture reagent comprises anti-IgG antibodies; a second capture reagent embedded in a second test area, wherein the second area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the second capture reagent comprises anti-IgM antibodies; a first detection reagent embedded in at least a portion of a first conjugate pad, wherein the first conjug
  • the first capture reagent comprises an anti-human IgG antibody.
  • the second capture reagent comprises an anti-human IgM antibody.
  • the flavivirus non- structural protein 1 and the flavivirus precursor membrane (PrM) protein is from a flavivirus strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
  • DV1 Dengue virus serotype 1
  • DV2 Dengue virus serotype 2
  • DV3 Dengue virus serotype 4
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8.
  • PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%,
  • the IgG antibody is a human IgG antibody capable of specifically recognizing at least one virus protein.
  • the at least one virus protein is a flavivirus non- structural protein 1 (NS1).
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%,
  • the at least one virus protein is a flavivirus precursor membrane (PrM) protein.
  • PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
  • IgG antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgG antibodies to PrM only or IgG antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
  • the IgM antibody is a human IgM antibody capable of specifically recognizing at least one virus protein.
  • the at least one virus protein is a flavivirus non- structural protein 1 (NS1).
  • NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
  • the at least one virus protein is a flavivirus precursor membrane (PrM) protein.
  • PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%,
  • IgM antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgM antibodies to PrM only or IgM antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
  • the visualization reagent comprises a species of nanoparticles with a structure and an emission spectrum. In some embodiments, the visualization reagent is conjugated to the first and second detection reagents. In some embodiments, the species of nanoparticles comprises 80 nm Gold NanoUrchins nanoparticles. In some embodiments, the species of nanoparticles comprises 40 nm InnovaCoat® Gold nanoparticles. In some embodiments, the control reagent comprises an anti-human Fc antibody. [0161] In some embodiments, the subject is suspected of having or is diagnosed with a flavivirus infection.
  • the flavivirus infection is selected from the strains consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
  • DV1 Dengue virus serotype 1
  • DV2 Dengue virus serotype 2
  • DV3 Dengue virus serotype 3
  • Dengue virus serotype 4 DV4
  • Zika virus ZIKV
  • Yellow Fever YFV
  • IHV Ilheus Virus
  • POWA Japanese encephalitis
  • JE Japanese
  • the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof.
  • the at least one visualizing agent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
  • Lateral flow immunoassays are a cost-effective methodology for detecting a target protein in a biological sample. Tests based on this methodology can be used at a point-of- care center by medical professionals or for at home testing by individuals without any medical or technical training.
  • the technology takes advantage of the capillary properties of materials such as nitrocellulose, which have the capacity to transport fluids spontaneously. Liquids display capillary action, which is their ability to flow through narrow spaces even in opposition to external forces. Capillary action can be observed in the drawing up of liquids into materials such as fibers or cellulose. It occurs due to intermolecular forces between the liquid and surrounding solid surfaces. If the diameter of the space is sufficiently small, then the combination of surface tension of the liquid and the adhesive forces between the liquid and the surrounding solid surface act to propel the liquid into the narrow space.
  • lateral flow immunoassays There are two main types of lateral flow immunoassays: a “sandwich assay” and a “competitive assay”.
  • the subject matter disclosed herein relates to “sandwich assays.”
  • a migrating sample can first come into contact with a detection reagent, which can specifically bind to a target protein or viral antigen.
  • the detection reagent is conjugated to a visualizing reagent. If the target protein is present in the sample, the conjugated detection reagent can bind to it and subsequently flow laterally to a test line (area).
  • the test line (area) may also contain a capture reagent such as proteins or antibodies specific to the target protein. A visual signal or readout can be produced when the target protein reaches the test line (area).
  • the target protein is “sandwiched” between two proteins such as antibodies.
  • the detection reagent is not directly labeled and both the capture reagent and the detection reagent are antibodies, then these antibodies must be from different species (i.e., if the capture antibody is a rabbit antibody, the detection antibody can be from goat, chicken, etc., but not rabbit). This will allow the use of a secondary antibody directed to the species heavy chain of the detection antibody. If the detection antibody is directly labeled, then the capture and detection antibodies can be from the same species.
  • an immunoassay sensitivity usually the protein or antibody binding affinity for the target antigen. As the target antigen concentration increases, the amount of detection reagent accumulated in the test line (area) increases, leading to a higher measured response or readout.
  • Lateral flow immunoassays are incorporated in low-cost, simple, rapid and portable detection devices. These devices are a paper-based platform for the detection and quantification of particular analytes in mixtures, where the sample is placed on a test device and the results are displayed within 2-30 min. A variety of biological samples can be tested using these devices including but not limited to serum, plasma, whole blood, urine, saliva, sweat, and other fluids.
  • the device can include a lateral flow test strip.
  • the test strip is paper- based.
  • the test strip consists of overlapping membranes that can be mounted on a backing card for enhanced stability and handling.
  • the sample can be applied at one end of the strip, on the adsorbent sample pad, which can be impregnated with buffer salts and/or surfactants suitable for interaction with the detection reagent.
  • the sample can then migrate laterally through the pad.
  • the pad contains antibodies that are specific to the target analyte.
  • at least one antibody or protein conjugated to colored or fluorescent particles such as colloidal gold or latex microspheres.
  • the sample, together with the conjugated antibody or protein bound to the target analyte, can laterally migrate along the strip into the detection zone.
  • the detection zone is a porous membrane pre-loaded with specific components.
  • the detection zone is composed of nitrocellulose.
  • the specific components are antibodies, antigens, or proteins.
  • the specific components are immobilized in lines.
  • the specific components react with the analyte bound to the conjugated antibody.
  • the presence of the target analyte can result in an observable response on the test line.
  • a response on the control line indicates that proper liquid flow through the text strip took place.
  • the read-out, represented by the lines can appear in different colors or with different intensities.
  • multiple analytes can be tested simultaneously and under the same conditions.
  • additional test lines of antibodies specific to different analytes can be immobilized on the test strip.
  • multiple test lines pre-loaded with the same antibody can be utilized for semi- quantitative analysis of the target analyte.
  • an absorbent pad is attached at one end of the strip.
  • the absorbent pad is on the opposite end of the test strip compared to the sample pad where the sample is initially applied.
  • the absorbent pad wicks the excess reagents to prevent backflow of the liquid across the test strip.
  • the label incorporated in the detection method is colloidal gold. In some embodiments, gold particles are red in color due to localized surface plasmon resonance.
  • the label incorporated in the detection method latex, which can be tagged with a variety of detector reagents such as colored or fluorescent dyes, and magnetic or paramagnetic components. In some embodiments, latex is blue. In some embodiments, the latex can be produced in multiple colors discriminating between numerous lines.
  • the label incorporated in the detection method is carbon.
  • the label incorporated in the detection method are fluorescent labels. In some embodiments, the label incorporated in the detection method comprises enzymatic modifications of the labels. In some embodiments, the label incorporated in the detection method is carbon nanotubes. In some embodiments, the label incorporated in the detection method is fluorescent nanoparticles such as quantum dots.
  • the conjugate pad holds the detector particles and ensures they are functionally stable.
  • the conjugate pad includes buffers.
  • the buffers contain carbohydrates to preserve and resolubilize conjugated particles.
  • Antibodies are glycoproteins capable of binding specific antigens. They are produced by a subject’s immune system in response to a foreign antigen invasion in the body. Antibodies exist as one or more copies of a Y-shaped unit, composed of four polypeptide chains. Each antibody contains two identical copies of a heavy chain, and two identical copies of a light chain. The heavy and light chains differ in their polypeptide sequence and length. The variable region binds tightly and specifically to an epitope on the antigen.
  • the light chains of an antibody can be classified as either kappa (K) or lambda (l) type based on differences in polypeptide sequence.
  • Antibodies are classified into five isotypes (IgM, IgD, IgG, IgA, and IgE) according to their heavy chains, which provide each isotype with distinct characteristics and roles in the immune response.
  • IgG antibodies are the most abundant antibody isotype in the blood (plasma). They account for 70-75% of human immunoglobulins. IgG antibodies counteract harmful antigens and are important in the recognition of antigen-antibody complexes by leukocytes and macrophages.
  • IgM antibodies circulate in the blood and account for about 10% of human immunoglobulins.
  • IgM antibodies have a characteristic pentameric structure in which five basic Y-shaped molecules are linked together.
  • IgM B cells produce IgM first in response to microbial infection/antigen invasion.
  • IgM antibodies have a lower affinity for antigens than IgG antibodies, IgM have higher avidity for antigens because of the pentameric/hexameric structure.
  • IgM are also capable of activating particular cell signaling pathways by binding to the cell surface receptors.
  • IgA antibodies are abundant in serum, nasal mucus, saliva, breast milk, and intestinal fluid. They account for 10- 15% of human immunoglobulins. IgA antibodies form dimers (i.e., two IgA monomers joined together).
  • IgE antibodies are generally present in minute amounts, accounting for no more than 0.001% of human immunoglobulins. Their original role is to protect against parasites but can be involve in allergic responses.
  • IgD antibodies account for less than 1% of human immunoglobulins. They can be involved in the induction of antibody production in B cells, however, their exact function is still unknown.
  • Antibody heavy chains differ in size and composition and can be approximately 450 amino acids to approximately 550 amino acids. Each heavy chain includes two regions, the constant region and the variable region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. The variable region of the heavy chain differs depending on the B cell that produced it, but is the same for all antibodies produced by a single B cell or B cell clone. The variable region of each heavy chain is approximately 110 amino acids long and is composed of a single Ig domain. [0177] Mammals have only two types of light chains, l and K. A light chain has two successive domains: one constant domain and one variable domain. The approximate length of a light chain is 211-217 amino acids. Each antibody contains two light chains that are always identical.
  • An antibody can be divided into three sections: two F(ab) regions and an Fc region.
  • the F(ab) regions contain the variable domain that binds to antigens.
  • the Fc fragment provides a binding site for endogenous Fc receptors on the surface of lymphocytes. It is also the site of binding for secondary antibodies.
  • dyes and enzymes can be covalently linked to antibodies on the Fc portion of the antibody to provide visualization.
  • Nanoparticles can also be conjugated to the Fc position of antibodies.
  • Viruses are small parasites that cannot reproduce on their own. Once a virus infects a cell, however, a virus can direct the cell machinery to produce more viruses. Most viruses utilize either RNA or DNA as their genetic material.
  • the nucleic acid may be single- or double-stranded.
  • the infectious virus particle or a virion includes a nucleic acid and an outer shell of protein. The simplest viruses contain only enough RNA or DNA to encode four proteins. On the other hand, the most complex ones can encode up to 200 proteins.
  • the nucleic acid of a virion is enclosed within a protein coat called a capsid.
  • the capsid id composed of multiple copies of one protein or a few different proteins, each of which is encoded by a single viral gene. Because of this structure, a virus is able to encode all the information for making a relatively large capsid in a small number of genes.
  • Coronaviruses are a large family of enveloped, positive-sense, single- stranded RNA viruses that infect a broad range of vertebrates. They are extensive in bats but can be found in many other birds and mammals including humans. In humans, CoVs tend to cause mild to moderate upper respiratory tract infections such as the common cold.
  • the Spike protein is a large type I transmembrane protein ranging from 1,160 amino acids to up to 1,400 amino acids. Spike proteins are highly glycosylated with 21 to 35 N- glycosylation sites. Spike proteins assemble into trimers on the virion surface to form the distinctive "corona", or crown-like appearance, of a coronavirus.
  • the ectodomain of all CoV Spike proteins is organized into an N-terminal domain named SI, responsible for receptor binding, and a C-terminal S2 domain, responsible for viral fusion.
  • Coronaviruses can infect the respiratory epithelial cells through interaction with the ACE2 receptor on the cell surface. Recombinant Spike protein can bind with recombinant ACE2 proteins.
  • the coronavirus Nucleocapsid is a structural protein capable of forming complexes with genomic RNA. Nucleocapsid proteins also interact with the viral membrane protein(s) during virion assembly and play a critical role in enhancing the efficiency of virus transcription and assembly.
  • DENV exists as four antigenic serotypes, DENV1 to DENV4. These viruses have a wide geographic distribution, with approximately 390 million infections annually and more than a quarter of the world’s population at risk.
  • ZIKV Prior to 2015, ZIKV was considered obscure and was known to circulate in Africa and Southeast Asia as two separate viral lineages, African and Asian. While most are asymptomatic, the clinical presentation of ZIKV infection resembles that of dengue, including fever, rash, conjunctivitis, arthralgia, and myalgia.
  • DENV contains a positive-sense, single-stranded RNA genome of about 10.6 kilobases.
  • the single open reading frame of the DENV encodes a polyprotein precursor, which is cleaved by cellular and viral protease into three structural proteins, the capsid (C), precursor membrane (PrM), and envelope (E), as well as seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5.
  • the E protein a glycoprotein of approximately 55 kDa, contains 12 strictly conserved cysteine residues forming six disulfide bridges and is present as a heterodimer with PrM protein before the maturation of the virion. E protein participates in virus entry and is the major target of both neutralizing and enhancing antibodies.
  • the PrM protein is a glycoprotein of about 19 kDa. It contains six highly conserved cysteine residues forming three disulfide bridges.
  • the NS1 protein is also a glycoprotein of about 40 kDa. It contains 12 highly conserved cysteine residues forming six disulfide bridges.
  • NS1 is present intracellularly, on the cell surface, and outside of the cells.
  • a primary infection is the first time a subject is exposed to an antigen. During a primary infection, the body has no innate defenses, such as antibodies, against the invading virus.
  • a secondary infection is an infection that occurs during or after treatment for a previous infection. The secondary infection may be caused by the first treatment, by changes in the immune system, increased susceptibility due to the primary infection, or it may be unrelated.
  • the primary and the secondary infection are caused by different viruses.
  • the primary and the secondary infection are caused by different viruses from the same family. In some embodiments, the primary and the secondary infection are caused by the same virus strain.
  • a primary immune response occurs when a subject’s immune system encounters an antigen for the first time. At this time the immune system has to learn to recognize the antigen and produce antibodies against it.
  • a secondary immune response occurs when a subject’s immune system encounters the same antigen for the second time. At this point it is expected that immunological memory has been established and the immune system can start making antibodies immediately.
  • IgG antibodies are primarily involved in the secondary immune response, while IgM antibodies are the main antibodies involved in the primary response.
  • the subject matter disclosed herein relates to determining if a viral infection is primary or secondary infection. In some embodiments, the subject matter disclosed herein relates to determining if a viral infection is primary or secondary infection based on antibodies raised to PrM and NS1 proteins.
  • a primary flavivirus infection leads to IgG and/or IgM antibodies raised only to the NS1 protein.
  • the rates of antibody responses to PrM and NS1 proteins are higher in patients with secondary virus infection than in those with primary infection.
  • Gold nanoparticles have many applications in biology and medicine due to their unique optical and physical properties. They can be used for biosensor development, as cellular probes, as drug delivery vehicles, or as optical contrast agents among others. The distinct optical and physical properties of gold nanoparticles are dependent upon their size (diameter), shape, surface structure and agglomeration state.
  • SEQ ID NO: 1 is the polypeptide sequence of the coronavirus SARS-CoV-2 Spike protein per gene bank accession NC 045512 and including polymorphisms already reported MT077125 and ancestor QHR63300 RaTG13.
  • SEQ ID NO: 2 is the polypeptide sequence of the coronavirus Nucleocapsid protein (NCBI Reference Sequence: YP_009724397.2)
  • SEQ ID NO: 3 is the polypeptide sequence of the Dengue virus 1 NS1 protein (NCBI Reference Sequence: NP_722461.1)
  • SEQ ID NO: 4 is the polypeptide sequence of the Dengue virus 2 NS1 protein (NCBI Reference Sequence: NP_739584.2)
  • SEQ ID NO: 5 is the polypeptide sequence of the Dengue vims 3 NS1 protein (NCBI Reference Sequence: YP 001531169.2)
  • SEQ ID NO: 6 is the polypeptide sequence of the Dengue vims 4 NS1 protein (NCBI Reference Sequence: NP_740318.1)
  • SEQ ID NO: 7 is the polypeptide sequence of the Asian strain of Zika vims NS1 protein (NCBI Reference Sequence: YP 009430301.1).
  • SEQ ID NO: 8 is the polypeptide sequence of the African strain of Zika vims NS1 protein (NCBI Reference Sequence: YP 009227199.1).
  • D GC S
  • VDF SKKETRCGT GVFIYND VE AWRDRYKYHPD SPRRL AAAVKQ AWEEGIC
  • VRAAKTNN SF
  • VVDGDTLKECPLEHRAWN SFLVEDHGF
  • SLECDP AVIGT AVKGREAAHSDLGYWIESEKNDTWRLKRAHLIEMKT CEWPKSHTLWTDGVEESDLIIPKSLAGPLSHHNTREGYRTQVKGPWHSEELEIRFEEC PGTKVYVEETCGTRGPSLRSTTASGRVIEEWCCRECTMPPLSFRAKDGCWY
  • SEQ ID NO: 9 is the polypeptide sequence of the Dengue vims 1 PrM protein (NCBI Reference Sequence: NP_733807.2)
  • SEQ ID NO: 10 is the polypeptide sequence of the Dengue vims 2 PrM protein
  • SEQ ID NO: 11 is the polypeptide sequence of the Dengue vims 3 PrM protein
  • SEQ ID NO: 12 is the polypeptide sequence of the Dengue vims 4 PrM protein
  • SEQ ID NO: 13 is the polypeptide sequence of the Asian strain of the Zika vims
  • PrM protein (NCBI Reference Sequence: YP 009430297.1)
  • SEQ ID NO: 14 is the polypeptide sequence of the African strain of the Zika virus PrM protein (NCBI Reference Sequence: YP 009227197.1)
  • Example 1 Two color technology for coronavirus detection in an infected subject
  • FIG. 1 shows two-color technology to determine coronavirus convalescence (IgG) infection based on detection of Spike (S) and Nucleocapsid (N) in a coronavirus-infected subject.
  • IgG coronavirus convalescence
  • S Spike
  • N Nucleocapsid
  • FIG. 2 shows two-color technology to determine coronavirus acute (IgM) infection based on detection of Spike (S) and Nucleocapsid (N) in a coronavirus-infected subject.
  • IgM coronavirus acute
  • S Spike
  • N Nucleocapsid
  • Example 2 Two color technology for coronavirus detection in a non- infected subject
  • FIG. 3 shows two-color technology to determine coronavirus convalescence (IgG) infection based on detection of Spike (S) and Nucleocapsid (N) in a coronavirus-non-infected subject.
  • IgG coronavirus convalescence
  • S Spike
  • N Nucleocapsid
  • FIG. 4 shows two-color technology to determine coronavirus acute (IgM) infection based on detection of Spike (S) and Nucleocapsid (N) in a coronavirus-non-infected subject.
  • IgM coronavirus acute
  • S Spike
  • N Nucleocapsid
  • Example 3 single-color technology for coronavirus infection in an infected subject
  • FIG. 5 shows single-color technology to determine coronavirus acute (IgM) and convalescence (IgG) infection based on detection of antibodies against Spike (S) protein in a coronavirus-infected subject.
  • This Spike antibody test shows that the coronavirus-infected subject developed both IgG and IgM antibodies to Spike protein.
  • subjects develop either IgG antibodies or IgM antibodies to Spike protein but not both.
  • FIG. 6 shows single-color technology to determine coronavirus acute (IgM) and convalescence (IgG) infection based on detection of antibodies against Nucleocapsid (N) in a coronavirus- infected subject.
  • This Nucleocapsid antibody test shows that an infected subject only developed IgM antibodies to Nucleocapsid.
  • subject develop both IgG and IgM antibodies.
  • FIG. 7 shows two-color technology to determine primary flavivirus infection based on IgG and IgM antibodies against NS1 and PrM proteins. There are two colors observed on the test strips. Red color in the test area of the strips showing that the biological sample contained IgG antibodies (top strip) and IgM antibodies (bottom strip) against only NS1 protein. Red color for the NS1 protein in the control area confirming successful lateral flow along the strip. Blue color for the PrM protein, also confirming the successful lateral flow. This test shows that the biological sample (in this case from a Dengue virus-infected subject) contained IgG but not IgM antibodies against NS1 protein only.
  • primary flavivirus infected subjects develop both IgG and IgM antibodies to NS1. In some embodiments, primary flavivirus infected subjects develop only IgG or only IgM antibodies to NS1.
  • FIG. 8 shows two-color technology to determine secondary flavivirus infection based on IgG and IgM antibodies against NS1 and PrM proteins. There are three colors observed on the test strips. Blue color for the NS1 protein in the control area confirming successful lateral flow along the strip. Red color for the PrM protein also confirming the successful lateral flow. Purple color in the test area of the strip shows that the biological sample (in this case from a Dengue virus infected subject) contained IgG antibodies (top strip) and IgM antibodies (bottom strip) to both PrM and NS1 flavivirus proteins. This result shows that the subject presents with a secondary flavivirus infection.
  • secondary flavivirus infected subjects develop IgG and IgM antibodies to NS1 and PrM proteins. In some embodiments, secondary flavivirus infected subjects develop only IgG or only IgM antibodies to NS1 and PrM proteins.
  • FIG. 9 shows single-color technology to determine primary flavivirus infection based on IgG and IgM antibodies against NS1 and PrM proteins.
  • a primary flavivirus- infected subject in this case Dengue virus-infected
  • PrM PrM
  • the absence of antibodies to PrM protein suggests that the subject presents with a primary flavivirus infection.
  • a primary infected patients only develops IgG or IgM antibodies to NS1.
  • FIG. 10 shows single color technology to determine secondary flavivirus infection based on IgG and IgM antibodies against NS1 and PrM proteins. As shown in FIG.
  • a secondary infected subject in this case a subject infected with Dengue virus develops IgG and IgM antibodies to both NS1 and PrM.
  • secondary infected subject develops only either IgG or IgM antibodies to NS1 and PrM.
  • a secondary flavivirus-infected subject presents only IgG antibodies to both NS1 and PrM.
  • a secondary flavivirus-infected subjects presents only IgM antibodies to both NS1 and PrM.
  • Example 6 Reagents for Experiments described in Example 1-5 are provided below in Tables 1-8.

Abstract

The subject matter disclosed herein relates to an immunoassay for detecting antibodies in a biological sample from a subject comprising a capture reagent immobilized on a solid support; a detection reagent; and a visualizing reagent.

Description

RAPID DIAGNOSTIC IMMUNOASSAY FOR PRESENCE OF ANTIBODIES
[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/018,318, filed on April 30, 2020, the content of which is hereby incorporated by reference in its entirety.
[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application.
[0003] This patent disclosure contains material that is subject to copyright protection.
The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.
BACKGROUND OF THE INVENTION
[0004] A viral infection is the proliferation of a harmful virus inside a subject’s body. Viruses depend on host cells for their reproduction. Coronavirus (CoV) disease COVID-19 is a fast spreading worldwide pandemic caused by a coronavirus strain. Symptoms can appear 2-14 days after exposure and include fever, cough, and shortness of breath. Flavivirus strains cause widespread morbidity and mortality throughout the world. They are transmitted by ticks and are responsible of encephalitis and hemorrhagic diseases.
SUMMARY OF THE INVENTION
[0005] In certain aspects, the invention provides an immunoassay for detecting a first and a second antigen in a biological sample from a subject, the immunoassay comprising: a first and a second capture reagent, wherein the first and the second capture reagents are immobilized on a solid support; a first and a second detection reagent; and an at least one visualizing reagent.
[0006] In some embodiments, the first capture reagent comprises an anti-human IgG antibody. In some embodiments, the second capture reagent comprises an anti-human IgM antibody. In some embodiments, the first detection reagent is at least a portion of a first virus protein. In some embodiments, the first virus protein is coronavirus Spike protein. [0007] In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
[0008] In some embodiments, the second detection reagent is at least a portion of a second virus protein. In some embodiments, the second virus protein is coronavirus Nucleocapsid protein. In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63,
OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0009] In some embodiments, the first virus protein is flavivirus non-structural protein 1 (NS1). In some embodiments, the second virus protein is flavivirus precursor membrane (PrM) protein. In some embodiments, the first and second virus protein is from a flavivirus non-structural protein (NS1) strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4),
Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
[0010] In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
[0011] In some embodiments, the first antigen is a human IgG antibody capable of specifically recognizing at least one virus protein. In some embodiments, the at least one virus protein is coronavirus Spike protein. In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
[0012] In some embodiments, the at least one virus protein is coronavirus Nucleocapsid protein. In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0013] In some embodiments, the at least one virus protein is a flavivirus non- structural protein 1 (NS1). In some embodiments, the at least one virus protein is a flavivirus precursor membrane (PrM) protein. In some embodiments, (1) IgG antibodies recognizing only NS1 are present in the sample, indicating a primary viral infection of the subject; or (2) IgG antibodies to PrM only or IgG antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
[0014] In some embodiments, the flavivirus non- structural protein 1 and flavivirus precursor membrane (PrM) protein is from a flavivirus strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
[0015] In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, PrM 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
[0016] In some embodiments, the second antigen is a human IgM antibody capable of specifically recognizing at least one virus protein. In some embodiments, the at least one virus protein is coronavirus Spike protein. In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
[0017] In some embodiments, the at least one virus protein is coronavirus Nucleocapsid protein. In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0018] In some embodiments, the at least one virus protein is a flavivirus non- structural protein 1 (NS1). In some embodiments, the at least one virus protein is a flavivirus precursor membrane (PrM) protein. In some embodiments, (1) IgM antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgM antibodies to PrM only or IgM antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject. In some embodiments, the flavivirus non- structural protein 1 and flavivirus precursor membrane (PrM) protein is from a flavivirus strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2),
Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV). [0019] In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
[0020] In some embodiments, the first detection reagent comprises an anti-human IgG antibody. In some embodiments, the second detection reagent comprises an anti-human IgM antibody. In some embodiments, the first capture reagent is at least a portion of a first virus protein.
[0021] In some embodiments, the first virus protein is coronavirus Spike protein. In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
[0022] In some embodiments, the second capture reagent is at least a portion of a second virus protein. In some embodiments, the second virus protein is coronavirus Nucleocapsid protein. In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0023] In some embodiments, the first virus protein is a flavivirus non-structural protein 1 (NS1). In some embodiments, the second virus protein is a flavivirus precursor membrane (PrM) protein. In some embodiments, the flavivirus non-structural protein 1 and flavivirus precursor membrane (PrM) protein is from a flavivirus strain is selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
[0024] In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
[0025] In some embodiments, the at least one visualizing reagent is at least one nanoparticle with a structure and an emission spectrum. In some embodiments, the at least one nanoparticle is conjugated to the first and second detection reagents. In some embodiments, the visualizing reagent comprises: (1) at least one first nanoparticle with a first structure and a first emission spectrum; and (2) at least one second nanoparticle with a second structure and a second emission spectrum; wherein the first and second emission spectra are visually distinct. In some embodiments, the first nanoparticle is conjugated to the first detection reagent and the second nanoparticle is conjugated to the second detection reagent. [0026] In some embodiments, the subject is suspected of having or is diagnosed with a viral infection. In some embodiments, the viral infection is a coronavirus (CoV) infection. In some embodiments, the coronavirus (CoV) is selected from the coronavirus (CoV) strains consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2.
[0027] In some embodiments, the viral infection is a flavivirus infection. In some embodiments, the flavivirus strain is selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
[0028] In some embodiments, the subject is suspected of having or is diagnosed with a coronavirus (CoV) disease. In some embodiments, the coronavirus disease is COVID-19. In some embodiments, the subject is suspected of having or is diagnosed with asymptomatic COVID-19. In some embodiments, the subject is suspected of having or is diagnosed with symptomatic COVID-19.
[0029] In some embodiments, the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof.
[0030] In some embodiments, the at least one visualizing reagent comprises a colorimetric readout.
[0031] In some embodiments, the at least one visualizing reagent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
[0032] In certain aspects, the invention provides a lateral flow immunoassay device for detecting a first and a second antigen in a biological sample from a subject, the device comprising: an elongated holder having at least one aperture for sample application and at least one aperture for readout viewing; a first and a second immunoassay test strips secured within the holder, wherein the first and second test strips are parallel to each other along a long edge; a first capture reagent embedded in a first test area, wherein the first test area comprises at least a portion of the first test strip; a second capture reagent embedded in a second test area, wherein the second area comprises at least a portion of the second test strip; a first detection reagent embedded in at least a portion of a first conjugate pad, wherein the first conjugate pad comprises at least a portion of the first test strip and at least a portion of the second test strip; a second detection reagent embedded in at least a portion of a second conjugate pad, wherein the second conjugate pad comprises at least a portion of the first test strip and at least a portion of the second test strip; a first control reagent embedded in at least a portion of a first control area, wherein the first control area comprises at least a portion of the first test strip and at least a portion of the second test strip; a second control reagent embedded in at least a portion of a second control area, wherein the second control area comprises at least a portion of the first test strip and at least a portion of the second test strip; and an at least one visualization reagent conjugated to at least one detection reagent; wherein the test areas, the conjugate pads, and the control areas each comprises a different portion of the test strip from the others; and wherein: (1) if the first antigen is present in the sample the first test area and the first and second control areas produce a signal; (2) if the second antigen is present in the sample the second test area and the first and second control areas produce a signal; (3) if the first antigen is not present in the sample the first test area does not produce a signal the first and second control areas produce a signal; (4) if the second antigen is not present in the sample the second test area does not produce a signal and the first and second control areas produce a signal.
[0033] In some embodiments, the first capture reagent comprises an anti-human IgG antibody. In some embodiments, the second capture reagent comprises an anti-human IgM antibody. In some embodiments, the first detection reagent is at least a portion of a coronavirus Spike protein. In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
[0034] In some embodiments, the second detection reagent is at least a portion of a coronavirus Nucleocapsid protein. In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RFl, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0035] In some embodiments, the first antigen is a human IgG antibody capable of specifically recognizing at least one virus protein. In some embodiments, the at least one virus protein is coronavirus Spike protein. In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus ITKU4/ITK/04/2005 (Ty-BatCoV ITKU4/ITK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
[0036] In some embodiments, the at least one virus protein is coronavirus Nucleocapsid protein. In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0037] In some embodiments, the second antigen is a human IgM antibody capable of specifically recognizing at least one virus protein. In some embodiments, the at least one virus protein is a coronavirus Spike protein. In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RFl, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
[0038] In some embodiments, the at least one virus protein is coronavirus Nucleocapsid protein. In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/ITK/04/2005 (Ty-BatCoV ITKU4/ITK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0039] In some embodiments, the first detection reagent comprises an anti-human IgG antibody. In some embodiments, the second detection reagent comprises an anti-human IgM antibody. In some embodiments, the first capture reagent is at least a portion of a coronavirus Spike protein. In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
[0040] In some embodiments, the second capture reagent is at least a portion of coronavirus Nucleocapsid protein. In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RFl, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0041] In some embodiments, the at least one visualizing reagent is at least one nanoparticle with a structure and an emission spectrum. In some embodiments, the at least nanoparticle is conjugated to the first and second detection reagents. In some embodiments, the visualizing reagent comprises (1) an at least one first nanoparticle with a first structure and a first emission spectrum; and (2) an at least one second nanoparticle with a second structure and a second emission spectrum; wherein the first and second emission spectra are visually distinct. In some embodiments, the at least one first nanoparticle is conjugated to the first detection reagent and the at least one second nanoparticle is conjugated to the second detection reagent.
[0042] In some embodiments, the first control reagent comprises an anti-coronavirus Spike protein antibody. In some embodiments, the second control reagent comprises an anti- coronavirus Nucleocapsid protein antibody.
[0043] In some embodiments, the subject is suspected of having or is diagnosed with a viral infection. In some embodiments, the viral infection is a coronavirus (CoV) infection. In some embodiments, the coronavirus (CoV) is selected from the coronavirus (CoV) strains consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2.
[0044] In some embodiments, the subject is suspected of having or is diagnosed with a coronavirus (CoV) disease. In some embodiments, the coronavirus disease is COVID-19. In some embodiments, the subject is suspected of having or is diagnosed with asymptomatic COVID-19. In some embodiments, the subject is suspected of having or is diagnosed with symptomatic COVID-19.
[0045] In some embodiments, the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof. [0046] In some embodiments, the at least one visualizing reagent comprises a colorimetric readout. In some embodiments, the at least one visualizing agent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
[0047] In certain aspects, the invention provides a lateral flow immunoassay device for detecting a first and a second antigen in a biological sample from a subject, the device comprising: an elongated holder having at least one aperture for sample application and at least one aperture for readout viewing; a first and a second immunoassay test strip secured within the holder, wherein the first and second test strips are parallel to each other along a long edge; a first capture reagent embedded in a first test area, wherein the first area comprises at least a portion of the first test strip; a second capture reagent embedded in a second test area, wherein the second area comprises at least a portion of the second test strip; a first detection reagent embedded in at least a portion of a first conjugate pad, wherein the first conjugate pad comprises at least a portion of the first test strip and at least a portion of the second test strip; a second detection reagent embedded in at least a portion of a second conjugate pad, wherein the second conjugate pad comprises at least a portion of the first test strip and at least a portion of the second test strip; a first control reagent embedded in at least a portion of a first control area, wherein the first control area comprises at least a portion of the first test strip and at least a portion of the second test strip; a second control reagent embedded in at least a portion of a second control area, wherein the second control area comprises at least a portion of the first test strip and at least a portion of the second test strip; and an at least one visualization reagent conjugated to at least one detection reagent; wherein the test areas, the conjugate pads, and the control areas each comprises a different portion of the test strip from the others; and wherein: (1) if the first antigen is present in the sample the first test area and the first and second control areas produce a signal; (2) if the second antigen is present in the sample the second test area and the first and second control areas produce a signal; (3) if the first antigen is not present in the sample the first test area does not produce a signal and the first and second control areas produce a signal; (4) if the second antigen is not present in the sample the second test area does not produce a signal and the first and second control areas produce a signal. [0048] In some embodiments, the first capture reagent comprises an anti-human IgG antibody. In some embodiments, the second capture reagent comprises an anti-human IgM antibody. In some embodiments, the first detection reagent is at least a portion of a flavivirus non- structural protein 1 (NS1). In some embodiments, the second virus protein is at least a portion of a flavivirus precursor membrane (PrM) protein. In some embodiments, the flavivirus non- structural protein 1 and the flavivirus precursor membrane (PrM) protein is from a flavivirus strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
[0049] In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, PrM comprises is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
[0050] In some embodiments, the first antigen is a human IgG antibody capable of specifically recognizing at least one virus protein. In some embodiments, the at least one virus protein is a flavivirus non- structural protein 1 (NS1). In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, the at least one virus protein is a flavivirus precursor membrane (PrM) protein. In some embodiments, PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 99% identical to SEQ ID NO: 14. In some embodiments, (1) IgG antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgG antibodies to PrM only or IgG antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
[0051] In some embodiments, the second antigen is a human IgM antibody capable of specifically recognizing at least one virus protein. In some embodiments, the at least one virus protein is a flavivirus non- structural protein 1 (NS1). In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, the at least one virus protein is a flavivirus precursor membrane (PrM) protein. In some embodiments, PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
[0052] In some embodiments, (1) IgM antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgM antibodies to PrM only or IgM antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject. [0053] In some embodiments, the first detection reagent comprises an anti-human IgG antibody. In some embodiments, the second detection reagent comprises an anti-human IgM antibody. In some embodiments, the first capture reagent is at least a portion of a flavivirus non- structural protein 1 (NS1). In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, the second capture reagent is at least a portion of is a flavivirus precursor membrane (PrM) protein. In some embodiments, PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 99% identical to SEQ ID NO: 14.
[0054] In some embodiments, the at least one visualizing reagent is at least one nanoparticle with a structure and an emission spectrum. In some embodiments, the at least one nanoparticle is conjugated to the first and second detection reagents. In some embodiments, the visualizing reagent comprises (1) at least one first nanoparticle with a first structure and a first emission spectrum; and (2) at least one second nanoparticle with a second structure and a second emission spectrum; wherein the first and second emission spectra are visually distinct. In some embodiments, the first nanoparticle is conjugated to the first detection reagent and the second nanoparticle is conjugated to the second detection reagent. [0055] In some embodiments, the first control reagent comprises an anti -human immunoglobulin or an anti-human IgG and IgM. In some embodiments, the second control reagent comprises an anti-PrM antibody or an NS1 antibody.
In some embodiments, the subject is suspected of having or is diagnosed with a viral infection. In some embodiments, the viral infection is a flavivirus infection. In some embodiments, the flavivirus infection is selected from the strains consisting of Dengue virus serotype 1 (DVl), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
[0056] In some embodiments, the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof. In some embodiments, the at least one visualizing reagent comprises a colorimetric readout.
[0057] In some embodiments, the at least one visualizing agent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
[0058] In certain aspects, the invention provides a lateral flow immunoassay device for detecting IgG and IgM antibodies in a biological sample from a subject, the device comprising: an elongated holder having at least one aperture for sample application and at least one aperture for readout viewing; a first and a second immunoassay test strips secured within the holder, wherein the first and second test strips are parallel to each other along a long edge; a first capture reagent embedded in a first test area, wherein the first test area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the first capture reagent comprises anti-IgG antibodies; a second capture reagent embedded in a second test area, wherein the second area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the second capture reagent comprises anti-IgM antibodies; a first detection reagent embedded in at least a portion of a first conjugate pad, wherein the first conjugate pad comprises at least a portion of the first test strip and wherein the first detection reagent comprises at least a portion of a coronavirus Spike protein; a second detection reagent embedded in at least a portion of a second conjugate pad, wherein the second conjugate pad comprises at least a portion of the second test strip and wherein the second detection reagent comprises at least a portion of a coronavirus Nucleocapsid protein; a control reagent embedded in at least a portion of a control area, wherein the control area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the control reagent is an anti-Fc antibody; and an a visualization reagent conjugated to at least one detection reagent; wherein the test areas, the conjugate pads, and the control areas each comprises a different portion of the test strip from the others; and wherein: (1) if the IgG antibody is present in the sample, the first test area and the control area produce a signal; (2) if the IgM antibody is present in the sample, the second test area and the first and second control areas produce a signal; (3) if the IgG antibody is not present in the sample, the first test area does not produce a signal and control area produces a signal; (4) if the IgM antibody is not present in the sample, the second test area does not produce a signal and the control area produces a signal. [0059] In some embodiments, the first capture reagent comprises an anti-human IgG antibody. In some embodiments, the second capture reagent comprises an anti-human IgM antibody. In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
[0060] In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/ITK/04/2005 (Ty-BatCoV ITKU4/ITK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0061] In some embodiments, the IgG is a human IgG antibody capable of specifically recognizing at least one coronavirus protein. In some embodiments, the at least one coronavirus protein is coronavirus Spike protein. In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS- CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh- BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1. [0062] In some embodiments, the at least one coronavirus protein is coronavirus Nucleocapsid protein. In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RFl, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0063] In some embodiments, the IgM antibody is a human IgM antibody capable of specifically recognizing at least one virus protein. In some embodiments, the at least one virus protein is a coronavirus Spike protein. In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus ITKU4/ITK/04/2005 (Ty-BatCoV ITKU4/ITK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
[0064] In some embodiments, the at least one virus protein is coronavirus a Nucleocapsid protein. In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0065] In some embodiments, the visualization reagent comprises a species of nanoparticles with a structure and an emission spectrum. In some embodiments, the visualization reagent is conjugated to the first and second detection reagents. In some embodiments, the species of nanoparticles comprises 80 nm Gold NanoUrchins nanoparticles. In some embodiments, wherein the species of nanoparticles comprises 40 nm InnovaCoat® Gold nanoparticles. In some embodiments, the control reagent is an anti-human Fc antibody.
[0066] In some embodiments, the subject is suspected of having or is diagnosed with a coronavirus (CoV) infection. In some embodiments, the coronavirus (CoV) is selected from the coronavirus (CoV) strains consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2.
[0067] In some embodiments, the subject is suspected of having or is diagnosed with a coronavirus (CoV) disease. In some embodiments, the coronavirus disease is COVID-19. In some embodiments, the subject is suspected of having or is diagnosed with asymptomatic COVID-19. In some embodiments, the subject is suspected of having or is diagnosed with symptomatic COVID-19.
[0068] In some embodiments, the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof. In some embodiments, the visualization reagent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
[0069] In certain aspects, the invention provides a lateral flow immunoassay device for detecting IgG and IgM antibodies in a biological sample from a subject, the device comprising: an elongated holder having at least one aperture for sample application and at least one aperture for readout viewing; a first and a second immunoassay test strip secured within the holder, wherein the first and second test strips are parallel to each other along a long edge; a first capture reagent embedded in a first test area, wherein the first area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the first capture reagent comprises anti-IgG antibodies; a second capture reagent embedded in a second test area, wherein the second area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the second capture reagent comprises anti-IgM antibodies; a first detection reagent embedded in at least a portion of a first conjugate pad, wherein the first conjugate pad comprises at least a portion of the first test strip and wherein the first detection reagent comprises at least a portion of a flavivirus non- structural protein 1 (NS1) protein; a second detection reagent embedded in at least a portion of a second conjugate pad, wherein the second conjugate pad comprises at least a portion of the second test strip and wherein the second detection reagent comprises at least a portion of a flavivirus precursor membrane (PrM) protein; a control reagent embedded in at least a portion of a control area, wherein the control area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the control reagent is an anti-Fc antibody; and an a visualization reagent conjugated to at least one detection reagent; wherein the test areas, the conjugate pads, and the control areas each comprises a different portion of the test strip from the others; and wherein: (1) if the IgG antibody is present in the sample, the first test area and the control area produce a signal; (2) if the IgM antibody is present in the sample, the second test area and the control area produce a signal; (3) if the IgG antibody is not present in the sample the first test area does not produce a signal and the control area produces a signal; (4) if the IgM is not present in the sample, the second test area does not produce a signal and control areas produces a signal. [0070] In some embodiments, the first capture reagent comprises an anti-human IgG antibody. In some embodiments, the second capture reagent comprises an anti-human IgM antibody. In some embodiments, the flavivirus non- structural protein 1 and the flavivirus precursor membrane (PrM) protein is from a flavivirus strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
[0071] In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14. In some embodiments, the IgG antibody is a human IgG antibody capable of specifically recognizing at least one virus protein. In some embodiments, the at least one virus protein is a flavivirus non- structural protein 1 (NS1). In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, the at least one vims protein is a flavivirus precursor membrane (PrM) protein. In some embodiments, PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14. In some embodiments, (1) IgG antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgG antibodies to PrM only or IgG antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject. In some embodiments, the IgM antibody is a human IgM antibody capable of specifically recognizing at least one vims protein. In some embodiments, the at least one vims protein is a flavivirus non-stmctural protein 1 (NS1). In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, the at least one vims protein is a flavivirus precursor membrane (PrM) protein. In some embodiments, PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14. In some embodiments, (1) IgM antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgM antibodies to PrM only or IgM antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject. [0072] In some embodiments, the visualization reagent comprises a species of nanoparticles with a structure and an emission spectrum. In some embodiments, the visualization reagent is conjugated to the first and second detection reagents. In some embodiments, the species of nanoparticles comprises 80 nm Gold NanoUrchins nanoparticles. In some embodiments, the species of nanoparticles comprises 40 nm InnovaCoat® Gold nanoparticles. In some embodiments, the control reagent comprises an anti-human Fc antibody.
[0073] In some embodiments, the subject is suspected of having or is diagnosed with a flavivirus infection. In some embodiments, the flavivirus infection is selected from the strains consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
[0074] In some embodiments, the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof. In some embodiments, the at least one visualizing agent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
BRIEF DESCRIPTION OF FIGURES
[0075] The patent or application file contains at least one drawing originally in color. To conform to the requirements for PCT patent applications, many of the figures presented herein are black and white representations of images originally created in color.
[0076] FIG. 1 shows two-color technology to determine coronavirus convalescence (IgG) infection based on detection of Spike (S) and Nucleocapsid (N) in a coronavirus-infected subject.
[0077] FIG. 2 shows two-color technology to determine coronavirus acute (IgM) infection based on detection of Spike (S) and Nucleocapsid (N) in a coronavirus-infected subject. [0078] FIG. 3 shows two-color technology to determine coronavirus convalescence (IgG) infection based on detection of Spike (S) and Nucleocapsid (N) in a coronavirus-non-infected subject.
[0079] FIG. 4 shows two-color technology to determine coronavirus acute (IgM) infection based on detection of Spike (S) and Nucleocapsid (N) in a coronavirus-non-infected subject.
[0080] FIG. 5 shows single-color technology to determine coronavirus acute (IgM) and convalescence (IgG) infection based on detection of antibodies against Spike (S) protein in a coronavirus-infected subject.
[0081] FIG. 6 shows single-color technology to determine coronavirus acute (IgM) and convalescence (IgG) infection based on detection of antibodies against Nucleocapsid (N) in a coronavirus-infected subject.
[0082] FIG. 7 shows two-color technology to determine primary flavivirus infection based on IgG and IgM antibodies against NS1 and PrM proteins.
[0083] FIG. 8 shows two-color technology to determine secondary flavivirus infection based on IgG and IgM antibodies against NS1 and PrM proteins.
[0084] FIG. 9 shows single-color technology to determine primary flavivirus infection based on IgG and IgM antibodies against NS1 and PrM proteins.
[0085] FIG. 10 shows single-color technology to determine secondary flavivirus infection based on IgG and IgM antibodies against NS1 and PrM proteins.
DETAILED DESCRIPTION OF THE INVENTION [0086] Definitions
[0087] The following are definitions of terms used in the present specification. The initial definition provided for a group or term herein applies to that group or term throughout the present specification individually or as part of another group, unless otherwise indicated. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
[0088] The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” [0089] As used herein the term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).
[0090] As used herein, the term “subject” refers to a vertebrate animal. In one embodiment, the subject is a mammal or a mammalian species. In one embodiment, the subject is a human. In one embodiment, the subject is a healthy human adult. In other embodiments, the subject is a non-human vertebrate animal, including, without limitation, non-human primates, laboratory animals, livestock, racehorses, domesticated animals, and non-domesticated animals. In one embodiment, the term “human subjects” means a population of healthy human adults.
[0091] As used herein, the term “patient” refers to a human or animal.
[0092] The term “mammal” includes, but is not limited to, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, such as a monkey, chimpanzee, baboon or rhesus. In one embodiment, the mammal is a human.
[0093] Non-limiting Embodiments of the Subject Matter
[0094] In certain aspects, the invention provides an immunoassay for detecting a first and a second antigen in a biological sample from a subject, the immunoassay comprising: a first and a second capture reagent, wherein the first and the second capture reagents are immobilized on a solid support; a first and a second detection reagent; and an at least one visualizing reagent.
[0095] In some embodiments, the first capture reagent comprises an anti-human IgG antibody. In some embodiments, the second capture reagent comprises an anti-human IgM antibody. In some embodiments, the first detection reagent is at least a portion of a first virus protein. In some embodiments, the first virus protein is coronavirus Spike protein.
[0096] In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
[0097] In some embodiments, the second detection reagent is at least a portion of a second virus protein. In some embodiments, the second virus protein is coronavirus Nucleocapsid protein. In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RFl, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0098] In some embodiments, the first virus protein is flavivirus non-structural protein 1 (NS1). In some embodiments, the second virus protein is flavivirus precursor membrane (PrM) protein. In some embodiments, the first and second virus protein is from a flavivirus non-structural protein (NS1) strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4),
Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
[0099] In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
[0100] In some embodiments, the first antigen is a human IgG antibody capable of specifically recognizing at least one virus protein. In some embodiments, the at least one virus protein is coronavirus Spike protein. In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavims Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus ITKU4/ITK/04/2005 (Ty-BatCoV ITKU4/ITK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
[0101] In some embodiments, the at least one virus protein is coronavirus Nucleocapsid protein. In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0102] In some embodiments, the at least one virus protein is a flavivirus non- structural protein 1 (NS1). In some embodiments, the at least one virus protein is a flavivirus precursor membrane (PrM) protein. In some embodiments, (1) IgG antibodies recognizing only NS1 are present in the sample, indicating a primary viral infection of the subject; or (2) IgG antibodies to PrM only or IgG antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
[0103] In some embodiments, the flavivirus non- structural protein 1 and flavivirus precursor membrane (PrM) protein is from a flavivirus strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
[0104] In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14. [0105] In some embodiments, the second antigen is a human IgM antibody capable of specifically recognizing at least one virus protein. In some embodiments, the at least one virus protein is coronavirus Spike protein. In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus ITKU4/ITK/04/2005 (Ty-BatCoV ITKU4/ITK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
[0106] In some embodiments, the at least one virus protein is coronavirus Nucleocapsid protein. In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0107] In some embodiments, the at least one virus protein is a flavivirus non- structural protein 1 (NS1). In some embodiments, the at least one virus protein is a flavivirus precursor membrane (PrM) protein. In some embodiments, (1) IgM antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgM antibodies to PrM only or IgM antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject. In some embodiments, the flavivirus non- structural protein 1 and flavivirus precursor membrane (PrM) protein is from a flavivirus strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2),
Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
[0108] In some embodiments, the first detection reagent comprises an anti-human IgG antibody. In some embodiments, the second detection reagent comprises an anti-human IgM antibody. In some embodiments, the first capture reagent is at least a portion of a first virus protein. [0109] In some embodiments, the first virus protein is coronavirus Spike protein. In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
[0110] In some embodiments, the second capture reagent is at least a portion of a second virus protein. In some embodiments, the second virus protein is coronavirus Nucleocapsid protein. In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0111] In some embodiments, the first virus protein is a flavivirus non-structural protein 1 (NS1). In some embodiments, the second virus protein is a flavivirus precursor membrane (PrM) protein. In some embodiments, the flavivirus non-structural protein 1 and flavivirus precursor membrane (PrM) protein is from a flavivirus strain is selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
[0112] In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14. [0113] In some embodiments, the at least one visualizing reagent is at least one nanoparticle with a structure and an emission spectrum. In some embodiments, the at least one nanoparticle is conjugated to the first and second detection reagents. In some embodiments, the visualizing reagent comprises: (1) at least one first nanoparticle with a first structure and a first emission spectrum; and (2) at least one second nanoparticle with a second structure and a second emission spectrum; wherein the first and second emission spectra are visually distinct. In some embodiments, the first nanoparticle is conjugated to the first detection reagent and the second nanoparticle is conjugated to the second detection reagent. [0114] In some embodiments, the subject is suspected of having or is diagnosed with a viral infection. In some embodiments, the viral infection is a coronavirus (CoV) infection. In some embodiments, the coronavirus (CoV) is selected from the coronavirus (CoV) strains consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2.
[0115] In some embodiments, the viral infection is a flavivirus infection. In some embodiments, the flavivirus strain is selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
[0116] In some embodiments, the subject is suspected of having or is diagnosed with a coronavirus (CoV) disease. In some embodiments, the coronavirus disease is COVID-19. In some embodiments, the subject is suspected of having or is diagnosed with asymptomatic COVID-19. In some embodiments, the subject is suspected of having or is diagnosed with symptomatic COVID-19.
[0117] In some embodiments, the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof.
[0118] In some embodiments, the at least one visualizing reagent comprises a colorimetric readout.
[0119] In some embodiments, the at least one visualizing reagent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
[0120] In certain aspects, the invention provides a lateral flow immunoassay device for detecting a first and a second antigen in a biological sample from a subject, the device comprising: an elongated holder having at least one aperture for sample application and at least one aperture for readout viewing; a first and a second immunoassay test strips secured within the holder, wherein the first and second test strips are parallel to each other along a long edge; a first capture reagent embedded in a first test area, wherein the first test area comprises at least a portion of the first test strip; a second capture reagent embedded in a second test area, wherein the second area comprises at least a portion of the second test strip; a first detection reagent embedded in at least a portion of a first conjugate pad, wherein the first conjugate pad comprises at least a portion of the first test strip and at least a portion of the second test strip; a second detection reagent embedded in at least a portion of a second conjugate pad, wherein the second conjugate pad comprises at least a portion of the first test strip and at least a portion of the second test strip; a first control reagent embedded in at least a portion of a first control area, wherein the first control area comprises at least a portion of the first test strip and at least a portion of the second test strip; a second control reagent embedded in at least a portion of a second control area, wherein the second control area comprises at least a portion of the first test strip and at least a portion of the second test strip; and an at least one visualization reagent conjugated to at least one detection reagent; wherein the test areas, the conjugate pads, and the control areas each comprises a different portion of the test strip from the others; and wherein: (1) if the first antigen is present in the sample the first test area and the first and second control areas produce a signal; (2) if the second antigen is present in the sample the second test area and the first and second control areas produce a signal; (3) if the first antigen is not present in the sample the first test area does not produce a signal the first and second control areas produce a signal; (4) if the second antigen is not present in the sample the second test area does not produce a signal and the first and second control areas produce a signal.
[0121] In some embodiments, the first capture reagent comprises an anti-human IgG antibody. In some embodiments, the second capture reagent comprises an anti-human IgM antibody. In some embodiments, the first detection reagent is at least a portion of a coronavirus Spike protein. In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1. [0122] In some embodiments, the second detection reagent is at least a portion of a coronavirus Nucleocapsid protein. In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0123] In some embodiments, the first antigen is a human IgG antibody capable of specifically recognizing at least one virus protein. In some embodiments, the at least one virus protein is coronavirus Spike protein. In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
[0124] In some embodiments, the at least one virus protein is coronavirus Nucleocapsid protein. In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RFl, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0125] In some embodiments, the second antigen is a human IgM antibody capable of specifically recognizing at least one virus protein. In some embodiments, the at least one virus protein is a coronavirus Spike protein. In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
[0126] In some embodiments, the at least one virus protein is coronavirus Nucleocapsid protein. In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0127] In some embodiments, the first detection reagent comprises an anti-human IgG antibody. In some embodiments, the second detection reagent comprises an anti-human IgM antibody. In some embodiments, the first capture reagent is at least a portion of a coronavirus Spike protein. In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RFl, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
[0128] In some embodiments, the second capture reagent is at least a portion of coronavirus Nucleocapsid protein. In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005) In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0129] In some embodiments, the at least one visualizing reagent is at least one nanoparticle with a structure and an emission spectrum. In some embodiments, the at least nanoparticle is conjugated to the first and second detection reagents. In some embodiments, the visualizing reagent comprises (1) an at least one first nanoparticle with a first structure and a first emission spectrum; and (2) an at least one second nanoparticle with a second structure and a second emission spectrum; wherein the first and second emission spectra are visually distinct. In some embodiments, the at least one first nanoparticle is conjugated to the first detection reagent and the at least one second nanoparticle is conjugated to the second detection reagent.
[0130] In some embodiments, the first control reagent comprises an anti-coronavirus Spike protein antibody. In some embodiments, the second control reagent comprises an anti- coronavirus Nucleocapsid protein antibody.
[0131] In some embodiments, the subject is suspected of having or is diagnosed with a viral infection. In some embodiments, the viral infection is a coronavirus (CoV) infection. In some embodiments, the coronavirus (CoV) is selected from the coronavirus (CoV) strains consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV ITKU4/ITK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2.
[0132] In some embodiments, the subject is suspected of having or is diagnosed with a coronavirus (CoV) disease. In some embodiments, the coronavirus disease is COVID-19. In some embodiments, the subject is suspected of having or is diagnosed with asymptomatic COVID-19. In some embodiments, the subject is suspected of having or is diagnosed with symptomatic COVID-19.
[0133] In some embodiments, the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof.
[0134] In some embodiments, the at least one visualizing reagent comprises a colorimetric readout. In some embodiments, the at least one visualizing agent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation. [0135] In certain aspects, the invention provides a lateral flow immunoassay device for detecting a first and a second antigen in a biological sample from a subject, the device comprising: an elongated holder having at least one aperture for sample application and at least one aperture for readout viewing; a first and a second immunoassay test strip secured within the holder, wherein the first and second test strips are parallel to each other along a long edge; a first capture reagent embedded in a first test area, wherein the first area comprises at least a portion of the first test strip; a second capture reagent embedded in a second test area, wherein the second area comprises at least a portion of the second test strip; a first detection reagent embedded in at least a portion of a first conjugate pad, wherein the first conjugate pad comprises at least a portion of the first test strip and at least a portion of the second test strip; a second detection reagent embedded in at least a portion of a second conjugate pad, wherein the second conjugate pad comprises at least a portion of the first test strip and at least a portion of the second test strip; a first control reagent embedded in at least a portion of a first control area, wherein the first control area comprises at least a portion of the first test strip and at least a portion of the second test strip; a second control reagent embedded in at least a portion of a second control area, wherein the second control area comprises at least a portion of the first test strip and at least a portion of the second test strip; and an at least one visualization reagent conjugated to at least one detection reagent; wherein the test areas, the conjugate pads, and the control areas each comprises a different portion of the test strip from the others; and wherein: (1) if the first antigen is present in the sample the first test area and the first and second control areas produce a signal; (2) if the second antigen is present in the sample the second test area and the first and second control areas produce a signal; (3) if the first antigen is not present in the sample the first test area does not produce a signal and the first and second control areas produce a signal; (4) if the second antigen is not present in the sample the second test area does not produce a signal and the first and second control areas produce a signal.
[0136] In some embodiments, the first capture reagent comprises an anti-human IgG antibody. In some embodiments, the second capture reagent comprises an anti-human IgM antibody. In some embodiments, the first detection reagent is at least a portion of a flavivirus non- structural protein 1 (NS1). In some embodiments, the second virus protein is at least a portion of a flavivirus precursor membrane (PrM) protein. In some embodiments, the flavivirus non- structural protein 1 and the flavivirus precursor membrane (PrM) protein is from a flavivirus strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
[0137] In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
[0138] In some embodiments, the first antigen is a human IgG antibody capable of specifically recognizing at least one virus protein. In some embodiments, the at least one virus protein is a flavivirus non- structural protein 1 (NS1). In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, the at least one virus protein is a flavivirus precursor membrane (PrM) protein. In some embodiments, PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 99% identical to SEQ ID NO: 14. In some embodiments, (1) IgG antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgG antibodies to PrM only or IgG antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
[0139] In some embodiments, the second antigen is a human IgM antibody capable of specifically recognizing at least one virus protein. In some embodiments, the at least one virus protein is a flavivirus non- structural protein 1 (NS1). In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, the at least one virus protein is a flavivirus precursor membrane (PrM) protein. In some embodiments, PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 99% identical to SEQ ID NO: 14.
[0140] In some embodiments, (1) IgM antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgM antibodies to PrM only or IgM antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
[0141] In some embodiments, the first detection reagent comprises an anti-human IgG antibody. In some embodiments, the second detection reagent comprises an anti-human IgM antibody. In some embodiments, the first capture reagent is at least a portion of a flavivirus non- structural protein 1 (NS1). In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, the second capture reagent is at least a portion of is a flavivirus precursor membrane (PrM) protein. In some embodiments, PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 99% identical to SEQ ID NO: 14.
[0142] In some embodiments, the at least one visualizing reagent is at least one nanoparticle with a structure and an emission spectrum. In some embodiments, the at least one nanoparticle is conjugated to the first and second detection reagents. In some embodiments, the visualizing reagent comprises (1) at least one first nanoparticle with a first structure and a first emission spectrum; and (2) at least one second nanoparticle with a second structure and a second emission spectrum; wherein the first and second emission spectra are visually distinct. In some embodiments, the first nanoparticle is conjugated to the first detection reagent and the second nanoparticle is conjugated to the second detection reagent. [0143] In some embodiments, the first control reagent comprises an anti -human immunoglobulin or an anti-human IgG and IgM. In some embodiments, the second control reagent comprises an anti-PrM antibody or an NS1 antibody.
In some embodiments, the subject is suspected of having or is diagnosed with a viral infection. In some embodiments, the viral infection is a flavivirus infection. In some embodiments, the flavivirus infection is selected from the strains consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
[0144] In some embodiments, the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof. In some embodiments, the at least one visualizing reagent comprises a colorimetric readout.
[0145] In some embodiments, the at least one visualizing agent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
[0146] In certain aspects, the invention provides a lateral flow immunoassay device for detecting IgG and IgM antibodies in a biological sample from a subject, the device comprising: an elongated holder having at least one aperture for sample application and at least one aperture for readout viewing; a first and a second immunoassay test strips secured within the holder, wherein the first and second test strips are parallel to each other along a long edge; a first capture reagent embedded in a first test area, wherein the first test area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the first capture reagent comprises anti-IgG antibodies; a second capture reagent embedded in a second test area, wherein the second area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the second capture reagent comprises anti-IgM antibodies; a first detection reagent embedded in at least a portion of a first conjugate pad, wherein the first conjugate pad comprises at least a portion of the first test strip and wherein the first detection reagent comprises at least a portion of a coronavirus Spike protein; a second detection reagent embedded in at least a portion of a second conjugate pad, wherein the second conjugate pad comprises at least a portion of the second test strip and wherein the second detection reagent comprises at least a portion of a coronavirus Nucleocapsid protein; a control reagent embedded in at least a portion of a control area, wherein the control area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the control reagent is an anti-Fc antibody; and an a visualization reagent conjugated to at least one detection reagent; wherein the test areas, the conjugate pads, and the control areas each comprises a different portion of the test strip from the others; and wherein: (1) if the IgG antibody is present in the sample, the first test area and the control area produce a signal; (2) if the IgM antibody is present in the sample, the second test area and the first and second control areas produce a signal; (3) if the IgG antibody is not present in the sample, the first test area does not produce a signal and control area produces a signal; (4) if the IgM antibody is not present in the sample, the second test area does not produce a signal and the control area produces a signal. [0147] In some embodiments, the first capture reagent comprises an anti-human IgG antibody. In some embodiments, the second capture reagent comprises an anti-human IgM antibody. In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
[0148] In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0149] In some embodiments, the IgG is a human IgG antibody capable of specifically recognizing at least one coronavirus protein. In some embodiments, the at least one coronavirus protein is coronavirus Spike protein. In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS- CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RFl, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh- BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1. [0150] In some embodiments, the at least one coronavirus protein is coronavirus Nucleocapsid protein. In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0151] In some embodiments, the IgM antibody is a human IgM antibody capable of specifically recognizing at least one virus protein. In some embodiments, the at least one virus protein is a coronavirus Spike protein. In some embodiments, the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
[0152] In some embodiments, the at least one virus protein is coronavirus a Nucleocapsid protein. In some embodiments, the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi- BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome- related coronavirus, SARS-related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RFl, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS- related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS- related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2. In some embodiments, the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
[0153] In some embodiments, the visualization reagent comprises a species of nanoparticles with a structure and an emission spectrum. In some embodiments, the visualization reagent is conjugated to the first and second detection reagents. In some embodiments, the species of nanoparticles comprises 80 nm Gold NanoUrchins nanoparticles. In some embodiments, wherein the species of nanoparticles comprises 40 nm InnovaCoat® Gold nanoparticles. In some embodiments, the control reagent is an anti-human Fc antibody.
[0154] In some embodiments, the subject is suspected of having or is diagnosed with a coronavirus (CoV) infection. In some embodiments, the coronavirus (CoV) is selected from the coronavirus (CoV) strains consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS-related human coronavirus, SARS- related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rf 1/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rm 1/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh-BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS-related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005). In some embodiments, the coronavirus strain is SARS-CoV-2.
[0155] In some embodiments, the subject is suspected of having or is diagnosed with a coronavirus (CoV) disease. In some embodiments, the coronavirus disease is COVID-19. In some embodiments, the subject is suspected of having or is diagnosed with asymptomatic COVID-19. In some embodiments, the subject is suspected of having or is diagnosed with symptomatic COVID-19.
[0156] In some embodiments, the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof. In some embodiments, the visualization reagent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
[0157] In certain aspects, the invention provides a lateral flow immunoassay device for detecting IgG and IgM antibodies in a biological sample from a subject, the device comprising: an elongated holder having at least one aperture for sample application and at least one aperture for readout viewing; a first and a second immunoassay test strip secured within the holder, wherein the first and second test strips are parallel to each other along a long edge; a first capture reagent embedded in a first test area, wherein the first area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the first capture reagent comprises anti-IgG antibodies; a second capture reagent embedded in a second test area, wherein the second area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the second capture reagent comprises anti-IgM antibodies; a first detection reagent embedded in at least a portion of a first conjugate pad, wherein the first conjugate pad comprises at least a portion of the first test strip and wherein the first detection reagent comprises at least a portion of a flavivirus non- structural protein 1 (NS1) protein; a second detection reagent embedded in at least a portion of a second conjugate pad, wherein the second conjugate pad comprises at least a portion of the second test strip and wherein the second detection reagent comprises at least a portion of a flavivirus precursor membrane (PrM) protein; a control reagent embedded in at least a portion of a control area, wherein the control area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the control reagent is an anti-Fc antibody; and an a visualization reagent conjugated to at least one detection reagent; wherein the test areas, the conjugate pads, and the control areas each comprises a different portion of the test strip from the others; and wherein: (1) if the IgG antibody is present in the sample, the first test area and the control area produce a signal; (2) if the IgM antibody is present in the sample, the second test area and the control area produce a signal; (3) if the IgG antibody is not present in the sample the first test area does not produce a signal and the control area produces a signal; (4) if the IgM is not present in the sample, the second test area does not produce a signal and control areas produces a signal. [0158] In some embodiments, the first capture reagent comprises an anti-human IgG antibody. In some embodiments, the second capture reagent comprises an anti-human IgM antibody. In some embodiments, the flavivirus non- structural protein 1 and the flavivirus precursor membrane (PrM) protein is from a flavivirus strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
[0159] In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14. In some embodiments, the IgG antibody is a human IgG antibody capable of specifically recognizing at least one virus protein. In some embodiments, the at least one virus protein is a flavivirus non- structural protein 1 (NS1). In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, the at least one virus protein is a flavivirus precursor membrane (PrM) protein. In some embodiments, PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14. In some embodiments, (1) IgG antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgG antibodies to PrM only or IgG antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject. In some embodiments, the IgM antibody is a human IgM antibody capable of specifically recognizing at least one virus protein. In some embodiments, the at least one virus protein is a flavivirus non- structural protein 1 (NS1). In some embodiments, NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8. In some embodiments, the at least one virus protein is a flavivirus precursor membrane (PrM) protein. In some embodiments, PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14. In some embodiments, (1) IgM antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgM antibodies to PrM only or IgM antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
[0160] In some embodiments, the visualization reagent comprises a species of nanoparticles with a structure and an emission spectrum. In some embodiments, the visualization reagent is conjugated to the first and second detection reagents. In some embodiments, the species of nanoparticles comprises 80 nm Gold NanoUrchins nanoparticles. In some embodiments, the species of nanoparticles comprises 40 nm InnovaCoat® Gold nanoparticles. In some embodiments, the control reagent comprises an anti-human Fc antibody. [0161] In some embodiments, the subject is suspected of having or is diagnosed with a flavivirus infection. In some embodiments, the flavivirus infection is selected from the strains consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
[0162] In some embodiments, the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof. In some embodiments, the at least one visualizing agent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
[0163] Lateral Flow Immunoassay
[0164] Lateral flow immunoassays are a cost-effective methodology for detecting a target protein in a biological sample. Tests based on this methodology can be used at a point-of- care center by medical professionals or for at home testing by individuals without any medical or technical training. The technology takes advantage of the capillary properties of materials such as nitrocellulose, which have the capacity to transport fluids spontaneously. Liquids display capillary action, which is their ability to flow through narrow spaces even in opposition to external forces. Capillary action can be observed in the drawing up of liquids into materials such as fibers or cellulose. It occurs due to intermolecular forces between the liquid and surrounding solid surfaces. If the diameter of the space is sufficiently small, then the combination of surface tension of the liquid and the adhesive forces between the liquid and the surrounding solid surface act to propel the liquid into the narrow space.
[0165] There are two main types of lateral flow immunoassays: a “sandwich assay” and a “competitive assay”. The subject matter disclosed herein relates to “sandwich assays.” In a “sandwich assay”, a migrating sample can first come into contact with a detection reagent, which can specifically bind to a target protein or viral antigen. In some embodiments, the detection reagent is conjugated to a visualizing reagent. If the target protein is present in the sample, the conjugated detection reagent can bind to it and subsequently flow laterally to a test line (area). The test line (area) may also contain a capture reagent such as proteins or antibodies specific to the target protein. A visual signal or readout can be produced when the target protein reaches the test line (area). Thus, the target protein is “sandwiched” between two proteins such as antibodies.
[0166] If the detection reagent is not directly labeled and both the capture reagent and the detection reagent are antibodies, then these antibodies must be from different species (i.e., if the capture antibody is a rabbit antibody, the detection antibody can be from goat, chicken, etc., but not rabbit). This will allow the use of a secondary antibody directed to the species heavy chain of the detection antibody. If the detection antibody is directly labeled, then the capture and detection antibodies can be from the same species.
[0167] Usually the main determination of an immunoassay sensitivity is the protein or antibody binding affinity for the target antigen. As the target antigen concentration increases, the amount of detection reagent accumulated in the test line (area) increases, leading to a higher measured response or readout.
[0168] Rapid Detection Devices
[0169] Lateral flow immunoassays are incorporated in low-cost, simple, rapid and portable detection devices. These devices are a paper-based platform for the detection and quantification of particular analytes in mixtures, where the sample is placed on a test device and the results are displayed within 2-30 min. A variety of biological samples can be tested using these devices including but not limited to serum, plasma, whole blood, urine, saliva, sweat, and other fluids.
The device can include a lateral flow test strip. In one embodiment the test strip is paper- based. In one embodiment the test strip consists of overlapping membranes that can be mounted on a backing card for enhanced stability and handling. The sample can be applied at one end of the strip, on the adsorbent sample pad, which can be impregnated with buffer salts and/or surfactants suitable for interaction with the detection reagent. The sample can then migrate laterally through the pad. In one embodiment, the pad contains antibodies that are specific to the target analyte. In one embodiment at least one antibody or protein conjugated to colored or fluorescent particles such as colloidal gold or latex microspheres. The sample, together with the conjugated antibody or protein bound to the target analyte, can laterally migrate along the strip into the detection zone. In one embodiment, the detection zone is a porous membrane pre-loaded with specific components. In one embodiment, the detection zone is composed of nitrocellulose. In one embodiment the specific components are antibodies, antigens, or proteins. In one embodiment, the specific components are immobilized in lines. In some embodiment, the specific components react with the analyte bound to the conjugated antibody.
[0170] The presence of the target analyte can result in an observable response on the test line. A response on the control line indicates that proper liquid flow through the text strip took place. In some embodiments the read-out, represented by the lines can appear in different colors or with different intensities. In some embodiment, multiple analytes can be tested simultaneously and under the same conditions. In some embodiment, additional test lines of antibodies specific to different analytes can be immobilized on the test strip. In some embodiments, multiple test lines pre-loaded with the same antibody can be utilized for semi- quantitative analysis of the target analyte. To maintain movement of the sample across the test strip, an absorbent pad is attached at one end of the strip. In some embodiments, the absorbent pad is on the opposite end of the test strip compared to the sample pad where the sample is initially applied. In some embodiment, the absorbent pad wicks the excess reagents to prevent backflow of the liquid across the test strip.
[0171] In some embodiments, the label incorporated in the detection method is colloidal gold. In some embodiments, gold particles are red in color due to localized surface plasmon resonance. In some embodiments, the label incorporated in the detection method latex, which can be tagged with a variety of detector reagents such as colored or fluorescent dyes, and magnetic or paramagnetic components. In some embodiments, latex is blue. In some embodiments, the latex can be produced in multiple colors discriminating between numerous lines. In some embodiments, the label incorporated in the detection method is carbon. In some embodiments, the label incorporated in the detection method are fluorescent labels. In some embodiments, the label incorporated in the detection method comprises enzymatic modifications of the labels. In some embodiments, the label incorporated in the detection method is carbon nanotubes. In some embodiments, the label incorporated in the detection method is fluorescent nanoparticles such as quantum dots.
[0172] In some embodiments, the conjugate pad holds the detector particles and ensures they are functionally stable. In some embodiments, the conjugate pad includes buffers. In some embodiments, the buffers contain carbohydrates to preserve and resolubilize conjugated particles.
[0173] Antibodies and Antibody Isotypes
[0174] Antibodies are glycoproteins capable of binding specific antigens. They are produced by a subject’s immune system in response to a foreign antigen invasion in the body. Antibodies exist as one or more copies of a Y-shaped unit, composed of four polypeptide chains. Each antibody contains two identical copies of a heavy chain, and two identical copies of a light chain. The heavy and light chains differ in their polypeptide sequence and length. The variable region binds tightly and specifically to an epitope on the antigen. The light chains of an antibody can be classified as either kappa (K) or lambda (l) type based on differences in polypeptide sequence.
[0175] Antibodies are classified into five isotypes (IgM, IgD, IgG, IgA, and IgE) according to their heavy chains, which provide each isotype with distinct characteristics and roles in the immune response. IgG antibodies are the most abundant antibody isotype in the blood (plasma). They account for 70-75% of human immunoglobulins. IgG antibodies counteract harmful antigens and are important in the recognition of antigen-antibody complexes by leukocytes and macrophages. IgM antibodies circulate in the blood and account for about 10% of human immunoglobulins. IgM antibodies have a characteristic pentameric structure in which five basic Y-shaped molecules are linked together. B cells produce IgM first in response to microbial infection/antigen invasion. Although IgM antibodies have a lower affinity for antigens than IgG antibodies, IgM have higher avidity for antigens because of the pentameric/hexameric structure. IgM are also capable of activating particular cell signaling pathways by binding to the cell surface receptors. IgA antibodies are abundant in serum, nasal mucus, saliva, breast milk, and intestinal fluid. They account for 10- 15% of human immunoglobulins. IgA antibodies form dimers (i.e., two IgA monomers joined together). IgE antibodies are generally present in minute amounts, accounting for no more than 0.001% of human immunoglobulins. Their original role is to protect against parasites but can be involve in allergic responses. IgD antibodies account for less than 1% of human immunoglobulins. They can be involved in the induction of antibody production in B cells, however, their exact function is still unknown.
[0176] Antibody heavy chains differ in size and composition and can be approximately 450 amino acids to approximately 550 amino acids. Each heavy chain includes two regions, the constant region and the variable region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. The variable region of the heavy chain differs depending on the B cell that produced it, but is the same for all antibodies produced by a single B cell or B cell clone. The variable region of each heavy chain is approximately 110 amino acids long and is composed of a single Ig domain. [0177] Mammals have only two types of light chains, l and K. A light chain has two successive domains: one constant domain and one variable domain. The approximate length of a light chain is 211-217 amino acids. Each antibody contains two light chains that are always identical.
[0178] An antibody can be divided into three sections: two F(ab) regions and an Fc region. The F(ab) regions contain the variable domain that binds to antigens. The Fc fragment provides a binding site for endogenous Fc receptors on the surface of lymphocytes. It is also the site of binding for secondary antibodies. For research and testing purposes, dyes and enzymes can be covalently linked to antibodies on the Fc portion of the antibody to provide visualization. Nanoparticles can also be conjugated to the Fc position of antibodies.
[0179] Virus Families
[0180] Viruses are small parasites that cannot reproduce on their own. Once a virus infects a cell, however, a virus can direct the cell machinery to produce more viruses. Most viruses utilize either RNA or DNA as their genetic material. The nucleic acid may be single- or double-stranded. The infectious virus particle or a virion includes a nucleic acid and an outer shell of protein. The simplest viruses contain only enough RNA or DNA to encode four proteins. On the other hand, the most complex ones can encode up to 200 proteins.
[0181] The nucleic acid of a virion is enclosed within a protein coat called a capsid. The capsid id composed of multiple copies of one protein or a few different proteins, each of which is encoded by a single viral gene. Because of this structure, a virus is able to encode all the information for making a relatively large capsid in a small number of genes.
[0182] Coronaviruses (CoVs) are a large family of enveloped, positive-sense, single- stranded RNA viruses that infect a broad range of vertebrates. They are extensive in bats but can be found in many other birds and mammals including humans. In humans, CoVs tend to cause mild to moderate upper respiratory tract infections such as the common cold.
However, in the past couple of decades, there have been outbreaks of severe, and sometimes fatal, respiratory illnesses that were later found to be caused by novel, human pathogenic CoVs. These strains exhibited stronger virulence and quickly passed from human to human. While infection with these CoVs typically produced mild symptoms, for certain individuals, responses were more severe. In extreme cases, death occurred due to gradual respiratory failure as the result of alveolar damage. The seven strain of CoVs found in humans include SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, and HKU1. [0183] The coronavirus entry into a cell is mediated by the viral Spike (S) glycoprotein. The Spike protein is a large type I transmembrane protein ranging from 1,160 amino acids to up to 1,400 amino acids. Spike proteins are highly glycosylated with 21 to 35 N- glycosylation sites. Spike proteins assemble into trimers on the virion surface to form the distinctive "corona", or crown-like appearance, of a coronavirus. The ectodomain of all CoV Spike proteins is organized into an N-terminal domain named SI, responsible for receptor binding, and a C-terminal S2 domain, responsible for viral fusion. Coronaviruses can infect the respiratory epithelial cells through interaction with the ACE2 receptor on the cell surface. Recombinant Spike protein can bind with recombinant ACE2 proteins.
[0184] The coronavirus Nucleocapsid (N) is a structural protein capable of forming complexes with genomic RNA. Nucleocapsid proteins also interact with the viral membrane protein(s) during virion assembly and play a critical role in enhancing the efficiency of virus transcription and assembly.
[0185] Aedes mosquitoes transmit globally relevant flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV). DENV exists as four antigenic serotypes, DENV1 to DENV4. These viruses have a wide geographic distribution, with approximately 390 million infections annually and more than a quarter of the world’s population at risk.
Prior to 2015, ZIKV was considered obscure and was known to circulate in Africa and Southeast Asia as two separate viral lineages, African and Asian. While most are asymptomatic, the clinical presentation of ZIKV infection resembles that of dengue, including fever, rash, conjunctivitis, arthralgia, and myalgia.
[0186] DENV contains a positive-sense, single-stranded RNA genome of about 10.6 kilobases. The single open reading frame of the DENV encodes a polyprotein precursor, which is cleaved by cellular and viral protease into three structural proteins, the capsid (C), precursor membrane (PrM), and envelope (E), as well as seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. The E protein, a glycoprotein of approximately 55 kDa, contains 12 strictly conserved cysteine residues forming six disulfide bridges and is present as a heterodimer with PrM protein before the maturation of the virion. E protein participates in virus entry and is the major target of both neutralizing and enhancing antibodies.
[0187] The PrM protein is a glycoprotein of about 19 kDa. It contains six highly conserved cysteine residues forming three disulfide bridges. The NS1 protein is also a glycoprotein of about 40 kDa. It contains 12 highly conserved cysteine residues forming six disulfide bridges. NS1 is present intracellularly, on the cell surface, and outside of the cells.
[0188] Primary and Secondary Viral Infections
[0189] A primary infection is the first time a subject is exposed to an antigen. During a primary infection, the body has no innate defenses, such as antibodies, against the invading virus. A secondary infection is an infection that occurs during or after treatment for a previous infection. The secondary infection may be caused by the first treatment, by changes in the immune system, increased susceptibility due to the primary infection, or it may be unrelated. In some embodiments, the primary and the secondary infection are caused by different viruses. In some embodiments, the primary and the secondary infection are caused by different viruses from the same family. In some embodiments, the primary and the secondary infection are caused by the same virus strain. Whether the subject presents with a primary or a secondary viral infection and whether the antigens are the same or even related greatly impacts the subject’s immune response to the antigens and, thus, the clinical outcome of the infection. A primary immune response occurs when a subject’s immune system encounters an antigen for the first time. At this time the immune system has to learn to recognize the antigen and produce antibodies against it.
A secondary immune response occurs when a subject’s immune system encounters the same antigen for the second time. At this point it is expected that immunological memory has been established and the immune system can start making antibodies immediately. IgG antibodies are primarily involved in the secondary immune response, while IgM antibodies are the main antibodies involved in the primary response.
[0190] In the field of flaviviruses, there is great interest in elucidating specific and cross reactive immune (T cell) responses to Zika and Dengue virus infections. Both the Zika and Dengue viruses belong to the same virus family, flaviviruses. T cell responses to Asian ZIKV infection are important for immune protection, and that previous Dengue virus (DENV) exposure amplifies the protective T cell response to Asian ZIKV. In some embodiments, the subject matter disclosed herein relates to determining if a viral infection is primary or secondary infection. In some embodiments, the subject matter disclosed herein relates to determining if a viral infection is primary or secondary infection based on antibodies raised to PrM and NS1 proteins. In some embodiments, a primary flavivirus infection leads to IgG and/or IgM antibodies raised only to the NS1 protein. In some embodiments, the rates of antibody responses to PrM and NS1 proteins are higher in patients with secondary virus infection than in those with primary infection.
[0191] Gold Nanoparticles
[0192] Gold nanoparticles have many applications in biology and medicine due to their unique optical and physical properties. They can be used for biosensor development, as cellular probes, as drug delivery vehicles, or as optical contrast agents among others. The distinct optical and physical properties of gold nanoparticles are dependent upon their size (diameter), shape, surface structure and agglomeration state.
[0193] Sequences
[0194] SEQ ID NO: 1 is the polypeptide sequence of the coronavirus SARS-CoV-2 Spike protein per gene bank accession NC 045512 and including polymorphisms already reported MT077125 and ancestor QHR63300 RaTG13.
MF VFLVLLPL V S SQC VNLTTRT QLPP AYTN SFTRGVYYPDKVFRS S VLHSTQDLFLPF F SN VTWFH AIH V S GTN GTKRFDNP VLPFNDGVYF AS TEK SNURGWIF GTTLD SKTQ S LLIVNNATNVVIKVCEF QFCNDPFLGVYYHKNNKSWMESEFRVY S S ANNCTFEYV S QPFLMDLEGKQGNFKNLREF VFKNIDGYFKIY SKHTPINL VRDLPQGF S ALEPL VDLP IGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDA VDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRF ASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRG DE VRQI APGQTGKI AD YNYKLPDDF T GC VI AWN SNNLD SK V GGNYNYL YRLFRK SN LKPFERDI S TEI Y Q AGS TPCN GVEGFN C YFPLQ S Y GF QPTN GV GY QP YRV VVL SFELL HAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTD AVRDPQTLEILDITPC SF GGV S VITPGTNTSNQ VAVL Y QDVNCTEVPVAIHADQLTPT WRV Y STGSNVF QTRAGCLIGAEHVNN S YECDIPIGAGIC AS Y QTQTN SPRRARS VAS QSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTEC SNLLLQ Y GSF C T QLNRALTGI A VEQDKNT QE VF AQ VKQI YKTPPIKDF GGFNF S QILP DPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLT DEMI AQYT S ALL AGTIT S GWTF GAGA ALQIPF AMQM A YRFN GIGVT QNVL YEN QKLI ANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDIL SRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSK RVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGV FVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEEL DKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYI KWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKG
VKLHYT
[0195] SEQ ID NO: 2 is the polypeptide sequence of the coronavirus Nucleocapsid protein (NCBI Reference Sequence: YP_009724397.2)
MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTAL
TQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYY
LGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPK
GF YAEGSRGGSQ AS SRS S SRSRNS SRNSTPGS SRGT SP ARM AGN GGD A AL ALLLLDR
LNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQ
TQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIK
LDDKDPNFKDQV
ILLNKHID AYKTFPPTEPKKDKKKK ADET Q ALPQRQKKQQT VTLLP AADLDDF SKQL QQSMSSADSTQA
[0196] SEQ ID NO: 3 is the polypeptide sequence of the Dengue virus 1 NS1 protein (NCBI Reference Sequence: NP_722461.1)
DSGCVINWKGRELKCGSGIFVTNEVHTWTEQYKFQADSPKRLSAAIGKAWEEGVCG IRSATRLENIMWKQISNELNHILLENDMKFTVVVGDVSGILAQGKKMIRPQPMEHKY SWKSWGKAKIIGADVQNTTFIIDGPNTPECPDNQRAWNIWEVEDYGFGIFTTNIWLK LRDSYTQVCDHRLMSAAIKDSKAVHADMGYWIESEKNETWKLARASFIEVKTCIWP KSHTLWSNGVLESEMIIPKIYGGPISQHNYRPGYFTQTAGPWHLGKLELDFDLCEGTT VVVDEHCGNRGPSLRTTTVTGKTIHEWCCRSCTLPPLRFKGEDGCWYGMEIRPVKE KEENLVKSMVSADSGCVINWKGRELKCGSGIFVTNEVHTWTEQYKFQADSPKRLSA AIGK AWEEGV C GIRS ATRLENIMWKQI SNELNHILLENDMKF T V V V GD V S GIL AQGK KMIRPQPMEHKYSWKSWGKAKIIGADVQNTTFIIDGPNTPECPDNQRAWNIWEVED Y GF GIFTTNIWLKLRD S YTQ VCDHRLMS A AIKD SK AVHADMGYWIESEKNETWKL A RASFIEVKT CIWPK SHTLW SN GVLE SEMIIPKI Y GGPI S QHN YRPGYF TQT AGPWHLG KLELDFDLCEGTT V VVDEHC GNRGP SLRTTT VT GKTIHEW C CRS C TLPPLRFKGEDG CWY GMEIRPVKEKEENLVKSMV S A
[0197] SEQ ID NO: 4 is the polypeptide sequence of the Dengue virus 2 NS1 protein (NCBI Reference Sequence: NP_739584.2)
D S GC V V S WKNKELKC GS GIFITDN VHTWTEQ YKF QPE SP SKL AS AIQK AHEEGICGIR SVTRLENLMWKQITPELNHILSENEVKLTIMTGDIKGIMQAGKRSLRPQPTELKYSW KTW GKAKML STESHNQTFLIDGPET AECPNTNRAWN SLEVED Y GF GVFTTNIWLKL KEKQDVFCDSKLMSAAIKDNRAVHADMGYWIESALNDTWKIEKASFIEVKNCHWP K SHTL W SN GVLE SEMIIPKNL AGP V S QHNYRPGYHT QIT GPWHLGKLEMDFDF CDG TTVVVTEDCGNRGPSLRTTTASGKLITEWCCRSCTLPPLRYRGEDGCWYGMEIRPLK EKEENLVNSLVTA
[0198] SEQ ID NO: 5 is the polypeptide sequence of the Dengue vims 3 NS1 protein (NCBI Reference Sequence: YP 001531169.2)
DMGCVINWKGKELKCGSGIFVTNEVHTWTEQYKFQADSPKRLATAIAGAWENGVC GIRSTTRMENLLWKQIANELNYILWENNIKLTVVVGDTLGVLEQGKRTLTPQPMELK Y S WKTW GKAKI VT AET QN S SFIIDGPNTPECP S ASRAWNVWEVED Y GF GVFTTNIWL KLREVYTQLCDHRLMSAAVKDERAVHADMGYWIESQKNGSWKLEKASLIEVKTCT WPK SHTL WTN GVLE SDMIIPK SL AGPI S QHNYRPGYHTQT AGP WHLGKLELDFN Y C EGTT V VITE SC GTRGP SLRTTT V S GKLIHEW C CRS C TLPPLRYMGED GC W Y GMEIRPI SEKEENMVK SL VS A
[0199] SEQ ID NO: 6 is the polypeptide sequence of the Dengue vims 4 NS1 protein (NCBI Reference Sequence: NP_740318.1)
DMGCVASWSGKELKCGSGIFVVDNVHTWTEQYKFQPESPARLASAILNAHKDGVC GIRSTTRLENVMWKQITNELNYVLWEGGHDLTVVAGDVKGVLTKGKRALTPPVSD LKY SWKTW GKAKIFTPEARNSTFLIDGPDTSECPNERRAWN SLEVED Y GFGMFTTNI WMKFREGS SEVCDHRLMS AAIKDQKAVHADMGYWIES SKNQTWQIEKASLIEVKT CL WPKTHTL W SN GVLES QMLIPK S Y AGPF S QHNYRQGY ATQT V GP WHLGKLEIDF G ECPGTTVTIQEDCDHRGPSLRTTTASGKLVTQWCCRSCTMPPLRFLGEDGCWYGMEI RPL SEKEENMVK S Q VT A
[0200] SEQ ID NO: 7 is the polypeptide sequence of the Asian strain of Zika vims NS1 protein (NCBI Reference Sequence: YP 009430301.1).
D V GC S VDF SKKETRCGT GVF VYND VE AWRDRYKYHPD SPRRL AA AVKQ AWEDGIC
GISSVSRMENIMWRSVEGELNAILEENGVQLTVVVGSVKNPMWRGPQRLPVPVNEL
PHGWKAW GKS YF VRAAKTNN SF VVDGDTLKECPLEHRAWN SFLVEDHGF GVFHTS
VWLKVRED Y SLECDP AVIGT AVKGKE AVHSDLGYWIESEKNDTWRLKRAHLIEMK
TCEWPKSHTLWADGIEESDLIIPKSLAGPLSHHNTREGYRTQMKGPWHSEELEIRFEE
CPGTKVHVEETCGTRGPSLRSTTASGRVIEEWCCRECTMPPLSFRAKDGCWYGMEIR
PRKEPESNLVRSVVTA
[0201] SEQ ID NO: 8 is the polypeptide sequence of the African strain of Zika vims NS1 protein (NCBI Reference Sequence: YP 009227199.1). D V GC S VDF SKKETRCGT GVFIYND VE AWRDRYKYHPD SPRRL AAAVKQ AWEEGIC GISSVSRMENIMWKSVEGELNAILEENGVQLTVVVGSVKNPMWRGPQRLPVPVNEL PHGWKAW GKS YF VRAAKTNN SF VVDGDTLKECPLEHRAWN SFLVEDHGF GVFHTS VWLKVRED Y SLECDP AVIGT AVKGREAAHSDLGYWIESEKNDTWRLKRAHLIEMKT CEWPKSHTLWTDGVEESDLIIPKSLAGPLSHHNTREGYRTQVKGPWHSEELEIRFEEC PGTKVYVEETCGTRGPSLRSTTASGRVIEEWCCRECTMPPLSFRAKDGCWYGMEIRP RKEPESNL VRSMVT A
[0202] SEQ ID NO: 9 is the polypeptide sequence of the Dengue vims 1 PrM protein (NCBI Reference Sequence: NP_733807.2)
FHLTTRGGEPHMIVSKQERGKSLLFKTSAGVNMCTLIAMDLGELCEDTMTYKCPRIT ETEPDD VDCW CNATETW VT Y GT C SQTGEHRRDKRS VAL APHV GLGLETRTETWMS SEGAWKQIQKVETWALRHPGFTVIALFLAHAIGTSITQKGIIFILLMLVTPSMA [0203] SEQ ID NO: 10 is the polypeptide sequence of the Dengue vims 2 PrM protein
(NCBI Reference Sequence: NP_739582.2).
FHLTTRNGEPHMIVSRQEKGKSLLFKTEDGVNMCTLMAMDLGELCEDTITYKCPLL RQNEPEDIDC W CN S T ST W VT Y GT C TTMGEHRREKRS V AL VPH V GMGLETRTET WM SSEGAWKHVQRIETWILRHPGFTMMAAILAYTIGTTHFQRALIFILLTAVTPSMT [0204] SEQ ID NO: 11 is the polypeptide sequence of the Dengue vims 3 PrM protein
(NCBI Reference Sequence: YP 001531166.1)
FHLTSRDGEPRMIVGKNERGKSLLFKTASGINMCTLIAMDLGEMCDDTVTYKCPHIT EVEPEDIDCWCNLTSTWVTYGTCNQAGEHRRDKRSVALAPHVGMGLDTRTQTWMS AEGAWRQVEKVETWALRHPGFTILALFLAHYIGTSLTQKVVIFILLMLVTPSMT [0205] SEQ ID NO: 12 is the polypeptide sequence of the Dengue vims 4 PrM protein
(NCBI Reference Sequence: NP_740315.1)
FSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLGEMCEDTVTYKCPLLV NTEPEDIDCWCNLTSTWVMYGTCTQSGERRREKRSVALTPHSGMGLETRAETWMSS EGAWKHAQRVESWILRNPGFALLAGFMAYMIGQTGIQRTVFFVLMMLVAPSYG [0206] SEQ ID NO: 13 is the polypeptide sequence of the Asian strain of the Zika vims
PrM protein (NCBI Reference Sequence: YP 009430297.1)
AEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMSYECPM LDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQ T WLE SRE YTKHLIRVENWIFRNPGF AL A A A AI AWLLGS STS QK VI YL VMILLI AP A Y S [0207] SEQ ID NO: 14 is the polypeptide sequence of the African strain of the Zika virus PrM protein (NCBI Reference Sequence: YP 009227197.1)
AEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMSYECPM LDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQ T WLE SRE YTKHLIK VENWIFRNPGF AL V A V AI AWLLGS STS QK VI YL VMILLI AP A Y S
[0208] References
[0209] Herrera BB, Tsai W-Y, Brites C, Luz E, Pedroso C, Drexler JF, Wang W-K, Kanki PJ. 2018. T cell responses to nonstructural protein 3 distinguish infections by dengue and Zika viruses. mBio 9:e00755-18.
[0210] Herrera BB, Tsai W-Y, Chang CA, Hamel DJ, Wang W-K, Lu Y, Mboup S,
Kanki PJ. 2018. Sustained specific and cross-reactive T cell responses to Zika and dengue virus NS3 in
West Africa. J Virol 92:e01992-17.
[0211] Lai C-Y, Tsai W-Y, Lin S-R, Kao C-L, Hu H-P, King C-C, Wu H-C, Chang G-J, Wang W-H. 2008. Antibodies to Envelope Glycoprotein of Dengue Virus during the Natural Course of Infection Are Predominantly Cross-Reactive and Recognize Epitopes Containing Highly Conserved Residues at the Fusion Loop of Domain IIT. J Virology, p. 6631-6643.
EXAMPLES
[0212] Example 1 - Two color technology for coronavirus detection in an infected subject
[0213] FIG. 1 shows two-color technology to determine coronavirus convalescence (IgG) infection based on detection of Spike (S) and Nucleocapsid (N) in a coronavirus-infected subject. There are three colors observed on the test strip. Purple for the presence of IgG antibodies in the test sample to coronavirus S and N proteins. Blue color for the control N protein, confirming the successful lateral flow. Red color for the control S protein, also confirming successful lateral flow along the strip. In some embodiments, subjects develop IgG antibodies to only S or N but not both proteins.
[0214] FIG. 2 shows two-color technology to determine coronavirus acute (IgM) infection based on detection of Spike (S) and Nucleocapsid (N) in a coronavirus-infected subject. There are two colors observed on the test strip. Blue for the presence of IgM antibodies in the test sample to coronavirus S protein. Red color for the control N protein confirming successful lateral flow along the strip. Blue color for the control S protein, also confirming the successful lateral flow. In some embodiments, subjects develop IgM antibodies to only S or N but not both proteins.
[0215] Example 2 - Two color technology for coronavirus detection in a non- infected subject
[0216] FIG. 3 shows two-color technology to determine coronavirus convalescence (IgG) infection based on detection of Spike (S) and Nucleocapsid (N) in a coronavirus-non-infected subject. There are two colors observed in the control areas of the strip. Red color for the S protein confirming successful lateral flow along the strip. Blue color for the N protein, also confirming the successful lateral flow. There is no signal observed in the test area of the strip because the subject is not infected with coronavirus. FIG. 4 shows two-color technology to determine coronavirus acute (IgM) infection based on detection of Spike (S) and Nucleocapsid (N) in a coronavirus-non-infected subject. There are two colors observed in the control areas of the strip. Red color for the N protein confirming successful lateral flow along the strip. Blue color for the S protein, also confirming the successful lateral flow. There is no signal observed in the test area of the strip because the subject is not infected with coronavirus.
[0217] Example 3 - single-color technology for coronavirus infection in an infected subject
[0218] FIG. 5 shows single-color technology to determine coronavirus acute (IgM) and convalescence (IgG) infection based on detection of antibodies against Spike (S) protein in a coronavirus-infected subject. This Spike antibody test shows that the coronavirus-infected subject developed both IgG and IgM antibodies to Spike protein. In some embodiments, subjects develop either IgG antibodies or IgM antibodies to Spike protein but not both. FIG.
6 shows single-color technology to determine coronavirus acute (IgM) and convalescence (IgG) infection based on detection of antibodies against Nucleocapsid (N) in a coronavirus- infected subject. This Nucleocapsid antibody test shows that an infected subject only developed IgM antibodies to Nucleocapsid. In some embodiments, subject develop both IgG and IgM antibodies.
[0219] Example 4 - two-color technology for flavivirus infections [0220] FIG. 7 shows two-color technology to determine primary flavivirus infection based on IgG and IgM antibodies against NS1 and PrM proteins. There are two colors observed on the test strips. Red color in the test area of the strips showing that the biological sample contained IgG antibodies (top strip) and IgM antibodies (bottom strip) against only NS1 protein. Red color for the NS1 protein in the control area confirming successful lateral flow along the strip. Blue color for the PrM protein, also confirming the successful lateral flow. This test shows that the biological sample (in this case from a Dengue virus-infected subject) contained IgG but not IgM antibodies against NS1 protein only. The absence of antibodies to PrM protein suggests that the subject presents with a primary flavivirus infection. In some embodiments, primary flavivirus infected subjects develop both IgG and IgM antibodies to NS1. In some embodiments, primary flavivirus infected subjects develop only IgG or only IgM antibodies to NS1.
[0221] FIG. 8 shows two-color technology to determine secondary flavivirus infection based on IgG and IgM antibodies against NS1 and PrM proteins. There are three colors observed on the test strips. Blue color for the NS1 protein in the control area confirming successful lateral flow along the strip. Red color for the PrM protein also confirming the successful lateral flow. Purple color in the test area of the strip shows that the biological sample (in this case from a Dengue virus infected subject) contained IgG antibodies (top strip) and IgM antibodies (bottom strip) to both PrM and NS1 flavivirus proteins. This result shows that the subject presents with a secondary flavivirus infection. In some embodiments, secondary flavivirus infected subjects develop IgG and IgM antibodies to NS1 and PrM proteins. In some embodiments, secondary flavivirus infected subjects develop only IgG or only IgM antibodies to NS1 and PrM proteins.
[0222] Example 5 - single-color technology for flavivirus infections [0223] FIG. 9 shows single-color technology to determine primary flavivirus infection based on IgG and IgM antibodies against NS1 and PrM proteins. A primary flavivirus- infected subject (in this case Dengue virus-infected), developed IgG and IgM antibodies against NS1 (top strip), but not PrM (bottom strip). The absence of antibodies to PrM protein suggests that the subject presents with a primary flavivirus infection. In some embodiment, a primary infected patients only develops IgG or IgM antibodies to NS1. FIG. 10 shows single color technology to determine secondary flavivirus infection based on IgG and IgM antibodies against NS1 and PrM proteins. As shown in FIG. 10, a secondary infected subject (in this case a subject infected with Dengue virus) develops IgG and IgM antibodies to both NS1 and PrM. However, in some embodiments, secondary infected subject develops only either IgG or IgM antibodies to NS1 and PrM. However, in some embodiments, a secondary flavivirus-infected subject presents only IgG antibodies to both NS1 and PrM. In another embodiment, a secondary flavivirus-infected subjects presents only IgM antibodies to both NS1 and PrM.
[0224] Example 6 - Reagents for Experiments described in Example 1-5 are provided below in Tables 1-8.
Table 1. Test line antibodies
Figure imgf000080_0001
Table 2. Control line antibodies
Figure imgf000080_0002
Table 3. Spike antigens for coronaviruses for conjugation to nanoparticles
Figure imgf000080_0003
Figure imgf000081_0001
Table 4. Nucleoprotein antigens for conjugation to nanoparticles
Figure imgf000081_0002
Table 5. Flavivirus antigen for conjugation to nanoparticles
Figure imgf000081_0003
Figure imgf000082_0001
Table 6. 40ng nanoparticles = gold conjugate
Figure imgf000082_0002
Table 7. Quencher is supplied in the gold conjugate kit
Figure imgf000082_0003
Table 8. Nitrocellulose paper
Figure imgf000082_0004

Claims

What is claimed is:
1. An immunoassay for detecting a first and a second antigen in a biological sample from a subject, the immunoassay comprising: a first and a second capture reagent, wherein the first and the second capture reagents are immobilized on a solid support; a first and a second detection reagent; and an at least one visualizing reagent.
2. The immunoassay of claim 1, wherein the first capture reagent comprises an anti human IgG antibody.
3. The immunoassay of claim 1, wherein the second capture reagent comprises an anti human IgM antibody.
4. The immunoassay of claim 1, wherein the first detection reagent is at least a portion of a first virus protein.
5. The immunoassay of claim 4, wherein the first virus protein is coronavirus Spike protein.
6. The immunoassay of claim 5, wherein the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKE11, Pipistrellus bat coronavirus HKE15, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
7. The immunoassay of claim 6, wherein the coronavirus strain is SARS-CoV-2.
8. The immunoassay of claim 5, wherein the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
9. The immunoassay of claim 1, wherein the second detection reagent is at least a portion of a second virus protein.
10. The immunoassay of claim 9, wherein the second virus protein is coronavirus Nucleocapsid protein.
11. The immunoassay of claim 10, wherein the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
12. The immunoassay of claim 11, wherein the coronavirus strain is SARS-CoV-2.
13. The immunoassay of claim 10, wherein the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
14. The immunoassay of claim 4, wherein the first virus protein is flavivirus non- structural protein 1 (NS1).
15. The immunoassay of claim 9, wherein the second virus protein is flavivirus precursor membrane (PrM) protein.
16. The immunoassay of any one of claims 14-15, wherein the first and second virus protein is from a flavivirus non-structural protein (NS1) strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
17. The immunoassay of claim 14, wherein NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8.
18. The immunoassay of claim 15, wherein PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
19. The immunoassay of claim 1, wherein the first antigen is a human IgG antibody capable of specifically recognizing at least one virus protein.
20. The immunoassay of claim 19, wherein the at least one virus protein is coronavirus Spike protein.
21. The immunoassay of claim 20, wherein the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
22. The immunoassay of claim 21, wherein the coronavirus strain is SARS-CoV-2.
23. The immunoassay of claim 20, wherein the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
24. The immunoassay of claim 19, wherein the at least one virus protein is coronavirus Nucleocapsid protein.
25. The immunoassay of claim 24, wherein the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKE11, Pipistrellus bat coronavirus HKE15, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
26. The immunoassay of claim 25, wherein the coronavirus strain is SARS-CoV-2.
27. The immunoassay of claim 24, wherein the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
28. The immunoassay of claim 19, wherein the at least one virus protein is a flavivirus non- structural protein 1 (NS1).
29. The immunoassay of claim 19, wherein the at least one virus protein is a flavivirus precursor membrane (PrM) protein.
30. The immunoassay of any one of claims 27-29, wherein (1) IgG antibodies recognizing only NS1 are present in the sample, indicating a primary viral infection of the subject; or (2) IgG antibodies to PrM only or IgG antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
31. The immunoassay of any one of claims 28-29, wherein the flavivirus non-structural protein 1 and flavivirus precursor membrane (PrM) protein is from a flavivirus strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
32. The immunoassay of claim 28, wherein NS1 is 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID
NO: 8.
33. The immunoassay of claim 29, wherein PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
34. The immunoassay of claim 1, wherein the second antigen is a human IgM antibody capable of specifically recognizing at least one virus protein.
35. The immunoassay of claim 34, wherein the at least one virus protein is coronavirus Spike protein.
36. The immunoassay of claim 35, wherein the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
37. The immunoassay of claim 35, wherein the coronavirus strain is SARS-CoV-2.
38. The immunoassay of claim 35, wherein the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
39. The immunoassay of claim 34, wherein the at least one virus protein is coronavirus Nucleocapsid protein.
40. The immunoassay of claim 39, wherein the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
41. The immunoassay of claim 40, wherein the coronavirus strain is SARS-CoV-2.
42. The immunoassay of claim 39, wherein the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
43. The immunoassay of claim 34, wherein the at least one virus protein is a flavivirus non- structural protein 1 (NS1).
44. The immunoassay of claim 34, wherein the at least one virus protein is a flavivirus precursor membrane (PrM) protein.
45. The immunoassay of any one of claims 43-44, wherein (1) IgM antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgM antibodies to PrM only or IgM antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
46. The immunoassay of any one of claims 43-44, wherein the flavivirus non-structural protein 1 and flavivirus precursor membrane (PrM) protein is from a flavivirus strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
47. The immunoassay of claim 43, wherein NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8.
48. The immunoassay of claim 44, wherein PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
49. The immunoassay of claim 1, wherein the first detection reagent comprises an anti human IgG antibody.
50. The immunoassay of claim 1, wherein the second detection reagent comprises an anti human IgM antibody.
51. The immunoassay of claim 1 , wherein the first capture reagent is at least a portion of a first virus protein.
52. The immunoassay of claim 51, wherein the first virus protein is coronavirus Spike protein.
53. The immunoassay of claim 52, wherein the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus ITKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
54. The immunoassay of claim 53, wherein the coronavirus strain is SARS-CoV-2.
55. The immunoassay of claim 52, wherein the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
56. The immunoassay of claim 1, wherein the second capture reagent is at least a portion of a second virus protein.
57. The immunoassay of claim 56, wherein the second virus protein is coronavirus Nucleocapsid protein.
58. The immunoassay of claim 57, wherein the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
59. The immunoassay of claim 58, wherein the coronavirus strain is SARS-CoV-2.
60. The immunoassay of claim 57, wherein the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
61. The immunoassay of claim 51, wherein the first virus protein is a flavivirus non- structural protein 1 (NS1).
62. The immunoassay of claim 56, wherein the second virus protein is a flavivirus precursor membrane (PrM) protein.
63. The immunoassay of any one of claims 49-50, wherein the flavivirus non-structural protein 1 and flavivirus precursor membrane (PrM) protein is from a flavivirus strain is selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
64. The immunoassay of claim 61, wherein NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID
NO: 8.
65. The immunoassay of claim 62, wherein PrM is 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID
NO: 14.
66. The immunoassay of any one of claims 1-65, wherein the at least one visualizing reagent is at least one nanoparticle with a structure and an emission spectrum.
67. The immunoassay of claim 66, wherein the at least one nanoparticle is conjugated to the first and second detection reagents.
68. The immunoassay of any one of claims 1-65, wherein the visualizing reagent comprises: (1) at least one first nanoparticle with a first structure and a first emission spectrum; and (2) at least one second nanoparticle with a second structure and a second emission spectrum; wherein the first and second emission spectra are visually distinct.
69. The immunoassay of claim 68, wherein the first nanoparticle is conjugated to the first detection reagent and the second nanoparticle is conjugated to the second detection reagent.
70. The immunoassay of any one of claims 1-69, wherein the subject is suspected of having or is diagnosed with a viral infection.
71. The immunoassay of claim 70, wherein the viral infection is a coronavirus (CoV) infection.
72. The immunoassay of claim 71, wherein the coronavirus (CoV) is selected from the coronavirus (CoV) strains consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
73. The immunoassay of claim 72, wherein the coronavirus strain is SARS-CoV-2.
74. The immunoassay of claim 70, wherein the viral infection is a flavivirus infection.
75. The immunoassay of claims 74, wherein the flavivirus strain is selected from the group consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OITFV).
76. The immunoassay of any one of claims 1-69, wherein the subject is suspected of having or is diagnosed with a coronavirus (CoV) disease.
77. The immunoassay of claim 76, wherein the coronavirus disease is COVID-19.
78. The immunoassay of claim 77, wherein the subject is suspected of having or is diagnosed with asymptomatic COVID-19.
79. The immunoassay of claim 77, wherein the subject is suspected of having or is diagnosed with symptomatic COVID-19.
80. The immunoassay of any one of claims 1-79, wherein the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof.
81. The immunoassay of any one of claims 1-80, wherein the at least one visualizing reagent comprises a colorimetric readout.
82. The immunoassay of any one of claims 1-81, wherein the at least one visualizing reagent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
83. A lateral flow immunoassay device for detecting a first and a second antigen in a biological sample from a subject, the device comprising: an elongated holder having at least one aperture for sample application and at least one aperture for readout viewing; a first and a second immunoassay test strips secured within the holder, wherein the first and second test strips are parallel to each other along a long edge; a first capture reagent embedded in a first test area, wherein the first test area comprises at least a portion of the first test strip; a second capture reagent embedded in a second test area, wherein the second area comprises at least a portion of the second test strip; a first detection reagent embedded in at least a portion of a first conjugate pad, wherein the first conjugate pad comprises at least a portion of the first test strip and at least a portion of the second test strip; a second detection reagent embedded in at least a portion of a second conjugate pad, wherein the second conjugate pad comprises at least a portion of the first test strip and at least a portion of the second test strip; a first control reagent embedded in at least a portion of a first control area, wherein the first control area comprises at least a portion of the first test strip and at least a portion of the second test strip; a second control reagent embedded in at least a portion of a second control area, wherein the second control area comprises at least a portion of the first test strip and at least a portion of the second test strip; and an at least one visualization reagent conjugated to at least one detection reagent; wherein the test areas, the conjugate pads, and the control areas each comprises a different portion of the test strip from the others; and wherein: (1) if the first antigen is present in the sample the first test area and the first and second control areas produce a signal; (2) if the second antigen is present in the sample the second test area and the first and second control areas produce a signal; (3) if the first antigen is not present in the sample the first test area does not produce a signal the first and second control areas produce a signal; (4) if the second antigen is not present in the sample the second test area does not produce a signal and the first and second control areas produce a signal.
84. The device of claim 83, wherein the first capture reagent comprises an anti-human IgG antibody.
85. The device of claim 83, wherein the second capture reagent comprises an anti-human IgM antibody.
86. The device of claim 83, wherein the first detection reagent is at least a portion of a coronavirus Spike protein.
87. The device of claim 86, wherein the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU 1 , Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
88. The device of claim 87, wherein the coronavirus strain is SARS-CoV-2.
89. The device of claim 86, wherein the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
90. The device of claim 83, wherein the second detection reagent is at least a portion of a coronavirus Nucleocapsid protein.
91. The device of claim 90, wherein the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
92. The device of claim 91, wherein the coronavirus strain is SARS-CoV-2.
93. The device of claim 90, wherein the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
94. The device of claim 83, wherein the first antigen is a human IgG antibody capable of specifically recognizing at least one virus protein.
95. The device of claim 94, wherein the at least one virus protein is coronavirus Spike protein.
96. The device of claim 95, wherein the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKUl , Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
97. The device of claim 96, wherein the coronavirus strain is SARS-CoV-2.
98. The device of claim 95, wherein the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
99. The device of claim 94, wherein the at least one virus protein is coronavirus Nucleocapsid protein.
100. The device of claim 99, wherein the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
101. The device of claim 100, wherein the coronavirus strain is SARS-CoV-2.
102. The device of claim 99, wherein the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
103. The device of claim 83, wherein the second antigen is a human IgM antibody capable of specifically recognizing at least one virus protein.
104. The device of claim 103, wherein the at least one virus protein is a coronavirus Spike protein.
105. The device of claim 104, wherein the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RFl, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
106. The device of claim 105, wherein the coronavirus strain is SARS-CoV-2.
107. The device of claim 104, wherein the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
108. The device of claim 103, wherein the at least one virus protein is coronavirus Nucleocapsid protein.
109. The device of claim 108, wherein the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RFl, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
110. The device of claim 109, wherein the coronavirus strain is SARS-CoV-2.
111. The device of claim 108, wherein the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
112. The device of claim 83, wherein the first detection reagent comprises an anti-human IgG antibody.
113. The device of claim 83, wherein the second detection reagent comprises an anti human IgM antibody.
114. The device of claim 83, wherein the first capture reagent is at least a portion of a coronavirus Spike protein.
115. The device of claim 114, wherein the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus ITKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
116. The device of claim 115, wherein the coronavirus strain is SARS-CoV-2.
117. The device of claim 114, wherein the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
118. The device of claim 83, wherein the second capture reagent is at least a portion of coronavirus Nucleocapsid protein.
119. The device of claim 118, wherein the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
120. The device of claim 119, wherein the coronavirus strain is SARS-CoV-2.
121. The device of claim 118, wherein the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
122. The device of any one of claims 83-121, wherein the at least one visualizing reagent is at least one nanoparticle with a structure and an emission spectrum.
123. The device of claim 122, wherein the at least nanoparticle is conjugated to the first and second detection reagents.
124. The device of any one of claims 69-121, wherein the visualizing reagent comprises (1) an at least one first nanoparticle with a first structure and a first emission spectrum; and (2) an at least one second nanoparticle with a second structure and a second emission spectrum; wherein the first and second emission spectra are visually distinct.
125. The device of claim 124, wherein the at least one first nanoparticle is conjugated to the first detection reagent and the at least one second nanoparticle is conjugated to the second detection reagent.
126. The device of any one of claims 83-125, wherein the first control reagent comprises an anti-coronavirus Spike protein antibody.
127. The device of any one of claims 83-125, wherein the second control reagent comprises an anti-coronavirus Nucleocapsid protein antibody.
128. The device of claims 83, wherein the subject is suspected of having or is diagnosed with a viral infection.
129. The device of claim 128, wherein the viral infection is a coronavirus (CoV) infection.
130. The device of claim 129, wherein the coronavirus (CoV) is selected from the coronavirus (CoV) strains consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus ITKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
131. The device of claim 130, wherein the coronavirus strain is SARS-CoV-2.
132. The device of any one of claims 83-127, wherein the subject is suspected of having or is diagnosed with a coronavirus (CoV) disease.
133. The device of claim 132, wherein the coronavirus disease is COVID-19.
134. The device of claim 133, wherein the subject is suspected of having or is diagnosed with asymptomatic COVID-19.
135. The device of claim 133, wherein the subject is suspected of having or is diagnosed with symptomatic COVID-19.
136. The device of any one of claims 83-135, wherein the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof.
137. The device of any one of claims 83-136, wherein the at least one visualizing reagent comprises a colorimetric readout.
138. The device of any one of claims 83-137, wherein the at least one visualizing agent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
139. A lateral flow immunoassay device for detecting a first and a second antigen in a biological sample from a subject, the device comprising: an elongated holder having at least one aperture for sample application and at least one aperture for readout viewing; a first and a second immunoassay test strip secured within the holder, wherein the first and second test strips are parallel to each other along a long edge; a first capture reagent embedded in a first test area, wherein the first area comprises at least a portion of the first test strip; a second capture reagent embedded in a second test area, wherein the second area comprises at least a portion of the second test strip; a first detection reagent embedded in at least a portion of a first conjugate pad, wherein the first conjugate pad comprises at least a portion of the first test strip and at least a portion of the second test strip; a second detection reagent embedded in at least a portion of a second conjugate pad, wherein the second conjugate pad comprises at least a portion of the first test strip and at least a portion of the second test strip; a first control reagent embedded in at least a portion of a first control area, wherein the first control area comprises at least a portion of the first test strip and at least a portion of the second test strip; a second control reagent embedded in at least a portion of a second control area, wherein the second control area comprises at least a portion of the first test strip and at least a portion of the second test strip; and an at least one visualization reagent conjugated to at least one detection reagent; wherein the test areas, the conjugate pads, and the control areas each comprises a different portion of the test strip from the others; and wherein: (1) if the first antigen is present in the sample the first test area and the first and second control areas produce a signal; (2) if the second antigen is present in the sample the second test area and the first and second control areas produce a signal; (3) if the first antigen is not present in the sample the first test area does not produce a signal and the first and second control areas produce a signal; (4) if the second antigen is not present in the sample the second test area does not produce a signal and the first and second control areas produce a signal.
140. The device of claim 139, wherein the first capture reagent comprises an anti-human IgG antibody.
141. The device of claim 139, wherein the second capture reagent comprises an anti human IgM antibody.
142. The device of claim 139, wherein the first detection reagent is at least a portion of a flavivirus non- structural protein 1 (NS1).
143. The device of claim 139, wherein the second virus protein is at least a portion of a flavivirus precursor membrane (PrM) protein.
144. The device of any one of claims 142-143, wherein the flavivirus non- structural protein 1 and the flavivirus precursor membrane (PrM) protein is from a flavivirus strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
145. The device of claim 142, wherein NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8.
146. The device of claim 143, wherein PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
147. The device of claim 139, wherein the first antigen is a human IgG antibody capable of specifically recognizing at least one virus protein.
148. The device of claim 147, wherein the at least one virus protein is a flavivirus non- structural protein 1 (NS1).
149. The device of claim 148, wherein NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8.
150. The device of claim 147, wherein the at least one virus protein is a flavivirus precursor membrane (PrM) protein.
151. The device of claim 150, wherein PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
152. The device of any one of claims 148-151, wherein (1) IgG antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgG antibodies to PrM only or IgG antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
153. The device of claim 139, wherein the second antigen is a human IgM antibody capable of specifically recognizing at least one virus protein.
154. The device of claim 153, wherein the at least one virus protein is a flavivirus non- structural protein 1 (NS1).
155. The device of claim 154, wherein NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8.
156. The device of claim 153, wherein the at least one virus protein is a flavivirus precursor membrane (PrM) protein.
157. The device of claim 156, wherein PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
158. The device of any one of claims 154-157, wherein (1) IgM antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgM antibodies to PrM only or IgM antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
159. The device of claim 139, wherein the first detection reagent comprises an anti-human IgG antibody.
160. The device of claim 139, wherein the second detection reagent comprises an anti human IgM antibody.
161. The device of claim 139, wherein the first capture reagent is at least a portion of a flavivirus non- structural protein 1 (NS1).
162. The device of claim 161, wherein NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8.
163. The device of claim 139, wherein the second capture reagent is at least a portion of is a flavivirus precursor membrane (PrM) protein.
164. The device of claim 163, wherein PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
165. The device of any one of claims 139-164, wherein the at least one visualizing reagent is at least one nanoparticle with a structure and an emission spectrum.
166. The device of claim 165, wherein the at least one nanoparticle is conjugated to the first and second detection reagents.
167. The device of any one of claims 139-164, wherein the visualizing reagent comprises (1) at least one first nanoparticle with a first structure and a first emission spectrum; and (2) at least one second nanoparticle with a second structure and a second emission spectrum; wherein the first and second emission spectra are visually distinct.
168. The device of claim 167, wherein the first nanoparticle is conjugated to the first detection reagent and the second nanoparticle is conjugated to the second detection reagent.
169. The device of any one of claims 139-168, wherein the first control reagent comprises an anti -human immunoglobulin or an anti-human IgG and IgM.
170. The device of any one of claims 139-169, wherein the second control reagent comprises an anti-PrM antibody or an NS1 antibody.
171. The device of any one of claims 139-170, wherein the subject is suspected of having or is diagnosed with a viral infection.
172. The device of claim 171, wherein the viral infection is a flavivirus infection.
173. The device of claim 172, wherein the flavivirus infection is selected from the strains consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2),
Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
174. The device of any one of claims 139-173, wherein the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof.
175. The device of any one of claims 139-174, wherein the at least one visualizing reagent comprises a colorimetric readout.
176. The device of any one of claims 139-175, wherein the at least one visualizing agent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
177. A lateral flow immunoassay device for detecting IgG and IgM antibodies in a biological sample from a subject, the device comprising: an elongated holder having at least one aperture for sample application and at least one aperture for readout viewing; a first and a second immunoassay test strips secured within the holder, wherein the first and second test strips are parallel to each other along a long edge; a first capture reagent embedded in a first test area, wherein the first test area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the first capture reagent comprises anti-IgG antibodies; a second capture reagent embedded in a second test area, wherein the second area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the second capture reagent comprises anti-IgM antibodies; a first detection reagent embedded in at least a portion of a first conjugate pad, wherein the first conjugate pad comprises at least a portion of the first test strip and wherein the first detection reagent comprises at least a portion of a coronavirus Spike protein; a second detection reagent embedded in at least a portion of a second conjugate pad, wherein the second conjugate pad comprises at least a portion of the second test strip and wherein the second detection reagent comprises at least a portion of a coronavirus Nucleocapsid protein; a control reagent embedded in at least a portion of a control area, wherein the control area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the control reagent is an anti-Fc antibody; and a visualization reagent conjugated to at least one detection reagent; wherein the test areas, the conjugate pads, and the control areas each comprises a different portion of the test strip from the others; and wherein: (1) if the IgG antibody is present in the sample, the first test area and the control area produce a signal; (2) if the IgM antibody is present in the sample, the second test area and the first and second control areas produce a signal; (3) if the IgG antibody is not present in the sample, the first test area does not produce a signal and control area produces a signal; (4) if the IgM antibody is not present in the sample, the second test area does not produce a signal and the control area produces a signal.
178. The device of claim 83, wherein the first capture reagent comprises an anti-human IgG antibody.
179. The device of claim 83, wherein the second capture reagent comprises an anti-human IgM antibody.
180. The device of claim 177, wherein the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
181. The device of claim 180, wherein the coronavirus strain is SARS-CoV-2.
182. The device of claim 177, wherein the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
183. The device of claim 177, wherein the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RFl, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
184. The device of claim 183, wherein the coronavirus strain is SARS-CoV-2.
185. The device of claim 184, wherein the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
186. The device of claim 177, wherein the IgG is a human IgG antibody capable of specifically recognizing at least one coronavirus protein.
187. The device of claim 186, wherein the at least one coronavirus protein is coronavirus Spike protein.
188. The device of claim 187, wherein the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus ITKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
189. The device of claim 188, wherein the coronavirus strain is SARS-CoV-2.
190. The device of claim 187, wherein the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
191. The device of claim 186, wherein the at least one coronavirus protein is coronavirus Nucleocapsid protein.
192. The device of claim 191, wherein the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavims Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
193. The device of claim 192, wherein the coronavirus strain is SARS-CoV-2.
194. The device of claim 191, wherein the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
195. The device of claim 177, wherein the IgM antibody is a human IgM antibody capable of specifically recognizing at least one virus protein.
196. The device of claim 195, wherein the at least one virus protein is a coronavirus Spike protein.
197. The device of claim 196, wherein the coronavirus Spike protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rml/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
198. The device of claim 197, wherein the coronavirus strain is SARS-CoV-2.
199. The device of claim 196, wherein the coronavirus Spike protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 1.
200. The device of claim 195, wherein the at least one virus protein is coronavirus a Nucleocapsid protein.
201. The device of claim 200, wherein the coronavirus Nucleocapsid protein is from a coronavirus strain selected from SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RFl, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
202. The device of claim 201, wherein the coronavirus strain is SARS-CoV-2.
203. The device of claim 200, wherein the coronavirus Nucleocapsid protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 2.
204. The device of claim 177, wherein the visualization reagent comprises a species of nanoparticles with a structure and an emission spectrum.
205. The device of claim 204, wherein the visualization reagent is conjugated to the first and second detection reagents.
206. The device of claim 204, wherein the species of nanoparticles comprises 80 nm Gold NanoUrchins nanoparticles.
207. The device of claim 204, wherein the species of nanoparticles comprises 40 nm InnovaCoat® Gold nanoparticles.
208. The device of claim 177, wherein the control reagent is an anti-human Fc antibody.
209. The device of claim 177, wherein the subject is suspected of having or is diagnosed with a coronavirus (CoV) infection.
210. The device of claim 209, wherein the coronavirus (CoV) is selected from the coronavirus (CoV) strains consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43, HKU1, Pipistrellus bat coronavirus HKU5, Pipistrellus bat coronavirus HKU5/HK/03/2005 (Pi-BatCoV HKU5/HK/03/2005), Rousettus bat coronavirus HKU9, Rousettus bat coronavirus HKU9/GD/005/2005 (Ro-BatCoV HKU9/GD/005/2005), Severe acute respiratory syndrome-related coronavirus, SARS- related human coronavirus, SARS-related human coronavirus Urbani (SARS CoV Urbani), SARS-related Rhinolophus bat coronavirus RF1, SARS-related Rhinolophus bat coronavirus Rfl/2004 (SARSr-Rh-BatCoV RF 1/2004), SARS-related Rhinolophus bat coronavirus Rml, SARS-related Rhinolophus bat coronavirus Rml/2005 (SARSr-Rh-BatCoV Rm 1/2005), SARS-related Rhinolophus bat coronavirus Rp3, SARS-related Rhinolophus bat coronavirus Rp3/2004 (SARSr-Rh- BatCoV Rp3/2004), SARS-related Rhinolophus bat coronavirus HKU3, SARS- related Rhinolophus bat coronavirus HKU3/HK/24/2005 (SARSr-Rh-BatCoV HKU3/HK/24/2005), SARS-related palm civet coronavirus, SARS-related palm civet coronavirus SZ3/2003 (SARSr-CiCoV SZ3/2003), SARS-related Chinese ferret badger coronavirus, SARS-related Chinese ferret badger coronavirus CFB/SZ/94/03 (SARSr CoV CFB/SZ/94/03), Tylonycteris bat coronavirus HKU4, and Tylonycteris bat coronavirus HKU4/HK/04/2005 (Ty-BatCoV HKU4/HK/04/2005).
211. The device of claim 210, wherein the coronavirus strain is SARS-CoV-2.
212. The device of claim 177, wherein the subject is suspected of having or is diagnosed with a coronavirus (CoV) disease.
213. The device of claim 212, wherein the coronavirus disease is COVID-19.
214. The device of claim 213, wherein the subject is suspected of having or is diagnosed with asymptomatic COVID-19.
215. The device of claim 213, wherein the subject is suspected of having or is diagnosed with symptomatic COVID-19.
216. The device of any one of claims 177-215, wherein the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof.
217. The device of any one of claims 177-216, wherein the visualization reagent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
218. A lateral flow immunoassay device for detecting IgG and IgM antibodies in a biological sample from a subject, the device comprising: an elongated holder having at least one aperture for sample application and at least one aperture for readout viewing; a first and a second immunoassay test strip secured within the holder, wherein the first and second test strips are parallel to each other along a long edge; a first capture reagent embedded in a first test area, wherein the first area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the first capture reagent comprises anti-IgG antibodies; a second capture reagent embedded in a second test area, wherein the second area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the second capture reagent comprises anti-IgM antibodies; a first detection reagent embedded in at least a portion of a first conjugate pad, wherein the first conjugate pad comprises at least a portion of the first test strip and wherein the first detection reagent comprises at least a portion of a flavivirus non- structural protein I (NS I) protein; a second detection reagent embedded in at least a portion of a second conjugate pad, wherein the second conjugate pad comprises at least a portion of the second test strip and wherein the second detection reagent comprises at least a portion of a flavivirus precursor membrane (PrM) protein; a control reagent embedded in at least a portion of a control area, wherein the control area comprises at least a portion of the first test strip and at least a portion of the second test strip and wherein the control reagent is an anti-Fc antibody; and a visualization reagent conjugated to at least one detection reagent; wherein the test areas, the conjugate pads, and the control areas each comprises a different portion of the test strip from the others; and wherein: (1) if the IgG antibody is present in the sample, the first test area and the control area produce a signal; (2) if the IgM antibody is present in the sample, the second test area and the control area produce a signal; (3) if the IgG antibody is not present in the sample the first test area does not produce a signal and the control area produces a signal; (4) if the IgM is not present in the sample, the second test area does not produce a signal and control areas produces a signal.
219. The device of claim 218, wherein the first capture reagent comprises an anti-human IgG antibody.
220. The device of claim 218, wherein the second capture reagent comprises an anti human IgM antibody.
221. The device of claim 218, wherein the flavivirus non- structural protein 1 and the flavivirus precursor membrane (PrM) protein is from a flavivirus strain selected from Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
222. The device of claim 218, wherein NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8.
223. The device of claim 218, wherein PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
224. The device of claim 218, wherein the IgG antibody is a human IgG antibody capable of specifically recognizing at least one virus protein.
225. The device of claim 224, wherein the at least one virus protein is a flavivirus non- structural protein 1 (NS1).
226. The device of claim 225, wherein NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8.
227. The device of claim 224, wherein the at least one virus protein is a flavivirus precursor membrane (PrM) protein.
228. The device of claim 227, wherein PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
229. The device of any one of claims 225-228, wherein (1) IgG antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgG antibodies to PrM only or IgG antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
230. The device of claim 218, wherein the IgM antibody is a human IgM antibody capable of specifically recognizing at least one virus protein.
231. The device of claim 230, wherein the at least one virus protein is a flavivirus non- structural protein 1 (NS1).
232. The device of claim 231, wherein NS1 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 3, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 4, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 8.
233. The device of claim 230, wherein the at least one virus protein is a flavivirus precursor membrane (PrM) protein.
234. The device of claim 233, wherein PrM is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 9, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 10, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 11, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 12, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 13, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 14.
235. The device of any one of claims 231-234, wherein (1) IgM antibodies recognizing only NS1 are present in the sample indicating a primary viral infection of the subject; or (2) IgM antibodies to PrM only or IgM antibodies to both NS1 and PrM are present in the sample indicating a secondary viral infection of the subject.
236. The device of any one of claims 218-235, wherein the visualization reagent comprises a species of nanoparticles with a structure and an emission spectrum.
237. The device of claim 236, wherein the visualization reagent is conjugated to the first and second detection reagents.
238. The device of claim 236, wherein the species of nanoparticles comprises 80 nm Gold NanoUrchins nanoparticles.
239. The device of claim 236, wherein the species of nanoparticles comprises 40 nm InnovaCoat® Gold nanoparticles.
240. The device of claim 218, wherein the control reagent comprises an anti-human Fc antibody.
241. The device of claim 218, wherein the subject is suspected of having or is diagnosed with a flavivirus infection.
242. The device of claim 241, wherein the flavivirus infection is selected from the strains consisting of Dengue virus serotype 1 (DV1), Dengue virus serotype 2 (DV2), Dengue virus serotype 3 (DV3), Dengue virus serotype 4 (DV4), Zika virus (ZIKV), Yellow Fever (YFV), Ilheus Virus (ILHV), Powassan (POWA), Japanese encephalitis (JE) virus, West Nile virus (WNV), Deer tick (DTV) tick-borne encephalitis virus (TBEV), Usuto (USTV), Saint Louis Encephalitis (SLEV), and Omsk hemorrhagic fever virus (OHFV).
243. The device of any one of claims 218-242, wherein the biological sample is nasopharyngeal swab content, oropharyngeal swab, sputum, serum, plasma, whole blood, saliva, urine, feces, pleural fluid, bodily fluids, a mixture thereof, or a derivative thereof.
244. The device of any one of claims 218-243, wherein the at least one visualizing agent provides a readout in less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes following application of a last component of the visualizing reagent, and wherein the time until readout does not invalidate the test’s correct interpretation.
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