WO2018218355A1

WO2018218355A1 - Method of diagnosing flavivirus infection

Info

Publication number: WO2018218355A1
Application number: PCT/CA2018/050637
Authority: WO
Inventors: Walter L. SIQUEIRA; Maria A. M. MACHADO; Debora HELLER
Original assignee: Western University; University Of São Paulo
Priority date: 2017-05-30
Filing date: 2018-05-30
Publication date: 2018-12-06

Abstract

A method of detecting Flaviviral peptides in a mammalian saliva sample is provided comprising: obtaining a saliva sample from the mammal; and detecting in the sample the presence of at least two peptides selected from the group consisting of: MDHFSLGVLVILLMVQEGLKKRMTTKIIISTSMAVLVAMILGGFSMSDLAKLAILMGATFAEMNTGGDVAHL; NIVRLKSGVDVFHMAAEPCDTLLCDIGESSSSPEVEE; WCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTK; VEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMN; LEIRFEECPGTKVHVEETCGTRGPSLRSTTASGRVIEE; EEALRGLPVRYMTTAVNVTHSGTEIVDLMCHATFTS; RVEMGEAAAIFMTATPPGTRDAFPDSNSPIMDTEVEVPERAWSSGFDW; and GQVLLIAVAVSSAILSRTAWGWGEAGALITAATSTLWEGSPNKYWNSSTATSLCNIF.

Description

METHOD OF DIAGNOSING FLAVIVIRUS INFECTION

Field of the Invention

[0001] The present invention generally relates to methods of diagnosis and treatment, and in particular, to methods useful to diagnose, monitor and treat infection by a Flavivirus, including Zika viras.

Background of the Invention

[0002] Zika viras (ZIKV; family Flaviviridae, genus Flavivirus) is a mosquito-borne virus that was first isolated from a sentinel rhesus monkey in the Zika forest of Uganda in 1947. After a few case reports in Asia and Africa, the first ZIKV outbreak occurred in 2007 affecting Yap Island in Micronesia. In 2013-2014, a new ZIKV outbreak was reported in French Polynesia that spread through the Pacific Islands. In 2015, the viras was detected in Brazil before spreading rapidly, starting a pandemic in the Americas in which 48 countries reported active ZIKV transmission, with an emerging epidemic in Singapore and the threat of further expansion into southeast Asia.

[0003] ZIKV, like other Flaviviruses, is a single-stranded positive-sense RNA viras that encodes a single polyprotein which is cleaved into 3 structural proteins - capsid (C), precursor of membrane (prM), and envelope (E) proteins, forming new virus particles - and 7 non- structural proteins necessary for intracellular replication (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5). The primary vectors of ZIKV are the Aedes mosquitoes, specifically, Ae. aegypti andAe. albopictus. Possible additional transmission routes of ZIKV, such as sexual transmission, have been reported and are the subject of continued investigations. ZIKV has been detected in blood, semen, urine and saliva during symptomatic disease, suggesting that the virus could be transmitted through these corporal fluids. However, there has been no evidence to support infection of ZIKV through the transfer of human saliva.

[0004] Clinical symptoms of Zika fever or infection are non-specific; the most common are rash, fever, arthralgia, myalgia, asthenia and conjunctivitis. Currently, the preferred diagnostic test for the acute stage of Zika fever is RT-qPCR, since viral RNA can often be identified in serum during the initial phase of disease. Laboratory detection of Zika viras infection is challenging as presently there is no "gold standard" diagnostic assay with sensitivity and specificity for both acute and convalescent stages. In addition, there is significant cross-reactivity of antibodies among Flaviviruses that limits the use of serology and antibody-based assays.

[0005] Therefore, it would be desirable to develop new diagnostic strategies to identify infection by a Flavivirus, as well as strategies to discriminate between closely related Flaviviral species.

Summary of the Invention

[0006] Novel methods of diagnosing Flaviviral infection, including Zika infection, have now been developed which are based on detection of specific viral peptides in a patient saliva sample.

[0007] Thus, in its broadest aspect, the present invention provides a method of detecting

Flaviviral peptides in a mammalian saliva sample. The method comprises detecting in a saliva sample obtained from the mammal the resence of at least two e tides selected from the group consisting of:

[0008] In another aspect of the invention, a method of diagnosing Flaviviral infection in a mammal is provided comprising the steps of:

i) detecting in a saliva sample obtained from the mammal at least two peptides selected from the group consisting of:

ii) diagnosing the mammal with Flaviviral infection when the presence of at least two of the viral peptides is detected.

[0009] In another aspect of the invention, a method of diagnosing and treating a Flaviviral infection in a mammal is provided comprising the steps of:

MDHFSLGVLVILLMVQEGLKKmTTKinSTSMAVLVAMILGGFSMSDLAKLMLMGATFAEMNTGGDVAHL (SEQ ID NO: 1); NIVRLKSG VD VFHMA AEPCDTLLCDIGES S S SPEVEE (SEQ ID NO:2);

WCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTK (SEQ ID NO:3);

VEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMN (SEQ ID NO:4);

LEIRFEECPGTKVHVEETCGTRGPSLRSTTASGRVIEE (SEQ ID NO:5);

EEALRGLPVRYMTTAVNVTHSGTEIVDLMCHATFTS (SEQ ID NO:6);

RVEMGEAAAIFMTATPPGTRDAFPDSNSPIMDTEVEVPERAWSSGFDW (SEQ ID NO:7); and

GQVLLIAVAVSSAILSRTAWGWGEAGALITAATSTLWEGSPNKYWNSSTATSLCNIF (SEQ ID NO:8),

ii) diagnosing the mammal with Flaviviral infection when the presence of at least two of the viral peptides is detected; and

iii) administering to the mammal an effective amount of a medication that treats pain and/or fever.

[0010] In a further aspect, a method of detecting Zika viral peptides in a saliva sample from a mammal is provided comprising the steps of:

i) generating a peptide profile of a saliva sample obtained from the mammal, wherein the profile identifies viral peptides by the amino acid region within a Zika polyprotein from which the peptide is generated and the number of peptides generated from each region of the Zika polyprotein; and

iv) diagnosing the mammal with a Zika infection when the peptide profile of the sample comprises at least 10 viral peptides generated from the amino acid regions of: 95-180, 185-250, 305-395, 400-495, 575-675, 740-825, 1070-1115, 1150-1230, 1280-1355, 1730-1780, 1825-1900, 2270-2350, 2430-2500, 2635-2700, 2720-2775 and 2815-2875 of the Zika polyprotein, and comprises no more than about 4 viral peptides in each of amino acid regions of: 1-90, 500-550, 875-975, 1445-1505, 1555-1630, 1930-2050, 2200-2265, 2510-2640, 2915-3025 and 3140-3300 of the Zika polyprotein. [0011] These and other aspects of the invention will become apparent in view of the detailed description that follows by reference to the Figures.

Brief Description of the Figures

[0012] Figure 1 graphically illustrates the distribution of identified peptides relative to the ZIKV polyprotein: (A) when peptidomics was carried out in saliva of patients A, B, and C. Peak height represents the abundance of peptides identified in a specific region; and (B) when classical proteomics was carried out in saliva of patients A, B, and C. Symbols over the ZIKV polyprotein represent the area of peptides identified in specific region. Note the structural region of ZIKV includes C - Capsid; prM - pre-Membrane; and E - Envelope; and NSl, 2A/B, 3, 4A/B, 5 is the non-structural region.

[0013] Figure 2 graphically illustrates the distribution of tryptic peptides identified by the classical proteomic approach from patients according to the number of amino acid residues.

[0014] Figure 3 graphically illustrates the distribution of peptides identified using the peptidomic approach according to the number of amino acids. Note. Each patient is represented by a different shade on the histogram.

[0015] Figure 4 illustrates the distribution of peptides identified using the peptidomic approach according to the Zika polyprotein region strain Mr766. Note. The structural region of the ZIKV polyprotein includes C - capsid, prM - pre-Membrane and E - Envelope; and NSl- Non-structural 1; NS2A/B, NS3, NS4A/B, and NS5 are non-structural regions.

[0016] Figure 5 graphically illustrates the distribution of unique ZIKV peptides from a mother, baby-A and baby-B, identified using the peptidomics approach, aligned to the Zika virus polyprotein. The peptides from the mother, baby-A and baby-B cover 67%, 84% and 45%) of the entire viral polyprotein, respectively. Note, structural region on ZIKV polyprotein includes C - Capsid, prM - pre- Membrane, and E - Envelope; and the non-structural region includes NSl, NS2A/B, NS3, NS4A/B and NS5.

[0017] Figure 6 graphically illustrates the distribution of peptides identified using the peptidomic approach according to the number of amino acids residues. [0018] Figure 7 illustrates the distribution of peptides identified using the peptidomic approach according to the Zika polyprotein region strain Mr 766 for the mother (A), baby-A (B) and baby-B (C) of Fig. 5. Note. The structural region of the ZIKV polyprotein includes C - capsid, prM - pre-Membrane and E - Envelope; and NS1- Non-structural 1 ; NS2A/B, NS3, NS4A/B, and NS5 are non-structural regions.

[0019] Figure 8 illustrates amino acid substitutions identified in peptides of baby-A and baby-B based on the ZIKV proteome identified in their mother as a comparison. A total of 22 mutations were observed; where 13 mutations were observed between mother and baby-A (with microcephaly) and 9 mutations were observed between mother and baby-B (without microcephaly). Note. The first letter represents the ZIKV amino acid identified in the mother ZIKV proteome; the number represents the amino acid location in the ZIKV polyprotein region; and the second letter represents the ZIKV amino acid identified in baby-A or baby-B.

[0020] Figure 9 graphically summarizes the abundance of peptides in patient saliva samples generated from regions of the Zika polyprotein.

Detailed Description of the Invention

[0021] In one aspect of the present invention, a method of detecting Flaviviral peptides in a mammal is provided. The method comprises detecting in a saliva sample obtained from the mammal the presence of at least two peptides selected from the group consisting of:

MDHFSLGVLVILLMVQEGLKKRMTTKIIISTSMAVLVAMILGGFSMSDLAKLAILMGATFAEMNTGGDVAHL (SEQ ID NO: l); NIVRLKSGVDVFHMAAEPCDTLLCDIGESSSSPEVEE (SEQ ID NO:2);

WCNTTSTWNWGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTK (SEQ ID NO:3);

VEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMN (SEQ ID NO:4);

LEIRFEECPGTKVHVEETCGTRGPSLRSTTASGRVIEE (SEQ ID NO:5);

EEALRGLPVRYMTTAVNVTHSGTEIVDLMCHATFTS (SEQ ID NO:6);

RVEMGEAAAIFMTATPPGTRDAFPDSNSPIMDTEVEVPERAWSSGFDW (SEQ ID NO:7); and

GQVLLIAVAVSSAILSRTAWGWGEAGALITAATSTLWEGSPNKYWNSSTATSLCNIF (SEQ ID NO:8).

[0022] The saliva sample may be obtained from the mammal using well-established techniques including passive drool collection, or the use of suction devices, capillary tubes, plastic pipettes, or absorbent materials such as cotton swabs or pads. Preferably, materials used to collect and store the saliva sample are inert so as not to adversely affect target peptide analytes in the sample. Polypropylene is generally an appropriate material for saliva collection and storage. If required, salivary stimulants may be used to promote saliva formation, including, for example, chewing parafilm, wax, or other inert substances that do not adversely affect target peptide analytes. For use in the present method, the volume of saliva sample required may vary with the method used to detect peptides therein. Generally, a volume in the range of about 10 to 100 μΐ is sufficient to detect peptides therein; however, the required volume may vary from this range.

[0023] Once a suitable saliva sample is collected, it may be processed to render it more suitable for analysis. For example, the saliva sample may be subjected to techniques such as filtration or centrifugation to remove solids therefrom that may interfere with peptide analysis.

[0024] The saliva sample is analyzed for the presence of at least two viral peptides selected from the group consisting of:

MDHFSLGVLVILLMVQEGLKKRMTTKIIISTSMAVLVAMILGGFSMSDLAKLAILMGATFAEMNTGGDVAHL (SEQ ID NO: 1 ); NIVRLKSGVDVFHMAAEPCDTLLCDIGESSSSPEVEE (SEQ ID NO:2);

WCNTTSTWVYGTCHffla-CGEAPvRSRRAVTLPSHSTFJCLQTRSQTWLESREYTK (SEQ ID NO:3);

VEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMN (SEQ ID NO:4);

LEIRFEECPGTKVHVEETCGTRGPSLRSTTASGRVIEE (SEQ ID NO:5);

EEALRGLPVRYMTTAVNVTHSGTEIVDLMCHATFTS (SEQ ID NO:6)J

RVEMGEAAAIFMTATPPGTRDAFPDSNSPIMDTEVEVPERAWSSGFDW (SEQ ID NO:7); and

GQVLLIAVAVSSAILSRTAWGWGEAGALITAATSTLWEGSPNKYWNSSTATSLCNIF (SEQ ID NO:8).

[0025] In one embodiment, the viral peptides may be detected using a reagent that specifically binds thereto. For example, the viral peptides may be detected via an immunoassay using antibodies that specifically bind to an epitope within the target viral peptides. Antibodies that specifically bind to a target viral peptide may be labelled either prior to or subsequent to target peptide binding, with a label that is itself detectable, or with a label suitable to generate a detectable signal. Detectable labels include, but are not limited to, radioactive, fluorescent, phosphorescent and luminescent (e.g. chemiluminescent or bioluminescent) compounds, dyes, particles such as colloidal gold and enzyme labels. The term "antibody" is used herein to refer to monoclonal or polyclonal antibodies, or antigen-binding fragments thereof, e.g. an antibody fragment that retains specific binding affinity for the target peptide. Antibodies to the target peptides may be prepared using techniques conventional in the art. For example, antibodies may be made by injecting a host animal, e.g. a mouse or rabbit, with the antigen (target peptide), and then isolating antibody from a biological sample taken from the host animal following a sufficient period of time for the host animal to generate antibodies. [0026] Different types of immunoassay may be used to detect the target viral peptides in a saliva sample, including indirect immunoassay in which the target peptide is non-specifically immobilized on a surface; and sandwich immunoassay in which the target peptide is specifically immobilized on a surface by linkage to a capture antibody bound to the surface. For example, Enzyme Linked Immunosorbent Assays (ELISAs) or Enzyme ImmunoAssays (EIA) may be used to detect the viral peptides. In this assay, the peptide to be detected is complexed with an antibody which is linked (either before or following formation of the complex) to an enzyme. Detection may then be accomplished by incubating this enzyme-complex with a substrate, for example, that produces a detectable product. The enzyme may be bound directly to the antibody or bound via a linker, such as a secondary antibody that recognizes the first or primary antibody, or a protein such as streptavidin if the primary antibody is biotin labeled. Examples of suitable enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, acetylcholinesterase and catalase. A large selection of substrates is available for performing the ELISA with an HRP or AP conjugate. Examples of commonly used substrates for HRP include chromogenic substrates, 3,3',5,5'-Tetramethylbenzidine, 3,3'- Diaminobenzidine and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), and chemiluminescent substrates such as luminol. Examples of commonly used substrates for AP include chromogenic substrates, 4-nitrophenyl phosphate and 4-methylumbelliferyl phosphate. Useful substrates depend on the level of detection required and the detection instrumentation used, e.g. spectrophotometer, fluorometer or luminometer.

[0027] It has been determined that identification of at least 4 consecutive amino acids from a target peptide indicates the presence of the target peptide. Thus, techniques sufficient to identify at least 4 consecutive amino acids in a target peptide may be utilized in the present method. In this regard, it is noted that antibodies suitable for the detection of target viral peptides may be directed to epitopes of at least 4 consecutive amino acids within a target viral peptide. Generally, target epitopes are at least about 8 amino acids in length, and may be longer, e.g. 10, 12, 15 or more amino acids in length. Thus, suitable antibodies for use in the present methods need not bind to the full-length of a target viral peptide.

[0028] Tandem mass spectrometry (MS/MS) sequencing of peptides may also be used. This involves ionization of the peptides, generally using electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI). MS/MS then measures peptide fragmentation spectra resulting from collision-induced dissociation of peptide fragments, which may be compared with predicted fragments of peptides from ZIKV proteins. As one of skill in the art will appreciate, a "top-down" approach may be utilized in which intact proteins are ionized for MS/MS sequencing, or a "bottom-up" approach may be used in which the proteins are first enzymatically digested into smaller peptides using a protease such as trypsin. The latter approach, thus, identifies peptides that infer the existence of proteins, as the analysis of the smaller peptide fragments provide increased accuracy. In order to improve accuracy of MS/MS sequencing in samples comprising a complex mixture of multiple proteins and molecules, methods of one- or two-dimensional gel electrophoresis, or high performance liquid chromatography may be used to separate proteins within the sample.

[0029] The present method is useful to identify the presence of Flaviviral peptides in a mammalian saliva sample, and is, therefore, useful to diagnose infection in a mammal by a Flavivirus. The term "mammal" is used herein to refer to human and non-human mammals, e.g. non-human primate species that may be susceptible to infection by a Flavivirus. Flaviviruses are characterized as enveloped viruses with icosahedral and spherical geometries having a diameter of about 40-65 nm, and a linear positive-sense RNA genome which is non-segmented and about 10-1 lkb in length. Examples of Flaviviruses that may be detected using the present method include, but are not limited to, West Nile vims, Dengue vims, tick-borne encephalitis vims, yellow fever virus and Zika vims.

[0030] To specifically distinguish Zika viral infection from infection by other closely related

Flaviviruses (i.e. West Nile and Dengue vimses), the saliva sample is analyzed as described above. Zika infection is confirmed by detection of at least one peptide selected from a first peptide group consisting of:

MDHFSLGVLVILLMVQEGLKKRMTTKIIISTSMAVLVAMILGGFSMSDLAI<:LAILMGATFAEMNTGGDVAHL (SEQ ID NO: l); and

NI VRLKSG VD VFHM A AEPCDTLLCDIGES S S SPEVEE (SEQ ID NO:2);

and detection of at least one peptide selected from a second peptide group consisting of:

WCNTTSTWYVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTK (SEQ ID NO:3);

VEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMN (SEQ ID NO:4);

LEIRFEECPGTKVHVEETCGTRGPSLRSTTASGRVIEE (SEQ ID NO:5);

EEALRGLPVRYMTTAVNVTHSGTEIVDLMCHATFTS (SEQ ID NO:6);

RVEMGEAAAIFMTATPPGTRDAFPDSNSPIMDTEVEVPERAWSSGFDW (SEQ ID NO:7); and

GQVLLIAVAVSSAILSRTAWGWGEAGALITAATSTLWEGSPNKYWNSSTATSLCNIF (SEQ ID NO:8).

The first peptide group consists of Zika peptides which are found in West Nile vims, but not found in Dengue virus, and the second peptide group consists of Zika peptides which are found in Dengue virus, but not in West Nile virus. Thus, identification of a peptide not found in each of these closely related Flaviviruses renders the method specific for Zika virus. [0031] In another aspect of the invention, the saliva sample may be analyzed to determine the presence of Flaviviral peptides by generating a peptide profile of the sample that indicates the abundance of peptides in the sample and the position of each peptide within the Flaviviral polyprotein, e.g. the N- terminal to C-terminal position of each peptide within the Flaviviral polyprotein. Such a peptide profile may be generated using a variety of techniques, including proteomic and peptidomic approaches that may be coupled with a mass spectrometry (MS)-based method useful to identify the peptide profile.

[0032] Suitable MS-based methods for use include all ionization techniques, particularly those techniques suitable for analyzing biological molecules including, but not limited to, direct infusion-mass spectrometry, electrospray ionization (ESI)-MS, desorption electrospray ionization (DESI)-MS, direct analysis in real-time (DART)-MS, atmospheric pressure chemical ionization (APCI)-MS, electron impact (EI) or chemical ionization (CI), matrix-assisted laser desorption/ionization (MALDI)-MS, as well as MS-based methods coupled with a separation technique, such as liquid chromatography (LC- MS), gas chromatography (GC-MS), or capillary electrophoresis (CE-MS) mass spectrometry, and MS- based methods coupled with sequencing techniques, including tandem MS (MS-MS).

[0033] Such an MS-based method is useful to generate a profile of the peptides in the saliva sample that can be compared to a reference peptide profile of the Flaviviral polyprotein sequence to determine the presence or absence and number of Flaviviral peptides in the sample. The presence of Flaviviral peptides resulting from degradation of the Flaviviral polyprotein in the oral cavity (e.g. enzymatic degradation) is confirmed when the sample peptide profile is detemiined to correspond to a reference Flaviviral peptide profile. In this regard, a Flaviviral peptide profile from saliva includes regions abundant in viral peptides (e.g. regions comprising at least about 10 viral peptides), and regions essentially void of viral peptides (e.g. comprising no more than about 4 Flaviviral peptides, e.g. no more than 4, 3, 2 or 1 viral peptide, or essentially no Flaviviral peptides).

[0034] To generate a viral peptide profile, including a Zika peptide profile, a proteomic approach may be used in which the sample is digested with a protease such as trypsin before generating a peptide profile. Alternatively, a peptidomic approach may be used to generate a viral peptide profile in which a low-molecular- weight proteome (<15 kDa) is generated using established techniques including but not limited to centrifugal filtration. Using either approach, viral peptides of about 5-50 amino acids are generated, and preferably, peptides of about 7-45 amino acids in size. A suitable peptide profile comprises greater than about 50 viral peptides, and preferably greater than 1000 viral peptides. [0035] For the diagnosis of a Zika virus, the peptide profile of a saliva sample will include abundant viral peptides (at least 10 peptides) generated from each of the amino acid regions of: 95-180, 185-250, 305-395, 400-495, 575-675, 740-825, 1070-1115, 1150-1230, 1280-1355, 1730-1780, 1825- 1900, 2270-2350, 2430-2500, 2635-2700, 2720-2775 and 2815-2875 of the ZIKV polyprotein, and essentially no viral peptides generated from each of the amino acid regions of: 1-90, 500-550, 875-975, 1445-1505, 1555-1630, 1930-2050, 2200-2265, 2510-2640, 2915-3025 and 3140-3300 of the ZIKV polyprotein. Half of the peptides of the Zika viral peptide profile originate from structural proteins of ZIKV polyprotein other than the capsid protein, and half of the peptides originate from non-structural proteins of the ZIKV polyprotein other than the NS2B protein. The greatest number of Zika viral peptides in the peptide profile originate from the E protein, NS2A, and E-NS1 interface of the ZIKV polyprotein.

[0036] The present diagnostic methods advantageously provide a non-invasive, rapid, and inexpensive means by which to identify Flaviviral infection, including Zika viral infection. Immunoassay-based methods may be readily conducted without requiring highly trained personnel. The use of saliva for diagnosis, in addition to providing a sample that may be obtained non-invasively, also provides a sample with multiple contributors (e.g. glandular secretions, serum and oral cell debris). Identification of viral peptides in saliva is much more sensitive than identification of viral nucleic acid due to high nuclease levels in saliva. The present methods may also be used to distinguish Zika infection from other closely related Flaviviras infections such as Dengue and West Nile. Further, since Zika vims persists in the oral cavity for relatively long periods of time replicating, Zika viral peptides can not only be detected in the saliva on infection (during the acute phase of infection) using the present methods, but can also be detected as late as about 9-months post infectious symptoms, i.e. during the convalescence phase.

[0037] A mammal diagnosed with a Flaviviral infection may then be appropriately treated, by either treating the infection, or minimizing the symptoms associated therewith such as fever, rash, headache and muscle or joint pain. For example, an effective amount of a medication that treats pain and/or fever associated with the infection may be administered to the mammal. Examples of suitable medications include, but are not limited to acetaminophen (Tylenol®), ibuprofen or paracetamol. If the Flaviviral infection may be Dengue fever, non-steroidal anti-inflammatory drugs are not recommended in order to reduce the risk of bleeding.

[0038] Importantly, Zika viral infection in a pregnant woman can be passed onto a fetus during pregnancy. Zika is a cause of microcephaly and other severe fetal brain defects. Thus, diagnosis of Zika infection in either women or men (since Zika may be sexually transmitted) is significant in preventing such fetal defects.

[0039] Embodiments of the present invention are described in the following specific examples which are not to be construed as limiting.

Example 1

[0040] Three women, described here as patients A, B, and C, were assessed at the Irmandade da

Santa Casa de Misericordia de Iacanga and State Hospital of Bauru, Sao Paulo, Brazil, in February 2016. Patients A, B, and C were 29, 50 and 72 years of age, respectively. All three patients presented the following signs and symptoms: headaches, skin irritation, and burning eyes, followed by extensive skin rash and redness associated with joint swelling and pain. Based on these symptoms and the prevalence of mosquito-borne flaviviruses in this community, Dengue and Zika fever were suspected. Serologic tests specific for ZIKV detection were not carried out due to budget limitations. NS1 antigen test for Dengue fever was negative for all three patients. Likewise, IgM tests for Dengue fever were also negative. Finally, a negative result for a Dengue-specific IgG test for patient A was also reported. All of the latter tests for Dengue were performed on patient sera. Haemograms within the first week after the initial clinical assessment showed elevated leucocyte counts, particularly lymphocyte levels, suggesting an ongoing viral infection for patients A and C (Table 1). No significant changes were detected for patient B. Based on clinical symptoms and additional laboratory tests summarized in Table 1, these three patients were diagnosed with Zika fever.

Methods

[0041] Mass spectrometry-based proteomics was conducted to detect and identify proteins/peptides originating from ZIKV in saliva from the three women diagnosed with Zika fever. Saliva was collected 21 days after the patients were diagnosed with Zika fever. Forproteomic analyses, saliva samples were subjected to a linear reversed-phase chromatogram gradient of 85-min ranging from 5% to 55% acetonitrile buffer tandem to mass spectrometry. Peptidomic analyses were carried out targeting only natural occurring ZIKV peptides with molecular weight below 10 kDa. MS/MS data were searched against a ZIKV protein database, and the identified ZIKV peptides were compared to those of different ZIKV strains.

[0042] Saliva collection - Saliva samples were collected at Irmandade da Santa Casa de

Misericordia de lacanga and State Hospital of Bauru, Sao Paulo, Brazil. The three subjects were asked to chew a piece of parafilm (Sigma, 5x5 centimeters, lg), with a frequency of 25-30 times per minute over a 5-min period. At every 30 sec interval for 5 minutes, the subjects expectorated into a tube on ice. Salivary flow rate (ml/min) was calculated at the end of saliva collection. The samples were kept on ice during the collection procedure, and immediately following collection, samples were centrifuged at 14,000 x g for 20 min at 4°C. Whole saliva supernatants (WSS) were separated from the pellet and lyophilized until further analyses. The total protein concentration of WSS was measured by the bicinchoninic acid (BCA) assay (Pierce Chemical, USA) with bovine serum albumin used as the standard. [0043] Classical Proteomic Approach - The equivalent of 20 μg of each WSS sample were dried by a rotary evaporator, denatured and reduced for 2 h by the addition of 50 μΐ of 4 M urea, 10 mM dithiothreitol (DTT), and 50 mM NH₄HC0₃, pH 7.8. The samples were diluted four-fold with 50 mM NH4HCO3, pH 7.8, and in-solution digestion with trypsin was carried out for 16 h at 37°C following the addition of 2% (w/w) sequencing-grade trypsin (Promega, USA). Finally, aliquots from each sample were dried again in a rotary evaporator, de-salted by C¹⁸ Pipette Tips (Millipore, USA) and subjected to mass spectrometry analyses.

[0044] Peptidomic Approach - Samples were also subjected to the peptidomic analysis. The equivalent of 20 μg of WSS samples from each subject were placed into a microcentrifuge tube to be filtered by centrifugal filtration using a 10 kDa molecular weight cut-off (MWCO) membrane (Pall Life Sciences, USA). The samples were centrifuged for 10 min at 14,000xg using a refrigerated eppendorf table-top centrifuge (Eppendorf, Parkway, USA). Next, the filtrates from each subject containing the proteins/peptides with molecular weights below 10 kDa were collected and dried in a rotary evaporator, de-salted by C-18 ZipTip Pipette Tips and subjected to mass spectrometry analyses.

[0045] Mass spectrometric analyses were carried out with an in-line liquid chromatography- containing C¹⁸ reversed-phase column linked to a mass spectrometer using electrospray ionization in a survey scan in the range of m/z values 400-2000 tandem MS/MS. Prior to in-line liquid chromatography, samples were resuspended in 20 μΕ of 97.5% H₂0/2.4% acetonitrile/0.1% formic acid and subjected to reversed-phase liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI- MS/MS). The nano-flow LC was run using a linear 85-minutes gradient ranging from 5% to 55% of a solution containing 80%) acetonitrile/19.9%> water and 0.1%> formic acid at a flow rate of 110 nL/min. The electrospray voltage and ion transfer capillary temperature was set at 1.8 kV and 230°C, respectively. This long-period gradient facilitated peptide/protein separation and consequently improved identification and characterization of ZIKV protein components in saliva. All samples were analyzed in triplicate.

[0046] RT-qPCR - RNA was extracted from saliva using the QiaAMP Viral RNA mini kit as per manufacturer's protocol. Reverse transcription reactions were performed with the Superscript III RT according to manufacturer's protocol using either a random hexamer DNA primer or the ZKV NS5 RT2.1 primer (5'-CCTGAGTTCTCTCTCCCCATCCA -3') (SEQ ID NO:9) specific for the NS5 coding region. cDNA was then subject to external-nested PCR amplification for the NS5 ZIKV coding region using the ZKV NS5 EXT IF (5'- AGGAGGCCCTGGTCATG -3') (SEQ ID NO: 10) and ZKV NS5 EXT1R (5 '-AGAAATCTAGCCCCTAGCCACATATAC -3') (SEQ ID NO:l l) primer pair in the first/external PCR reaction and the ZKV NS5 NST3F (5'- AGGTTCTGGGCTCTAGTGG -3') (SEQ ID NO: 12) and ZKV NS5 NST2R (5'- CCTTGTTTCTTTTCTCTTTTtCCC ATC ATG-3 ' ) (SEQ ID NO: 13) primer pair in the second/nested PCR reaction. An external/nested PCR amplification was also performed on the saliva cDNA using the JD-386 (5'- ACAACTTTGGTATCGTGGAAGG -3') (SEQ ID NO:14) and JD-387 (5'- GCCATCACGCCACAGTTTC -3') (SEQ ID NO:15) primer sets for the GAPDH housekeeping cellular mRNA. Approximately 5μ1 of sample was used for the RT step, external and nested PCR amplifications. All external and nested PCR reaction mixtures were electrophoresed on 0.8% agarose gel and any PCR NS5 and GAPDH products were stained with SybrSafe and visualized with Alphaimager EP. If ZIKV PCR products had been detected, these analyses would have been repeated using real time PCR.

[0047] Database Searches - The acquired MS/MS spectra were searched against a specific ZIKV protein database (Swiss PROT and TREMBL, http://ca.expasy.org) using SEQUEST and Proteome Discoverer 1.3 software (Thermo, USA). Parameters XCorr and ACn were used to validate the presence of a peptide within the sample. The SEQUEST score filter criteria applied to MS/MS spectra were: XCorr score > 1.9, 2.2 or 3.5 for Z = 1 , 2, or 3, respectively, for tryptic peptides. ACn was set to 0.1. A sequence reversed protein database was used as a decoy to evaluate the false positive rate during the search. Furthermore, each result was judged and validated by manual inspection of the MS/MS spectra to confirm and ensure that the fragment ions (e.g. a, b and y ions) were above the background and abundant fragments were assigned by the search.

[0048] Bioinformatics - The identified ZIKV peptides from both proteomic and peptidomic approaches were aligned and mapped to the ZIKV reference polyprotein sequence using the web-based tool Clustal Omega, and visualized with Jalview version 2.8.2. For phylogenetic analysis, patient- specific ZIKV amino acid consensus sequences were generated from the proteomic and peptidomic data with a custom Python script to parse alignment outputs from the database queries and used to filter the MS-derived peptide sequence data. A maximum likelihood phylogeny was reconstructed from the consensus sequences using RAxML.

Results

[0049] Clinical parameters of each patient, salivary flow rates, and total salivary protein concentrations are shown in Table 1. No traces of ZIKV RNA were detected in the saliva supernatant samples by RT-qPCR using two sets of primers/probe specific for ZIKV. Also, 18S rRNA and transcripts for GAPDH were not detected via PCR, suggesting that RNA was not present in the saliva samples. Whereas proteases in saliva may aid in the detection of ZIKV peptides by MS, high levels of extracellular nucleases (e.g. onconase) are antiviral in native saliva and can degrade both viral and cellular RNA during lysis/extraction. It should be noted that samples were collected for these analyses using protocols aimed at preserving proteins.

[0050] The base-peak chromatogram for proteomic analysis showed consistent elution of peptides from 20-45 minutes, demonstrating similarity among peptides present in samples from the three patients. Proteomic analysis identified a total of 68 ZIKV tryptic peptides (51 unique peptides) from the saliva samples (Table 2). Sequence varied in length from 9 to 41 residues (Figure 2). The alignment of these 51 unique peptides indicated that they originated from all regions of the Zika polyprotein, except the capsid and NS2B proteins (Figure 1).

[0051] In contrast to proteomics, the peptidomic approach yielded a much greater number of peptides, which amplified the coverage of the ZIKV polyprotein. A total of 14,335 ZIKV peptides were identified in the saliva samples, of which 2876 peptides were unique (Table 3). Again, identified peptides varied in length, ranging from 7 to 44 amino acid residues, a finding which was very similar and consistent among all patients (Figure 3). Alignment of identified peptides from patients A (623), B (1128), and C (1125) to the ZIKV reference sequence revealed that peptides mapped to all structural and non-structural proteins resulting in an impressive coverage of 77%, 85% and 84% of the viral polyprotein, respectively (Figure 4). Notably, specific regions in the polyprotein exhibited higher frequency of peptides than others, for example, regions of the E protein, NS2A, and E-NS1 interface. As expected, peptides identified by both proteomic and peptidomic approaches shared the same locations on the ZIKV polyprotein. Interestingly, peptidomic analysis revealed that all patients shared similar regions on the polyprotein, which exhibited low/absent coverage (Figure 1). Lastly, consensus sequences were constructed with unique peptides for each patient using JalView, and further aligned to 74 ZIKV strains using SeaView to generate phylogenetic trees. These phylogenetic analyses revealed that the ZIKV present in all three patients was closely related to the ZIKV strain from Nigeria.

Discussion

[0052] The foregoing study shows that ZIKV peptides are detectable in patient saliva 21 days- post symptoms by MS, whereas ZIKV RNA was undetectable. Natively cleaved ZIKV peptides, which represent large segments of the proteome in multiple patients, have been identified. Thus, peptide analyses can be used to test and monitor Zika infection during both the acute and convalescent phases of disease. Second, the native proteomic approach with MS (i.e. peptidomics) yields a ZIKV peptide pool in saliva that essentially covers the complete viral proteome. Third, approximately half of the peptides originated from the structural proteins found in free virus particles, while the other half definitively aligned with non-structural ZIKV proteins, which are found in infected cells.

[0053] The discovery of ZIKV protein/peptides in patient saliva provides a powerful diagnostic approach that can overcome the challenge of cross-reactivity with other Flaviviruses. The identification of multiple peptides from across the ZIKV proteome provides greater specificity and sensitivity for the detection of ZIKV and its discrimination from, in particular, Dengue virus. The prospect of identifying ZIKV peptides in saliva also opens the door for earlier diagnosis, monitoring the progression of disease, and compliance to treatment modalities.

Example 2

Materials and Methods

[0054] A 25-year-old woman (mother) in her first trimester of pregnancy presented clinical symptoms of ZIKV infection, including generalized redness and itching. After medical assessment by an infectious disease physician at the Santa Casa de Misericordia Hospital in Santos, Brazil, she was clinically diagnosed with ZIKV infection. No laboratorial test was performed. Past 6 months from infection onset, she gave birth to dizygotic twins: a female (baby- A) with microcephaly and a male (baby- B) without microcephaly. At the time the twins were 3 -months old, all three patients were assessed, and saliva samples were collected in Santos, Brazil, on March 2016 (9 months after initial infection onset). The patients had no signs and symptoms related to ZIKV infection when saliva was collected.

[0055] Saliva collection from the mother was conducted as described in Example 1. For both baby-A and B, saliva was collected for 3 minutes using a suction device (Siqueira et al. 2005). The salivary flow rate (ml/min) was calculated at the end of saliva collection. Whole saliva supernatants (WSS) were separated from the pellet by centrifugation (14,000 x g for 20 min at 4°C) and lyophilized until further analyses. The total protein concentration of WSS was measured by the bicinchoninic acid assay (Pierce Chemical, USA).

[0056] Samples were subjected to the peptidomic analysis as described in Example 1.

[0057] Samples were applied to a nano-flow reversed-phase HPLC capillary column connected to a LTQ-Velos mass spectrometry as described in Example 1.

[0058] RT-qPCR was conducted as described in Example 1.

[0059] Bioinformatics Analysis - A ZIKV protein database was specifically created for use in this proteomic study. All 74 ZIKV strains isolated from humans were included in this protein database (Swiss PROT and TREMBL). The acquired MS/MS spectra data were searched against the ZIKV protein database using SEQUEST and Proteome Discoverer 1.3 software (Thermo, USA). XCorr and ACn parameters were used to validate the existence of a ZIKV peptide within the sample. A sequence reversed protein database was used as a decoy to evaluate the false positive rate during the search. [0060] Phylogenetic Tree Construction - The identified ZIKV peptides from the peptidomic approach were aligned and mapped to the ZIKV reference polyprotein sequence using the web-based tool Clustal Omega (Sievers et al. 2011. Mol Syst Biol. 7:539), and visualized with Jalview version 2.8.2 (Waterhouse et al. 2009. Bioinformatics. 25(9):1189-1191). For phylogenetic analysis, patient-specific ZIKV amino acid consensus sequences were generated from the peptidomic data using a custom Python script to parse alignment outputs from the database queries used to filter the MS-derived peptide sequence data. A maximum likelihood phylogeny was reconstructed from the consensus sequences using RAxML (Stamatakis 2014. Bioinformatics. 30(9):1312-1313).

Results

[0061] The salivary flow rate was 1.1 ml/min for the mother, 0.2 ml/min for baby-A, and 0.6 ml/min for baby-B. The salivary total protein concentration was 0.8 ± 0.1 mg/ml for the mother, 1.2 ± 0.3 mg/ml for baby-A and 0.9 ± 0.1 mg/ml for baby-B.

[0062] No traces of ZIKV RNA were detected in saliva supernatant samples by RT-qPCR using both sets of primers/probe specific for ZIKV. Also, transcripts of 18S rRNA and GAPDH were not detected via PCR, suggesting that no available RNA was present. It is important to note that samples were collected to preserve proteins first and foremost for the peptidomic analysis.

[0063] Lyophilized saliva samples were subjected to MS/MS as described above for ZIKV peptidomic analyses. A total of 2331, 2630 and 859 ZIKV peptides were detected and mapped on the proteome of which 420, 670 and 183 were unique for the mother, baby-A and baby-B, respectively (Table 4). This extraordinary number of identified peptides were identified in saliva at 9 months following ZIKV acute infection in the mother, which represents 6 months of gestation and 3 months post- delivery for the baby twins. The ZIKV peptides covered 67% (mother), 84% (baby-A), 45% (baby-B) of the ZIKV polyprotein with similar peptide coverage in all the structural and nonstructural proteins (Figure 5). Interestingly, the length and number of peptides found in the saliva in baby-A with microcephaly was significantly greater than observed in the "healthy" baby-B and the mother (Figure 6). The crude difference between baby-A and B may suggest increased viral load associated with microcephaly.

[0064] When a more comprehensive characterization of these peptides were carried out, in terms of the structural vs. non-structural peptides origin; both mother and baby-A demonstrated similar structural/non- structural ratio, whereas baby-B showed a slightly reduced ratio of structural peptides. Specifically, pertaining to the structural peptides (C, PrM and E), a relatively similar distribution in mother and baby-A were noted, whereas there was a reduction in the amount of expressed prM in baby-B (3% in baby-B vs. 7-8% in baby-A and mother). For the non-structural peptides, baby-B had an increase in NS5 (24% vs. 14% mother and 18% baby-A), but with similar values in the other non-structural peptides when compared with mother and baby-A (Figure 7). Despite these slight differences, similar detection of structural and NS proteins could only be related to ongoing ZIKV replication in the vertical transmission group.

[0065] Based on the extensive proteome coverage, robust amino acid alignments and bootstrapped maximum likelihood trees were performed using a 1306 amino acid shared between mother and babies along with 74 reference ZIKV sequences. These analyses showed that the vertical transmission group showed the highest sequence homology (only x amino acid substitutions) and closer in similarity than any other two (or three) ZIKV sequences in the database. The ZIKV sequence from this transmission group was most closely related to the KU963574 (Protein ID AMR68906) strain from Nigeria.

[0066] All amino acid substitutions between the members in this vertical transmission group were mapped and found to be scattered throughout the proteome (Figure 8). Twelve of the 21 mutations observed between mother and one of the two babies were relatively conservative (e.g. Leucine to Isoleucine substitutions) or involved a mutation from mother to babies-A or B to an amino residue found conserved in the ZIKV consensus or was the wild type sequence in the closely related Nigerian strain. Mutations were also mapped where mother and "healthy" baby-B had a sequence different than baby-A with microcephaly, i.e. assuming the mother was infected with a non-neurovirulent strain that mutated in baby-A. Likewise, sequences were scanned for mutations that only appeared in the healthy baby-B assuming the neurovirulent strain was infecting mother and baby-A. Based on these assumptions, two clusters of mutations appearing in the structural Envelope glycoprotein and in the NS4B were identified.

Discussion

[0067] As described herein, ZIKV peptides were detected in the saliva of the mother and her babies 9- months post symptoms by MS whereas ZIKV RNA was undetectable. Thus, peptide analyses could be used to test and monitor Zika infection during both acute and convalescent phases of the disease. Second, the native proteomic approach with MS, i.e. peptidomic, yields a ZIKV peptide pool in saliva that nearly covered the complete viral proteome. Third, approximately half of the peptides originate from the structural proteins found in free virus particles while the other half definitively align with non-structural ZIKV proteins, which are only present during intracellular replication or released from lysed infected cells. [0068] Of course, initial in utero transmission infection of these dizygotic twins hinges on the phylogenetic analyses which shows a closer relationship between the ZIKV proteome sequences of this vertical transmission group than any other ZIKV sequences identified in the database. Furthermore, these sequences were nearly isogenic aside from 21 amino acid substitutions, which often toggled between the mother, babies and the consensus sequence. These findings suggest that virus in the babies did not originate from a subsequent infection post-delivery. There was sufficient divergent evolution in the mother and each baby to suggest an early in utero transmission but does not rule out ongoing transmission within the transmission group, e.g. in utero or during breast feeding. Of greatest interest was the position of mutations between mother/healthy baby-B and the baby-A with microcephaly. Two clusters of mutations were found, one in the Envelope glycoprotein which is the protein most responsible for cell binding and virus entiy and may be associated with a neurotropism. The other cluster of mutations appeared in NS4B. In other related flaviviruses such as Dengue and West Nile viruses, NS4B appears to antagonize intracellular viral immunity by counteracting (i) type I IFN signaling; (ii) RNA interference; and (iii) formation of stress granules. NS4B appears to be a functional homolog to NS5A in hepatitis C virus which is major target of antiviral drugs such as Ledipasvir. Any enhancement of the ZIKV NS4B activity could relate to increased viral replication as well as enhanced neuroviralence.

Example 3 - Identifying Zika peptides in Saliva

[0069] Peptides (generated using the peptidomic method) identified in the saliva samples of several patients infected with ZIKV (from Examples 1 and 2) were aligned and mapped to the ZIKV reference polyprotein sequence using the web-based tool Clustal Omega, and visualized with Jalview version 2.8.2.

[0070] The results are shown in Figure 9 which illustrates abundance of peptides present in the samples originating from the ZIKV polyprotein sequence based on a comparison with a ZIKV reference. As shown, there are regions of the ZIKV polyprotein which generate a relatively high number of peptides, e.g. regions comprising at least 10 peptides originating from the viral polyprotein due to degradation (such as enzymatic degradation) of the polyprotein within the oral cavity, and regions of the ZIKV polyprotein which did not generate any peptides (or essentially no peptides). Amino acid regions of the polyprotein that generate a high level of peptides are listed in Tables 5A/B (see Coverage column), and amino acid regions of the polyprotein that did not generate peptides are identified as No Coverage in the Tables.

Example 4 - Distinguishing between Zika, and West Nile and Dengue viruses

[0071] The Zika peptide sequences identified herein in saliva samples were compared against

West Nile and Dengue polypeptide sequences. A summary of the comparison is provided in Table 6. As can be seen, there are two Zika peptide sequences with no similarity in West Nile peptide sequences, and 5 peptide sequences with no similarity in Dengue peptide sequences. Dengue West Nile

[0072] Thus, identification of a sequence from Group A (not found in West Nile as indicated in

Table 6) and a sequence from Group B (not found in Dengue as indicated in Table 6) will positively identify Zika from these similar Flaviviruses. Position is the position of the sequence in the Zika polypeptide.

Claims

1. A method of detecting Flaviviral peptides in a mammal comprising detecting in a saliva sample obtained from the mammal the presence of at least two peptides selected from the group consisting of:

MDHFSLGVLVILLMVQEGLKKRMTTKIIISTSMAVLVAMILGGFSMSDLAKLAILMGATFAEMNTGGDVAHL;

NIVRLKSGVDVFHMAAEPCDTLLCDIGESSSSPEVEE;

WCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTPvKLQTRSQTWLESREYTK;

VEITPNSPPvAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMN;

LEIRFEECPGTKVHVEETCGTRGPSLRSTTASGRVIEE;

EEALRGLPVRYMTTAVNVTHSGTEIVDLMCHATFTS;

RVEMGEAAAIFMTATPPGTRDAFPDSNSPIMDTEVEVPERAWSSGFDW; and

GQVLLIAVAVSSAILSRTAWGWGEAGALITAATSTLWEGSPNKYWNSSTATSLCNIF.

2. The method of claim 1, wherein the peptides are Zika viral peptides and at least one peptide is detected from the group consisting of:

MDHFSLGVLVILLMVQEGLKKRMTTKIIISTSMAVLVAMILGGFSMSDLAKLAILMGATFAEMNTGGDVAHL; and

NIVRLKSGVDVFHMAAEPCDTLLCDIGESSSSPEVEE;

and at least one peptide is detected from the group consisting of:

WCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTK;

VEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMN;

LEIRFEECPGTKVHVEETCGTRGPSLRSTTASGRVIEE;

EEALRGLPVRYMTTAVNVTHSGTEIVDLMCHATFTS;

RVEMGEAAAIFMTATPPGTRDAFPDSNSPIMDTEVEVPERAWSSGFDW; and

GQVLLIAVAVSSAILSRTAWGWGEAGALITAATSTLWEGSPNKYWNSSTATSLCNIF.

3. The method of claim 1 , wherein the sample is obtained during the acute infection phase or during convalescence phase of infection.

4. The method of claim 1 , wherein the viral peptides are detected by detecting at least 4 consecutive amino acids thereof.

5. A method of diagnosing Flaviviral infection in a mammal comprising the steps of:

i) detecting in a saliva sample obtained from the mammal the presence of at least two viral peptides selected from the group consisting of:

MDHFSLGVLVILLMVQEGLKKRMTTKIIISTSMAVLVAMILGGFSMSDLAKLAILMGATFAEMNTGGDVAHL; NIVRLKSGVDVFHMAAEPCDTLLCDIGESSSSPEVEE;

WCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTK; VEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMN;

LEIRFEECPGTKVHVEETCGTRGPSLRSTTASGRVIEE;

EEALRGLPVRYMTTAVNVTHSGTEIVDLMCHATFTS;

RVEMGEAAAIFMTATPPGTRDAFPDSNSPIMDTEVEVPERAWSSGFDW; and

GQVLLIAVAVSSAILSRTAWGWGEAGALITAATSTLWEGSPNKYWNSSTATSLCNIF,

wherein the peptides are detected using a reagent that specifically binds to at least 4 consecutive amino acids of the viral peptides; and ii) diagnosing the mammal with Flaviviral infection when the presence of the at least two viral peptides is detected.

6. The method of claim 5, wherein the reagent that specifically binds to the peptides is an antibody, or antigen-binding fragment thereof.

7. The method of claim 5, wherein the mammal is diagnosed with a Zika infection when at least one peptide is detected from the group consisting of:

MDHFSLGVLVILLMVQEGLKKRMTTKin^ and

NIVRLKSGVDVFHMAAEPCDTLLCDIGESSSSPEVEE;

and at least one peptide is detected from the group consisting of:

WCNTTSTWWYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTK;

VEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMN;

LEIRFEECPGTKVHVEETCGTRGPSLRSTTASGRVIEE;

EEALRGLPVRYMTTAVNVTHSGTEIVDLMCHATFTS;

RVEMGEAAAIFMTATPPGTRDAFPDSNSPIMDTEVEVPERAWSSGFDW; and

GQVLLIAVAVSSAILSRTAWGWGEAGALITAATSTLWEGSPNKYWNSSTATSLCNIF.

8. A method of diagnosing and treating a Flaviviral infection in a mammal comprising the steps of: i) detecting in a saliva sample obtained from the mammal the presence of at least two viral peptides selected from the group consisting of:

MDHFSLGVLVILLMVQEGLiaCRMTTKIIISTSMAVLVAMILGGFSMSDLAKLAILMGATFAEMNTGGDVAHL; NI VRLKSG VDVFHMA AEPCDTLLCDIGES SS SPEVEE;

WCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTK;

VEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMN;

LEIRFEECPGTKVHVEETCGTRGPSLRSTTASGRVIEE;

EEALRGLPVRYMTTAVNVTHSGTEIVDLMCHATFTS;

RVEMGEAAAIFMTATPPGTRDAFPDSNSPIMDTEVEVPERAWSSGFDW;

GQVLLIAVAVSSAILSRTAWGWGEAGALITAATSTLWEGSPNKYWNSSTATSLCNIF;

ii) diagnosing the mammal with Flaviviral infection when the presence of at least 4 consecutive amino acids from at least two of the viral peptides is detected; and iii) administering to the mammal an effective amount of a medication that treats pain and/or fever.

9. The method of claim 8, wherein the medication is selected from acetaminophen, ibuprofen or paracetamol.

10. The method of claim 8, wherein the mammal is diagnosed with Zika infection when at least one peptide is detected from the group consisting of:

MDHFSLGVLVILLMVQEGLKKRMTTKIIISTSMAVLVAMILGGFSMSDLAKLAILMGATFAEMNTGGDVAHL; and

NI VRLKSG VD VFHMA AEPCDTLLCDIGES S S SPEVEE;

and at least one peptide is detected from the group consisting of:

WCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRI<:LQTRSQTWLESREYTK;

VEITPNSPRAEATLGGFGSLGLDCEPPVTGLDFSDLYYLTMN;

LEIRFEECPGTKVHVEETCGTRGPSLRSTTASGRVIEE;

EEALRGLPVRYMTTAVNVTHSGTEIVDLMCHATFTS;

RVEMGEAAAIFMTATPPGTRDAFPDSNSPIMDTEVEVPERAWSSGFDW; and

GQVLLIAVAVSSAILSRTAWGWGEAGALITAATSTLWEGSPNKYWNSSTATSLCNIF.

11. A method of detecting Zika viral peptides in a mammalian saliva sample comprising the steps of: i) generating a peptide profile of the sample, wherein the profile identifies viral peptides by the amino acid region within a Zika viral polyprotein from which each peptide is generated and the number of peptides generated from each region of the Zika viral polyprotein; and

ii) detecting the presence of Zika viral peptides in the sample when the peptide profile of the sample comprises at least 10 viral peptides generated from each of the amino acid regions of: 95-180, 185-250, 305-395, 400-495, 575-675, 740-825, 1070-1115, 1150-1230, 1280-1355, 1730-1780, 1825- 1900, 2270-2350, 2430-2500, 2635-2700, 2720-2775 and 2815-2875 of the Zika viral polyprotein, and is essentially void of viral peptides in each of amino acid regions of: 1-90, 500-550, 875-975, 1445-1505, 1555-1630, 1930-2050, 2200-2265, 2510-2640, 2915-3025 and 3140-3300 of the Zika viral polyprotein.

12. The method of claim 11 , which is a mass spectrometry (MS)-based method.

13. The method of claim 12, wherein the MS-based method is tandem MS .

14. The method of claim 12, wherein the MS-based method is selected from the group consisting of direct infusion-mass spectrometry, electrospray ionization (ESI)-MS, desorption electrospray ionization (DESI)-MS, direct analysis in real-time (DART)-MS, atmospheric pressure chemical ionization (APCI)- MS, electron impact (EI) or chemical ionization (CI) MS, matrix-assisted laser desorption/ionization (MALDI)-MS, and MS-based methods coupled with one of liquid chromatography (LC-MS), gas chromatography (GC-MS), or capillary electrophoresis (CE-MS) mass spectrometry.

15. The method of claim 11, wherein the sample is obtained during the acute infection phase.

16. The method of claim 11 , wherein the sample is obtained during convalescence phase of infection.

17. The method of claim 11, where half of the peptides originated from the structural proteins of ZIKV polyprotein, and half of the peptides originated from the ZIKV polyprotein.

18. The method of claim 17, wherein the Zika viral peptides originate from the structural proteins of ZIKV other than the capsid protein and from the non-structural proteins of ZIKV other than the NS2B protein.

19. The method of claim 17, wherein the greatest number of Zika viral peptides originate from the E protein, NS2A, and E-NS1 interface of the ZIKV polyprotein.

20. The method of claim 11, wherein a proteomic analysis is used in which the sample is digested with a protease before generating a peptide profile.

21. The method of claim 20, wherein the protease is trypsin.

22. The method of claim 11 , wherein a peptidomic analysis is used in which the sample is subjected to centrifugal filtration using a 10 kDa molecular weight cut-off before generating a peptide profile.

23. The method of claim 11, wherein the viral peptides of the peptide profile are about 5-50 amino acids in size.

24. The method of claim 23, wherein the viral peptides are about 7-45 amino acids in size.

25. The method of claim 11, wherein the peptide profile comprises greater than about 50 viral peptides, and preferably greater than 1000 viral peptides.