WO2021173597A1

WO2021173597A1 - Dengue serotype specific rt-pcr multiplex assay

Info

Publication number: WO2021173597A1
Application number: PCT/US2021/019306
Authority: WO
Inventors: Beth A. Arnold; Joseph M. ANTONELLO; Brad R. EVAN; Sabrina GOZLAN KELNER; Kate L. HICKS; Sanjiv Ghanshani; Nathalie Lauren A. MORALES
Original assignee: Merck Sharp & Dohme Corp.
Priority date: 2020-02-28
Filing date: 2021-02-24
Publication date: 2021-09-02

Abstract

The present invention provides an RT-PCR multiplex assay to detect, differentiate and quantify dengue vaccine and wild type dengue viremia by serotype. The invention further provides methods, kits, primers and probes.

Description

TITLE OF THE INVENTION

DENGUE SEROTYPE SPECIFIC RT-PCR MULTIPLEX ASSAY

FIELD OF INVENTION

The present invention provides an RT-PCR multiplex assay to detect, quantify and differentiate dengue virus vaccine and wild type dengue viremia by serotype. The invention further provides methods, kits, primers and probes.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of the present application is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name 24945-WO-PCT- SEQLIST-04FEB2021.txt, creation date of February 4, 2021, and a size of 5.47 kb. This sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

The family Flaviviridae includes the prototype yellow fever virus (YF), the four serotypes of dengue virus (DENV1, DENV2, DENV3, and DENV4), Japanese encephalitis virus (JE), tick-borne encephalitis virus (TBE), West Nile virus (WN), Saint Louis encephalitis virus (SLE), and about 70 other disease causing viruses. Flaviviruses are small, enveloped viruses containing a single, positive-strand RNA genome. Ten gene products are encoded by a single open reading frame and are translated as a polyprotein organized in the order: capsid (C), “preMembrane” (prM, which is processed to “Membrane” (M) just prior to virion release from the cell), “envelope” (E), followed by non-structural (NS) proteins NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5 (reviewed in Chambers, T. J. et al. , Annual Rev Microbiol (1990) 44:649-688; Henchal, E. A. and Putnak, J. R., Clin Microbiol Rev. (1990) 3:376-396). Individual flaviviral proteins are then produced through precise processing events mediated by the host as well as virally encoded proteases.

The envelope of flaviviruses is derived from the host cell membrane and contains the virally-encoded membrane anchored membrane (M) and envelope (E) glycoproteins. The E glycoprotein is the largest viral structural protein and contains functional domains responsible for cell surface attachment and intra-endosomal fusion activities. It is also a major target of the host immune system, inducing the production of virus neutralizing antibodies, which are associated with protective immunity.

Dengue viruses are transmitted to humans by mosquitoes of the genus Aedes, primarily A. aegypti and A. albopictus. Infection by dengue viruses leads to a diverse clinical picture ranging from an inapparent or mild febrile illness, through classical dengue fever (DF), to dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS).

DF is characterized by high fever, headache, joint and muscle pain, rash, lymphadenopathy and leucopenia (Gibbons, R. V. and D. W. Vaughn, British Medical Journal (2002) 324: 1563-1566). DHF/DSS is a more severe form of infection more common in children, marked by vascular permeability and/or severe hemorrhagic manifestations ranging from the presence of petechiae and ecchymosis to spontaneous severe hemorrhage and profound shock. Without diagnosis and prompt medical intervention, the sudden onset and rapid progression of DHF/DSS can be fatal if untreated.

Dengue viruses are the most significant group of arthropod-transmitted viruses in terms of global morbidity and mortality with an estimated one hundred million dengue infections occurring annually including at least 36 million cases of DF and 250,000 to 500,000 cases of DHF/DSS (Gubler, D. I, Clin. Microbiol. Rev. (1998)

11:480-496; Gibbons, supra). With the global increase in population, urbanization of the population especially throughout the tropics, and the lack of sustained mosquito control measures, the mosquito vectors of dengue have expanded their distribution throughout the tropics, subtropics, and some temperate areas, bringing the risk of dengue infection to over half the world’s population. Modem jet travel and human emigration have facilitated global distribution of dengue serotypes, such that multiple serotypes of dengue are now endemic in many regions. There has been an increase in the frequency of dengue epidemics and the incidence of DHF/DSS in the last 20 or more years. For example, in Southeast Asia, DHF/DSS is a leading cause of hospitalization and death among children (Gubler, supra,· Gibbons and Vaughn, supra).

To date, the development of flavivirus vaccines has been met with mixed success. There are four basic approaches that have been implemented in an effort to produce vaccine candidates to protect against disease caused by flaviviruses: live- attenuated, inactivated whole virus, recombinant subunit protein, and DNA-based vaccines. A live-attenuated vaccine for yellow fever virus has been available for decades and more recently a live attenuated vaccine for Japanese encephalitis has been registered in various countries around the world. The use of inactivated whole virus vaccines has been demonstrated for TBE and JE viruses with several registered products available (Heinz et al. Flavivirus and flavivirus vaccines. Vaccine 30: 4301-06 (2012)).

Despite the successes of the YF, JE, and TBE vaccines highlighted above, the use of live-attenuated virus and inactivated virus methods to develop vaccines for dengue virus has been met with significant challenges. There are four serotypes of dengue virus (DENV1, DENV2, DENV3, and DENV4) and strains of each serotype are found circulating throughout the dengue endemic regions of the world. Natural infection confers long lasting immunity to the infecting serotype but not to other dengue serotypes. The more severe forms of the disease (DHF/DSS) occur most often after secondary dengue infection, when infection with one serotype of dengue virus is followed by a second infection with another serotype. The more frequent association of DHF and DSS with secondary dengue infection has been hypothesized to be due to non-neutralizing antibodies induced by infection with one virus type enhancing infectivity of a second dengue virus type (antibody-dependent enhancement - ADE).

To date, the majority of the vaccines tested clinically are live, attenuated vaccines. The use of non-replicating vaccine candidates is also being explored. For example, Ivy et al. (US 6432411) disclose atetravalent subunit vaccine comprising DENI-4 80% E (the peptide region of DEN 1-4 corresponding to amino acids 1-395 of the DENV2 envelope polypeptide) proteins. Ivy et al, supra, also report compositions comprising DENV1-4 80% E and ISCOMATRIX® adjuvant. Coller et al. (WO 2012/154202) disclose tetravalent formulations comprising DENI -4 80% E of DENI -4. Inactivated viruses may also be used as potential vaccine candidates or as components of an effective vaccine (Putnak et al.

Vaccine 23: 4442-4452 (2005), US 6190859, US 6254873 and Sterner et al. WO 2007/002470). Compositions comprising a live attenuated dengue virus vaccine and a non- replicating dengue vaccine are also disclosed (WO 2014/204892).

More severe disease in a subset of patients following secondary dengue infection led to the development of an ADE theory (S.B. Halstead, Yale J Biol Med., 42 (1970), pp. 350-362) According to the theory, infection with dengue virus induces the production of neutralizing homotypic IgG antibodies which provide lifelong immunity against the infecting virus serotype but also produces some cross-protective immunity against the other three virus serotypes. In subsequent infections, cross-reactive antibodies facilitate uptake and replication of the virus leading to more severe Dengue, ultimately DHF/DSS. Therefore, an ideal dengue vaccine should generate high and balanced efficacy/immunity across all serotypes to minimize risk of ADE linked to vaccination. Previous studies showed that the presence of vaccine viremia may correlate with presence of type-specific virus neutralizing responses (G.D. Gromowski. el al. Vaccine, 36 (2018), pp. 2403-2410). Therefore, development of an assay to detect and quantify vaccine and wild type dengue viremia is not only important from a safety and/or diagnostic perspective but is an indicator of vaccine take and therefore could be an indication of potential efficacy.

SUMMARY OF THE INVENTION

An RT-PCR multiplex assay has been developed to distinguish dengue virus vaccine from wild-type dengue viruses as well as discriminate and quantify each of the four dengue virus serotypes, regardless of wild-type or dengue virus vaccine serotype, in sera from individuals. The assay consists of high-throughput total nucleic acid purification followed by four real-time reverse-transcription polymerase chain reactions. In one embodiment, viral nucleic acids are isolated from serum samples using a magnetic-bead based extraction procedure performed on the MagNA Pure 96 instrument. In a further embodiment, target amplification and detection are accomplished using TaqMan chemistry on the Applied Biosystems QuantStudio™ 6 Flex Real Time PCR System (QS6).

Dengue virus wild-type (DENV-WT) and dengue virus vaccine (DENV-Vacc) RT-PCR reactions are performed first. These reactions qualitatively determine if a test sample is positive for wild-type and/or dengue virus vaccine. The DENV-WT RT-PCR reaction targets a D30 sequence deleted in the 3’-UTR of dengue virus vaccines to selectively detect all wild-type serotypes. The DENV-Vacc RT-PCR reaction bridges the D30 deletion present only in dengue virus vaccines to selectively detect the four dengue virus vaccine serotypes. The DENV-WT and DENV-Vacc RT-PCR reactions are duplex assays in that they also detect a process control (MS2 RNA phage) spiked into each raw sample (i.e. a clinical specimen), to ensure successful nucleic acid extraction and RT-PCR amplification.

If a sample is determined to contain wild-type dengue virus and/or vaccine virus by the DENV-WT and DENV-Vacc RT-PCR reactions, the extracted nucleic acids are then subjected to a DENV1/3 and a DENV2/4 RT-PCR reaction. The DENV1/3 and DENV2/4 RT-PCR reactions determine serotype identity and quantity, and are used to quantify the total viral load of a sample by extrapolation from the appropriate external standard curve generated from each of the four wild-type dengue virus and vaccine calibrators run in these assays. The DENV1 and DENV3 serotype-specific primers and probes in the duplex DENV1/3 (dengue virus serotypes 1 and 3) RT-PCR reaction target RNA sequence encoding the nucleocapsid (C) and non-structural 2B (NS2B) proteins, respectively, and are designed for reliable quantification of DENV1 and DENV3 serotypes, irrespective of wild-type dengue virus or vaccine. The DENV2 and DENV4 serotype- specific primers and probes in the duplex DENV2/4 (dengue virus serotypes 2 and 4) RT- PCR reaction both target RNA sequence encoding the envelope (E) protein and are designed for reliable quantification of DENV2 and DENV4 serotypes, irrespective of wild-type dengue virus or vaccine.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an RT-PCR multiplex assay for detecting wild- type dengue virus and dengue virus vaccine or differentiating between wild-type dengue virus and dengue virus vaccine in a sample comprising: a) a high-throughput nucleic acid purification step; b) one or more amplification steps comprising real-time reverse- transcription polymerase chain reactions; and c) a detection step. In embodiments of the assay, the amplification step comprises a first reaction wherein DENV-Vacc and DENV-WT RT-PCR reactions are performed to determine if wild-type dengue virus and/or dengue virus vaccine is present. If wild-type dengue virus and/or dengue virus vaccine is detected, a second reaction is performed wherein extracted nucleic acids from the first reaction (or newly extracted and from a different reaction) are then subjected to DENV1/3 and DENV2/4 RT- PCR reactions to determine serotype identity and quantity, and are used to quantify the total viral load of a sample. In further embodiments of the assay, the DENV-Vacc and DENV-WT RT-PCR reactions comprise amplifying nucleic acid using DENV-Vacc forward primer (SEQ ID NO: 1), DENV-Vacc reverse primer (SEQ ID NO: 2), DENV-Vacc probe (SEQ ID NO:

3), DENV 1-WT forward primer (SEQ ID NO: 4), DENV 2-WT forward primer (SEQ ID NO: 5), DENV 3-WT forward primer (SEQ ID NO: 6), DENV 4-WT forward primer (SEQ ID NO: 7), DENV-WT reverse primer (SEQ ID NO: 8), and DENV-WT probe (SEQ ID NO: 9). In further embodiments of the assay, the DENV1/3 and DENV2/4 RT-PCR reactions comprise amplifying nucleic acid using DENV1 forward primer (SEQ ID NO: 10), DENV1 reverse primer (SEQ ID NO: 11), DENV1 probe (SEQ ID NO: 12), DENV3 forward primer (SEQ ID NO: 13), DENV 3 reverse primer (SEQ ID NO: 14), DENV 3 probe (SEQ ID NO: 15), DENV2 forward primer (SEQ ID NO: 16), DENV2 reverse primer (SEQ ID NO: 17), DENV2 probe (SEQ ID NO: 18), DENV4 forward primer (SEQ ID NO: 19), DENV4 reverse primer (SEQ ID NO: 20), and DENV4 probe (SEQ ID NO: 21).

The present invention also provides methods of detecting wild-type dengue virus and dengue virus vaccine or differentiating between wild-type dengue virus and dengue virus vaccine in a sample utilizing a RT-PCR multiplex assay comprising: a) a high- throughput nucleic acid purification step; b) one or more amplification steps comprising real time reverse-transcription polymerase chain reactions; and c) a detection step. In an embodiment, the amplification step comprises a first reaction wherein DENV-Vacc and DENV-WT RT-PCR reactions are performed to determine if wild-type dengue virus and/or dengue virus vaccine is present. If wild-type dengue virus and/or dengue virus vaccine is detected, a second reaction is performed wherein extracted nucleic acids from the first reaction are then subjected to DENV1/3 and DENV2/4 RT-PCR reactions to determine serotype identity and quantity, and are used to quantify the total viral load of a sample. In another embodiment, the DENV-Vacc and DENV-WT RT-PCR reactions comprise amplifying nucleic acid using DENV-Vacc forward primer (SEQ ID NO: 1), DENV-Vacc reverse primer (SEQ ID NO: 2), DENV-Vacc probe (SEQ ID NO: 3), DENV 1-WT forward primer (SEQ ID NO: 4), DENV 2-WT forward primer (SEQ ID NO: 5), DENV 3-WT forward primer (SEQ ID NO: 6), DENV 4-WT forward primer (SEQ ID NO: 7), DENV-WT reverse primer (SEQ ID NO: 8), and DENV-WT probe (SEQ ID NO: 9). In another embodiment, the DENV1/3 and DENV2/4 RT-PCR reactions comprise amplifying nucleic acid using DENV1 forward primer (SEQ ID NO: 10), DENV1 reverse primer (SEQ ID NO:

11), DENV1 probe (SEQ ID NO: 12), DENV 3 forward primer (SEQ ID NO: 13), DENV 3 reverse primer (SEQ ID NO: 14), DENV3 probe (SEQ ID NO: 15), DENV2 forward primer (SEQ ID NO: 16), DENV2 reverse primer (SEQ ID NO: 17), DENV2 probe (SEQ ID NO: 18), DENV4 forward primer (SEQ ID NO: 19), DENV4 reverse primer (SEQ ID NO: 20), and DENV4 probe (SEQ ID NO: 21).

In another embodiment the invention provides a RT-PCR multiplex assay for differentiating between wild-type dengue virus and dengue virus vaccine in a sample comprising: a) a high-throughput nucleic acid purification step; b) one or more amplification steps comprising real-time reverse-transcription polymerase chain reactions; and c) a detection step. In another embodiment, the amplification step comprises a first reaction wherein DENV-Vacc and DENV-WT RT-PCR reactions are performed to determine if wild- type dengue virus and/or dengue virus vaccine is present. If wild-type dengue virus and/or dengue virus vaccine is detected, a second reaction is performed wherein extracted nucleic acids from the first reaction (or newly extracted from a different reaction) are then subjected to DENV1/3 and DENV2/4 RT-PCR reactions to determine serotype identity and quantity, and are used to quantify the total viral load of a sample. In another embodiment, the DENV- Vacc and DENV-WT RT-PCR reactions comprise amplifying nucleic acid using DENV- Vacc forward primer (SEQ ID NO: 1), DENV-Vacc reverse primer (SEQ ID NO: 2), DENV- Vacc probe (SEQ ID NO: 3), DENV 1-WT forward primer (SEQ ID NO: 4), DENV 2-WT forward primer (SEQ ID NO: 5), DENV 3-WT forward primer (SEQ ID NO: 6), DENV 4- WT forward primer (SEQ ID NO: 7), DENV-WT reverse primer (SEQ ID NO: 8), and DENV-WT probe (SEQ ID NO: 9). In another embodiment, the DENV1/3 and DENV2/4 RT-PCR reactions comprise amplifying nucleic acid using DENV1 forward primer (SEQ ID NO: 10), DENV1 reverse primer (SEQ ID NO: 11), DENV1 probe (SEQ ID NO: 12),

DENV3 forward primer (SEQ ID NO: 13), DENV3 reverse primer (SEQ ID NO: 14),

DENV 3 probe (SEQ ID NO: 15), DENV2 forward primer (SEQ ID NO: 16), DENV2 reverse primer (SEQ ID NO: 17), DENV2 probe (SEQ ID NO: 18), DENV4 forward primer (SEQ ID NO: 19), DENV4 reverse primer (SEQ ID NO: 20), and DENV4 probe (SEQ ID NO: 21).

In another embodiment, the invention provides a method of differentiating between wild-type dengue virus and dengue virus vaccine in a sample utilizing a RT-PCR multiplex assay comprising: a) a high-throughput nucleic acid purification step; b) one or more amplification steps comprising real-time reverse-transcription polymerase chain reactions; and c) a detection step. In another embodiment, the amplification step comprises a first reaction wherein DENV-Vacc and DENV-WT RT-PCR reactions are performed to determine if wild-type dengue virus and/or dengue virus vaccine is present. If wild-type dengue virus and/or dengue virus vaccine is detected, a second reaction is performed wherein extracted nucleic acids from the first reaction are then subjected to DENV1/3 and DENV2/4 RT-PCR reactions to determine serotype identity and quantity, and are used to quantify the total viral load of a sample. In a further embodiment, the DENV-Vacc and DENV-WT RT- PCR reactions comprise amplifying nucleic acid using DENV-Vacc forward primer (SEQ ID NO: 1), DENV-Vacc reverse primer (SEQ ID NO: 2), DENV-Vacc probe (SEQ ID NO: 3), DENV 1-WT forward primer (SEQ ID NO: 4), DENV 2-WT forward primer (SEQ ID NO:

5), DENV 3-WT forward primer (SEQ ID NO: 6), DENV 4-WT forward primer (SEQ ID NO: 7), DENV-WT reverse primer (SEQ ID NO: 8), and DENV-WT probe (SEQ ID NO: 9). In another embodiment, the DENV1/3 and DENV2/4 RT-PCR reactions comprise amplifying nucleic acid using DENV1 forward primer (SEQ ID NO: 10), DENV1 reverse primer (SEQ ID NO: 11), DENV1 probe (SEQ ID NO: 12), DENV 3 forward primer (SEQ ID NO: 13), DENV 3 reverse primer (SEQ ID NO: 14), DENV 3 probe (SEQ ID NO: 15), DENV2 forward primer (SEQ ID NO: 16), DENV2 reverse primer (SEQ ID NO: 17), DENV2 probe (SEQ ID NO: 18), DENV4 forward primer (SEQ ID NO: 19), DENV4 reverse primer (SEQ ID NO: 20), and DENV4 probe (SEQ ID NO: 21). In another embodiment, the present invention provides a kit for detecting wild- type dengue virus and/or dengue virus vaccine or differentiating between wild-type dengue virus and dengue virus vaccine in a sample, the kit comprising the primers and probes selected from (SEQ ID NOs: 1-21) and instructions for use. In another embodiment, the present invention provides a kit for detecting and quantifying wild-type dengue virus and/or dengue virus vaccine serotypes DENV1, DENV2, DENV3 and/or DENV4 in a sample, the kit comprising the primers and probes selected from (SEQ ID NOs: 1-21) and instructions for use.

In another embodiment the invention provides an RT-PCR multiplex assay and methods of using thereof wherein the DENV-WT RT-PCR reaction targets a D30 sequence, deleted in the 3’-UTR of dengue virus vaccines, to selectively detect all wild-type dengue virus serotypes.

In another embodiment the invention provides an RT-PCR multiplex assay and methods of using thereof wherein the DENV-Vacc RT-PCR reaction bridges the D30 deletion present only in dengue virus vaccines to selectively detect the four dengue virus vaccine serotypes.

In another embodiment the invention provides an RT-PCR multiplex assay and methods of using thereof wherein the DENV1/3 RT-PCR reaction contains DENV1 and DENV3 serotype-specific primers and probes which target RNA sequence encoding the nucleocapsid (C) and non-structural 2B (NS2B) proteins present in wild-type dengue virus or dengue virus vaccine.

In another embodiment the invention provides an assay wherein the DENV2/4 RT-PCR reaction contains DENV2 and DENV4 serotype-specific primers and probes which target RNA sequence encoding the envelope (E) protein present in wild-type dengue virus or dengue virus vaccine.

In another embodiment of the invention, the serotype-specific primers and probes are fluorescently labeled. Preferred labeling includes 6-carboxyfluorescein (FAM), ABY, 2'-chloro-7'phenyl-l,4-dichloro-6-carboxy-fluorescein (VIC) and tetrachlorofluorescein (TET) with tetramethylrhodamine (TAMRA) or QSY quenchers or structure modifications such as minor groove binder (MGB) with nonfluorescent quencher (NFQ) probes.

In another embodiment the invention provides for alternative fluorescent dyes attached to DENV1, DENV2, DENV3 and/or DENV4 serotype specific probes and multiplex dye combinations, as well as alternate quenchers or structural modifications to mask fluorescence while the probe is intact.

In another embodiment the invention provides an RT-PCR multiplex assay that comprises a DENV-Vacc and a DENV-WT RT-PCR reaction, wherein the DENV-WT RT-PCR reaction targets a 30 base pair region present in the 3’-UTR of wild-type dengue virus RNA sequences, which has been deleted in the 3’-UTR of dengue virus vaccines, to selectively detect wild-type dengue virus serotypes; and wherein the DENV-Vacc RT-PCR reaction bridges the D30 deletion present only in a dengue virus vaccine to selectively detect the four dengue virus vaccine serotypes.

In another embodiment the assay further comprises a DENV1/3 and a DENV2/4 RT-PCR reaction.

In another embodiment, the DENV1/3 RT-PCR reaction contains DENV1 and DENV3 serotype-specific primers and probes which target RNA sequences encoding the nucleocapsid (C) and non-structural 2B (NS2B) proteins.

In another embodiment, the DENV2/4 RT-PCR reaction contains DENV2 and DENV4 serotype-specific primers and probes which target RNA sequences encoding the envelope (E) protein.

In another embodiment, the DENV-Vacc and DENV-WT RT-PCR reactions comprise the following primers and probes: DENV-Vacc forward primer (SEQ ID NO: 1), DENV-Vacc reverse primer (SEQ ID NO: 2), DENV-Vacc probe (SEQ ID NO: 3), DENV 1- WT forward primer (SEQ ID NO: 4), DENV 2-WT forward primer (SEQ ID NO: 5), DENV 3-WT forward primer (SEQ ID NO: 6), DENV 4-WT forward primer (SEQ ID NO: 7), DENV-WT reverse primer (SEQ ID NO: 8), and DENV-WT probe (SEQ ID NO: 9).

In another embodiment, the DENV1/3 and DENV2/4 RT-PCR reactions comprise the following primers and probes: DENV 1 forward primer (SEQ ID NO: 10), DENV 1 reverse primer (SEQ ID NO: 11), DENV 1 probe (SEQ ID NO: 12), DENV 3 forward primer (SEQ ID NO: 13), DENV 3 reverse primer (SEQ ID NO: 14), DENV 3 probe (SEQ ID NO: 15), DENV 2 forward primer (SEQ ID NO: 16), DENV 2 reverse primer (SEQ ID NO: 17), DENV 2 probe (SEQ ID NO: 18), DENV 4 forward primer (SEQ ID NO: 19), DENV 4 reverse primer (SEQ ID NO: 20), and DENV 4 probe (SEQ ID NO: 21).

In another embodiment, the invention provides an assay which comprises the following steps: (a) a nucleic acid purification step comprising purifying nucleic acid from a sample; (b) one or more amplification steps comprising amplifying the nucleic acid purified in step (a) using reverse-transcription polymerase chain reactions (RT-PCR reactions); (c) a detection step comprising detecting and differentiating wild-type dengue virus and/or dengue virus vaccine in the sample; and (d) an optional differentiation and quantification step comprising differentiating and quantifying between DENV1, DENV2, DENV3 and/or DENV4 in the sample; wherein the RT-PCR reactions in step (b) comprise DENV-Vacc and DENV-WT RT-PCR reactions, and step (d) comprises DENV1/3 and DENV2/4 RT-PCR reactions; wherein the DENV-WT RT-PCR reaction targets a 30 base pair region present in the 3’-UTR of wild-type dengue virus RNA sequences that has been deleted in the 3’-UTR of dengue virus vaccines to selectively detect wild-type dengue virus serotypes; and wherein the DENV-Vacc RT-PCR reaction bridges the D30 deletion present only in dengue virus vaccines to selectively detect the four dengue virus vaccine serotypes.

In another embodiment the invention provides a method of detecting wild-type dengue virus and/or dengue virus vaccine in a sample utilizing the assays described above.

In another embodiment the invention provides a method of differentiating between wild-type dengue virus and dengue virus vaccine in a sample utilizing the assays described above. In another embodiment the invention provides a method of quantifiying total dengue viral load of a sample utilizing the assays described above.

In another embodiment the invention provides a kit for detecting wild-type dengue virus and/or dengue virus vaccine in a sample, the kit comprising the primers and probes selected from (SEQ ID NOs: 1-21) and instructions for use.

In another embodiment the invention provides a kit for differentiating between wild-type dengue virus and dengue virus vaccine in a sample, the kit comprising the primers and probes selected from (SEQ ID NOs: 1-9) and instructions for use.

In another embodiment, the present invention provides a kit for detecting and quantifying wild-type dengue virus and/or dengue virus vaccine serotypes DENV1, DENV2, DENV3 and/or DENV4 in a sample, the kit comprising the primers and probes selected from (SEQ ID NOs: 1-21) and instructions for use.

The present invention also provides novel primers and probes:

DENV-Vacc RT-PCR:

Forward primer - DENV-Vacc (Example section reference: DVVaccSpecificFor2)

5 -MAR SGG GGC CCA AGA CT-3’ (SEQ ID NO: 1);

Reverse primer - DENV-Vacc (Example section reference: DVUnivRev2)

5’-RGA GAC AGC AGG ATC TCT GGT CT-3’ (SEQ ID NO: 2); and

Probe (TaqMan) - DENV-Vacc (Example section reference: DVUnivProbe2)

5 ’-F AM- AAA CAG CAT ATT GAC GCT GG-NFQ/MGB 3’ (SEQ ID NO: 3).

DENV-WT RT-PCR:

Forward primer - DENV1-WT (Example section reference: DVlWTSpecificFor)

5’-GGG AAG CTG TAC CCT GGT GGT-3’ (SEQ ID NO: 4);

Forward primer - DENV2-WT (Example section reference: DV2WTSpecificFor)

5’-CCA AGG YGA GAT GAA GCT GTA GTC TC-3’ (SEQ ID NO: 5);

Forward primer - DENV3-WT (Example section reference: DV3WTSpecificFor)

5 ’-GAG CAC TGA GGG AAG CTG TAC CTC-3’ (SEQ ID NO: 6);

Forward primer - DENV4-WT (Example section reference: DV4WTSpecificFor)

5 ’-CAG GAG GAA GCT GTA CTC CTG GTG-3’ (SEQ ID NO: 7);

Reverse primer - DENV-WT (Example section reference: DVUnivRevl)

5’-TCC CAG CGT CAA TAT GCT GTT-3’ (SEQ ID NO: 8); and

Probe (TaqMan) - DENV-WT (Example section reference: DVUnivProbel)

5’-FAM-ACT AGA GGT TAG AGG AGA CC-NFQ/MGB 3’ (SEQ ID NO: 9). DENY 1/3 RT-PCR:

Forward primer - DENV1 (Example section reference: DVlUnivFor3)

5 -TCT TTC AAT ATG CTG AAA CGC GC-3’ (SEQ ID NO: 10);

Reverse primer - DENV1 (Example section reference: DVlUnivRev3)

5’-TCA TGG GTC CTT GGC CTG A-3’ (SEQ ID NO: 11);

Probe (TaqMan) - DENV1 (Example section reference: DVlUnivProbe3)

5’-FAM-CGT GTC AAC TGT TTC AC-NFQ/MGB 3’ (SEQ ID NO: 12);

Forward primer - DENV3 (Example section reference: DV3UnivFor2)

5’-CAC GTC AGC AGA CCT CAC TGT AGA A-3’ (SEQ ID NO: 13);

Reverse primer - DENV3 (Example section reference: DV3UnivRev2)

5’-GTT CCA TCR TCA TCA ACT GTG ATC AT-3’ (SEQ ID NO: 14);

Probe (TaqMan) - DENV3 (Example section reference: DV3UnivProbe4)

5’-ABY-GCY GAG CAA ACA GGA GTG TCC CAC AAT TT -QSY-3’ (SEQ ID NO: 15).

DENV2/4 RT-PCR:

Forward primer - DENV2 (Example section reference: DV2UnivFor2)

5 ’-AGC AGA ACC TCC ATT YGG AGA C-3’ (SEQ ID NO: 16);

Reverse primer - DENV2 (Example section reference: DV2UnivRev2)

5’-YTG GCC GAT AGA ACT TCC TTT CT-3’ (SEQ ID NO: 17);

Probe (TaqMan) - DENV2 (Example section referenc: DV2UnivProbe2)

5 ’-F AM- ATC ATC ATA GGA GTA GAG CCG-NFQ/MGB-3’ (SEQ ID NO: 18);

Forward primer - DENV4 (Example section reference: DV4UnivFor4)

5 ’-GGA AGG AGT CTC AGG TGG AGC A-3’ (SEQ ID NO: 19);

Reverse primer - DENV4 (Example section reference: DV4UnivRev4)

5’-TCC AAG GTT GGT TTT CCC TGG-3’ (SEQ ID NO: 20); and

Probe (TaqMan) - DENV4 (Example section reference: DV4UnivProbe4)

5’-TET-GGA TGC GTC ACA ACC A-NFQ/MGB-3’ (SEQ ID NO: 21).

INTERNAL CONTROLS:

Forward primer - Internal Control (Example section reference: MS2-F2)

5 ’-GTT TGA CCT GTG CGA GCT TTT AGT A-3’ (SEQ ID NO: 22);

Reverse primer - Internal Control (Example section reference: MS2-R2)

5 ’-GTT AAA ACG ACS GGG AGT CCA-3’ (SEQ ID NO: 23); and

Probe (TaqMan) - Internal Control (Example section reference: MS2-P1)

5’-[VIC]-AGA GAA TGA GTT ATC TTC AGT CTC ACC GTC CGC-[TAMRA]-3’ (SEQ

ID NO: 24). Fluorophores: VIC: 2'-chloro-7'phenyl-l,4-dichloro-6-carboxy-fluorescein (fluorophore); FAM: 6-carboxyfluorescein; TET: tetrachlorofluorescein.

Quenchers: TAMRA: tetramethylrhodamine; NFQ/MGB: nonfluorescent quenchers/minor groove binder; QSY : Carboxylic Acid, Siccinimidyl Ester. Table 1 Code of degenerated primers:

The term “RT-PCR multiplex assay” means an assay wherein amplification of multiple specific nucleic acid sequences from RNA template(s) by PCR occurs within a single assay reaction using unique primers and/or probes for each specific sequence. The RT-PCR assay comprises purification, amplification, detection and discrimination steps.

The term “purification” means manual, semi-automated, and/or fully automated multi-well plate processing methods to purify nucleic acid from a sample. In a preferred embodiment, the processing method utilizes the Roche MagNA Pure 96 reagents and instrument.

The term “amplification” means generation of replicate copies of a specific nucleic acid sequence present in the viral genome by providing target specific primer sequences within a PCR reaction that includes components needed for enzymatic replication, including dideoxynucleotides, magnesium, reverse transcriptase enzyme and amplicase enzyme in a suitable buffer. Amplification includes subjecting the reaction to repeated temperature cycles to allow for nucleic acid template denaturation, followed by primer annealing and extension from the primer sequences to generate replicate copies of the template.

The term “detection” means the method to measure amplification of a target sequence from a genetic template. Detection is achieved by the PCR instrument that is used for amplification. In one embodiment, the instrument used for detection is a real-time PCR detection system made by any manufacturer. In a more preferred embodiment, the instrument used for detection is the Applied Biosystems® QuantStudio™ 6 Flex Real-Time PCR System.

The term “discrimination” means the use of target sequence-specific primers and probes which allow selective amplification and detection of a specific wild-type dengue virus or dengue virus vaccine DENV1, DENV2, DENV3, or DENV4 serotype sequence preferentially over other nonspecific dengue virus sequences.

The term “wild-type dengue virus” means any non-vaccine dengue virus, including the mosquito-bome dengue viruses found in nature as well as any laboratory generated strains that embody the characteristics of naturally-found dengue virus, and consisting of four serotypes, DENV1, DENV2, DENV3, and DENV4.

The term “dengue vaccine” or “dengue virus vaccine” means a live attenuated tetravalent dengue virus vaccine comprising a DEN1A30 virus, a DEN2/4A30 virus, a DEN3A30 virus and/or a DEN4A30 virus (see WO 03/092592 and Whitehead el al, US Patent No. 8,337,860). In another embodiment, the live attenuated tetravalent dengue virus vaccine comprises rDENlA30-1545 virus, rDEN2/4A30 (ME)-1495,7163 virus, rDEN 3 D30/31-7164 virus, and rDEN4A30-7132, 7163, 8308 virus (see WO 03/092592 and Whitehead et al, US Patent No. 8,337,860).

The designation “rDENlA30-1545” refers to a recombinant dengue 1 virus wherein the viral genome comprises (1) a 30 nucleotide (nt) deletion of the TL2 stem-loop structure of the 3’-UTR and (2) a substitution at nucleotide position 1545 to G.

The designation “rDEN2/4 D 30(ME)-1495,7163” refers to a recombinant chimeric dengue 2/4 virus, wherein the viral genome comprises: (1) a dengue 4 virus backbone (C, NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5 genes) comprising (i) a 30 nt deletion of the TL2 stem-loop structure of the 3’-UTR, and (ii) substitutions at nucleotide positions 1495 to U and 7163 to C, and (2) dengue 2 virus prM and E genes.

The designation “rDEN3A30/31-7164” refers to a recombinant dengue 3 virus wherein the viral genome comprises: (1) a 30 nt deletion of the TL2 stem-loop structure of the 3’-UTR, (2) a separate, 31 nt deletion in the 3’-UTR, upstream of the D30 mutation, that deletes the TL-3 structure and (3) a substitution at nucleotide position 7164 to C.

The designation “rDEN4A 30-7132,7163,8308” refers to a recombinant dengue 4 virus wherein the viral genome comprises: (1) a 30 nt deletion of the TL2 stem-loop structure of the 3’-UTR and (2) substitutions at nucleotide positions 7132 to U, 7163 to C and 8308 to G.

The term “sample” means human sample types including but not limited to serum, blood, saliva, semen, perspiration, urine, tissue, synovial fluids, and/or tears. In a preferred embodiment, the term “sample” means human serum and/or blood. In a more preferred embodiment, the term “sample” means human serum.

The term “reverse-transcription polymerase chain reaction” or “RT-PCR” means executing real-time amplification in the presence of a fluorescently-labeled target- specific probe that binds the genetic template between the target-specific primers. The intact probe does not emit any fluorescent signal. Cleavage of the fluorescent dye-labeled probe during target amplification removes the presence of probe structural components or attached quencher dye that quenches the fluorescent dye signal on the intact probe. Upon probe cleavage, a detectable fluorescent signal is released which is measured by the PCR instrument. Result is reported as the PCR cycle where fluorescent dye is detectable above a threshold level. This PCR cycle can be read from a reference standard curve to generate a quantifiable template copy number in the original sample.

The term “DENV-Vacc RT-PCR reaction(s)” means RT-PCR reaction(s) that contain specific primers and probes that target dengue virus vaccine RNA sequences. In an embodiment, the specific primers and probes bridge the D30 RNA deletion present only in the dengue virus vaccine constructs to selectively detect the four DENV vaccine serotypes.

The term “DENV-WT RT-PCR reaction(s)” means RT-PCR reaction(s) that contain specific primers and probes that target wild-type dengue viral RNA sequences. In an embodiment, the specific primers and probes target the 30 base pair region present in the 3’- UTR of wild-type dengue RNA sequence, that has been deleted in the 3’-UTR of vaccines, to selectively detect all wild-type dengue serotypes.

The term “DENV1/3 RT-PCR reaction(s)” means RT-PCR reaction(s) that contain DENV1 and DENV3 serotype specific primers and probes that target DENV1 and DENV3 RNA sequences. In an embodiment, the DENV1 and DENV3 serotype specific primers and probes target DENV1 and DENV3 RNA sequences that encode the nucleocapsid (C) and non-structural 2B (NS2B) proteins, respectively, and are designed for reliable quantification of DENV 1 and DENV3 serotypes, irrespective of wild-type or vaccine in origin.

The term “DENV2/4 RT-PCR reaction(s)” means RT-PCR reaction(s) that contain DENV2 and DENV4 serotype specific primers and probes that target DENV2 and DENV4 RNA sequences. In an embodiment, the DENV2 and DENV4 serotype specific primers and probes both target DENV2 and DENV4 RNA sequences that encode the envelope (E) protein, and are designed for reliable quantification of DENV2 and DENV4 serotypes, irrespective of wild-type or vaccine in origin.

The term “wild-type strain(s)” means any non-vaccine dengue virus, including the mosquito-borne dengue viruses found in nature as well as any laboratory -generated strains that embody the characteristics of naturally-found dengue virus, and consisting of four serotypes, DENV1, DENV2, DENV3, and DENV4.

The term “DENV vaccine serotype(s)” or “dengue virus vaccine serotype(s)” means a live, attenuated tetravalent dengue virus vaccine, consisting of four serotypes, DENV1, DENV2, DENV3, and DENV4, with genetically-altered sequence intended to reduce viral virulence.

Having described various embodiments of the invention with reference to the accompanying description and tables, it is to be understood that the invention is not limited to those precise embodiments, and that various changes and modifications may be effected therein by one skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

The following examples illustrate, but do not limit the invention.

EXAMPLES GENERAL RT-PCR AMPLIFICATION AND DETECTION Real-time RT-PCR reactions are well known in the art and there are numerous methods in the literature (S.A. Bustin, J Mol Endocrinol, 25 (2000), pp. 169-193; S.A.

Bustin, J Mol Endocrinol, 29 (2002), pp. 23-39; M.L. Wong and J.F. Medrano, BioTechniques, 39 (2005), pp. 75-85; S.A Bustin, Expert Rev Mol Diagn, 5 (2005), pp. 493 - 498; M. Arya et al, Expert Rev Mol Diagn, 5 (2005), pp. 209-219).

EXAMPLE 1

Nucleic Acid Extraction

Total nucleic acids (DNA and RNA) were extracted from serum samples and assay controls using the Roche MagNA Pure 96 DNA and Viral NA Small Volume kit and the Roche MagNA Pure 96 instrument following manufacturer’s instructions, laboratory procedures, and MagNA Pure96 instrument instructions. The sample volume used for extraction was 200 pL, and the elution volume was 50 pL.

200 pL of each sample or control was placed in the correct position in the MagNA Pure Processing Cartridge. The Processing Cartridge was transferred to the MagNA Pure 96 Automated Nucleic Acid extractor and the extraction run initiated. MS2 internal process control was added to each sample and extraction control prior to addition of sample lysis buffer. The process control was prepared by adding 2 pL of MS2 internal control to 10.0 mL PBS, or equivalent ratio. The MP96 instrument draws 20 pL of PBS/MS2 mixture to add to individual sample wells. After nucleic acid extraction, the purified RNA is stored at 2°C to 8°C if used on the same day. If the RT-PCR reactions are not to be set up immediately, the plate is stored at < -60°C until use.

DENV-Vacc or DENV-WT Real-time RT-PCR reaction set-up

Fresh aliquots of DENV-Vacc master-mix (“MMX”) or DENV-WT MMX were used for each assay. 20 pL of RT-PCR MMX, containing appropriate target primers and probes and Superscript III RT/Platinum Taq, was dispensed into each well of a 96-Well Fast Optical Reaction Plate. The 96-well Fast Optical Reaction Plate containing MMX was transferred to the PCR Setup Room where 10 pL of the extracted RNA (samples and controls) were added to the appropriate wells of the 96-well Optical Reaction Plate. The plate was covered with an Optical Adhesive Cover and briefly centrifuged to collect the reactions at the bottom of the wells and to eliminate any air bubbles. The plate was placed on the QS6 instrument in the correct orientation. The PCR reaction was performed using the following cycling parameters in a final reaction volume of 30 pL per sample: (Hold Stage: 48°C for 30 minutes followed by 95°C for 2 minutes and PCR Stage: 95°C for 15 sec followed by 62°C for 1 minute; 45 cycles).

Analyzing the Run Data for DENV-Vacc or DENV-WT reactions

At the completion of the run, the run data was analyzed. The Manual Baseline was selected (Start at 3 cycles, End at 15 cycles). The Target thresholds for analysis in each assay were set as follows: A) DENV-Vacc assay: “DENV-Vacc” at 0.15 and “MS2” at 0.10; and B) DENV-WT assay: “DENV-WT” at 0.20 and “MS2 at 0.10. Analysis settings were applied and the analysis executed. After the instrument completed the analysis, the Amplification plot and Multicomponent plot for each sample were reviewed to check for the accuracy of each target result. Samples with a DENV-WT CT < 45 were considered to contain wild-type dengue virus and samples with a DENV-Vacc CT < 45 were considered to contain dengue vaccine candidate. If either the DENV-WT or DENV-Vacc RT-PCR reaction generates a CT < 45, then the sample is tested in the DENVl/3and DENV2/4 RT-PCR reactions to determine the virus serotype and copies/mL result. Samples that are negative in both the DENV-Vacc and DENV-WT RT-PCR reactions (target CT = 45 or “Undetermined” or Nonspecific Fluorescence (“NSF”) for both RT-PCR reactions) are not tested in the DENV1/3 or DENV2/4 RT-PCR reactions since the samples do not contain dengue viruses. Setting Up Real-time RT-PCR Reactions for DENY 1/3 and DENV2/4 Quantitative assays Fresh aliquots of DENV1/3 MMX or DENV2/4 MMX were used for each assay. 20 pL of RT-PCR MMX, containing appropriate target primers and probes and Fast Virus MMX enzymes, were dispensed into each well of a 96-Well Fast Optical Reaction Plate. The 96-well Fast Optical Reaction Plate containing MMX was transferred to the PCR Setup Room where 10 pL of the extracted RNA (samples and controls) were added to the appropriate wells of the 96-well Optical Reaction Plate. The plate was covered with an Optical Adhesive Cover and briefly centrifuged to collect the reactions at the bottom of the wells and to eliminate any air bubbles. The plate was placed on the QS6 instrument in the correct orientation. The PCR reaction was performed using the following cycling parameters in a final reaction volume of 30 pL per sample: (Hold Stage: 48°C for 30 minutes followed by 95°C for 2 minutes and PCR Stage: 95°C for 15 sec followed by 62°C for 1 minute; 45 cycles).

Analyzing the Run Data for DENV1/3 and DENV2/4 Quantitative assays

At the completion of the run, the run data was analyzed. The Manual Baseline should be selected (Start at 3 cycles, End at 15 cycles). Target thresholds for analysis in each assay were as follows: A) DENV1/3 assay: “DENV1” at 0.10 and “DENV3” at 0.07; and B) DENV2/4 assay: “DENV2” at 0.10 and “DENV4” at 0.07. Analysis settings were applied and the analysis executed. After the instrument completed the analysis, the Amplification plot and Multicomponent plot for each sample were reviewed to check for the accuracy of each target result. Samples with a DENV1 CT < 45 were considered to contain DENV1 serotype virus. Similarly, samples with a DENV2, DENV3 or DENV4 CT < 45 were considered to contain DENV2, DENV3 or DENV4 serotype virus, respectively.

Evaluation ofRT-PCR results

All assay controls must be examined prior to interpretation of sample results.

If the controls are not valid, the sample results cannot be interpreted. A CT value will be assigned for each amplification reaction occurring in a reaction well. The CT value indicates the cycle at which the fluorescence increase in the well exceeds the set threshold. A CT value can be the result of amplification of the desired target or can be the result of non-specific fluorescence (“NSF”). If the result is reported as “Undetermined” it means that no amplification of the target was achieved (CT= 45). Amplification plots and Multicomponent data should be examined for all samples and controls. If the Amplification plot shows an exponential increase in fluorescence crossing the threshold and the Multicomponent plot shows an increase in fluorescence of the detector, the target has been amplified. If the amplification plot does not exhibit an exponential increase crossing the threshold or the Multicomponent plot does not show an increase in fluorescence, amplification of the target has not occurred. If a CT value has been assigned to a well but the amplification plot does not exhibit an exponential increase crossing the threshold or the Multicomponent plot does not show an increase in fluorescence, the CT value is the result of non-specific fluorescence (mark the CT value as “NSF” on the results report). “NSF” results are considered equivalent to “Undetermined” results in that no amplification of the target was achieved.

The RNA process control (MS2 phage), that is introduced into every test sample prior to extraction and run in the DENV-Vacc and DENV-WT assays, is used to verify successful RNA extraction for a sample. All negative samples must have a MS2 CT value that is no greater than 3 CT’S above the MS2 CT value of the Negative Control. This criterion must be met in order for the result to be valid. A value greater than 3 CT’S above the Negative Control or non-amplification of the MS2 process control indicates possible inefficient RNA extraction or inhibition of the RT-PCR reaction (due to presence of inhibitors co-purified with the nucleic acids). When sufficient clinical sample is available, this particular sample must be re-extracted and re-tested. It is acceptable for the CT value of a negative test sample to be lower by more than 3 CT’S below the MS2 CT value of the Negative Control.

The Negative Control should have a CT value = 45 or “Undetermined” or reveal an NSF (non-specific fluorescent signal) in each RT-PCR reaction ( DENV-Vacc, DENV-WT, DENV1/3 and DENV2/4, as appropriate). Both positive controls (HPC and LPC) must yield a CT value within the acceptable ranges in each RT-PCR reaction (DENV- Vacc, DENV-WT, DENV1/3, and DENV2/4, as appropriate).

DENV-WT and DENV-Vacc Results

The DENV-WT and DENV-Vacc assays are reported as qualitative results. A CT value = 45 is a “Not Detected” result, while a CT value <45 is a “Detected” result. The results of the DENV-WT and DENV-Vacc RT-PCR reactions are evaluated in combination with the DENV1/3, DENV2/4, and MS2 RT-PCR results to determine the final sample reported result as described in Table 1 below.

DENV1/3 and DENV2/4 Results

The DENV1/3 and DENV2/4 RT-PCR assays are quantitative. Using an external standard curve for each of the four wild-type and dengue vaccine serotypes, the linear regression equation of each curve is used to calculate virus logio copies/mL (x value) in test samples based on the observed CT values (y value) for any result with a detected CT value (CT < 45) in the DENV1/3 and DENV2/4 quantitative assays. Inverse of this Logio copies/mL value will result in the reportable copies/mL result. The specific standard curve used to determine the viral load of a test sample will depend on whether it is wild-type or dengue vaccine positive and its serotype. The results of the DENV1/3 and DENV2/4 RT- PCR reactions are evaluated in combination with the DENV-WT, DENV-Vacc, and MS2 RT-PCR results to determine the final sample reported result as described in Table 1 below. Cross-reactive results in DENV-Vacc assay

Cross-reactivity was observed for some high concentration DENV-WT samples in the DENV-Vacc assay. As a result, an evaluation of mixed populations of DENV-WT/DENV-Vacc samples at varying percentages was performed to define reporting rules for mixed population samples.

If both of the DENV-Vacc and DENV-WT RT-PCR reactions are positive (CT values < 45) and the DENV-WT CT value is > 12 CT’S lower than the DENV-Vacc CT value, the DENV-Vacc CT value is considered to be the result of cross-reactivity and the DENV- Vacc result is reported as “Not Detected”. Viral load concentrations are calculated from the wild-type external curve(s) for the dengue virus serotype(s) identified in the DENV1/3 and DENV2/4 RT-PCR reactions.

If both of the DENV-Vacc and DENV-WT RT-PCR reactions are positive (CT values < 45) and the DENV-WT CT value is < 12 CT’S lower than the DENV-Vacc CT value, the DENV-WT is reported as “Detected” whereas the DENV-Vacc result is reported as “Inconclusive”. Viral load concentrations are calculated from the external curve(s) for the dengue virus serotype(s) that are positive and give the lowest calculated concentration in the DENV1/3 and DENV2/4 RT-PCR reactions.

If both of the DENV-Vacc and DENV-WT RT-PCR reactions are positive (CT values < 45) and the DENV-Vacc CT value is lower than the DENV-WT CT value, the DENV-Vacc result is reported as “Detected”, and the DENV-WT is also reported as “Detected”. Viral load concentrations are calculated from the external curve(s) for the dengue virus serotype(s) that are positive and give the lowest calculated concentration in the DENV1/3 and DENV2/4 RT-PCR reactions. Final RT-PCR Result Reporting combining results for all 4 RT-PCR assays

Final scenario results are highlighted in Table 1.

Table 1

¹ Viral load concentrations are calculated from the external curve(s) for the dengue virus serotype(s) that are positive and give the lowest calculated concentration in the DENV1/3 and

DENV2/4 RT-PCR reactions

EXAMPLE 2 Assay Qualification Results Linearity

The linearity of the RT-PCR assay was assessed by spiking each of the four DENV vaccine viruses (DENV1, DENV2, DENV3, and DEV4) or each of the four dengue wild-type viruses (DENV1, DENV2, DENV3, and DEV4) separately in triplicate into negative human pooled serum to a final concentration of 1 x 10⁶, lx 10⁵, 1 x 10⁴, 1000, 500, 250, 125 or 62.5 copies/mL and evaluated in the DENV-Vacc or DENV-WT RT-PCR reaction, respectively. These samples were further tested in the DENV1/3 and DENV2/4 RT- PCR reactions. The slope and coefficient of determination (R²) obtained from least square regression where the observed CT values were plotted against the logio concentrations of the samples were used to evaluate linearity for each dengue virus in each RT-PCR reaction.

EXAMPLE 3

Assay Qualification Results Sensitivity (Lower Limit of Detection and Lower Limit of Quantitation)

The sensitivity of the DENV-Vacc and DENV-WT RT-PCR assays was assessed by spiking each of the four DENV vaccine viruses (DENV1, DENV2, DENV3 and DENV4) and each of the four DENV wild-type viruses DENV1, DENV2, DENV3 and DENV4) independently in triplicate into negative human pooled serum to a final concentration of 250, 125 or 62.5 copies/mL. The DENV vaccine viruses were tested in a single run of the DENV-Vacc RT-PCR and the DENV wild-type viruses were tested in a single run of the DENV-WT RT-PCR. The lowest concentration at which all three replicates were detected was treated as the tentative LLOD for each virus. The LLOD of each virus was confirmed by testing 20 replicates at 250 and 125 copies/mL in a single run if the tentative LLOD was 250 copies/mL, or by testing 20 replicates at 125 and 62.5 copies/mL in a single run if the tentative LLOD was <125 copies/mL.

The sensitivity of the DENV1/3 and DENV2/4 RT-PCR assays was assessed by spiking individual DENV vaccine viruses and DENV wild-type viruses separately into negative human pooled serum in triplicate to a final concentration of 250, 125 or 62.5 copies/mL. DENV1 and DENV3 samples (vaccine and wild-type) were tested in a single run of the DENV1/3 RT-PCR. DENV2 and DENV4 samples (dengue vaccine and wild-type) were tested in a single run of the DENV2/4 RT-PCR. The lowest concentration at which all three replicates were detected was treated as the tentative LLOD for each virus tested. The concentration of each virus at which all replicates had CT values within 4 units of each other was treated as the tentative LLOQ. The LLOD of each virus was confirmed by testing 20 replicates at 250 and 125 copies/mL in a single run if the tentative LLOD was 250 copies/mL, or by testing 20 replicates at 125 and 62.5 copies/mL in a single run if the tentative LLOD was <125 copies/mL.The LLOQ of each virus was confirmed by testing 20 replicates at 250 and 125 copies/mL in a single run if the tentative LLOQ was 250 copies/mL, or by testing 20 replicates at 125 and 62.5 copies/mL in a single run if the tentative LLOQ was <125 copies/mL.

The LLOD was defined for a DENV virus in an RT-PCR reaction as the concentration at which at least 19 out of the 20 replicates (> 95%) were detected. The LLOQ was defined for a DENV virus as the concentration at which all replicates (20/20) were detected and the standard deviation of their CT values was < 1.5. The sensitivity established during the assay qualification is at least as follows:

DENV-Vacc RT-PCR - LLOD DENV1 Vaccine: 125 copies/mL DENV2 Vaccine: 125 copies/mL DENV3 Vaccine: 125 copies/mL DENV4 Vaccine: 125 copies/mL DENV-WT RT-PCR - LLOD DENV1 WT: 62.5 copies/mL DENV2 WT: 62.5 copies/mL DENV 3 WT: 62.5 copies/mL DENV4 WT: 62.5 copies/mL DENV 1/3 RT-PCR - LLOD DENV1 WT: 62.5 copies/mL DENV1 Vaccine: 62.5 copies/mL DENV 3 WT: 62.5 copies/mL DENV3 Vaccine: 62.5 copies/mL DENV 1/3 RT-PCR - LLOQ DENV1 WT: 62.5 copies/mL DENV1 Vaccine: 62.5 copies/mL DENV 3 WT: 125 copies/mL DENV3 Vaccine: 62.5 copies/mL DENV2/4 RT-PCR-LLOD

DENV2 WT: 62.5 copies/mL DENV2 Vaccine: 62.5 copies/mL

DENV4 WT: 125 copies/mL DENV4 Vaccine: 62.5 copies/mL DENV2/4 RT-PCR-LLOO DENV2 WT: 62.5 copies/mL DENV2 Vaccine: 62.5 copies/mL DENV4 WT: 125 copies/mL DENV4 Vaccine: 62.5 copies

EXAMPLE 4

Assay Qualification Results Intermediate Precision and Repeatability

The intermediate precision of the RT-PCR assay was assessed by spiking each of the four DENV vaccine viruses (DENV1, DENV2, DENV3 and DENV4) and each of four DENV wild type viruses (DENV1, DENV2, DENV3 and DENV4) separately into negative human pooled serum at high, medium and low concentrations. Three aliquots at each concentration level were tested in the DENV-Vacc or DENV-WT RT-PCR, respectively, in three independent runs performed on three separate days by two different operators. The appropriate DENV-Vacc and DENV-WT samples were further tested in the DENV1/3 and DENV2/4 assays in three independent runs performed on three separate days by two different operators. The overall variability (that takes into consideration operator to operator variance, run to run variance for the same operator, as well as the intra-assay precision) was determined from the calculated viral loads at low, medium, and high concentration levels for each DENV virus tested in each of the four RT-PCR reactions.

The repeatability of the the RT-PCR assay was assessed by spiking each of the four DENV vaccine viruses (DENV1, DENV2, DENV3 and DENV4) and each of four DENV wild type viruses (DENV1, DENV2, DENV3 and DENV4) separately into negative human pooled serum at high, medium and low concentrations. Six replicates at each concentration level were tested in the DENV-Vacc or DENV-WT RT-PCR, respectively, in a single run by a single operator. The appropriate DENV-Vacc and DENV-WT samples were further tested in the DENV1/3 and DENV2/4 assays in a single run by a single operator. The repeatability (% CV) of high, medium and low concentrations for each DENV strain tested in each of the four RT-PCR reactions was determined from the calculated viral loads. EXAMPLE 5

Assay Qualification Results Analytical Specificity

The analytical specificity of the RT-PCR assay was assessed by testing genomic RNA from West Nile Virus, Yellow Fever virus, Zika Virus, the four DENV vaccine viruses and the four DENV wild-type virus serotypes in the DENV-Vacc RT-PCR reaction, the DENV-WT RT-PCR reaction, the DENV1/3 RT-PCR reaction and the DENV2/4 RT-PCR reaction. Samples were tested in a single run by a single operator.

Source genomic material was determined to be positive or negative in each assay from the observed CT values and used to establish assay specificity.

Since there is low level cross-reactivity observed in the DENV-Vacc assay against DENV wild-type virus when present at very high viral loads, a mixing experiment was performed to establish a ACT threshold which allows wild-type and vaccine strains to be distinguished in the presence of potential cross-reactivity. DENV1 parent wild-type and DENV1 vaccine viruses were mixed to a final concentration of lxlO⁸ copies/mL and lxlO⁶ copies/mL at defined % wild-type to vaccine virus ratios (100:0, 99.995:0.005, 99.95:0.05, 99.5:0.5, 95:5, 75:25, 50:50, and 25:75) and tested in triplicate in the DENV-WT RT-PCR reaction and the DENV-Vacc RT-PCR reaction in a single run by a single operator.

EXAMPLE 6

Sample Results Verification of Discrimination, Serotype Identification and Quantitation of Wild Type and Vaccine Viral Load

The ability of the DENV RT-PCR assays to accurately detect and quantitate Dengue WT and Dengue Vaccine strains was evaluated using a panel of 52 samples which included 1) samples constructed by spiking negative serum with varying known amounts of WT or vaccine DENV1, DENV2, DENV3, and/or DENV4 and 2) clinical samples previously determined to be positive for wild type DENV (serotype and quantity unknown). The sample panel test results are included in Table 2. The observed outcome of the verification showed 100% correlation (54 out of 54 samples) based on dengue virus status, and 95% correlation (38 out of 40 samples) based on serotype/wild-type/vaccine virus identity. A regression plot of observed versus expected virus concentrations resulted in a slope of 0.9693.

Table 2

Claims

WHAT IS CLAIMED IS:

1. An RT-PCR multiplex assay that comprises a DENV-Vacc and a DENV-WT RT-PCR reaction, wherein the DENV-WT RT-PCR reaction targets a 30 base pair region present in the 3’-UTR of wild-type dengue virus RNA sequences, which has been deleted in the 3’-UTR of dengue virus vaccines, to selectively detect wild-type dengue virus serotypes; and wherein the DENV-Vacc RT-PCR reaction bridges the D30 deletion present only in a dengue virus vaccine to selectively detect the four dengue virus vaccine serotypes.

2. The assay of claim 1, further comprising a DENV1/3 and a DENV2/4 RT-PCR reaction.

3. The assay of claim 2, wherein the DENV1/3 RT-PCR reaction contains DENV1 and DENV3 serotype-specific primers and probes which target RNA sequences encoding the nucleocapsid (C) and non-structural 2B (NS2B) proteins.

4. The assay of claims 2 or 3, wherein the DENV2/4 RT-PCR reaction contains DENV2 and DENV4 serotype-specific primers and probes which target RNA sequences encoding the envelope (E) protein.

5. The assay of claims 1-4 wherein the DENV-Vacc and DENV-WT RT- PCR reactions comprise the following primers and probes: DENV-Vacc forward primer (SEQ ID NO: 1), DENV-Vacc reverse primer (SEQ ID NO: 2), DENV-Vacc probe (SEQ ID NO: 3), DENV 1-WT forward primer (SEQ ID NO: 4), DENV 2-WT forward primer (SEQ ID NO: 5), DENV 3-WT forward primer (SEQ ID NO: 6), DENV 4-WT forward primer (SEQ ID NO: 7), DENV-WT reverse primer (SEQ ID NO: 8), and DENV-WT probe (SEQ ID NO: 9).

6. The assay of claims 2-5, wherein the DENV1/3 and DENV2/4 RT- PCR reactions comprise the following primers and probes: DENV 1 forward primer (SEQ ID NO: 10), DENV 1 reverse primer (SEQ ID NO: 11), DENV 1 probe (SEQ ID NO: 12),

DENV 3 forward primer (SEQ ID NO: 13), DENV 3 reverse primer (SEQ ID NO: 14),

DENV 3 probe (SEQ ID NO: 15), DENV 2 forward primer (SEQ ID NO: 16), DENV 2 reverse primer (SEQ ID NO: 17), DENV 2 probe (SEQ ID NO: 18), DENV 4 forward primer (SEQ ID NO: 19), DENV 4 reverse primer (SEQ ID NO: 20), and DENV 4 probe (SEQ ID NO: 21).

7. An assay which comprises the following steps: (a) a nucleic acid purification step comprising purifying nucleic acid from a sample; (b) one or more amplification steps comprising amplifying the nucleic acid purified in step (a) using reverse- transcription polymerase chain reactions (RT-PCR reactions); (c) a detection step comprising detecting and differentiating wild-type dengue virus and/or dengue virus vaccine in the sample; and (d) an optional differentiation and quantification step comprising differentiating and quantifying between DENV1, DENV2, DENV3 and/or DENV4 in the sample; wherein the RT-PCR reactions in step (b) comprise DENV-Vacc and DENV-WT RT-PCR reactions, and step (d) comprises DENV1/3 and DENV2/4 RT-PCR reactions; wherein the DENV-WT RT-PCR reaction targets a 30 base pair region present in the 3’-UTR of wild-type dengue virus RNA sequences that have been deleted in the 3’-UTR of dengue virus vaccines to selectively detect wild-type dengue virus serotypes; and wherein the DENV-Vacc RT-PCR reaction bridges the D30 deletion present only in dengue virus vaccines to selectively detect the four dengue virus vaccine serotypes.

8. The assay of claim 7, wherein the DENV1/3 RT-PCR reaction contains DENV1 and DENV3 serotype-specific primers and probes which target RNA sequences encoding the nucleocapsid (C) and non-structural 2B (NS2B) proteins.

9. The assay of claims 7 or 8, wherein the DENV2/4 RT-PCR reaction contains DENV2 and DENV4 serotype-specific primers and probes which target RNA sequences encoding the envelope (E) protein.

10. The assay of claims 7-9, wherein the DENV-Vacc and DENV-WT RT- PCR reactions comprise the following primers and probes: DENV-Vacc forward primer (SEQ ID NO: 1), DENV-Vacc reverse primer (SEQ ID NO: 2), DENV-Vacc probe (SEQ ID NO: 3), DENV 1-WT forward primer (SEQ ID NO: 4), DENV 2-WT forward primer (SEQ ID NO: 5), DENV 3-WT forward primer (SEQ ID NO: 6), DENV 4-WT forward primer (SEQ ID NO: 7), DENV-WT reverse primer (SEQ ID NO: 8), and DENV-WT probe (SEQ ID NO: 9).

11. The assay of claims 7-10 wherein the DENV1/3 and DENV2/4 RT- PCR reactions comprise the following primers and probes: DENV 1 forward primer (SEQ ID NO: 10), DENV 1 reverse primer (SEQ ID NO: 11), DENV 1 probe (SEQ ID NO: 12),

DENV 3 forward primer (SEQ ID NO: 13), DENV 3 reverse primer (SEQ ID NO: 14),

12. A method of detecting wild-type dengue virus and/or dengue virus vaccine in a sample utilizing the assay of claims 1-11.

13. A method of differentiating between dengue virus serotypes 1-4

(DENV1, DENV2, DENV 3 and DENV4.

14. A method of differentiating between wild-type dengue virus and dengue virus vaccine in a sample utilizing the assay of claims 1-11.

15. A method of quantifiying total dengue viral load of a sample utilizing the assay of claims 1-11.

16. A kit for detecting wild-type dengue virus and/or dengue virus vaccine in a sample, the kit comprising the primers and probes selected from (SEQ ID NOs: 1-9) and instructions for use.

17. A kit for detecting and quantifying wild-type dengue virus and/or dengue virus vaccine serotypes DENV1, DENV2, DENV3 and/or DENV4 in a sample, the kit comprising the primers and probes selected from (SEQ ID NOs: 1-21) and instructions for use.

18. A kit for differentiating between wild-type dengue virus and dengue virus vaccine in a sample, the kit comprising the primers and probes selected from (SEQ ID NOs: 1-21) and instructions for use.