Multiplexed Biomarker Panels Discriminate Zika and Dengue Virus Infection in Humans

Abstract

Zika virus (ZIKV) and dengue virus (DENV) are closely related flaviviruses that cause widespread, acute febrile illnesses, notably microcephaly for fetuses of infected pregnant women. Detecting the viral cause of these illnesses is paramount to determine risks to patients, counsel pregnant women, and help fight outbreaks. A combined diagnostic algorithm for ZIKV and DENV requires Reverse transcription polymerase chain reaction (RT-PCR) and IgM antibody detection. RT-PCR differentiates between DENV and ZIKV infections during the acute phases of infection, but differentiation based on IgM antibodies is currently nearly impossible in endemic areas. We have developed a ZIKV/DENV protein array and tested it with serum samples collected from ZIKV- and DENV-infected patients and healthy subjects in Puerto Rico. Our analyses reveal a biomarker panel that are capable of discriminating ZIKV and DENV infections with high accuracy, including Capsid protein from African ZIKV strain MR766, and other 5 pair of proteins (NS1, NS2A, NS3, NS4B and NS5) from ZIKV and DENV respectively. Both sensitivity and specificity of the test for ZIKV from DENV are around 90%. We propose that the ZIKV/DENV protein array will be used in future studies to discriminate patients infected with ZIKV from DENV. Zika virus (ZIKV) and dengue virus (DENV) are closely related flaviviruses that cause widespread, acute febrile illnesses, notably microcephaly for fetuses of infected pregnant women. Detecting the viral cause of these illnesses is paramount to determine risks to patients, counsel pregnant women, and help fight outbreaks. A combined diagnostic algorithm for ZIKV and DENV requires Reverse transcription polymerase chain reaction (RT-PCR) and IgM antibody detection. RT-PCR differentiates between DENV and ZIKV infections during the acute phases of infection, but differentiation based on IgM antibodies is currently nearly impossible in endemic areas. We have developed a ZIKV/DENV protein array and tested it with serum samples collected from ZIKV- and DENV-infected patients and healthy subjects in Puerto Rico. Our analyses reveal a biomarker panel that are capable of discriminating ZIKV and DENV infections with high accuracy, including Capsid protein from African ZIKV strain MR766, and other 5 pair of proteins (NS1, NS2A, NS3, NS4B and NS5) from ZIKV and DENV respectively. Both sensitivity and specificity of the test for ZIKV from DENV are around 90%. We propose that the ZIKV/DENV protein array will be used in future studies to discriminate patients infected with ZIKV from DENV. The rapid, global spread of Zika virus (ZIKV) 1The abbreviations used are: ZIKV, Zika virus; DENV, Dengue virus; GBS, Guillain-Barré syndromes; RT-PCR, Reverse transcription polymerase chain reaction; ELISA, Enzyme-linked immunosorbent assay; PRNT, Plaque reduction neutralization test; ORFs, Open reading frames; GST, Glutathione S-transferases; ROC, Receiver operating characteristic; MAC-ELISA, IgM antibody capture enzyme-linked immunosorbent assay.., 1The abbreviations used are: ZIKV, Zika virus; DENV, Dengue virus; GBS, Guillain-Barré syndromes; RT-PCR, Reverse transcription polymerase chain reaction; ELISA, Enzyme-linked immunosorbent assay; PRNT, Plaque reduction neutralization test; ORFs, Open reading frames; GST, Glutathione S-transferases; ROC, Receiver operating characteristic; MAC-ELISA, IgM antibody capture enzyme-linked immunosorbent assay. its association with congenital ZIKV and Guillain-Barré syndromes (GBS), and the demonstration that it is sexually transmitted, have raised unprecedented public health concerns (1.Lazear H.M. Diamond M.S. Zika Virus: New Clinical Syndromes and Its Emergence in the Western Hemisphere.J. Virol. 2016; 90: 4864-4875Crossref PubMed Scopus (336) Google Scholar). Indeed, ZIKV infection is now known to be a cause of microcephaly and severe brain abnormalities and has been linked to other birth defects (https://www.cdc.gov/zika/pdfs/zika-key-messages.pdf). It has become one of the greatest concerns for pregnant women, who can pass ZIKV to their developing fetus if infected during pregnancy (2.Petersen L.R. Jamieson D.J. Powers A.M. Honein M.A. Zika virus.N. Engl. J. Med. 2016; 374: 1552-1563Crossref PubMed Scopus (885) Google Scholar). Therefore, it is critical to determine the potentially causal virus in an individual who presents with suspicion of infection. However, the arrival of ZIKV in the Americas was preceded by decades of endemic transmission of dengue virus (DENV), which has a similar clinical presentation that makes it difficult for clinicians to accurately distinguish (3.Priyamvada L. Quicke K.M. Hudson W.H. Onlamoon N. Sewatanon J. Edupuganti S. Pattanapanyasat K. Chokephaibulkit K. Mulligan M.J. Wilson P.C. Ahmed R. Suthar M.S. Wrammert J. Human antibody responses after dengue virus infection are highly cross-reactive to Zika virus.Proc. Natl. Acad. Sci. U.S.A. 2016; 113: 7852-7857Crossref PubMed Scopus (408) Google Scholar). Diagnostic algorithms for ZIKV and DENV often include RNA nucleic acid testing (e.g. RT-PCR) and IgM detection tests, according to guidelines that can be found in references for physicians (4.Organization, P. A. H., (accessed may 5th 2017) Dengue: guidelines for patient care in the Region of the Americas. 2. ed. http://iris.paho.org/xmlui/handle/123456789/31207,Google Scholar, 5.Announcement: Guidance for U.S. laboratory testing for Zika virus infection: Implications for health care providers.MMWR Morb. Mortal. Wkly. Rep. 2016; 65: 1304Crossref PubMed Scopus (0) Google Scholar, 6.Shukla S. Hong S.Y. Chung S.H. Kim M. Rapid detection strategies for the global threat of Zika virus: Current state, new hypotheses, and limitations.Front. Microbiol. 2016; 7: 1685Crossref PubMed Scopus (31) Google Scholar). RT-PCR can detect and differentiate ZIKV and DENV early in infection (7.Santiago G.A. Vergne E. Quiles Y. Cosme J. Vazquez J. Medina J.F. Medina F. Colon C. Margolis H. Munoz-Jordan J.L. Analytical and clinical performance of the CDC real time RT-PCR assay for detection and typing of dengue virus.PLoS Negl. Trop. Dis. 2013; 7: e2311Crossref PubMed Scopus (239) Google Scholar). However, later in infection when RT-PCR assays are less sensitive, or in asymptomatic pregnant women, differentiating these two diseases requires specific serology-based diagnostic methods, which is challenging because of immunologic cross-reactivity among the flaviviruses (3.Priyamvada L. Quicke K.M. Hudson W.H. Onlamoon N. Sewatanon J. Edupuganti S. Pattanapanyasat K. Chokephaibulkit K. Mulligan M.J. Wilson P.C. Ahmed R. Suthar M.S. Wrammert J. Human antibody responses after dengue virus infection are highly cross-reactive to Zika virus.Proc. Natl. Acad. Sci. U.S.A. 2016; 113: 7852-7857Crossref PubMed Scopus (408) Google Scholar). Enzyme-linked immunosorbent assay (ELISA) tests of IgM have recently become available for determining recent or remote ZIKV exposure, but do not completely differentiate ZIKV from DENV (8.U.S. Food and Drug Administration (accessed may 5th 2017) Emergency Use Authorizations https://www.fda.gov/MedicalDevices/Safety/EmergencySituations/ucm161496.htm - zika,Google Scholar). ZIKV IgM positive results by these tests require confirmation by a plaque reduction neutralization test (PRNT), a cumbersome and lengthy laboratory method with some utility for differentiating flaviviruses in populations with low exposure (9.Shan C. Xie X. Ren P. Loeffelholz M.J. Yang Y. Furuya A. Dupuis 2nd, A.P. Kramer L.D. Wong S.J. Shi P.Y. A rapid Zika diagnostic assay to measure neutralizing antibodies in patients.EBioMedicine. 2017; 17: 157-162Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), but not in populations with high incidence of DENV and ZIKV, such as Puerto Rico (5.Announcement: Guidance for U.S. laboratory testing for Zika virus infection: Implications for health care providers.MMWR Morb. Mortal. Wkly. Rep. 2016; 65: 1304Crossref PubMed Scopus (0) Google Scholar, 10.Rabe I.B. Staples J.E. Villanueva J. Hummel K.B. Johnson J.A. Rose L. Mts Hills S. Wasley A. Fischer M. Powers A.M. interim guidance for interpretation of Zika virus antibody test results.MMWR Morb. Mortal. Wkly. Rep. 2016; 65: 543-546Crossref PubMed Scopus (209) Google Scholar). To counsel potentially infected pregnant women, establish the cause of congenital syndrome, or make informed decisions to treat patients with GBS or hemorrhagic disease, it is important to determine the pathogen responsible for the infection. Recently, a DENV protein microarray were fabricated through gene synthesis and successfully applied to discriminate patients infected by DENV from other related pathogens, or uninfected patients (11.Qi H. Zhou H. Czajkowsky D.M. Guo S. Li Y. Wang N. Shi Y. Lin L. Wang J. Wu Tao S.C. Rapid production of virus protein microarray using protein microarray fabrication through gene synthesis (PAGES).Mol. Cell. Proteomics. 2017; 16: 288-299Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar), suggesting a protein array containing proteomes of ZIKV and DENV would be an effective method to differentiate patients infected by ZIKV or DENV. In this study, we developed a ZIKV/DENV protein array and applied it to discover a biomarker panel that can specifically distinguish ZIKV and DENV infections in humans. By comparing the IgM serum profiles obtained with our protein array with “gold standard” IgM-confirmed RT-PCR ZIKV-positive and DENV-positive samples, we discovered and validated a biomarker panel that could distinguish ZIKV from DENV infections with high accuracy. All Serum samples were obtained from the Dengue Surveillance System sample collection at Puerto Rico under guidelines approved by the CDC institutional review board (IRB). Patients did not provide verbal or written consent for this study. Samples were de-linked from patient identifiers according to CDC's IRB. Serum samples used for elaboration in the discovery stage and validation panels were listed in supplemental Fig. S1. ZIKV specimens were collected during the 2016 epidemic. DENV specimens were collected in years 2010–2012, before the arrival of ZIKV in Puerto Rico. Of the 49 dengue IgM positive samples by CDC IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA), 33 were from patients with confirmed DENV RT-PCR positive results by the CDC DENV-RT-PCR (7.Santiago G.A. Vergne E. Quiles Y. Cosme J. Vazquez J. Medina J.F. Medina F. Colon C. Margolis H. Munoz-Jordan J.L. Analytical and clinical performance of the CDC real time RT-PCR assay for detection and typing of dengue virus.PLoS Negl. Trop. Dis. 2013; 7: e2311Crossref PubMed Scopus (239) Google Scholar). The 33 Zika IgM positive samples were dengue IgM negative and collected from patients with Zika RT-PCR positive results by the CDC TRIOPLEX test (8.U.S. Food and Drug Administration (accessed may 5th 2017) Emergency Use Authorizations https://www.fda.gov/MedicalDevices/Safety/EmergencySituations/ucm161496.htm - zika,Google Scholar). Serum samples were obtained by standard procedures (12.Paz-Bailey G. Rosenberg E.S. Doyle K. Munoz-Jordan J. Santiago G.A. Klein L. Perez-Padilla J. Medina F.A. Waterman S.H. Gubern C.G. Alvarado L.I. Sharp T.M. Persistence of Zika virus in body fluids - Preliminary report.N. Engl. J. Med. 2017; Crossref PubMed Scopus (295) Google Scholar) and stored at −20 °C until use. To fabricate a ZIKV/DENV protein array, two ZIKV strains (i.e. African MR766 and Cambodia strain FSS13025), and two DENV sero-types (i.e. DENV-1 TH-S-man Hawaii strain and DENV-2 NGC Thailand strain) were selected, respectively. Lysates of virus-infected mosquito cells were used to extract total RNA and 1 μg of total RNA was converted to cDNA using Superscript III (Thermo Fisher Scientific) for PCR templates. Primer sets were designed for amplifying the full-length ORFs and attB1 and attB2 sequences at the 5′and 3′-ends of each primer were added to clone PCR amplicons into Gateway Entry vector pDONR221 (Thermo Fisher Scientific) by gateway recombination. To use the expression vector pEGHA for expression and purification of N-terminal GST fusion proteins, a stop codon (TAA) was added between the gene and the attB2 gene specific sequence in the reverse primers. Four ORFs, 2K, NS2B, NS3, and NS4A, were highly conserved among ZIKV strains, so those were only cloned from the MR-766 strain to minimize the redundancy of viral proteins on the arrays. Regarding capsid proteins encoded by ZIKV, we also included two capsid protein C genes (C) with an additional propeptide of 14 amino acids in addition to the capsid protein C (prC). The list of all primers used in this experiment can be found in supplemental Table S3. PCR amplification was carried out with 1 μl of cDNA in 50 μl PCR reaction using Phusion High-Fidelity DNA polymerase (New England BioLabs, Ipswich, MA), and a final concentration of 10 pmol for each primer using the following PCR conditions: 98 °C for 30 s; 5 cycles of 98 °C for 10 s, 50–55 °C for 30 s, 72 °C for 30–100 s; 20 cycles of 98 °C for 10 s, 65 °C for 30 s, 72 °C for 30–100 s; 72 °C for 5 min. 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The biomarker panel in this ZIKV from DENV Therefore, this array may be applied to that can distinguish ZIKV- or DENV-infected patients from healthy of the of protein we could this array by additional proteins encoded by other such as virus and to the Guidance for for Dengue and Zika G. L. and Scholar, L. G. Scholar), Dengue and Zika can be using The in discriminating these in testing specimens in the stage RT-PCR is and such as testing for antibodies is but in the tests is Diagnostic for these is used in patients that at or 1 after the of of and patients tested for to have IgM antibody (12.Paz-Bailey G. Rosenberg E.S. Doyle K. Munoz-Jordan J. Santiago G.A. Klein L. Perez-Padilla J. Medina F.A. Waterman S.H. Gubern C.G. Alvarado L.I. Sharp T.M. Persistence of Zika virus in body fluids - Preliminary report.N. Engl. J. Med. 2017; Crossref PubMed Scopus (295) Google Scholar), and according to new guidance G. L. and Scholar). it has been that IgM can be in infected patients for to after infection IgM in with Zika Virus: Implications for for Scholar). all of the specimens used in our test were to be IgM positive in tests by the for the a samples from patients with time (i.e. the other DENV and ZIKV are RNA and highly but their to the of tests for their the ZIKV strain in Puerto Rico during a to the ZIKV strain discovered in in is less of a at the amino acid that when using an such as that virus the of the test for years at the sensitivity is high the of these Our to assay 14 which can be by the to with and by the to because proteins will be for the the standard is the can be which will in and these biomarker panels can be potentially into a to of ZIKV and DENV We for with