Detection of Specific ZIKV IgM in Travelers Using a Multiplexed Flavivirus Microsphere Immunoassay.
ABSTRACT: Zika virus (ZIKV) has spread widely in the Pacific and recently throughout the Americas. Unless detected by RT-PCR, confirming an acute ZIKV infection can be challenging. We developed and validated a multiplexed flavivirus immunoglobulin M (IgM) microsphere immunoassay (flaviMIA) which can differentiate ZIKV-specific IgM from that due to other flavivirus infections in humans. The flaviMIA bound 12 inactivated flavivirus antigens, including those from ZIKV and yellow fever virus (YFV), to distinct anti-flavivirus antibody coupled beads. These beads were used to interrogate sera from patients with suspected ZIKV infection following travel to relevant countries. FlaviMIA results were validated by comparison to the ZIKV plaque reduction neutralization test (PRNT). The results highlight the complexity of serological ZIKV diagnosis, particularly in patients previously exposed to or vaccinated against other flaviviruses. We confirmed 99 patients with ZIKV infection by a combination of RT-PCR and serology. Importantly, ZIKV antibodies could be discriminated from those ascribed to other flavivirus infections. Serological results were sometimes confounded by the presence of pre-existing antibodies attributed to previous flavivirus infection or vaccination. Where RT-PCR results were negative, testing of appropriately timed paired sera was necessary to demonstrate seroconversion or differentiation of recent from past infection with or exposure to ZIKV.
Project description:BACKGROUND:Zika virus (ZIKV) and Dengue virus (DENV) are often co-endemic. The high protein-sequence homology of flaviviruses renders IgG induced by and directed against them highly cross-reactive against their antigen(s), as observed on a large set of sera, leading to poorly reliable sero-diagnosis. METHODS:We selected Domain III of the ZIKV Envelope (ZEDIII) sequence, which is virus specific. This recombinant domain was expressed and purified for the specific detection of ZEDIII-induced IgG by ELISA from ZIKV-RT-PCR-positive, ZIKV-IgM-positive, flavivirus-positive but ZIKV-negative, or flavivirus-negative sera. We also assessed the reactivity of ZEDIII-specific human antibodies against EDIII of DENV serotype 4 (D4EDIII) as a specific control. Sera from ZEDIII-immunized mice were also tested. RESULTS:Cross-reactivity of IgG from 5,600 sera against total inactivated DENV or ZIKV was high (71.0% [69.1; 72.2]), whereas the specificity and sensitivity calculated using a representative cohort (242 sera) reached 90% [84.0; 95.8] and 92% [84.5; 99.5], respectively, using a ZEDIII-based ELISA. Moreover, purified human IgG against D2EDIII or D4EDIII did not bind to ZEDIII and we observed no D4EDIII reactivity with ZIKV-induced mouse polyclonal IgGs. CONCLUSIONS:We developed a ZEDIII-based ELISA that can discriminate between past or current DENV and ZIKV infections, allowing the detection of a serological scar from other flaviviruses. This could be used to confirm exposure of pregnant women or to follow the spread of an endemic disease.
Project description:The recent outbreak of Zika virus (ZIKV) in the Americas has challenged diagnostic laboratory testing strategies. At the Wadsworth Center, ZIKV serological testing was performed for over 10,000 specimens, using a combination of an enzyme-linked immunosorbent assay (ELISA) for IgM antibodies (Abs) to ZIKV, a polyvalent microsphere immunoassay (MIA) to detect Abs broadly reactive with flaviviruses, and a plaque reduction neutralization test (PRNT) for further testing. Overall, 42% of patients showed serological evidence of flavivirus infection (primarily past dengue virus [DENV] infection), while 7% possessed IgM Abs to ZIKV and/or DENV. ZIKV IgM Abs typically arose within 3 to 4 days, with only one instance of duration beyond 100 days after reported symptoms. PRNT analysis of 826 IgM-positive specimens showed 7% positive neutralization to ZIKV alone, 9% to DENV alone, and 85% to both ZIKV and DENV. Thus, the extensive Ab cross-reactivity among flaviviruses significantly reduced the value of performing PRNT analysis, especially when a traditional paired serum algorithm with viral neutralization titering was used. Nevertheless, the finding of a negative ZIKV result by PRNT was invaluable for reassuring both physicians and patients. The MIA detected both IgM and IgG, which enabled us to identify patients who presented without IgM anti-ZIKV Abs but still had ZIKV-specific neutralizing Abs. On the basis of these results, a new algorithm, which included an IgM Ab capture (MAC)-ELISA to detect recent infection, a flavivirus MIA to identify patients no longer producing IgM, and a single-dilution PRNT for ZIKV exclusion and occasional discrimination of ZIKV and DENV, was implemented.
Project description:Zika virus (ZIKV) is a mosquito-borne flavivirus that emerged recently as a global health threat, causing a pandemic in the Americas. ZIKV infection mostly causes mild disease, but is linked to devastating congenital birth defects and Guillain-Barré syndrome in adults. The high level of cross-reactivity among flaviviruses and their cocirculation has complicated serological approaches to differentially detect ZIKV and dengue virus (DENV) infections, accentuating the urgent need for a specific and sensitive serological test. We previously generated a ZIKV nonstructural protein 1 (NS1)-specific human monoclonal antibody, which we used to develop an NS1-based competition ELISA. Well-characterized samples from RT-PCR-confirmed patients with Zika and individuals exposed to other flavivirus infections or vaccination were used in a comprehensive analysis to determine the sensitivity and specificity of the NS1 blockade-of-binding (BOB) assay, which was established in laboratories in five countries (Nicaragua, Brazil, Italy, United Kingdom, and Switzerland). Of 158 sera/plasma from RT-PCR-confirmed ZIKV infections, 145 (91.8%) yielded greater than 50% inhibition. Of 171 patients with primary or secondary DENV infections, 152 (88.9%) scored negative. When the control group was extended to patients infected by other flaviviruses, other viruses, or healthy donors (n = 540), the specificity was 95.9%. We also analyzed longitudinal samples from DENV-immune and DENV-naive ZIKV infections and found inhibition was achieved within 10 d postonset of illness and maintained over time. Thus, the Zika NS1 BOB assay is sensitive, specific, robust, simple, low-cost, and accessible, and can detect recent and past ZIKV infections for surveillance, seroprevalence studies, and intervention trials.
Project description:Zika virus (ZIKV) is a mosquito-borne flavivirus that is responsible for recent explosive epidemics in the Americas. Notably, ZIKV infection during pregnancy has been found to cause congenital birth defects, including microcephaly, and ZIKV has been associated with Guillain-Barré syndrome in adults. Diagnosis and surveillance of Zika in the Americas have been challenging due to similar clinical manifestations and extensive antibody cross-reactivity with endemic flaviviral diseases, such as dengue. We evaluated four serological and two reverse transcription-PCR (RT-PCR) methods in acute-phase (mean day, 1.8), early-convalescent-phase (mean day, 16.7), and late-convalescent-phase (mean, ~7 months) samples from the same individuals in a long-term pediatric cohort study in Nicaragua. Well-characterized samples from 301 cases of Zika, dengue, or non-Zika, nondengue febrile illnesses were tested. Compared to a composite reference, an in-house IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) and the NIAID-Biodefense and Emerging Infections (BEI) MAC-ELISA measuring IgM yielded sensitivities of 94.5% and 70.1% and specificities of 85.6% and 82.8%, respectively. The NS1 blockade-of-binding ELISA measuring anti-ZIKV NS1 antibody levels yielded sensitivities of 85.0% and 96.5% and specificities of 91.4% and 92.6% at early and late convalescence, respectively. An inhibition ELISA detecting total anti-ZIKV antibodies had sensitivity and specificity values of 68.3% and 58.3% for diagnosis and 94.0% and 98.6% for measuring annual infection incidence. Finally, the ZCD and Trioplex real-time RT-PCR assays detecting Zika, chikungunya, and dengue viruses both yielded a sensitivity of 96.1% and specificity of 100%. Together, these assays resolve the urgent need for diagnostic and surveillance tools for countries affected by Zika virus infections.
Project description:The recent spread of Zika virus (ZIKV) in the Americas and Asia necessitates an increased preparedness for improved maternal and perinatal health and blood safety. However, serological cross-reactions, especially to Dengue virus (DENV), complicate ZIKV antibody serodiagnosis. A novel "pan-Flavi" suspension multiplex immunoassay (PFSMIA) using 25 antigens, whole virus (WV), non-structural protein 1 (NS1), and envelope (E) proteins, from 7 zoonotic flaviviruses for specific detection of ZIKV and DENV IgM and IgG was developed. Patterns of antibody cross-reactivity, avidity, and kinetics were established in 104 sera from returning travelers with known ZIKV and DENV infections. PFSMIA gave IgM- and IgG-sensitivities for both viruses of 96-100%, compared to an immunofluorescence assay. Main IgM cross-reactions were to NS1, for IgG to the E and WV antigens. Infecting virus yielded reactivity to several antigens of the homologous virus, while cross-reactions tended to occur only to a single antigen from heterologous virus(es). A specificity-enhancing computer procedure took into account antibody isotype, number of antibody-reactive antigens per virus, avidity, average degree of cross-reactivity to heterologous flavivirus antigens, and reactivity changes in serial sera. It classified all 50 cases correctly. Applied to sera from 200 pregnant women and 173 blood donors from Sweden, one blood donor was found ZIKV NS1 IgM positive, and another as ZIKV NS1 IgG positive. These samples did not react with other ZIKV antigens and were thereby judged as false-positives. PFSMIA provided sensitive and specific ZIKV and DENV serology, warranting high-throughput serological surveillance and a minimized need for laborious and expensive virus neutralization assays.
Project description:The recent outbreaks of Zika virus (ZIKV) in flavivirus-endemic regions highlight the need for sensitive and specific serological tests. Previously we and others reported key fusion loop (FL) residues and/or BC loop (BCL) residues on dengue virus (DENV) envelope protein recognized by flavivirus cross-reactive human monoclonal antibodies and polyclonal sera. To improve ZIKV serodiagnosis, we employed wild type (WT) and FL or FL/BCL mutant virus-like particles (VLP) of ZIKV, DENV1 and West Nile virus (WNV) in enzyme linked immunosorbent assays (ELISA), and tested convalescent-phase serum or plasma samples from reverse-transcription PCR-confirmed cases with different ZIKV, DENV and WNV infections. For IgG ELISA, ZIKV WT-VLP had a sensitivity of 100% and specificity of 52.9%, which was improved to 83.3% by FL/BCL mutant VLP and 92.2% by the ratio of relative optical density of mutant to WT VLP. Similarly, DENV1 and WNV WT-VLP had a sensitivity/specificity of 100%/70.0% and 100%/56.3%, respectively; the specificity was improved to 93.3% and 83.0% by FL mutant VLP. For IgM ELISA, ZIKV, DENV1 and WNV WT-VLP had a specificity of 96.4%, 92.3% and 91.4%, respectively, for primary infection; the specificity was improved to 93.7-99.3% by FL or FL/BCL mutant VLP. An algorithm based on a combination of mutant and WT-VLP IgG ELISA is proposed to discriminate primary ZIKV, DENV and WNV infections as well as secondary DENV and ZIKV infection with previous DENV infections; this could be a powerful tool to better understand the seroprevalence and pathogenesis of ZIKV in regions where multiple flaviviruses co-circulate.
Project description:Diseases caused by the genus Flavivirus, including dengue virus (DENV) and Zika virus (ZIKV), have a serious impact on public health worldwide. Due to serological cross-reactivity among flaviviruses, current enzyme-linked immunosorbent assay (ELISA) for IgM/G cannot reliably distinguish between infection by different flaviviruses. In this study, we developed a reporter-based neutralization assay using single-round infectious particles (SRIPs) derived from representative flaviviruses. SRIPs were generated by transfection of human embryonic kidney 293?T cells with a plasmid encoding premembrane and envelope (prME) proteins from DENV1-4, ZIKV, Japanese encephalitis virus, West Nile virus, yellow fever virus, Usutu virus, and tick-borne encephalitis virus, along with a plasmid carrying DENV1 replicon containing the luciferase gene and plasmid for expression of DENV1 capsid. Luciferase activity of SRIPs-infected cells was well correlated with number of infected cells, and each reporter SRIP was specifically neutralized by sera from mice immunized with each flavivirus antigen. Our high-throughput reporter SRIP-based neutralization assay for multiple flaviviruses is a faster, safer, and less laborious diagnostic method than the conventional plaque reduction neutralization test to screen the cause of primary flavivirus infection. The assay may also contribute to the evaluation of vaccine efficacy and assist in routine surveillance and outbreak response to flaviviruses.
Project description:Zika virus (ZIKV) is an emerging flavivirus that can cause birth defects and neurologic complications. Molecular tests are effective for diagnosing acute ZIKV infection, although the majority of infections produce no symptoms at all or present after the narrow window in which molecular diagnostics are dependable. Serology is a reliable method for detecting infections after the viremic period; however, most serological assays have limited specificity due to cross-reactive antibodies elicited by flavivirus infections. Since ZIKV and dengue virus (DENV) widely cocirculate, distinguishing ZIKV infection from DENV infection is particularly important for diagnosing individual cases or for surveillance to coordinate public health responses. Flaviviruses also elicit type-specific antibodies directed to non-cross-reactive epitopes of the infecting virus; such epitopes are attractive targets for the design of antigens for development of serological tests with greater specificity. Guided by comparative epitope modeling of the ZIKV envelope protein, we designed two recombinant antigens displaying unique antigenic regions on domain I (Z-EDI) and domain III (Z-EDIII) of the ZIKV envelope protein. Both the Z-EDI and Z-EDIII antigens consistently detected ZIKV-specific IgG in ZIKV-immune sera but not cross-reactive IgG in DENV-immune sera in late convalescence (>12 weeks postinfection). In contrast, during early convalescence (2 to 12 weeks postinfection), secondary DENV-immune sera and some primary DENV-immune sera cross-reacted with the Z-EDI and Z-EDIII antigens. Analysis of sequential samples from DENV-immune individuals demonstrated that Z-EDIII cross-reactivity peaked in early convalescence and declined steeply over time. The Z-EDIII antigen has much potential as a diagnostic antigen for population-level surveillance and for detecting past infections in patients.
Project description:Zika virus (ZIKV), transmitted by Aedes species mosquitoes, was first isolated in Uganda in 1947. From February 2014 to October 2017, the Uganda Virus Research Institute, in collaboration with the US Centers for Diseases Control and Prevention, conducted arbovirus surveillance in acute febrile illness (AFI) patients at St Francis hospital in Nkonkonjeru. Three hundred and eighty-four serum samples were collected and tested for IgM antibodies to yellow fever virus (YFV), West Nile virus (WNV), dengue virus (DENV), chikungunya virus (CHIKV) and ZIKV. Of the 384 samples, 5 were positive for ZIKV IgM. Of these five, three were confirmed by plaque reduction neutralization test (PRNT) to be ZIKV infections. Of the remaining two, one was determined to be a non-specific flavivirus infection and one was confirmed to be alphavirus-positive by reverse transcriptase polymerase chain reaction (RT-PCR). This study provides the first evidence of laboratory-confirmed ZIKV infection in Uganda in five decades, and emphasizes the need to enhance sentinel surveillance.
Project description:Zika virus (ZIKV) is a mosquito-borne flavivirus associated with a febrile illness as well as severe complications, including microcephaly and Guillain-Barré Syndrome. Antibody cross-reactivity between flaviviruses has been documented, and in regions where ZIKV is circulating, dengue virus (DENV) is also endemic, leaving the potential that previous exposure to DENV could alter clinical features of ZIKV infection. To investigate this, we performed a retrospective case-control study in which we compared Canadian travellers who had been infected with ZIKV and had serological findings indicating previous DENV or other flavivirus exposure (n = 16) to those without any previous exposure (n = 44). Patient samples were collected between February 2016 and September 2017 and submitted to Public Health Ontario for testing. ZIKV infection was determined using real-time RT-PCR and antibodies against DENV were identified by the plaque-reduction neutralization test. The mean time from symptom onset to sample collection was 5 days for both groups; the magnitude of viremia was not statistically different (Ct values: 35.6 vs. 34.9, p-value = 0.2). Clinical scores were also similar. Our findings indicate that previous DENV or other flavivirus exposure did not result in greater viremia or a higher illness score.