Evaluation of a Diagnostic Algorithm Using Immunoglobulin M Enzyme-Linked Immunosorbent Assay To Differentiate Human West Nile Virus and St. Louis Encephalitis Virus Infections during the 2002 West Nile Virus Epidemic in the United States

A diagnostic algorithm was developed to differentiate between human infections of West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) using positive-to-negative (P/N) ratios derived from the immunoglobulin M capture enzyme-linked immunosorbent assay (MAC-ELISA). To validate this algorithm, we tested 1,418 serum and cerebrospinal fluid (CSF) samples from confirmed WNV and SLEV infections collected during the WNV epidemic of 2002 in the United States. WNV P/N-to-SLEV P/N ratios (W/S ratios) were calculated and used to identify the infecting virus. These results were compared to results from the plaque reduction neutralization test (PRNT), which is currently the standard assay used to discriminate between closely related flavivirus infections. If the W/S ratio was ≥1, the predictive value positive (PNP) for WNV was 97.8%, where 95% of flavivirus cases were due to WNV infection and only 3.7% of specimens would require PRNT to differentiate WNV from SLEV infection. Use of the W/S ratio as part of the testing algorithm to interpret MAC-ELISA results generates reportable probable cases quickly, alleviating the need for PRNT in most instances.     

Evaluation of a diagnostic algorithm using immunoglobulin M enzyme-linked immunosorbent assay to differentiate human West Nile Virus and St. Louis Encephalitis virus infections during the 2002 West Nile Virus epidemic in the United States

A diagnostic algorithm was developed to differentiate between human infections of West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) using positive-to-negative (P/N) ratios derived from the immunoglobulin M capture enzyme-linked immunosorbent assay (MAC-ELISA). To validate this algorithm, we tested 1,418 serum and cerebrospinal fluid (CSF) samples from confirmed WNV and SLEV infections collected during the WNV epidemic of 2002 in the United States. WNV P/N-to-SLEV P/N ratios (W/S ratios) were calculated and used to identify the infecting virus. These results were compared to results from the plaque reduction neutralization test (PRNT), which is currently the standard assay used to discriminate between closely related flavivirus infections. If the W/S ratio was > or =1, the predictive value positive (PNP) for WNV was 97.8%, where 95% of flavivirus cases were due to WNV infection and only 3.7% of specimens would require PRNT to differentiate WNV from SLEV infection. Use of the W/S ratio as part of the testing algorithm to interpret MAC-ELISA results generates reportable probable cases quickly, alleviating the need for PRNT in most instances.     

Development of Monoclonal Antibodies to West Nile Virus and Their Application in Immunohistochemistry

West Nile virus (WNV) is endemic throughout Africa, Eurasia, America, and Australia and has important implications for avian, horse, and human health. In these regions, dead birds are monitored for the presence of WNV through immunohistochemistry (IHC) and PCR. However, a number of the tools for IHC are inadequate owing to their cross-reactivity to other Japanese encephalitis serogroup viruses. Here we have established eight monoclonal antibodies (MAbs) to WNV. Four of them bound to the envelope protein, three of them bound to nonstructural protein 1 (NS1), and one bound to precursor membrane protein (prM), as shown by Western blot analysis. The anti-NS1 MAbs and the anti-prM MAb did not cross-react with Japanese encephalitis virus (JEV), Murray valley encephalitis virus, or St. Louis encephalitis virus in an indirect enzyme-linked immunosorbent assay. One NS1-specific MAb, SHW-32B1, and the previously reported NS1-specific MAb, SHW-7A11, were shown by IHC to specifically detect the cytoplasm of degenerated cells in the heart and brain of a WNV-infected goose. Neither of these MAbs were shown by IHC to cross-react with degenerated cells in the brain of a JEV-infected pig. These MAbs are the first reported anti-NS1 MAbs that can be used for WNV-specific IHC using formalin-fixed, paraffin-embedded sections. They may be useful for WNV research and surveillance.     

Comparative analysis of immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay using virus-like particles or virus-infected mouse brain antigens to detect IgM antibody in sera from patients with evident flaviviral infections

The use of immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) serves as a valuable tool for the diagnosis of acute flaviviral infections, since IgM antibody titers are detectable early, peak at about 2 weeks postinfection, and subsequently decline to lower levels over the next few months. Traditionally, virus-infected tissue culture or suckling mouse brain (SMB) has been the source of viral antigens used in the assay. In an effort to provide a reliable source of standardized viral antigens for serodiagnosis of the medically important flaviviruses, we have developed a eukaryotic plasmid vector to express the premembrane/membrane and envelope proteins which self-assemble into noninfectious virus-like particles (VLPs). In addition to the plasmids for Japanese encephalitis virus, West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and dengue virus type 2 (DENV-2) reported earlier, we recently constructed the DENV-1, -3, and -4 VLP expression plasmids. Three blind-coded human serum panels were assembled from patients having recent DENV, SLEV, and WNV infections to assess the sensitivity and specificity of the MAC-ELISA using VLPs or SMB antigens. In addition, serum specimens from patients infected with either Powassan virus or La Crosse encephalitis virus were used to evaluate the cross-reactivity of seven mosquito-borne viral antigens. The results of the present studies showed higher sensitivity when using SLEV and WNV VLPs and higher specificity when using SLEV, WNV, and the mixture of DENV-1 to -4 VLPs in the MAC-ELISA than when using corresponding SMB antigens. Receiver operating characteristic (ROC) curve analysis, a plot of the sensitivity versus false positive rate (100 - specificity), was applied to discriminate the accuracy of tests comparing the use of VLPs and SMB antigen. The measurement of assay performance by the ROC analysis indicated that there were statistically significant differences in assay performance between DENV and WNV VLPs and the respective SMB antigens. Additionally, VLPs had a lower cutoff positive/negative ratio than corresponding SMB antigens when employed for the confirmation of current infections. The VLPs also performed better than SMB antigens in the MAC-ELISA, as indicated by a higher positive prediction value and positive likelihood ratio test. Cell lines continuously secreting these VLPs are therefore a significantly improved source of serodiagnostic antigens compared to the traditional sources of virus-infected tissue culture or suckling mouse brain.     

Nonstructural Protein 1-Specific Immunoglobulin M and G Antibody Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Flaviviral Infections in Humans

IgM antibody- and IgG antibody-capture enzyme-linked immunosorbent assays (MAC/GAC-ELISAs) targeted at envelope protein (E) of dengue viruses (DENV), West Nile virus, and Japanese encephalitis virus (JEV) are widely used as serodiagnostic tests for presumptive confirmation of viral infection. Antibodies directed against the flavivirus nonstructural protein 1 (NS1) have been proposed as serological markers of natural infections among vaccinated populations. The aim of the current study is to optimize an IgM and IgG antibody-capture ELISA (MAC/GAC-ELISA) to detect anti-NS1 antibodies and compare it with anti-E MAC/GAC-ELISA. Plasmids to express premembrane/envelope (prM/E) or NS1 proteins of six medically important flaviviruses, including dengue viruses (DENV-1 to DENV-4), West Nile virus (WNV), and Japanese encephalitis virus (JEV), were constructed. These plasmids were used for the production of prM/E-containing virus-like particles (VLPs) and secreted NS1 (sNS1) from COS-1 cells. Archived clinical specimens from patients with confirmed DENV, JEV, and WNV infections, along with naive sera, were subjected to NS1-MAC/GAC-ELISAs before or after depletion of anti-prM/E antibodies by preabsorption with or without VLPs. Human serum specimens from previously confirmed DENV infections showed significantly enhanced positive-to-negative (P/N) ratios for NS1-MAC/GAC-ELISAs after the depletion of anti-prM/E antibodies. No statistical differences in sensitivities and specificities were found between the newly developed NS1- and VLP-MAC/GAC-ELISAs. Further application of the assays to WNV- and JEV-infected serum panels showed similar results. A novel approach to perform MAC/GAC-ELISAs for NS1 antibody detection was successfully developed with great potential to differentiate antibodies elicited by the tetravalent chimeric yellow fever-17D/dengue vaccine or DENV infection.     

Enzyme-linked immunosorbent assays using novel Japanese encephalitis virus antigen improve the accuracy of clinical diagnosis of flavivirus infections

The cross-reactive antibodies induced by flavivirus infections confound serodiagnosis and pathogenesis, especially in secondary infections caused by antigenically closely related yet distinct flaviviruses. The envelope (E) glycoprotein fusion peptide contains immunodominant cross-reactive determinants. Using a recombinant Japanese encephalitis virus (JEV) premembrane and E expression plasmid producing JEV virus-like particles (VLPs), dramatic reductions in cross-reactivity were produced by the G106K-L107D (KD) double-mutant VLP against a panel of flavivirus murine monoclonal antibodies. Human serum panels from patients with recent flavivirus infections were analyzed to compare the accuracy of JEV wild-type (WT) and KD VLPs as serodiagnostic antigens in enzyme-linked immunosorbent assays. Statistical analysis demonstrated significant differences in assay performances for accurate determination of current JEV infections between WT and KD antigens by detecting immunoglobulin M antibodies at a serum dilution of 1:4,000 (likelihood ratios = 2.74 [WT] and 22 [KD]). The application and continued development of cross-reactivity-reduced antigens should improve both flavivirus infection serodiagnosis and estimates of disease burden.     

Non-structural protein 1 (NS1) antibody-based assays to differentiate West Nile (WN) virus from Japanese encephalitis virus infections in horses: effects of WN virus NS1 antibodies induced by inactivated WN vaccine

Antibodies to non-structural protein 1 (NS1) of West Nile virus (WNV) have been used to differentiate WNV infection from infection by serologically cross-reactive flaviviruses, including Japanese encephalitis virus (JEV), in horses. However, since the inactivated West Nile (WN) vaccine has been reported to induce NS1 antibodies, there is concern about the reliability of using NS1-based assays for testing vaccinated horses. Therefore, the effect of inactivated WN vaccine-induced antibodies on an epitope-blocking ELISA and complement-dependent cytotoxicity (CDC) assay were investigated. Both assays are based on NS1 antibodies and were established previously to differentiate WNV from JEV infections in horses. Groups of three horses were vaccinated with two or three doses of a commercial inactivated WN vaccine and NS1 antibodies were detected by a conventional ELISA after the second vaccination. Vaccine-induced NS1 antibodies were also detected by blocking ELISA and a CDC assay and affected the ability of these assays to differentiate WNV from JEV infections. However, the effect was less significant in the CDC assay, where use of a low serum concentration ensured effective differentiation. The more efficient detection of infection-induced antibodies over vaccine-induced antibodies by the CDC assay was potentially attributable to the different IgG isotype profiles of these antibodies.     

Screening for T cell-eliciting proteins of <Emphasis Type="Italic">Japanese encephalitis virus</Emphasis> in a healthy JE-endemic human cohort using recombinant baculovirus-infected insect cell preparations

Summary. The analysis of cell-mediated immune responses in virus-exposed but healthy individuals may contribute to define the features of the T cell response associated with resistance. We report, for the first time, on adaptive T cell responses to 5 largest of the 10 proteins that together constitute 76% of the coding potential of the Japanese encephalitis virus (JEV) genome in a naturally exposed healthy JE-immune human cohort. Fixed and sonified whole cell preparations of insect cells individually expressing recombinant prM, E, NS1, NS3 and NS5 proteins of JEV were used in vitro to stimulate lymphocytes from individuals who had experienced subclinical JEV infections. NS3-specific memory T cells were detected in up to 86% of the JEV-infected cohort whereas prM, E and NS1 each elicited reactions in approximately 45% among individuals tested, suggesting that NS3 is an important target for JEV-specific cell-mediated immune responses. Responses to NS5, the largest viral protein were in contrast the poorest, seen in only 13% of the cohort. Moreover, NS3 stimulated interferon- production in both CD4⁺ and CD8⁺ T cells indicating that a Th1 immune response to the NS3 protein may be a critical determinant of immune control of JEV infection.Received July 26, 2002; accepted March 18, 2003 Published online June 2, 2003     

Comparison of Capture Immunoglobulin M (IgM) and IgG Enzyme-Linked Immunosorbent Assay (ELISA) and Nonstructural Protein NS1 Serotype-Specific IgG ELISA for Differentiation of Primary and Secondary Dengue Virus Infections

We have found that NS1 serotype-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) can be used to differentiate primary and secondary dengue virus infections. This is due to the fact that the NS1-specific IgG antibody cannot be detected before day 9 of illness for primary infection, so the NS1-specific IgG antibodies measured in acute-phase sera must come from previous infection. Comparison of NS1 serotype-specific IgG ELISA with envelope- and membrane-specific capture IgM and IgG ELISA in the differentiation of primary and secondary dengue virus infections showed good correlation (95.90% agreement). Most important, we have found that the serotype of the dengue virus from the majority of patients with primary infection could be correctly identified when convalescent-phase or postinfection sera were analyzed by NS1 serotype-specific IgG ELISA. These findings suggested that NS1 serotype-specific IgG ELISA could be reliably applied for serodiagnosis and seroepidemiological study of dengue virus infection.     

Characterization and application of monoclonal antibodies specific to West Nile virus envelope protein

Recent epidemics of West Nile virus (WNV) around the world have been associated with significant rates of mortality and morbidity in humans. To develop standard WNV diagnostic tools that can differentiate WNV from Japanese encephalitis virus (JEV), four monoclonal antibodies (MAbs) specific to WNV envelope (E) protein were produced and characterized by isotyping, reactivity with denatured and native antigens, affinity assay, immunofluorescence assay (IFA), and epitope competition, as well as cross-reactivity with JEV. Two of the MAbs (6A11 and 4B3) showed stronger reactivity with E protein than the others (2F5 and 6H7) in Western blot analysis. 4B3 could bind with denatured antigen, as well as native antigens in indirect ELISA, flow cytometry analysis, and IFA; whereas 2F5 showed highest affinity with native antigen. 4B3 and 2F5 were therefore used to establish an antigen capture-ELISA (AC-ELISA) detection system. The sensitivity of this AC-ELISA was 3.95 TCID(50)/0.1 ml for WNV-infected cell culture supernatant. Notably, these MAbs showed no cross-reactivity with JEV, which suggests that they are useful for further development of highly sensitive, easy handling, and less time-consuming detection kits/tools in WNV surveillance in areas where JEV is epidemic.     

Evaluation of an Epitope-Blocking Enzyme-Linked Immunosorbent Assay for the Diagnosis of West Nile Virus Infections in Humans

An epitope-blocking enzyme-linked immunosorbent assay (b-ELISA) was evaluated for the diagnosis of West Nile virus (WNV) infections in humans. Sera from patients diagnosed with WNV infections from an outbreak in 2003 in Colorado and from patients diagnosed with dengue virus infections from Mexico and Thailand were tested with the b-ELISA. The b-ELISAs were performed using the WNV-specific monoclonal antibody (MAb) 3.1112G and the flavivirus-specific MAb 6B6C-1. Although the WNV-specific b-ELISA was effective in diagnosing WNV infections in humans from Colorado, it was not efficacious for diagnosing WNV infections in serum specimens from Mexico and Thailand. In serum specimens from patients from Colorado, the WNV b-ELISA and the WNV plaque reduction neutralization test showed an overall agreement of 91%. The sensitivity and specificity of the WNV b-ELISA were 89% and 92%, respectively, with a false-positive rate of 5%, based on receiver operating characteristic analysis. In contrast, false-positive rate results in specimens from the countries of Mexico and Thailand, where flaviviruses are endemic, were 79% and 80%, presumably due to the presence of antibodies resulting from previous dengue virus infections in Mexico and/or Japanese encephalitis virus infections or vaccination in Thailand. Thus, in regions where people have experienced previous or multiple flavivirus infections, the use of the b-ELISA for WNV diagnosis is contraindicated.The most medically important flaviviruses include dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), tick-borne encephalitis virus (TBEV), and Saint Louis encephalitis virus (SLEV) (16, 31, 38). Flaviviruses are positive-strand RNA viruses with genomes of approximately 11 kb that encode three structural and seven nonstructural (NS) proteins in the gene order C (capsid), M (membrane), E (envelope), NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. WNV is a member of the JEV serocomplex within the genus Flavivirus, family Flaviviridae. The virus has been isolated in Africa, Australia, Eastern Europe, the Middle East, North America, and South America (7, 20, 24). WNV was first detected in the United States in July 1999 and spread rapidly throughout the country, causing large numbers of infections in humans, horses, and birds (19, 31).Prior to 1999, flavivirus infections in humans in the United States were infrequent, and most were attributed to sporadic cases of SLEV and travel-associated cases of DENV (41). In Thailand, all four DENV serotypes and JEV circulate (39), resulting in very high flavivirus transmission and seroprevalence rates. In the Yucatán Peninsula of Mexico, all four DENV serotypes circulate and seroprevalence rates are very high (8). Serological diagnosis of WNV infections is complicated by the high rates of both primary DENV infections and secondary DENV infections in inhabitants of Thailand and Yucatan, Mexico, with seroprevalence rates of >85% in Thailand (1) and 72% in the Yucatán in 1985 (12, 28). WNV introduction into the Yucatán in 2002 was revealed by detection of antibodies in horses (29) and then later in migratory and resident birds (10) and in zoo animals (11). However, no WNV infections of humans have been diagnosed in the Yucatán.The immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) is the preferred test used for diagnosis of WNV in humans in the United States (32). The test is used to detect antibodies to WNV in serum and/or cerebrospinal fluid. The plaque reduction neutralization test (PRNT) is the gold standard for serodiagnosis of flavivirus infections and for identifying the infecting agent (2). However, both of these tests can be confounded if patients have had previous flavivirus infections. Indeed, diagnosis of flavivirus infections in humans is very difficult in geographic areas where multiple flaviviruses are circulating and cause sequential infections. Because of “original antigenic sin” the highest antibody titer may be due to a previous flavivirus infection rather than to the etiologic agent (18, 26). Serological diagnosis of WNV, SLEV, and YFV infections is extremely difficult in patients from areas where DENV is hyperendemic.Previously, we exploited an epitope-blocking ELISA (b-ELISA) to detect antibodies to WNV in diverse species of birds and domestic mammals (3, 4). The WNV b-ELISA measures the ability of antibodies present in sera to block the binding of a monoclonal antibody (MAb) to a WNV-specific epitope on the NS1 protein (17). The WNV b-ELISA had not been previously evaluated for use in humans. In this study, a WNV-specific and a flavivirus broadly reactive b-ELISAs were evaluated for their abilities to detect antibodies against WNV in human serum specimens from countries with differing levels of flavivirus endemicity: the United States, Thailand, and Mexico. The objectives of this study were (i) to determine the ability of the b-ELISA to detect antibodies to WNV in human serum samples and (ii) to determine the effects of previous flavivirus infections of patients (e.g., DENV and JEV) on the diagnostic efficacy of the WNV b-ELISA.     

Epitope-blocking enzyme-linked immunosorbent assay to differentiate west nile virus from Japanese encephalitis virus infections in equine sera

West Nile virus (WNV) is now widely distributed worldwide, except in most areas of Asia where Japanese encephalitis virus (JEV) is distributed. Considering the movement and migration of reservoir birds, there is concern that WNV may be introduced in Asian countries. Although manuals and guidelines for serological tests have been created in Japan in preparedness for the introduction of WNV, differential diagnosis between WNV and JEV may be complicated by antigenic cross-reactivities between these flaviviruses. Here, we generated a monoclonal antibody specific for the nonstructural protein 1 (NS1) of WNV and established an epitope-blocking enzyme-linked immunosorbent assay that can differentiate WNV from JEV infections in horse sera. Under conditions well suited for our assay system, samples collected from 95 horses in Japan (regarded as negative for WNV antibodies), including those collected from horses naturally infected with JEV, showed a mean inhibition value of 8.2% and a standard deviation (SD) of 6.5%. However, inhibition values obtained with serum used as a positive control (obtained after 28 days from a horse experimentally infected with WNV) in nine separate experiments showed a mean of 54.4% and an SD of 7.1%. We tentatively determined 27.6% (mean + 3 x SD obtained with 95 negative samples) as the cutoff value to differentiate positive from negative samples. Under this criterion, two horses experimentally infected with WNV were diagnosed as positive at 12 and 14 days, respectively, after infection.     

Differentiation of West Nile and St. Louis encephalitis virus infections by use of noninfectious virus-like particles with reduced cross-reactivity

Differential diagnosis of St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) infections can be complicated due to the high degree of cross-reactivity observed in most serodiagnostic assays. In an effort to provide a more specific diagnostic test, we developed virus-like particle (VLP) antigens with reduced cross-reactivity for both SLEV and WNV by identifying and mutating envelope protein amino acids within the cross-reactive epitopes of VLP expression plasmids. To determine the serodiagnostic discriminatory ability of the antigens with reduced cross-reactivity, a panel of 134 human serum samples collected predominately from North American patients with SLEV or WNV infections was used to evaluate the performance of these novel antigens in imunoglobulin M antibody-capture enzyme-linked immunosorbent assays. Positive/negative ratios and the resulting diagnostic classifications were compared between the mutant and the wild-type (WT) VLPs. The mutant VLP antigens were more specific, with higher positive predictive values and higher likelihood ratios than the WT VLP antigens. Both the SLEV and WNV mutant VLPs greatly reduced the observed cross-reactivity, significantly increasing the specificity and sensitivity of the assay. The use of these novel VLP antigens with reduced cross-reactivity in these serodiagnostic assays and others should lead to more accurate diagnoses of current infections, thereby reducing the need for time-consuming and cumbersome confirmatory plaque-reduction neutralization tests to differentiate between SLEV and WNV infections in North America.     

Comparison of capture immunoglobulin M (IgM) and IgG enzyme-linked immunosorbent assay (ELISA) and nonstructural protein NS1 serotype-specific IgG ELISA for differentiation of primary and secondary dengue virus infections

We have found that NS1 serotype-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) can be used to differentiate primary and secondary dengue virus infections. This is due to the fact that the NS1-specific IgG antibody cannot be detected before day 9 of illness for primary infection, so the NS1-specific IgG antibodies measured in acute-phase sera must come from previous infection. Comparison of NS1 serotype-specific IgG ELISA with envelope- and membrane-specific capture IgM and IgG ELISA in the differentiation of primary and secondary dengue virus infections showed good correlation (95.90% agreement). Most important, we have found that the serotype of the dengue virus from the majority of patients with primary infection could be correctly identified when convalescent-phase or postinfection sera were analyzed by NS1 serotype-specific IgG ELISA. These findings suggested that NS1 serotype-specific IgG ELISA could be reliably applied for serodiagnosis and seroepidemiological study of dengue virus infection.     

Analyses of mutations selected by passaging a chimeric flavivirus identify mutations that alter infectivity and reveal an interaction between the structural proteins and the nonstructural glycoprotein NS1

We previously described a single-cycle dengue vaccine (RepliVAX D2) engineered from a capsid (C) gene-deleted West Nile virus (WNV) expressing dengue virus serotype 2 (DENV2) prM/E genes in place of the corresponding WNV genes. That work demonstrated that adaptation of RepliVAX D2 to grow in WNV C-expressing cells resulted in acquisition of non-synonymous mutations in the DENV2 prM/E and WNV NS2A/NS3 genes. Here we demonstrate that the prM/E mutations increase the specific infectivity of chimeric virions and the NS2A/NS3 mutations independently enhance packaging. Studies with the NS2A mutant demonstrated that it was unable to produce a larger form of NS1 (NS1'), suggesting that the mutation had been selected to eliminate a ribosomal frame-shift “slippage site” in NS2A. Evaluation of a synonymous mutation at this slippage site confirmed that genomes that failed to make NS1' were packaged more efficiently than WT genomes supporting a role for NS1/NS1' in orchestrating virion assembly.     

Use of immunoglobulin m cross-reactions in differential diagnosis of human flaviviral encephalitis infections in the United States

To define the virus specificity of the immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) among the medically important members of the Japanese encephalitis (JE) virus serocomplex of flaviviruses, 103 IgM-positive human serum samples from patients with confirmed West Nile (WN) virus, St. Louis encephalitis (SLE) virus, or JE virus infections were assembled and simultaneously tested against all three viral antigens in a standardized MAC-ELISA. Of the serum samples tested, 96 (93%) showed higher positive-to-negative absorbance ratios (P/Ns) with the infecting virus antigen compared to those obtained with the other two virus antigens. Of the seven specimens with higher P/Ns with heterologous virus antigens, six were from patients with SLE virus infections (the serum samples had higher levels of reactivity with WN virus antigen) and one was from a patient with a JE virus infection (this serum sample also had a higher level of reactivity with WN virus antigen). Not surprisingly, similar virus specificity was observed with WN virus-elicited IgM in cerebrospinal fluid. As shown in previous studies, a subset of these specimens was even less reactive in the MAC-ELISA with dengue virus, a member of a different flavivirus serocomplex. The degree of virus cross-reactivity did not appear to be related to days postonset, at least during the first 40 days of infection. Infections with WN virus could be correctly distinguished from infections with SLE virus on the basis of the observed anti-viral IgM cross-reactivities alone 92% of the time. Infections with SLE virus resulted in antibody that was more cross-reactive, so identification of SLE virus as the infecting agent by use of MAC-ELISA cross-reactivity alone was more problematic.     

Use of Immunoglobulin M Cross-Reactions in Differential Diagnosis of Human Flaviviral Encephalitis Infections in the United States

To define the virus specificity of the immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) among the medically important members of the Japanese encephalitis (JE) virus serocomplex of flaviviruses, 103 IgM-positive human serum samples from patients with confirmed West Nile (WN) virus, St. Louis encephalitis (SLE) virus, or JE virus infections were assembled and simultaneously tested against all three viral antigens in a standardized MAC-ELISA. Of the serum samples tested, 96 (93%) showed higher positive-to-negative absorbance ratios (P/Ns) with the infecting virus antigen compared to those obtained with the other two virus antigens. Of the seven specimens with higher P/Ns with heterologous virus antigens, six were from patients with SLE virus infections (the serum samples had higher levels of reactivity with WN virus antigen) and one was from a patient with a JE virus infection (this serum sample also had a higher level of reactivity with WN virus antigen). Not surprisingly, similar virus specificity was observed with WN virus-elicited IgM in cerebrospinal fluid. As shown in previous studies, a subset of these specimens was even less reactive in the MAC-ELISA with dengue virus, a member of a different flavivirus serocomplex. The degree of virus cross-reactivity did not appear to be related to days postonset, at least during the first 40 days of infection. Infections with WN virus could be correctly distinguished from infections with SLE virus on the basis of the observed anti-viral IgM cross-reactivities alone 92% of the time. Infections with SLE virus resulted in antibody that was more cross-reactive, so identification of SLE virus as the infecting agent by use of MAC-ELISA cross-reactivity alone was more problematic.     

Induction of virus-specific neutralizing immune response against West Nile and Japanese encephalitis viruses by chimeric peptides representing T-helper and B-cell epitopes

West Nile virus (WNV) and Japanese encephalitis virus (JEV), the members of JEV serocomplex group are pathogens of global health concern. The co-circulation of these viruses poses challenges in effective diagnostics due to antigenic similarity between the E-protein of these viruses. The present study aimed to design chimeric peptides and study the immune response against the same. B-cell epitopes were predicted on structural proteins of WNV and JEV based on bioinformatics tools. The peptides representing to these B-cell epitopes were synthesized and subjected to ELISA. Two peptides, one each from WNV (named WE147) and JEV (named JE40) E-protein, showed virus-specific and strong reactivity to the immune mice sera and human clinical samples. The chimeric peptides for WNV and JEV were constructed by synthesizing the B-cell epitope of WNV (WE147) or JEV (JE40) with T-helper epitope (JM17) separated by diglycine spacer in between. The immune response generated against these chimeric peptides was found to be specific to the respective B-cell epitopes. The anti-peptide sera showed virus-specific reactivity in ELISA and in immunofluorescence assay with no cross-reactivity. Also, the anti-peptide sera could neutralize JE and WN viruses in an in vitro virus neutralization assay. The B-cell epitopes identified in the present study may be used as diagnostic markers for differentiating between WN and JE virus infections. The present study can form a basis for future design of vaccines.     

Duplex microsphere-based immunoassay for detection of anti-West Nile virus and anti-St. Louis encephalitis virus immunoglobulin m antibodies

West Nile (WN) virus was introduced into the United States in 1999, when the first human cases of WN fever and encephalitis appeared in New York City. From there, the virus has spread throughout North America, in some areas cocirculating with the related flavivirus St. Louis encephalitis (SLE) virus. Public health laboratories currently use an immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) as a primary test for human serodiagnosis, followed by a confirmatory plaque-reduction neutralization test (PRNT). The MAC-ELISAs take 2 days to perform; therefore there is a need for a more rapid test. This report describes a duplex microsphere-based immunoassay (MIA) that shortens the test processing time to about 4.5 h. The assay employs two sets of microspheres coupled to a single flavivirus group-reactive antibody, which are used to capture the WN and SLE viral antigens independently. Immunoglobulin G-depleted serum is concurrently assayed for IgM antibodies to each of the viral antigens. The results are standardized and classified by using quadratic discriminant analysis so that a single result, anti-WN IgM-positive, anti-SLE IgM-positive, negative, or nonspecific, can be determined. The duplex MIA results compared favorably to those of the plaque-reduction neutralization test and MAC-ELISA. The assay proved to be reproducible, produced accurate classifications as to the infecting virus, and was specific.