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.     

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.     

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.     

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.Since its introduction into the United States in 1999, West Nile (WN) virus has spread throughout most of the country. Human disease cases have been reported in all states except Alaska, Hawaii, and Washington as of October 2004. A total of 9175 human disease cases were reported to Centers for Disease Control ArboNET for 2003, as reported in the Centers for Disease Control West Nile website (http://www.cdc.gov/ncidod/dvbid/westnile/index.htm). The related flavivirus St. Louis encephalitis (SLE) virus is endemic in the United States. A total of 4482 confirmed human disease cases of SLE have been documented between 1964 and 2000 (http://www.cdc.gov/ncidod/dvbid/arbor/pdf/cases-sle-1964to2000.pdf). The last major outbreak of SLE in the United States occurred in 1974 to 1977, when more than 2500 human SLE disease cases were reported.WN and SLE virus infections often present with similar clinical profiles. Symptoms common to both diseases may include sudden onset of fever, headache, and myalgia in mild cases and disorientation, meningitis, and encephalitis in severely affected patients. WN virus can produce a rash, and flaccid paralysis has been reported in some cases (5).Both WN and SLE viruses belong to the Japanese encephalitis virus serocomplex of viruses (12). Not only do they share many clinical manifestations but they are serologically similar. Immunoglobulin G (IgG) antibodies to viruses within the serocomplex exhibit extensive cross-reactivity, whereas immunoglobulin M (IgM) antibodies are less cross-reactive (10, 15). The traditional serological method for identifying the infecting virus is the time-consuming and technically difficult plaque-reduction neutralization test (PRNT) (8). The serological testing algorithm that has been adopted by most of the United States state health departments uses the IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) (9) and IgG-ELISA (6) as primary tests. A confirmatory PRNT for positive samples is often performed by laboratories that have this capability. The MAC-ELISA is a 2-day test that requires about 4 h of hands-on time for a 40-sample test. The advent of a more rapid yet equally sensitive, single test to replace the WN and SLE MAC-ELISAs would be of benefit.Microsphere-based immunoassays (MIAs) are becoming increasingly popular as a serological option for laboratory diagnosis of many diseases (4, 7). The technology involves the detection and analysis of a reaction attached to microspheres or beads. The detecting instrument is a simplified flow cytometer, and lasers simultaneously identify the microsphere sets (bead sets) and measure the fluorescence associated with the reaction. The speed at which these tests can be performed and the ability to multiplex make this methodology particularly attractive. MIAs have the potential to be especially applicable in arbovirus serology because tests for infection due to viruses of the same genus can share similar formats.MIAs using microspheres coupled to recombinant envelope and nonstructural 5 proteins of WN virus have been described recently by Wong et al. (16, 17). Here we describe a different format that utilizes an antibody that, when coupled to beadsets, can be used to assay for human IgM antibodies directed against any flavivirus, in this case WN and SLE viruses. The data transformation methodology constitutes a significant departure from those used in other serological methods for arbovirology.     

Detection of Japanese encephalitis virus immunoglobulin M antibodies in serum by antibody capture radioimmunoassay.

An assay for detecting human immunoglobulin M (IgM) antibodies to Japanese encephalitis (JE) virus was developed by using the antibody capture solid-phase radioimmunoassay approach (JE IgM ACRIAAA). Heavy-chain-specific goat antihuman IgM was first bound to the wells of a polyvinyl microtiter plate, and successive steps involved sequential binding of test sample IgM, acetone-extracted mouse brain JE antigen, and (125)I-labeled flavivirus hyperimmune human IgG. Among 20 patients hospitalized in Bangkok with clinical diagnoses of acute encephalitis, and with acute flavivirus infections proven by hemagglutination inhibition (HAI) serology, 16 had detectable (positive/negative [P/N] ratio, greater than 3.0) JE IgM ACRIA antibodies in the acute-phase serum specimen, and 19 had such antibodies in the convalescent-phase serum specimen. Convalescent patient sera regularly had higher P/N values than the corresponding acute-phase sera (mean +/- 1 standard deviation = 13.0 +/- 9.3 with acute-phase sera and 25.8 +/- 19.6 with convalescent-phase sera). JE virus-infected patients with HAI serological responses indicative of a primary flavivirus infection had higher JE IgM ACRI P/N responses than did those patients whose serological response indicated past exposure to other flaviviruses. None of 70 serum specimens from healthy Thai adults and children with serum JE HAI antibodies had detectable JE IgM ACRIA activity (P/N ratios all less than or equal to 3.0). Biological false-positives with low P/N ratios (range, 3 to 15) were found in sera from patients with acute or recent infections with flaviviruses other than JE virus but could be differentiated by the fact that these sera gave higher P/N ratios with homologous antigens than with JE virus. False-positive reactions with low P/N ratios (range, 3 to 6) due to serum rheumatoid factor activity were differentiated by testing with control antigen. The JE IgM ACRIA technique permits a rapid, accurate diagnosis of acute JE virus infections in both patients with and those without previous exposure to other flaviviruses.     

Antibody-capture ELISA for detection of immunoglobulin M antibodies in sera from Japanese encephalitis and dengue hemorrhagic fever patients

Immunoglobulin M (IgM) antibody titers in paired sera from 19 encephalitis and 44 dengue hemorrhagic fever (DHF) patients in Thailand and 42 Japanese encephalitis (JE) patients in Japan were measured by the antibody capture ELISA and applied to distinguish JE virus infection from dengue virus infection. Titer distribution and the ratio of the titers against JE and dengue antigens led to the following diagnostic criteria. The specimens can be considered as positive with JE when IgM-ELISA titer showed over 200 against JE and 4-fold or more higher than titers against any types of dengue antigens. The specimens can be considered as positive with dengue infection when IgM ELISA titer showed over 200 against one of the 4 types of dengue antigens and 4-fold or more higher than against JE antigen. Based on these criteria, 41 of 42 patients in Japan and 11 of 19 encephalitis patients in Thailand could be diagnosed as having JE virus infection while 2 of 19 encephalitis patients in Thailand and 26 of 44 DHF patients in Thailand could be diagnosed as having dengue virus infections.     

Establishment of an Algorithm Using prM/E- and NS1-Specific IgM Antibody-Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Japanese Encephalitis Virus and West Nile Virus Infections in Humans

The front-line assay for the presumptive serodiagnosis of acute Japanese encephalitis virus (JEV) and West Nile virus (WNV) infections is the premembrane/envelope (prM/E)-specific IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Due to antibody cross-reactivity, MAC-ELISA-positive samples may be confirmed with a time-consuming plaque reduction neutralization test (PRNT). In the present study, we applied a previously developed anti-nonstructural protein 1 (NS1)-specific MAC-ELISA (NS1-MAC-ELISA) on archived acute-phase serum specimens from patients with confirmed JEV and WNV infections and compared the results with prM/E containing virus-like particle-specific MAC-ELISA (VLP-MAC-ELISA). Paired-receiver operating characteristic (ROC) curve analyses revealed no statistical differences in the overall assay performances of the VLP- and NS1-MAC-ELISAs. The two methods had high sensitivities of 100% but slightly lower specificities that ranged between 80% and 100%. When the NS1-MAC-ELISA was used to confirm positive results in the VLP-MAC-ELISA, the specificity of serodiagnosis, especially for JEV infection, was increased to 90% when applied in areas where JEV cocirculates with WNV, or to 100% when applied in areas that were endemic for JEV. The results also showed that using multiple antigens could resolve the cross-reactivity in the assays. Significantly higher positive-to-negative (P/N) values were consistently obtained with the homologous antigens than those with the heterologous antigens. JEV or WNV was reliably identified as the currently infecting flavivirus by a higher ratio of JEV-to-WNV P/N values or vice versa. In summary of the above-described results, the diagnostic algorithm combining the use of multiantigen VLP- and NS1-MAC-ELISAs was developed and can be practically applied to obtain a more specific and reliable result for the serodiagnosis of JEV and WNV infections without the need for PRNT. The developed algorithm should provide great utility in diagnostic and surveillance activities in which test accuracy is of utmost importance for effective disease intervention.     

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

A microsphere-based immunoassay (MIA) was previously developed that is capable of determining the presence of anti-West Nile (WN) virus or anti-St. Louis encephalitis (SLE) virus immunoglobulin M (IgM) antibodies in human serum or cerebrospinal fluid. The original data set on which the classification rules were based comprised 491 serum specimens obtained from the serum bank at the Division of Vector-Borne Infectious Diseases of the Centers for Disease Control and Prevention (DVBID). The classification rules were used to provide a result and to determine whether confirmatory testing was necessary for a given sample. A validation study was coordinated between the DVBID and five state health laboratories to determine (i) the reproducibility of the test between different laboratories, (ii) the correlation between the IgM-enzyme-linked immunosorbent assay (MAC-ELISA) and the MIA, and (iii) whether the initial nonspecific parameters could be refined to reduce the volume of confirmatory testing. Laboratorians were trained in the method, and reagents and data analysis software developed at the DVBID were shipped to each validating laboratory. Validating laboratories performed tests on approximately 200 samples obtained from their individual states, the collections of which comprised approximately equal numbers of WN virus-positive and -negative samples, as determined by MAC-ELISA. In addition, 377 samples submitted to the DVBID for arbovirus testing were analyzed using the MIA and MAC-ELISA at the DVBID only. For the specimens tested at both the state and the DVBID laboratories, a correlation of results indicated that the technology is readily transferable between laboratories. The detection of IgM antibodies to WN virus was more consistent than detection of IgM antibodies to SLE virus. Some changes were made to the analysis software that resulted in an improved accuracy of diagnosis.     

Group B Arbovirus Structural and Nonstructural Antigens III. Serological Specificity of Solubilized Intracellular Viral Proteins

Solubilized nonstructural antigen III from Saint Louis encephalitis (SLE)-Japanese B encephalitis (JBE)-West Nile (WN) and dengue-2 arbovirus-infected pig kidney cells was purified by employing Brij-58 solubilization, organic solvent extraction, and column chromatography. Diethylaminoethyl-cellulose column peak C eluate contained only intracellular viral envelope protein designated antigen I. Antigen I of SLE virus absorbed homologous neutralizing antibody; however, the intracellular nonstructural protein designated antigen III did not. The antigenic relationships of solubilized antigens I and III prepared from SLE, JBE, and WN virus-infected cells were determined by complement-fixation (CF) and immunodiffusion analyses. Solubilized antigen I of each virus cross-reacted broadly with both homologous and heterologous antibody at high antigen concentrations in the CF test. Antigen III of each virus reacted with only homologous antibody by both CF and immunoprecipitation. This study demonstrates that during SLE, JBE, WN, and dengue-2 infections, virus-specific proteins containing both type-specific and group-reactive determinants are synthesized. Antigen III, a nonstructural protein, is serologically virus type specific and may be useful as a type-specific diagnostic reagent.     

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.     

Detection of anti-West Nile virus immunoglobulin M in chicken serum by an enzyme-linked immunosorbent assay

The emergence of West Nile (WN) virus in New York and the surrounding area in 1999 prompted an increase in surveillance measures throughout the United States, including the screening of sentinel chicken flocks for antibodies. An enzyme-linked immunosorbent assay (ELISA) for the detection of chicken immunoglobulin M (IgM) to WN virus was developed, standardized, and characterized as a rapid and sensitive means to detect WN viral antibodies in sentinel flocks. Serum specimens from experimentally infected chickens were analyzed by using this assay, and IgM was detected as early as 3 to 7 days postinfection. Persistence of IgM varied from at least 19 to more than 61 days postinfection, which indicates the need to bleed sentinel flocks at least every 2 weeks for optimal results if this method is to be used as a screening tool. The ELISA was compared to hemagglutination-inhibition and plaque reduction neutralization tests and was found to be the method of choice when early detection of WN antibody is required. House sparrows and rock doves are potential free-ranging sentinel species for WN virus, and the chicken WN IgM-capture ELISA was capable of detecting anti-WN IgM in house sparrow serum samples from laboratory-infected birds but not from rock dove serum samples. The chicken WN IgM-capture ELISA detected anti-WN antibodies in serum samples from naturally infected chickens. It also detected IgM in serum samples from two species of geese and from experimentally infected ring-necked pheasants, American crows, common grackles, and redwinged blackbirds. However, the test was determined to be less appropriate than an IgG (IgY)-based assay for use with free-ranging birds. The positive-to-negative ratios in the ELISA were similar regardless of the strain of WN viral antigen used, and only minimal cross-reactivity was observed between the WN and St. Louis encephalitis (SLE) IgM-capture ELISAs. A blind-coded serum panel was tested, and the chicken WN IgM-capture ELISA produced consistent results, with the exception of one borderline result. A preliminary test was done to assess the feasibility of a combined SLE and WN IgM-capture ELISA, and results were promising.     

Noninfectious recombinant antigen for detection of St. Louis encephalitis virus-specific antibodies in serum by enzyme-linked immunosorbent assay

Proper surveillance of virus activity and a timely response to viral outbreaks depend upon the rapid diagnosis of viral infections. The immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) is a fast, sensitive test routinely used for the diagnosis of the medically important West Nile and St. Louis encephalitis flaviviruses. However, the suckling mouse brain-derived (SMB) antigen used in this assay is tedious to prepare and has a risk of exposing personnel to live virus and hazardous chemicals. We report the development of a St. Louis encephalitis virus (SLEV) noninfectious recombinant antigen that is a safe and easily produced alternative antigen for use in diagnostic assays. The expression plasmid pCB8SJ2, containing the premembrane and envelope structural protein-encoding regions of SLEV, was constructed to express secreted extracellular virus-like particles (VLPs) from CHO cells. Blind-coded human serum panels were assembled from patients having recent SLEV, West Nile virus (WNV), Powassan virus, or La Crosse encephalitis virus infections to assess the sensitivity and specificity of assays with SLEV VLP or SMB antigen. MAC-ELISAs with either antigen had comparable sensitivity for the detection of IgM antibodies against SLEV. Importantly, when these two antigens were tested against a human serum panel from patients having recent WNV or Powassan virus infections, the SLEV VLPs were less likely than SMB antigen to detect flavivirus cross-reactive IgM antibodies. An optimized IgG antibody capture ELISA (GAC-ELISA) with both WNV and SLEV VLPs was developed to circumvent the frequently observed higher background in the antigen-capture IgG-ELISA (ACG-ELISA). For the detection of IgG antibodies against WNV, the GAC-ELISA resulted in a statistically significant higher performance accuracy (P = 0.003) than the ACG-ELISA when the WNV VLP antigen was used in both assays. However, no statistical difference was observed in the assay performance of the GAC-ELISA with SLEV VLP or the ACG-ELISA with SLEV SMB antigen.     

Antibody responses determined for Japanese dengue fever patients by neutralization and hemagglutination inhibition assays demonstrate cross-reactivity between dengue and Japanese encephalitis viruses

Titers of antibodies to infecting dengue virus serotypes determined by serum neutralization assay were higher than those of antibody to Japanese encephalitis (JE) virus in Japanese dengue patients after disease day 8. Titers of antibody to dengue virus antigens determined by hemagglutination inhibition (HI) assay were higher in only 1 of 23 serum specimens after disease day 11. Thus, the neutralization test is more reliable than the HI test for serological diagnosis of dengue in countries where JE vaccination is widely used or JE is endemic.     

Antibody Responses Determined for Japanese Dengue Fever Patients by Neutralization and Hemagglutination Inhibition Assays Demonstrate Cross-Reactivity between Dengue and Japanese Encephalitis Viruses

Titers of antibodies to infecting dengue virus serotypes determined by serum neutralization assay were higher than those of antibody to Japanese encephalitis (JE) virus in Japanese dengue patients after disease day 8. Titers of antibody to dengue virus antigens determined by hemagglutination inhibition (HI) assay were higher in only 1 of 23 serum specimens after disease day 11. Thus, the neutralization test is more reliable than the HI test for serological diagnosis of dengue in countries where JE vaccination is widely used or JE is endemic.     

Development of an enzyme-linked immunosorbent assay for serological diagnosis of tick-borne encephalitis using subviral particles

The similarity of symptoms produced by tick-borne encephalitis (TBE) and Japanese encephalitis (JE) and the high degree of cross-reactivity between TBE and JE viruses by serological tests make the development of a differential diagnostic test a priority. In this study, recombinant prM/E proteins of TBE virus strain Oshima 5-10 expressed in mammalian cells resulted in the release of subviral particles (SPs) into the culture medium. Using the SPs as antigens, enzyme-linked immunosorbent assay (ELISA) systems were developed to detect TBE virus-specific IgM and IgG antibodies, designated SP-IgG and SP-IgM ELISAs, respectively. Of 83 serum samples from encephalitis patients in Khabarovsk, Russia, which were positive with the neutralization test (NT), 82 were positive by the SP-IgG ELISA, for a sensitivity of 98.8%, which was higher than that of a commercial ELISA kit. All 12 NT-negative samples were also negative by the SP-IgG ELISA (specificity, 100%). Of 17 patient samples that were NT-positive, 16 (94.1%) were positive by the SP-IgM ELISA. Of 15 paired serum samples that yielded equivocal results by NT, 11 had positive results with the SP-IgM ELISA, indicating a diagnosis of TBE infection. The SP-IgG and SP-IgM ELISAs showed no cross-reactivity with antibodies to the JE virus. The results indicate that these ELISAs will be useful for the detection of TBE-specific antibodies.     

Study of combined foci of mosquito-transmitted arbovirus infections

A total of 5227 serum specimens from humans, horses and swine collected in the seasons of 1968--1976 in 15 administrative areas of the Primorskiy Kray were examined with antigens of a number of mosquito-borne arboviruses: Japanese encephalitis (JE), West Nile (WN), Getah, and Sindbis. Both independent and combined circulation of these viruses in the region was established. Sindbis virus was found to be circulating separately most frequently, West Nile virus the least frequently. According to the results of the serological analysis, the conditions for combined circulation are most closely related for JE and Getah, and JE and WN viruses. The interpretation of the results of examinations of the sera reacting simultaneously with JE and WN virus is most difficult because of close antigenic relationship of these viruses. A set of methods for serological differentiation of bivalent sera to JE and WN viruses and the criteria for the assessment of the results are recommended.     

Enzyme-linked immunosorbent assay using recombinant antigens expressed in mammalian cells for serodiagnosis of tick-borne encephalitis

A recombinant plasmid that expresses the tick-borne encephalitis (TBE) virus premembrane (prM) and envelope (E) proteins in mammalian cells was constructed. Recombinant proteins retained antigenic and conformational structures similar to those of native virus proteins, and transfected cells released virus-like particles (VLPs), which were 1.13-1.14 g/ml in density and 20-30 nm in diameter, into the culture medium. Recombinant E proteins were used for the development of an enzyme-linked immunosorbent assay (ELISA) to detect TBE virus-specific IgM and IgG antibodies in serum. The results of this ELISA correlated well with the results of commercial ELISA, when tested with 95 serum samples from clinically TBE-suspected patients. In addition, ELISA using recombinant antigens showed no cross-reactivity against serum from Japanese encephalitis (JE) patients, despite the cross-reactivity shown by commercial ELISA systems. These observations indicated that this newly developed ELISA system could distinguish tick-borne encephalitis from Japanese encephalitis infection, and that it constitutes a useful and safe alternative to conventional ELISA systems.     

MAC-ELISA and ELISA inhibition methods for detection of antibodies after yellow fever vaccination

The IgM antibody capture ELISA (MAC-ELISA) and ELISA inhibition methods for the detection of antibodies against dengue virus were modified to detect antibodies against yellow fever virus. Tests were carried out in 21 persons vaccinated with 17D and compared with the Plaque reduction neutralizing test. Of 17 naive subjects vaccinated, 16 (94%) seroconverted using the MAC-ELISA test and 14 (82%) seroconverted (or ≥fourfold titer increase) in the ELISA inhibition method. Cross-reactivity was evaluated by both tests and resulted in a high specificity to IgM antibodies against yellow fever, when all the samples from vaccinated individuals were negative by MAC-ELISA using dengue antigen. However, 10.7% of the positive dengue sera from the Santiago de Cuba epidemic cross-reacted by MAC-ELISA using yellow fever antigen. ELISA inhibition method showed high cross-reactivity when the 21 sera pairs were worked with yellow fever and dengue antigens. The MAC-ELISA and ELISA inhibition methods have become indispensable tools in our laboratory in order to maintain a surveillance system for dengue and dengue hemorrhagic fever. They are relatively rapid, simple, and they do not require sophisticated equipment. Both MAC-ELISA and ELISA inhibition methods for yellow fever could be useful for diagnosis, surveillance and yellow fever vaccine evaluation.     

Seroepidemiological study of infection with West Nile virus in Karachi, Pakistan, in 1983 and 1985

The prevalence of West Nile (WN) virus infection in Karachi, Pakistan, was unknown until 1982. It had been noticed that there were more than a few patients with encephalitides in Karachi, and it was supposed that Japanese encephalitis (JE) cases would be found among them. Therefore, a seroepidemiological study was conducted to define the prevalence of WN virus infection and the possible occurrence of JE virus infection in the Karachi area. Prevalences of haemagglutination inhibition (HI) and neutralization (NT) antibodies against WN virus were studied among 81 serum samples (in July, 33 samples; in September, 48) during 1983, and among 156 paired serum samples that were collected twice, in July and October of 1985. Nearly the same antibody-positive rates were obtained in July of both years (1983: HI 55%; 1985: HI 53%; NT 50%); the rates increased slightly during September/October (1983: HI 65%; 1985: HI 59%, NT 54%). Among 156 paired samples in 1985, 20 (13%) and 12 (8%) showed positive- or negative-antibody conversion between July and October. Two serum samples from each of 156 residents obtained in July had a significantly higher NT antibody titre against JE virus than against WN virus (in case 1, JE 1:80, WN less than 1:10; in case 2, JE 1:40, WN less than 1:10). This is the first report to show the prevalence of WN virus infection in Karachi, Pakistan.