Microneutralization test for rabies virus based on an enzyme immunoassay.

We have developed an enzyme immunoassay for rabies virus by using acetone-fixed infected cell cultures as the antigen. This test was used to demonstrate virus-neutralizing antibodies in human and animal sera and was as sensitive as and easier to perform than the rapid fluorescent-focus inhibition technique.     

Characterization of rabies virus isolates in Bolivia

In Latin America, rabies is still an important public health problem. Canine rabies, and wild animal rabies, especially transmitted by hematofagous and insectivorous bats, has become an emerging problem in the countries of this region.We received 363 samples with a laboratory-confirmed rabies diagnosis from Bolivia during l997-2001. From these, we could obtain 222 rabies virus isolates by intra-cerebral inoculation in mice. By antigenic characterization we could identify 147 isolates as variant 1, 2 isolates as variant 2, 3 isolates as variant 3, and 1 isolate as variant 5. Phylogenetic analysis of 84 isolates established that they segregated in 3 different branches, corresponding to 3 genetic variants, 78 isolates corresponding to antigenic variant 1 segregated in the same lineage as the antigenic variant 5, 2 isolates corresponding to antigenic variant 2 segregated in another lineage, and 3 isolates from antigenic variant 3 segregated in a different lineage.The genetic variant that mainly circulates in Bolivia is maintained in a cycle whose main reservoir are dogs, but it is not possible to discard the presence of other cycles, in which different species of bats or other wild mammals could be participating.     

Evaluation of enzyme immunoassay for hepatitis B virus DNA based on anti-double-stranded DNA.

We have evaluated a new enzyme immunoassay technology to detect the products of PCR-based amplification that may be applicable to routine testing of hepatitis B virus (HBV) DNA. Two hundred eight serum samples were studied: 73 were basal samples and 135 were sequential serum samples from patients with chronic hepatitis, some of whom were being treated with alpha interferon. We compared the new detection method (PCR-DNA enzyme immunoassay [DEIA]) with dot blot hybridization performed without prior PCR amplification and with two other methods for detection of PCR products: agarose gel electrophoresis with ethidium bromide staining (PCR-EB) and dot blot (PCR-dot blot). For hepatitis B-antigen-positive basal samples, HBV DNA was detected in 70.4% by dot blot, 74.1% by PCR-EB, and 100% by PCR-DEIA and PCR-dot blot; for anti-hepatitis B e-antigen basal samples, HBV DNA was found in 10.5% by dot blot and PCR-EB and in 42.1% by PCR-DEIA and PCR-dot blot. Chi-square tests showed a strong association between dot blot and PCR-EB and between PCR-DEIA and PCR dot blot. Using PCR-dot blot as the reference, dot blot shows a 56.9% sensitivity and a 100% specificity, PCR-EB shows a 55.0% sensitivity and a 100% specificity, and PCR-DEIA shows a 95.4% sensitivity and a 97% specificity. We conclude that the technical advantages of the DEIA method and its high sensitivity and specificity may facilitate the use of PCR in routine testing for HBV DNA in clinical microbiology laboratories.     

Typing Candida albicans oral isolates from human immunodeficiency virus-infected patients by multilocus enzyme electrophoresis and DNA fingerprinting.

A total of 189 Candida albicans isolates have been typed by multilocus enzyme electrophoresis. The results obtained confirm the clonal mode of reproduction of C. albicans. The C. albicans populations found in the oropharynx of human immunodeficiency virus (HIV)-infected patients, in the oropharynx of healthy carriers, or in association with invasive candidiasis could not be distinguished. No clone or group of clones could be associated with the appearance of clinical disorders or with a reduced in vitro susceptibility to the antifungal agent fluconazole. Multiple and sequential oral isolates from 24 HIV-infected patients were also typed by restriction enzyme analysis with the enzymes EcoRI and HinfI and by use of the Ca3 repetitive probe. The results obtained by the combination of all three typing methods show that all but one patient each carried a unique major C. albicans clone in their oropharynx. The 21 patients with sequential isolates had the same C. albicans clones in their throats during recurrent oropharyngeal candidiasis episodes, independently of clinical status or of changes of in vitro susceptibility to fluconazole. Finally, several isolates of the same C. albicans clone found simultaneously in the oropharynx of a patient may present different levels of susceptibility to fluconazole.     

Detection of Norwalk virus in stools by enzyme immunoassay

The development of a solid-phase microtiter enzyme immunoassay (EIA) for detection of Norwalk virus antigen in stool samples is described. The EIA was compared with a previously developed radioimmunoassay (RIA) for detection of Norwalk virus antigen in stools obtained from 30 volunteers who received Norwalk virus. The EIA detected viral antigen in stools from 17 of the volunteers and the RIA detected viral antigen in 15. Seroconversion was a more sensitive indicator of infection in some patients. However, two samples from volunteers who were clinically ill but did not show seroconversion to Norwalk virus were positive for Norwalk virus antigen by both immunoassays. This indicates that antigen detection may be important for use in epidemiological studies. Neither of the immunoassays gave positive reactions for stools known to contain enteric adenovirus, rotavirus, or Hawaii virus, or in stools from patients with acute diarrhea of unknown cause. The stability of the EIA reagents and ease of use should provide a means for more extensive testing for Norwalk virus in outbreaks of gastroenteritis.     

Molecular epidemiology of rabies virus isolates in India

In India, rabies is enzootic and is a serious public health and economic problem. India has a large population of stray dogs which, together with a lack of effective control strategies, might have led to the persistence of rabies virus (RV) in the canine population. Our objective was to study the molecular epidemiology of RV isolates in India based on nucleotide sequence analysis of 29 RV isolates originating from different species of animals in four states. Here we have analyzed two sets of sequence data based upon a 132-nucleotide region of the cytoplasmic domain (CD) of the G gene (G-CD) and a 549-nucleotide region (Psi-L) that combines the noncoding G-L intergenic region (Psi) and a fragment of the polymerase gene (L). Phylogenetic analysis revealed that the RV isolates belong to genotype 1 and that they were related geographically but were not related according to host species. Five different genetic clusters distributed among three geographical regions were identified. Comparison of the deduced amino acid sequences of G-CD between RV isolates revealed three amino acid changes (amino acid 462G [aa462G], aa465H, and aa468K) that distinguished the Indian RVs from RV isolates in other parts of the world. Analysis of the data indicated that the dog rabies virus variants are the major circulating viruses in India that transmit the disease to other domestic animals and humans as well.     

Quantitation of immunoglobulin to hepatitis E virus by enzyme immunoassay

We developed a quantitative enzyme immunoassay (EIA) for antibody to hepatitis E virus (HEV) by using truncated HEV capsid protein expressed in the baculovirus system to improve seroepidemiology, to contribute to hepatitis E diagnosis, and to enable vaccine evaluations. Five antigen lots were characterized; we used a reference antiserum to standardize antigen potency. We defined Walter Reed antibody units (WR U) with a reference antiserum by using the four-parameter logistic model, established other reference pools as assay standards, and determined the conversion factor: 1 WR U/ml = 0.125 World Health Organization unit (WHO U) per ml. The EIA performed consistently; median intra- and inter-test coefficients of variation were 9 and 12%, respectively. The accurate minimum detection limit with serum diluted 1:1,000 was 5.6 WR U/ml; the test could detect reliably a fourfold antibody change. In six people followed from health to onset of hepatitis E, the geometric mean antibody level rose from 7.1 WR U/ml to 1,924.6 WR U/ml. We used the presence of 56- and 180-kDa bands by Western blotting as a confirmatory test and to define true-negative and -positive serum specimens. A receiver-operating characteristics plot identified 30 WR U/ml as an optimum cut-point (sensitivity, 86%; specificity, 89%). The EIA detected antibody more sensitively than a commercially available test. The EIA was transferred to another laboratory, where four operators matched reference laboratory results for a panel of unknowns. Quantitation of antibody to HEV and confirmation of its specificity by Western blotting make HEV serology more meaningful.     

Detection and quantification of cell-free Epstein-Barr virus by polymerase chain reaction and subsequent DNA enzyme immunoassay.

Amplification by polymerase chain reaction and subsequent DNA enzyme immunoassay (DEIA) were employed to determine the number of genome equivalents of cell-free Epstein Barr virus (EBV) DNA in peripheral blood. The assay detected cell-free EBV DNA in the serum of 14 out of 18 patients with primary, productive EBV infection (sensitivity 77.7%) but not in healthy EBV carriers with latent infection (specificity 100%). Our assay has the potential for a clinical diagnostic tool to monitor patients at risk for EBV reactivation and productive infection with subsequent EBV-induced lymphoproliferative diseases.     

Postmortem enzyme immunoassay for human immunodeficiency virus

The reliability of postmortem enzyme immunoassay testing for antibody to human immunodeficiency virus (HIV) was assessed in blood and vitreous humor. Vitreous humor tested up to 34 hours post mortem and blood tested at least 58 days post mortem were consistently repeatedly positive for HIV antibody in patients with histopathologic criteria and premortem HIV test results that satisfied the Centers for Disease Control definition of acquired immunodeficiency syndrome (AIDS). There were no false-negative results in serum specimens, although one third of vitreous specimens that were tested 34 hours or more post mortem were negative in patients with AIDS. There were no false-positive results in control specimens from patients at autopsy without evidence of AIDS, despite prolonged postmortem intervals producing hemolysis and autolysis of the specimens. Postmortem enzyme immunoassay testing of blood and vitreous humor may be useful to screen for HIV infection in high-risk groups and for diagnosis of patients with histopathologic evidence of AIDS for medical, legal, and epidemiologic purposes.     

Neutralization enzyme immunoassay for influenza virus.

A neutralization enzyme immunoassay (N-EIA) was developed for the detection of antibody titer rises in sera of patients infected with influenza A (H3N2) virus. In this N-EIA, a selected strain of influenza A (H3N2) virus was added to monolayers of LLC-MK2 cells in microtiter plates. After 24 h, the replicated virus could be demonstrated with a virus-specific enzyme-labeled monoclonal antibody. Preincubation of the influenza virus with convalescent-phase sera of patients infected with influenza A (H3N2) virus resulted 1 day later in decreased absorbance values that could be used for calculation of neutralization titers. From use of paired serum samples from 10 patients with a history of flu-like symptoms, the results obtained with N-EIA correlated well (r = 0.83) with those of the standard hemagglutination inhibition test.     

Restriction enzyme cleavage maps of the DNA of two cauliflower mosaic virus isolates

To provide a physical basis for the functional mapping of the cauliflower mosaic virus (CaMV) genome, restriction enzyme cleavage maps of the DNA of CaMV isolates NY8153 and CM4-184-OS (a variant of CM4-184) were constructed. CM4-184-OS DNA contained five EcoRI, eight HindIII six BglIII, and two BamHI cleavage sites, while NY8153 DNA contained one additional HindIII site and one fewer EcoRI site. Fragments were ordered by examination of multienzyme and partial digests. The resulting maps revealed that the CM4-184-OS genome was 5.5% smaller than that of NY8153, because CM4-184-OS was deleted for 463 +/- 10 base pairs including the HindIII site at 0.427 map unit (relative to a common EcoR1 site) and the S1 site at 0.45 map unit on NY8153 DNA. Since the deleted region did not include the two BamHI sites, this variant of the CM4-184 isolate is different from the John Innes variant of CM4-184 (R. Hull, and S. H. Howell, Virology 86, 482-493, 1978) which is deleted for a different, but overlapping region of the CaMV genome.     

Detection of rubella virus antigen by one-step time-resolved fluoroimmunoassay and by enzyme immunoassay

A one-step time-resolved fluoroimmunoassay (TR-FIA) and a conventional two-step enzyme immunoassay (EIA) for the detection of rubella virus antigen were developed. Two noncompetitive mouse monoclonal antibodies reactive with epitopes on the E1 polypeptide of rubella virus served as immunoreagents. One of the monoclones (7A6) was used for coating the solid phase, and the other (2C3) was labeled with either Europium chelate or with horseradish peroxidase. For TR-FIA, the specimen was incubated simultaneously with the label at 4 degrees C overnight. EIA required an overnight incubation with the specimen and after washing another 1 hr of incubation at 37 degrees C with the conjugate. The sensitivity of TR-FIA was 10 pg in an assay volume of 100 microliters, and the sensitivity of EIA was between 50 and 100 pg. Antigens could be detected by TR-FIA in supernatant of cultures of Vero cells 48 hr after inoculation with approximately 1 TCID50, while cytopathogenic effect (CPE) at that time was detected only in cultures inoculated with 10(5) TCID50 or more. Virus mixed with human amniotic fluid containing antirubella-specific IgG was detectable after an incubation at 37 degrees C for 5 days. The assays may find applications in prenatal diagnosis of intrauterine rubella infection, in early identification of viral antigens in cell culture and in monitoring production, concentration, and purification of rubella antigen for antibody assays.     

Molecular characterization of rabies virus isolates in China during 2004

Human rabies cases have been on the rise during the past few years in China and a total of 2651 cases were reported in 2004. To better understand the current rabies epidemics in China, we isolated rabies viruses from dogs and humans from five provinces and characterized these isolates genetically by sequencing the entire nucleoprotein (N) gene. Comparison of the N genes among these isolates revealed 86.6-99.9% homology and these viruses can be grouped into three lineages. Phylogenetic analysis indicates that all the Chinese isolates have a close relationship with viruses circulating in Asian canine population. When compared with rabies viruses isolated previously, the three lineages were similar to three of the four groups. Thus, our data suggest that rabies viruses currently circulating in China were similar, if not identical, to those reported in the previous epidemics.     

Detection of bluetongue virus antigens in Culicoides variipennis by enzyme immunoassay.

A solid-phase enzyme immunoassay (EIA) was developed to detect bluetongue (BT) virus antigens in infected cell cultures and in suspensions of infected Culicoides variipennis midges. The technique was equally sensitive for detecting the five U.S. BT virus serotypes (2, 10, 11, 13, and 17) in cell cultures. EIA reliably detected about 3.8 log10 median tissue culture infective doses per ml of BT virus in infected cell culture lysates. The EIA readily detected virus antigens in pools of midges infected with BT serotypes 2, 10, 11, 13, and 17 and contained 2.3 to 4.8 log10 median tissue culture infective doses per ml of BT virus. The technique was sensitive enough to detect a single infected midge in a pool with 99 noninfected midges. The EIA may be a sensitive and rapid alternative to virus isolation for surveillance of BT viruses in vector populations.     

Typing of recent infectious bronchitis virus isolates causing nephritis in chicken

Summary Four isolates of infectious bronchitis viruses (IBV) from chickens with nephritis, were characterized by polymerase chain reaction (PCR) and restriction enzyme fragment length polymorphism (RFLP), and were found to be genetically different from the other twelve strains which we previously studied.     

Typing of Mycobacterium avium isolates by PCR.

A Mycobacterium avium typing method based on PCR amplification of genomic sequences located between the recently described repetitive elements IS1245 and IS1311 was developed. This method was applied to a set of epidemiologically related and unrelated strains and compared with restriction fragment length polymorphism analysis with IS1245 as the probe. This PCR typing consists of a rapid and simple technique, providing a reproducible M. avium characterization as discriminant as restriction fragment length polymorphism analysis.