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We evaluated the ability of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) to detect West Nile virus (WNV) antibodies in domestic mammals. Sera were collected from experimentally infected horses, cats, and pigs at regular intervals and screened in ELISAs and plaque reduction neutralization tests. The diagnostic efficacies of these techniques were similar.
Attempts were made to analyze the specificity of inhibitory activities of normal bovine and equine sera to the Mahoney strain of type 1 poliovirus. A total of five inhibitory factors were postulated to explain the complicated results. Two of the three bovine inhibitors were identical in specificity to certain equine inhibitors despite differences in their mode of virus inactivation and their molecular size. In addition to this, inhibitors that could inactivate certain resistant mutants, but not the parent virus, were newly detected in a number of normal bovine and equine sera. Antigenic variation of the resistant mutants against equine sera containing an inhibitory factor h-11 was determined by means of the kinetic neutralization test by using both anti-Mahoney and anti-M-H11 sera. These results offer evidence that some inhibitors, at least in part, are indistinguishable from specific antibody.
The results of identification of 115 enterovirus strains by means of counter immunoelectrophoresis and the neutralization tests in cell culture are presented. Even at a high rate of agreement of the two methods, counter immuno-electrophoresis was shown to be more rapid, economic, less labour-consuming but requiring concentrated viral antigens.
A reliable bovine viral diarrhea (BVD) viral antigen was prepared from BVD virus grown on Madin Darby bovine kidney (MDBK) cells by solubilizing the virus with detergent MEGA-10 (decanoyl-N-methylglucamide) followed by removal of hydrophobic proteins with Triton X-100 treatment. By these treatments, problems of high background associated with BVD viral antigen in the enzyme-linked immunosorbent assay (ELISA) were eliminated. With this new antigen, an ELISA was adapted to detect bovine serum antibody against BVD virus. The diagnostic specificity of the assay in 403 bovine sera collected from a BVD virus-free herd was 100%; in 296 bovine sera with serum neutralizing antibody titers of greater than or equal to 1:2, 289 sera were ELISA positive (relative sensitivity of 97.6%), two sera gave false negative reactions (0.7%) and five sera gave suspicious reactions (1.7%). These interpretations were based on positive/negative (P/N) ratio readings, i.e. a P/N ratio of less than 1.50, 1.50-1.99 and greater than or equal to 2.00 were interpreted as negative, suspicious and positive reactions, respectively. The ELISA results gave excellent agreement with serum neutralization in detecting both seropositive and seronegative animals (Kappa = 0.994). The ELISA assay was considered to be technically superior to the serum neutralization test for the routine detection of BVD viral antibody in bovine sera.
Five new phlebotomus fever virus serotypes (Bunyaviridae: Phlebovirus) are described. These viruses, designated Ambe, Ixcanal, Mariquita, Armero, and Durania, were isolated from sand flies (Diptera: Psychodidae) collected in Brazil, Colombia, and Guatemala. Two of the agents were recovered from pools of male sand flies. The new viruses are antigenically related to other members of the phlebotomus fever serogroup by immunofluorescence, but are distinct from the other 39 members of this serogroup by plaque reduction neutralization test.
From 1981 through 1989, serum samples from 855 white-tailed deer (Odocoileus virginianus) from Ossabaw Island, Georgia (USA), were tested for antibodies to bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV). During this period, prevalence of precipitating antibodies to BTV and EHDV as determined by agar gel immunodiffusion (AGID) tests decreased from 74% to 3% and from 34% to 1%, respectively. Antibodies were detected in serum samples from 0.5-yr-old deer only during 1981, 1982, and 1983, and with few exceptions, positive serological results after 1983 were restricted to older age classes. A decrease in prevalence of precipitating antibodies to BTV and EHDV in age classes exposed during 1981 indicates that AGID results from white-tailed deer populations underestimate the extent of previous exposure to these viruses. Serum neutralization test results from AGID-positive deer indicated that BTV 11 was the principal serotype responsible for infections during 1981. Since 1983, this serotype has been replaced by BTV 13; however, there has been a low level of transmission within the herd. Infection with EHDV 2 appeared most prevalent during 1982; as with BTV 13, there has been limited transmission in this high density deer population since 1983.
A cytolytic toxin (kentin) from the Indo-Pacific sea anemone, Stoichactis kenti, was purified to near homogeneity. The toxin is a basic polypeptide of molecular weight approximately 18,000. It broadly resembles cytotoxins from Stoichactis helianthus (helianthin), as well as similar toxins from a number of other anemones, namely Condylactis, Epiactis, Actinia, Pseudactinia, Tealia, Anthopleura, Radianthus and Gyrostoma. The amino acid composition of kentin shows considerable resemblance to that of helianthin, but there are also several significant differences. Neutralization tests indicate that kentin and helianthin are immunologically related but distinguishable. In contrast, no immunological relatedness was found between helianthin and cytolytic toxins from Condylactis gigantea and Epiactis prolifera.
A total of 912 cases of sleeping sickness have been recorded from the Lambwe Valley from 1959 to 1984. After a period of decreasing prevalence in the 1970s an outbreak of disease occurred between 1980 and 1984. The incidence of disease for this five-year period was highest in areas adjoining the Ruma National Park, reaching 54% in Area I. Attack rates were highest in the 50+ age group (125) and children had significantly lower attack rates (8%) in this area of peridomestic transmission. Sex ratios of patients (M/F) were near 1.0 in areas in closest proximity to the thickets in the National Park, while in distant areas the ratios rose to 6.0. The distribution of the number of patients within different households was studied; fewer households than expected had 0 or one patient, and more than expected had three or more patients. No difference in attack rates were found between Nilotic and Bantu groups. Twelve different zymodemes were found in 136 stocks of Trypanosoma brucei rhodesiense. Four new zymodemes appeared in 1980 in the latest outbreak and accounted for 73% of the stocks isolated from man during this outbreak. Neutralization tests indicated that each trypanosome zymodeme may also represent a different serodeme.
Twenty-five hybridoma cultures secreted monoclonal antibodies directed against the glycoprotein of rabies or rabies-related viruses. The antibodies had different specificities for the glycoproteins of eight rabies and rabies-related viruses. They could be classified into fourteen groups which probably correspond to different antigenic determinants on the glycoproteins. These hybridomas when used in either radioimmunoassay (RIA) or in neutralization tests allow differentiation of laboratory strains of rabies virus from each other as well as from the rabies-related viruses.
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Six recently isolated field strains of infectious bursal disease virus (IBDV) were compared to vaccine strains at the antigenic and genetic level to ascertain the level of heterogeneity among Australian IBDV strains. Five strains, 01/94, 02/95, 03/95, 04/95 and 08/95, isolated at four locations in the state of Victoria, were antigenic variants. They failed to react with monoclonal antibodies directed against two different epitopes on the VP2 protein which were present in vaccine strains and one field isolate (06/95) from the state of New South Wales. Serum neutralization tests confirmed that these strains were antigenic variants as they were of a different subtype to that of vaccine strains. Sequence comparison of the hypervariable region of the VP2 proteins showed that the five Victorian strains had between 13 and 16 amino acid substitutions in comparison with vaccine strains. Four to six of these substitutions were in the two hydrophilic domains previously identified as being of importance in the formation of protective virus neutralizing antibodies. Comparison of these five variants to those isolated previously in the USA revealed little similarity at both the antigenic and genetic level. Phylogenetic analysis showed that Australian IBDV strains belong to a separate and distinct genetic group which is considerably heterogeneous. Overall the results indicate that the current Australian IBDV situation resembles that seen in the USA, with the existence of classical and variant IBDV strains, but neither the classical nor the variant strains found in Australia are closely related to those prevalent in the USA.
A serological classification of five feline calicivirus (FCV) strains of Swiss origin and 13 isolates of Austrian origin was attempted. The antisera used had been prepared in rabbits against the five Swiss strains and in goats against six American strains. Homologous and heterologous neutralization tests were made in tube cultures using sera at dilutions that contained 20 antibody units, in unabsorbed sera at low dilution, and in sera after subjection to three consecutive absorptions with lyophilized feline liver powder. None of these procedures resulted in the delineation of serotypes. A study using 20 antibody units yeilded FCV strains with broad-spectrum antigenicity, which seem promising for vaccination trials and for covering global needs. In addition, readily neutralized strains suitable for epidemiological work on cat sera, and possibly also for measuring humoral response to vaccines, could be recognized. The extent of serum titer variance, after 10-fold variance of virus input, was established as being only two- to threefold. Five Swiss FCV strains have been shown to remain antigenically stable over 10 years of laboratory passage. FCV strains and the procedure using 20 antibody units offered a suitable approach for international comparative work. FCV strains with little cross-neutralization should be subjected to higher antibody concentrations of existing antisera before attempting to create serotypes. Liver powder absorptions, which reduced homologous and heterologous neutralization titers to similar extents, subsequently proved to be unsuitable for use in FCV classification.
Since the late 1980s, a birnaviral gill disease has been occurring in Japanese eels Anguilla japonica reared in warmwater ponds in western regions in Japan. Diseased eels mostly displayed marked formations of aneurysmal hematomas within gill lamellae and high mortalities. Histological examination revealed necrosis of pillar cells and subsequent aggregation of erythrocytes inside the lamellar capillaries, and proliferation of interlamellar epithelia onto the lamellae. Gastric gland cells were also necrotized. Electron microscopy revealed birnavirus infection in lamellar pillar cells. The causative birnavirus was isolated and cultured in fish cell lines and was found to be related to an infectious pancreatic necrosis virus (IPNV) Sp serotype by neutralization tests. The viral pathogenicity was confirmed by the results of histopathological examinations and infectivity experiments.
Wild and domestic animals of New Brunswick were tested serologically for antibodies to the California group (CAL) of arboviruses. Deer (Odocoileus virginianus) blood collected during 1976 showed 6 of 129 (4.7%) sera with neutralizing antibodies to the snowshoe hare strain (SSH) of CAL. Neutralization tests on moose (Alces alces americana Clinton) sera collected during 1979 indicated 94 of 127 (74%) with antibodies to SSH, 4 of 127 (3.2%) with antibodies to the Jamestown Canyon Strain (JC) of CAL, and 17 of 127 (13.4%) with equal antibody titers to SSH and JC. Hemagglutination inhibition tests on house blood collected during 1977 showed 54 of 204 (26.5%) with antibodies to SSH; of these, 36 also had neutralizing antibodies to SSH. This study is the first indication of CAL activity in New Brunswick and supports evidence of JC activity in northeastern North America.
Severe acute respiratory syndrome (SARS) is a recently discovered viral disease, characterized by fever, cough, acute fibrinous pneumonia and high infectivity. Specific pathogen-free (SPF) chickens were immunized with inactivated SARS coronavirus and their eggs were harvested at regular intervals. Yolk immunoglobulin (IgY) was extracted using the water dilution method, followed by further purification on a Sephadex G-75 column. SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot and neutralization test results showed that the IgY obtained was of a high purity and had a strong reactive activity with a neutralization titer of 1:640. Lyophilization and stability tests showed that lyophilized anti-SARS coronavirus IgY had promising physical properties, with no significant reduction in reactive activity and good thermal stability. All these data suggest that the anti-SARS coronavirus IgY could be a new useful biological product for specific antiviral therapy against SARS.
We investigated the antigenic and genetic characters of one of two human rotavirus strains 69M and 57M isolated in Indonesia, both of which showed a "super-short" RNA electrophoretic pattern (A. Hasegawa et al., Microbiol. Immunol. 28, 719-722, 1984). By an enzyme-linked immunosorbent assay with subgroup-specific monoclonal antibodies, one virus, strain 57M, was found to have subgroup II antigenicity. The cross-reaction of this strain, in a plaque neutralization test, with a serotype 4 strain was high whereas that of strain 69M was low. When radiolabeled RNA probes prepared from this virus were hybridized with RNAs from reference strains of different serotypes, treated with S-1 nuclease and then subjected to polyacrylamide gel electrophoresis, we found that (i) RNA segment 10 (the super-short segment) hybridized with that of another super-short pattern virus, strain 69M; (ii) segment 7, coding for a neutralization antigen, hybridized with that of the serotype 4 virus; (iii) segment 6, coding for a major inner-shell protein, hybridized with that of a serotype 1 virus; and (iv), some other segments hybridized with those of the reference viruses of serotypes other than 2 and 3. We suspect that this strain is possibly a naturally-occurring reassortant virus whose genetic segments are derived from different human rotaviruses.
Serums from broiler breeders of an integrated broiler operator were examined for precipitin antibodies using antigens prepared from preliminary reovirus serotypes represented by WVU-2937, Reo-25, Reo-59, and Fahey-Crawley virus. The precipitin antibodies were present in most of the flocks. Virus neutralization tests indicated that the reovirus responsible for viral arthritis represented by WVU-2937 was not present in the breeder flocks, but antibodies to Reo-25, Reo-59, and the Fahey-Crawley viruses were present.
Nine monoclonal antibodies (Mab) against a 40 Kd capsid protein of infectious bursal disease virus (IBDV) strain P3009 were isolated. They were characterized by enzyme-linked immunosorbent assay, indirect fluorescent antibody staining and virus neutralization. They were divided into two groups concerning virus neutralization. Group I Mabs were able to neutralize virus infectivity; however, group II Mabs were not. Competitive binding assays using these Mabs demonstrated the existence of two distinct antigenic regions (A and B) on the 40 Kd protein. Region A was recognized by group I Mabs and region B was by group II Mabs. The binding reaction with group I Mabs was affected by denaturing of the viral proteins, indicating that the antigenic region involving neutralization was conformation-dependent. The results of enzyme-linked immunosorbent assays and virus neutralization tests suggested that group I Mabs might react with one epitope within region A and group II Mabs with 2 or 3 epitopes within region B.