Preliminary studies on the use of solid-phase immunosorbent techniques for the rapid detection of Wesselsbron virus (WSLV) IgM by haemagglutination-inhibition.

Serum samples from 446 randomly selected persons belonging to different age groups and locations in Nigeria were tested for the presence of WSLV IgM using the flavivirus haemagglutination-inhibition (HI) test adopted to the solid-phase immunosorbent technique (SPIT). 61 (14%) persons had IgM to WSLV only, while 9 (2%) persons had heterologous IgM to WSLV and two other flaviviruses, namely yellow fever and Uganda S viruses. There was a high prevalence of IgM in people of younger age groups than those in older groups. The majority of the IgM positive sera (67 (96%) of the 70 positive sera reacted to high titres (>21:80). With the conventional HI tests, 314 (70%) of the total sera tested had HI antibodies to one or more flaviviruses (yellow fever, West Nile, Potiskum, Zika and Uganda S) out of which 305/314 (97%) had antibodies to 3 or more flaviviruses used in the tests. Although SPIT may not be as sensitive as the conventional HI test, it was found to be more specific and could be adopted for the detection of early WSLV infections in flavivirus hyperendemic environments.

Comparison of the genome ds RNA of human rotavirus strains shed in parts of Ibadan, Nigeria.

The incidence and genome electropherotypes of human rotavirus detected in a hospital and in a community within Ibadan, Nigeria were compared by polyacrylamide gel electrophoresis. On the whole, 13% (31/239) rotavirus was detected; 14.7% (15/102) from the community and 11.7% (16/137) from the hospital. The incidence was significantly higher (P < 0.01) in the community than in the hospital. There were 11 (80.0%) long and 3 (20.0%) short forms observed in the community, whereas 14 (87.5%) long and 2 (12.5%) short froms were detected in the hospital. On co-electrophoresis, however, only 4 and 6 distinct electropherotypes were demonstrated in the hospital and the community respectively. Three of these were common to both places with 1 and 3 electropherotypes being unique to the hospital and the community, respectively.

Evaluation of the effects of disinfectants on rotavirus RNA and infectivity by the polymerase chain reaction and cell-culture methods.

Rotaviruses have been linked to outbreaks of acute gastroenteritis of children in day-care centres and hospital paediatric wards. There is, therefore, the need for monitoring effective decontamination of such environments. We have evaluated the effects of seven different methods of disinfection/inactivation (four chemical and three physical) on rotavirus using the PCR and cell-culture methods. We observed that 6% H2O2, 2500 ppm chlorine, an ethano-phenolic disinfectant, u.v. irradiation and heat completely destroyed the infectivity of rotavirus as well as RNA amplifiable by PCR. On the other hand, treatment with 80% ethanol resulted in the loss of infectivity despite the fact that RNA was still amplifiable. Rotavirus subjected to drying over a 24 h period still retained amplifiable RNA but infectivity was reduced by 100-fold when compared to the control. This study demonstrated an agreement between PCR and cell-culture monitoring systems, however, PCR is a more rapid and sensitive assay.

Wesselsbron virus antibody in domestic animals in Nigeria: retrospective and prospective studies.

Retrospective and prospective serological surveys to determine the prevalence of Wesslsbron (WSL) virus infections in animal populations were carried out in different vegetational zones in Nigeria. Sera from 1,492 animals comprising 292 camels, 81 horses, 4 donkeys, 320 cattle, 235 sheep, 260 goats, 114 pigs, 101 dogs and 85 domestic fowls were assayed by haemagglutination-inhibition (HI) test for presence of antibodies to WSL virus and other flavivirus antigens: Yellow Fever (YF), Potiskum (POT), Banzi (BAN), Uganda S (UGS) and West Nile (WN) viruses. Four hundred and eighty one (32%) of the total sera tested were positive for the presence of flavivirus antibodies. The prevalence rates among animals varied with species and vegetational zones of the country. The highest prevalence was noted in animals from a swamp forest zone and was higher among camels, horses, donkeys and sheep when compared with goats, pigs and fowls in different zones. Although monotypic reactions with WSL virus antigen were observed in positive sera, the majority of the WSL virus positive sera cross-reacted with more than two other flavivirus antigens. Serological cross-reactions were most extensive in WSL virus positive horse sera. A ten month sentinel survey among 28 cattle, 68 sheep and 30 goats revealed considerable activity of WSL virus in Nigeria. Of these, 11 cattle and 12 sheep showed antibody conversion to WSL virus antigen. None of the goats seroconverted. Although, there are no records of outbreak of WSL disease in Nigeria, this study revealed that WSL virus is actively circulating among livestock populations in this environment. Flavivirus nucleotide data are needed for final determination of genetic relatedness in this group of viruses.

Subgroup determination of group A rotaviruses recovered from piglets in Nigeria.

Subgroup analysis using subgroup-specific monoclonal ELISA revealed a preponderance of subgroup 2-specific antigens of group A porcine rotaviruses over subgroup 1. Of 113 polyacrylamide gel electrophoresis-positive test samples, obtained from 4 States of Nigeria, 31 (27.4%) and 45 (39.8%) were determined to have subgroup 1 and 2 specificities, respectively. However, 37 (32.7%) test samples could not be classified into any of the known group A rotavirus subgroups. These "unclassifiable" samples probably had neither subgroup 1 nor 2 specificities in ELISA or could belong to a third subgroup unknown or not investigated in this study. In all age groups investigated, subgroup 1 and 2 specific antigens were prevalent. This was also observed after yearly analysis of subgroup specificity; however, a higher prevalence of subgroup 2 specificity was observed in 1990 and 1991. Subgroup 1 rotaviruses were found in all the 4 States sampled (Plateau, Benue, Oyo, and Cross River), whereas subgroup 2 rotaviruses were detected only in Plateau State. All rotaviruses recovered in this study had long genome electrophoretic migration patterns, irrespective of subgroup. Thus genomic diversity of group A rotaviruses does not necessarily reflect antigenic diversity, as there is no mandatory correlation between genome electropherotype pattern and subgroup specificity.

Isolation and characterisation of Newcastle disease virus strain in a feral dove (Stigmatopelia senegalensis) in Nigeria.

An isolate of Newcastle disease virus (NDV) was obtained from a feral dove, (Stigmatopelia senegalensis). The isolate was shown to have a mean-death-time of 96 h and an intracerebral pathogenicity index of 0.10. It was immunogenic but not pathogenic for 6-week old chicks on experimental infection. Based on these observations the isolate was classified as a lentogenic strain. The role of such isolates of NDV from wild birds on the Nigerian poultry industry is discussed.

Production and characterization of monoclonal antibodies to porcine group C rotaviruses cross-reactive with group A rotaviruses.

Five monoclonal antibodies (MAbs) to porcine group (gp) C rotaviruses (Cowden and Ah strains) reactive with both gp A and C rotaviruses in cell culture immunofluorescence (CCIF) tests were produced and characterized. These MAbs reacted with three strains of gp A and two strains of gp C rotaviruses in a CCIF test and were classified into two groups based on their CCIF titers. The MAbs also reacted to various degrees with cell-culture-propagated porcine gp C rotavirus (Cowden) and bovine gp A rotavirus (NCDV) in an enzyme-linked immunosorbent assay by using the MAbs as capture antibodies. Fecal samples containing human, bovine, and porcine strains of gp A and C rotaviruses were positive when tested using one of the MAbs in this assay. The MAbs recognized VP6 of gp A rotavirus and the VP6 counterpart (41-kDa protein) of gp C rotavirus in a Western blot assay. Results of competitive binding assays on four MAbs indicated that gp A and gp C rotaviruses share three overlapping epitopes within a single antigenic domain. These results suggest that gp A and C rotaviruses share a common antigen located on the VP6 protein, which is recognized by certain MAbs in various serologic assays.

Development of a biotin-streptavidin-enhanced enzyme-linked immunosorbent assay which uses monoclonal antibodies for detection of group C rotaviruses.

A biotin-streptavidin-enhanced enzyme-linked immunosorbent assay (ELISA) which uses monoclonal antibodies (MAbs) for the detection of group C rotaviruses was developed. An assay in which plates were coated with three pooled MAbs and biotinylated polyclonal immunoglobulin G (IgG) (polyclonal antibody [PAb]) was used as the detector (MAb capture-PAb detector) was found to be the most sensitive and specific of the assays when it was compared with assays in which plates were coated with polyclonal antiserum and detection was done with either biotinylated polyclonal antiserum (PAb capture-PAb detector) or biotinylated pooled MAbs (PAb capture-MAb detector). The MAb capture-PAb detector ELISA detected 83% of samples confirmed to be positive for group C rotaviruses, whereas the PAb capture-PAb detector assay detected 63% of positive samples and the PAb capture-MAb detector assay detected 65% of positive samples. All three procedures detected both of the bovine and the two human group C rotaviruses, but none of the three procedures detected fecal samples containing group A and B rotaviruses or fecal samples negative for group C rotaviruses used in this study. The sensitivity of the MAb capture-PAb detector ELISA was determined by serially diluting fecal group C rotaviruses; antigens were detected in maximal positive dilution ranges of 1:1,000 to 1:3,000 for the samples tested. On the basis of the cell culture immunofluorescence assay infectivity titer of semipurified cell culture-passaged Cowden group C rotavirus, the sensitivity of the MAb capture-PAb detection ELISA for detection of homologous group C rotavirus was 53 fluorescent focus units per ml. Epitope mapping by use of the biotinylated MAbs in competition assay suggested that our MAbs may bind to three different but overlapping epitopes. These results suggest that the MAb capture-PAb detector ELISA can be used to study the epidemiology of group C rotaviruses in humans and animals.

Molecular analysis of the gene 6 from a porcine group C rotavirus that encodes the NS34 equivalent of group A rotaviruses.

The complete nucleotide sequence of the gene 6 from the porcine group (Gp) C rotavirus strain Cowden was determined from gene 6-specific clones selected from a cDNA library and from viral transcript RNA. The gene is 1348 nucleotides in length with a potential initiation codon beginning at nucleotide 25 and a stop codon at nucleotide 1231. The deduced protein contains 402 amino acids. Comparison of the gene 6 from this Gp C strain with sequences in the GenBank data base indicated that this gene shared homology with gene 7 of Gp A rotavirus strain SA-11 (22.9%) and gene 9 of Gp A rotavirus strain UK (22.6%), both of which encode the NS34 protein. In vitro translation products produced from transcripts generated from a gene 6 clone containing the entire open reading frame were not immunoprecipitated with either hyperimmune serum specific for the Gp C Cowden strain or a monoclonal antibody directed against the group antigen (VP6) of the Cowden strain. However, products generated from a full-length gene 5 clone of the Cowden strain were immunoprecipitated by each of these antibodies. These data suggest that in contrast to the Gp A viruses in which the gene 6 encodes the major inner capsid protein VP6, the gene 6 of the Cowden Gp C strain encodes a nonstructural protein corresponding to the NS34 of Gp A rotaviruses.

Reactivity of monoclonal antibodies to the 41-kilodalton protein of porcine group C rotavirus with homologous and heterologous rotavirus serogroups in immunofluorescence tests.

Three monoclonal antibodies (MAbs) to porcine group C rotavirus immunoprecipitated the major inner capsid protein (41 kDa) but failed to precipitate group A rotavirus proteins. In immunofluorescence tests of rotavirus-infected cell cultures or pig intestines, the MAbs recognized porcine and bovine group C rotaviruses but not group A or B rotaviruses. These MAbs may recognize the group C rotavirus counterpart to VP6 of group A rotaviruses and may be useful as diagnostic reagents.

Detection of rotavirus antigen in diarrhoeic and non-diarrhoeic piglets in Nigeria.

Faecal samples from 96 piglets with acute gastroenteritis and 41 samples from non-diarrhoeic piglets were investigated for porcine rotavirus (PRV) antigens by enzyme-linked immunosorbent assay (ELISA). PRV was detected in 43 of 96 (44.8%) in the diarrhoeic group whilst none of the samples in the non-diarrhoeic group was positive for PRV (P less than 0.01). Age distribution of infection showed a higher infection rate of 30.2% in piglets aged 1-3 weeks, which was significantly higher (P less than 0.05) than in piglets aged 4-6 weeks, where the infection rate was 14.6 per cent. No PRV antigen was detected in diarrhoeic piglets above 6 weeks of age. Titration of 10 randomly selected ELISA-positive samples showed titres ranging from 512 to greater than or equal to 4.096.

Dot enzyme immunoassay for visual detection of peste-des-petits-ruminants virus antigen from infected caprine tissues.

An enzyme-linked immunosorbent microassay using nitrocellulose paper as the solid-phase support was developed for the detection of peste-des-petits-ruminants virus antigens in infected caprine tissue homogenates. Dots of tissue homogenates were applied to nitrocellulose papers, and any unreacted sites were blocked with 5% skim milk powder in triethanolamine-buffered saline. After incubation of the papers in tissue culture supernatant monoclonal antibody against the peste-des-petits-ruminants virus, the antigen-antibody reaction was detected with peroxidase-conjugated anti-mouse immunoglobulin G and the enzyme substrate 4-chloro-1-naphthol. Positive results were visualized as blue dots. Results of the dot enzyme immunoassay compared favorably with those of the standard enzyme-linked immunosorbent assay. Incorporation of Nonidet P-40 in the washing solution did not improve the sensitivity of the dot enzyme immunoassay, and pretreatment of homogenates with Nonidet P-40 before application to the nitrocellulose paper inhibited the binding of the antigen to the paper and reduced the intensity of the color development.

Bovine rotavirus serotypes and their significance for immunization.

Neutralization assays on calf fecal rotavirus with antisera to two previously described bovine rotavirus serotypes allowed the isolation of four rotaviruses belonging to a distinct third serotype. In a survey of 85 calf isolates, 80 rotaviruses belonged to serotype 1 (91%), 1 belonged to serotype 2 (1%), and 4 belonged to serotype 3 (5%). Serotypes 1 and 2 were shown to not cross-protect in a passive immunization experiment in gnotobiotic lambs. Ingestion of specific antiserum protected against infection with the homologous, but not heterologous, serotype. Rabbits with no previous exposure to rotavirus responded to sequential vaccination with bovine and human rotavirus serotypes with antibody specific to those serotypes, and the response did not broaden to include serotypes to which they had not been exposed. These factors suggested the need for multivalent rotavirus vaccines. By contrast, 47 adult cows on 11 farms had neutralizing antibodies to two bovine and three human rotavirus serotypes. After vaccination with one bovine rotavirus serotype, these cows produced a significant increase in antibody titers to these same five serotypes but not to two other serotypes to which they had no preexisting antibody. These results were interpreted to indicate that cows will respond heterotypically after monovalent vaccination to all rotavirus serotypes with which they have had experience and, therefore, that single serotype vaccination may be sufficient. This conclusion has practical importance for rotavirus immunization procedures.

Evidence for serotypic variation among bovine rotaviruses.

Eight field strains of calf rotavirus from the U.K. were compared by neutralisation tests, using convalescent and hyperimmune antisera. Seven of these strains cross-reacted and were considered to be of one serotype, while the 8th was distinguished by a greater than 20-fold two-way difference in neutralisation titre suggesting a second serotype. Three widely-distributed reference strains (U.K., Northern Ireland and Lincoln) cross-reacted with the strains in the dominant serotype, as did 33 of 42 other field calf rotavirus strains. Nine field strains failed to cross-react with either serotype, suggesting the existence of other potential serotypes in the calf population.

Rapid diagnosis of rotavirus infection by direct detection of viral nucleic acid in silver-stained polyacrylamide gels.

A rapid simple technique for the diagnosis of rotavirus has been developed based on the sensitive detection of rotavirus double-stranded RNA genome segments separated in polyacrylamide gels. The method utilizes a recently described ultrasensitive silver stain for polypeptides, which can also detect subnanogram amounts of nucleic acid. The sensitivity of the technique is comparable with that of electron microscopy or enzyme-linked immunosorbent assay.