Experimental infection of non-human primates with a human rotavirus isolate.

Several rotavirus candidate vaccines have been developed and are at various stages of evaluation. In order to assess the safety and efficacy of these candidate vaccines, an appropriate non-human primate model is desirable. In earlier studies, we reported the presence of naturally occurring anti-rotavirus antibodies in monkeys and demonstrated that parenteral vaccination of baboons led to production of specific rotavirus antibodies in their milk. In the present study, we assessed the possibility of developing the baboon and the vervet monkey as an animal model for rotavirus studies by inoculating them with a pathogenic human rotavirus isolate prepared from the fresh faeces obtained from a child suffering from rotavirus diarrhoea. Preliminary studies have showed excretion of rotavirus in the faeces of 5 of 5 vervets monkeys and 1 of 2 baboons, by antigen ELISA and SDS-PAGE. These results were confirmed by RT-PCR and electron microscopy. The animals also showed elevation of IgG and high titres of virus neutralising antibodies. These data indicate that baboon and vervet monkeys may be useful models for human rotavirus infection and for pre-clinical evaluation of rotavirus candidate vaccines.

Factors affecting the serological response of dogs and cats to rabies vaccination.

After being vaccinated against rabies some cats and dogs fail to show an antibody titre adequate to meet the requirements of the UK Pet Travel Scheme. To investigate this problem, the data derived from 16,073 serum samples submitted to the Veterinary Laboratories Agency for serological testing between 1999 and 2002, 1002 samples submitted to BioBest during March and April 2001, and 1264 samples associated with one make of vaccine submitted to BioBest between June 2001 and January 2003, were analysed. The probability of antibody titre failing to reach at least 0.5 iu/ml was analysed by logistic regression as a function of the choice of vaccine, the interval between vaccination and sampling, the sex and age of the animal, and its country of origin. In dogs, all these factors, except sex, had highly significant (P < 0.001) effects on the test failure rate, and in cats all the factors had a significant effect (P < 0.05).

Characterisation of the bovine enteric calici-like virus, Newbury agent 1.

The bovine enteric calici-like virus, Newbury agent 1 (NA1) was characterised to determine if it is a member of the Caliciviridae and to establish its antigenic relationship to the established bovine enteric calicivirus Newbury agent 2 (NA2). Solid phase immune electron microscopy (SPIEM) allowed quantification of NA1 virions and identification of faecal samples with optimal virus levels. NA1 particles were 36.6 nm in diameter, had an indefinite surface structure resembling that of human small round structured viruses (SRSVs), and a buoyant density of 1.34 g ml(-1). A single capsid protein of 49.4 kDa was detected by Western blotting in purified NA1 preparations prepared from post-infection but not pre-infection faecal samples and with post- but not pre-infection sera. NA1 was antigenically unrelated to the bovine enteric calicivirus NA2 by SPIEM. These properties were consistent with classification of NA1 within the Caliciviridae but demonstrated heterogeneity in the capsid composition of bovine enteric caliciviruses.

Effect of oral rotavirus/iscom vaccines on immune responses in gnotobiotic lambs.

A comparison of the effect on the immune responses in gnotobiotic lambs was made between an iscom vaccine prepared from recombinant rotavirus VP6 protein, an inactivated rotavirus/iscom-matrix vaccine and a vaccine comprising inactivated rotavirus alone. All three vaccines induced immunological priming and some degree of protection was observed after a single oral dose. However, different immune responses were induced in response to a virulent infection. The group vaccinated with the rotavirus/iscom-matrix vaccine showed a Th2-like response characterised by rotavirus-specific antibodies and a down-regulation of IFNgamma in jejunal Peyer's patches. Both Th1-like and Th2-like immune responses were induced in the group receiving the VP6 vaccine as seen by significantly increased expressions of IFNgamma and IL-6 in the jejunal Peyer's patch together with an increased percentage of CD8+ T cells in the intestine and rotavirus-specific antibodies at mucosal surfaces. Iscom vaccines given orally have the ability to induce both Th1-like and Th2-like immune responses in a ruminant model.

Characterisation of the primary local and systemic immune response in gnotobiotic lambs against rotavirus infection.

This study characterised the primary immune response in gnotobiotic lambs after infection with a lamb rotavirus (RV). Lambs were infected and killed over a 7 week period together with controls. RV-ELISA and neutralising antibodies were determined in serum, nasal secretions, and intestinal scrapings. RV-antibody secreting cells (ASC) were enumerated in blood. Lymphocyte proliferations were determined in blood and gut-associated lymphoid tissues and cytokine expression was analysed in jejunal Peyer's patches (JPPs) and mesenteric lymph nodes (MLNs). Infected lambs cleared the virus by 8-9 days after infection without showing any clinical signs. The first indication of a specific immune response to RV was an increased expression of IL-4 mRNA in the JPPs in the infected group compared to the control group 3 days after infection. Rotavirus-specific IgA ASC in blood and IgA antibodies in serum and nasal secretions were detected from 7 days after infection followed at 10 days after infection by RV-specific IgG ASC and antibodies. Rotavirus-specific IgA antibodies were not detected in intestinal scrapings in the first 10 days after infection, but were detected by 52 days after infection. No RV-specific neutralising antibodies were seen in the intestine during the course of the experiment.

Survey of equine rotaviruses shows conservation of one P genotype in background of two G genotypes.

DIG-labelled ssRNA probes were prepared from variable regions of VP4 and VP7 cognate genes, and used in hybridization assays for P and G genotyping of group A cell culture-adapted equine rotaviruses and fecal samples collected from foals with and without diarrhea. The probes confirmed known P and G serotypes of sixteen cell culture-adapted strains. From one-hundred and twenty-one rotavirus-positive samples, 83 reacted when tested for their P and G genotype specific probes. From these, 71 were found to contain G3 P12 genotypes, and 11 G14 P12 genotypes. No sample reacted with H1 or L338 P and G genotype probes. This suggests that the equine rotavirus population is conservative, containing predominantly one P genotype and two G genotypes. One isolate (26/94) whose dsRNA was visualized in an agarose gel did not react with any of the equine probes, and was found to belong to G8 and P1 genotypes. This is the fourth example of a single unique equine isolate (after H1, L338, and R-22). The remaining thirty-eight untypable field isolates had no detectable dsRNA after storage for 1 to 3 years.

Intestinal cellular immunity after primary rotavirus infection.

Infection of neonatal gnotobiotic lambs with a bovine strain of rotavirus was used to characterize the kinetics of the primary cellular intestinal immune response to this agent. At 2-3 days after infection virus was first detected in the faeces and increased numbers of CD45R+ cells were observed in peripheral blood. These cells persisted in significantly increased numbers in the circulation until 7-8 days after infection. At this time, virus was no longer detectable in the faeces. The increase in CD45R+ cells preceded the appearance of virus-neutralizing antibodies in the serum at 1 week after infection. Maximal antibody titres were reached 2 weeks after infection. Virus-primed cells were first observed 1 week after infection in the jejunal and ileal Peyer's patches, mesenteric lymph nodes and peripheral blood, and persisted in the mesenteric lymph nodes and jejunal Peyer's patches for a further 4 weeks. Analysis of lymphocyte surface antigens indicated that different sub-populations of lymphocytes were responding in the various lymphoid tissues; a majority of CD4+ cells was observed in the mesenteric lymph nodes, whereas B cells predominated in the ileal Peyer's patches.

Stimulation of rotavirus IgA, IgG and neutralising antibodies in baboon milk by parenteral vaccination.

A rhesus rotavirus vaccine adjuvanted with ISCOMs was injected intramuscularly to 5 pregnant baboons, with repeated doses 1-2 and 14 weeks after delivery. Maternal blood and milk samples and blood samples from their babies were collected at 2-weekly intervals until 26 weeks after parturition. Samples were assayed for rotavirus antibodies by ELISAs and neutralisation tests. Vaccination produced statistically significant increases in maternal serum IgG and neutralising antibodies, and in milk IgA, IgG, and neutralising antibodies. Control baboon mothers sampled from 12 weeks after delivery had lower serum and milk antibody titres, but responded to vaccination at 16 weeks by producing a similar antibody profile in serum and milk to those previously vaccinated. Because of the endemic nature of human rotaviral infections, similar maternal vaccinations have potential as a means of increasing milk antibodies to a level at which they may be protective to infants.

Serological and genomic characterisation of group A rotaviruses from lambs.

Four lamb rotaviruses were characterised serologically by reactions with monoclonal antibodies and genomically by hybridisation assays and sequencing. Each was found to be distinct. Three viruses belonged to the bovine genogroup and were of subgroup I. These viruses possessed serotypes G3, G6, and G10. Their corresponding P types were P1, P11, and P14 respectively. The only previous isolation of a rotavirus with VP4 of type P14 was also from lambs. The fourth isolate was G9P8, which is the first record of a G9 rotavirus from a species other than man.

Recognition of rotavirus antigens by mouse L3T4-positive T helper cells.

A lymphocyte proliferation assay was used to examine the helper T cell response to rotavirus in mice following parenteral immunization with the UK strain of bovine rotavirus. Mixed populations of lymphocytes prepared from spleen or peripheral lymph nodes were tested for proliferation in the presence of UK strain rotaviruses, prepared as cell culture lysates, ultracentrifuged (pelleted) lysates, sucrose-purified virus and caesium chloride-purified virus. Live rotavirus induced non-specific stimulation of lymphocytes, which was not observed in response to inactivated virus. Putative helper T cells of the L3T4+ phenotype were prepared as an enriched population from UK strain-immunized mice or grown in vitro as a polyclonal T cell line. The response of L3T4(+)-enriched cells from mice immunized with inactivated virus was dependent on antigen-presenting cells (APCs). Cells obtained following immunization with live virus did not require further addition of APCs. The response of the L3T4+ T cell line was wholly dependent on APCs. UK strain-specific L3T4+ cells responded to whole UK rotavirus and to isolated VP6 of both UK and C486 rotavirus strains. The results indicate that virus-specific L3T4+ T cells are induced following rotavirus immunization and can respond to epitopes on VP6. UK strain-primed L3T4+ cells also responded to an avian rotavirus strain, Ch2, which shares only minimal serological cross-reactivity with the UK strain. T cell recognition of rotavirus may thus be broadly cross-reactive.

Serological and genomic characterization of equine rotavirus VP4 proteins identifies three different P serotypes.

A series of viral reassortants was prepared between equine rotaviruses H1 (G5), H2 (G3), and L338 (G13) and human rotavirus ST3 (G4). All contained the VP4 cognate gene segment 4 from the equine parental virus and the VP7 cognate gene segment 9 from ST3. Using these viruses and antisera prepared to them, it was shown that each of the three equine viruses possessed a serologically distinct VP4 or P serotype with a > or = 16-fold difference in reciprocal cross-neutralization titers. H1 VP4 was closely related to that of porcine virus OSU, i.e., P7. L338 gene 4 was sequenced, and the sequence and serological data indicated that it constituted a novel P serotype L338. P serotype H2 was predominant among our cell culture-adapted equine rotavirus strains, but showed some serological cross-reactivity.

Identification of four VP4 serological types (P serotypes) of bovine rotavirus using viral reassortants.

A series of five reassortant viruses each containing the VP4 gene of a distinct bovine rotavirus and the VP7 gene of human rotavirus strain ST3 was prepared, and antisera to these were produced in rabbits. In neutralization tests, these antisera allowed the differentiation of the five original strains (from three different VP7 or G serotypes) into three or possibly four VP4 or P serotypes. All of a further seven bovine rotavirus strains adapted to cell culture were successfully typed by these antisera. There was a degree of cross-reaction between antiserum to the fourth bovine rotavirus P serotype and the predominant human rotavirus serotype. However, antisera raised in guinea-pigs to recombinant VP4 from this serotype showed the bovine serotype to be distinct. There was no significant serological relationship between these four bovine rotavirus P serotypes and previously described P serotypes from rotaviruses isolated from man and non-bovine animals. The predominant bovine rotavirus VP7 serotypes G6 and G10 tended to have distinct P serotypes also.

A liquid-hybridization method for typing the Vp4 and Vp7 genes of bovine rotaviruses.

A simple liquid-hybridization assay was developed which allows assessment of the degree of hybridization between the two serotype-determining genes of the bovine rotavirus strain UK and the homologous genes of the isolate under test. 32P-labelled transcription probes were produced from cloned complementary DNA (cDNA) copies of UK gene segments 4 and 8 and hybridized to double stranded RNA (dsRNA) extracted from rotavirus-positive field samples. Subsequent treatment with ribonuclease A (RNase A), separation of the RNase A-resistant hybrid fragments by polyacrylamide gel electrophoresis (PAGE) and autoradiography yielded a specific, reproducible banding pattern for each isolate. A total of 74 field samples was tested by both the hybridization assay and by an enzyme-linked immunosorbent assay (ELISA) using serotype-specific monoclonal antibodies (Mabs). The results obtained were in excellent agreement and confirmed that serotype G6 rotaviruses predominated. Hybridization of these G6 viruses with the gene 4 probe suggested that viruses with Vp4s related to that of UK rotavirus are also common. The hybridization assay was more sensitive than the ELISA.

Evidence for two serotype G3 subtypes among equine rotaviruses.

Ten cultivable equine rotavirus isolates, two of North American, six of British, and two of Irish origin, were compared with standard rotavirus strains and with each other by cross neutralization, neutralization with a panel of monoclonal antibodies (MAbs), hybridization to a simian rotavirus (SA-11) VP7 gene probe, and reaction with rotavirus subgrouping and serotyping MAbs in enzyme-linked immunosorbent assays. Six isolates, two of which had previously been serotyped as G3 by other workers, were found to be serotype G3; one was confirmed to be G5, and three were not related to serotypes G1 to G10. The serotype G3 strains were divisible into two subtypes, G3A and G3B, on the basis of cross neutralization. This division was also apparent in reactions with neutralizing VP7-specific MAbs and in the liquid hybridization assay. Two of the isolates were not bound by either subgroup MAb, six were bound by both subgroup I and II MAbs, and two were bound by only the subgroup I MAb. The assays used in this characterization provide a range of epidemiological information for use in future field investigations.

Rotavirus serotype G3 predominates in horses.

Foal fecal group A rotavirus strains were characterized by electropherotype, serotype, and subgroup and shown to be distinctly different from rotaviruses of other mammals. Of 86 strains that were electropherotyped, 98% had similar profiles, with gene segments 3 and 4 close together and segments 7, 8, and 9 widely spaced. Of 70 strains that had sufficient detectable VP7 antigen to be serotyped by enzyme-linked immunosorbent assays (ELISAs), 63% were serotype G3 (39% were subtype G3A and 24% were subtype G3B), 4% were serotype G13, and 33% were untypeable. Serotypes G1, G2, G4, G5, G6, G9, G10, and G14 were not detected, although G5 and G14 strains have been identified among cultivable equine strains. Of 50 strains that had sufficient detectable VP6 antigen to be subgrouped by ELISAs, only 12% were able to be assigned to either subgroup I or II, with the remaining 88% belonging to neither subgroup.

Molecular and serological analyses of two bovine rotaviruses (B-11 and B-60) causing calf scours in Australia.

Fecal specimens from 78 calves involved in outbreaks of calf diarrhea which occurred in three farms in Victoria, Australia, in 1988 were analyzed for rotaviruses. Thirty-eight samples were positive for group A virus antigen by enzyme-linked immunosorbent assay, and 20 of these contained viral double-stranded RNAs that could be detected by polyacrylamide gel electrophoresis. Two major electropherotypes could be observed, and a representative isolate of each electropherotype (isolates B-11 and B-60) was successfully adapted to grow in MA104 cells. Sequencing of the VP7 genes directly from RNA transcripts of fecal and cell culture-adapted viruses demonstrated that no base changes occurred in this gene upon adaptation to growth in MA104 cells. Sequencing also revealed that the VP7 protein of B-60 was closely related to G serotype 6 (G6) strains, whereas the B-11 sequence was significantly different from all previously published sequences except the recently reported VP7 sequences of bovine isolates 61A and B223, particularly across the antigenic regions A, B, and C. The other strains most closely related to B-11 by VP7 amino acid sequence analysis were G4 porcine strains BMI-1 and BEN-144 and G8 human strain 69M. Serotyping of B-11 and B-60 gave results that were in good agreement with the sequencing data. Hyperimmune typing sera clearly identified B-60 as a member of G6, whereas the B-11 strain reacted to moderate titers only with antisera to some G10 strains. Antiserum raised against B-11 neutralized some strains of G10 cross-reacted with porcine G4 type isolates BMI-1 and BEN-144 but not with other G4 strains or with rotaviruses of other mammalian G serotypes. Northern blot hybridization showed that B-11 was closely related to the recently reported bovine G10 strain B223, and they both possessed a similar segment 4 that was different from that of either UK bovine or NCDV rotavirus.

Human and bovine serotype G8 rotaviruses may be derived by reassortment.

The origin of, and relationship between human and bovine serotype G8 rotaviruses were investigated by genomic hybridisation. Radiolabelled mRNAs of human G8 rotaviruses 69M (isolated in Indonesia) and HAL1271 (isolated in Finland), and bovine rotaviruses KK3 (G10) and NCDV (G6), were used as probes. The products of liquid hybridisation between the probes and the genomic RNA of human and bovine rotaviruses, including bovine G8 rotavirus 678 (isolated in Scotland) and two other Finnish human G8 rotaviruses HAL1166 and HAL8590, were examined by separation in polyacrylamide gels. The genomes of Finnish human G8 rotaviruses were similar to those of bovine G6 and G10 rotaviruses. Neither Indonesian human G8 nor bovine G8 viruses had high levels of similarity to each other or to other bovine and human rotaviruses. Thus these three epidemiologically distinct G8 rotaviruses have different origins and may be derived by reassortment with rotaviruses of a third, as yet unknown, host species. The similarity between the Finnish isolates and the bovine isolate NCDV suggests that they have diverged recently and that these human G8 rotaviruses may be derived from a zoonotic infection, or alternatively, from the live rotavirus vaccine of bovine origin which has been used to vaccinate Finnish children.

Monoclonal antibodies differentiate between the haemagglutinating and the receptor-destroying activities of bovine coronavirus.

A relatively simple and sensitive method is described which enables the effect of monoclonal antibodies (MAbs) on the receptor-destroying enzyme (RDE) and the haemagglutination (HA) activity of bovine coronavirus (BCV) to be analysed in one assay. A lysate of HRT-18 cells infected with the L9 strain of BCV was found to have a higher RDE:HA ratio than purified virus. At 4 degrees C the lysate induced an HA pattern which completely disappeared upon raising of the temperature to 37 degrees C. This L9-infected cell lysate was used to determine the HA inhibition (HAI) titres of MAbs directed against the surface glycoproteins S and HE of BCV. Thereafter, the test plates were incubated at 37 degrees C to enable the ability of the MAbs to prevent elution of virus from BCV-erythrocyte complexes to be assessed. No inhibition of RDE was detectable with MAbs against glycoprotein S, which had HAI titres ranging from 1:16 to 1:128. On the other hand, MAbs directed against glycoprotein HE had similar HAI titres, but they inhibited elution of 8 HA units of BCV at titres of up to 1:65,000.