Comparison of the efficacy of rotavirus VLP vaccines to a live homologous rotavirus vaccine in a pig model of rotavirus disease.

Rotavirus-like particles (VLPs) have shown promise as rotavirus vaccine candidates in mice, rabbits and pigs. In pigs, VLP vaccines reduced rotavirus shedding and disease but only when used in conjunction with live attenuated human rotavirus. Using a porcine rotavirus pig model, rotavirus antigen shedding was reduced by up to 40% after vaccination with VLPs including the neutralizing antigens VP7 and VP8* when used in combination with the adjuvant polyphosphazene poly[di(carbozylatophenoxy)phoshazene] (PCPP). In contrast, complete protection from rotavirus antigen shedding and disease was induced by vaccination with the virulent porcine rotavirus PRV 4F. This is the first study to demonstrate some post-challenge reductions in rotavirus antigen shedding in a pig model of rotavirus disease after vaccination with VLPs without combining with infectious rotavirus.

Serotype 1 and 2 bovine noroviruses are endemic in cattle in the United kingdom and Germany.

The genomically and antigenically distinct bovine noroviruses Bo/Jena/1980/DE and Bo/Newbury2/1976/UK have been associated with calf diarrhea. In the present seroprevalence study, both were found to be endemic in cattle from Germany and the United Kingdom, a finding in contrast to previous virus prevalence studies. They were less common than group A rotaviruses, particularly in calves, suggesting a different epidemiology.

Retrospective study of noroviruses in samples of diarrhoea from cattle, using the Veterinary Laboratories Agency's Farmfile database.

A collaborative study was undertaken by the Veterinary Laboratories Agency (vla) and the Royal Veterinary College (rvc) to determine the prevalence of bovine noroviruses in cattle with diarrhoea. Samples of bovine diarrhoea were provided by the vla from routine diagnostic submissions and a reverse transcription-pcr was used by the rvc to detect the viruses. Epidemiological information about the samples was provided retrospectively by the Farmfile database. Noroviruses were detected in 44 (11 per cent) of the 398 samples tested, and Farmfile data were used to investigate the differences between the positive and negative animals.

Complete genomic characterization and antigenic relatedness of genogroup III, genotype 2 bovine noroviruses.

Bovine enteric noroviruses form a genogroup, III, distinct from the 2 human norovirus genogroups, I and II. Two genogroup III genotypes were suggested by partial genomic analyses. In the present study, analysis of the full-length genome sequence of Bo/Newbury2/76/UK and the more contemporary Newbury2-like virus, Bo/Dumfries/1994/UK, showed that both were 7311 nucleotides in length and had three open reading frames (ORFs), amino acids motifs typical of noroviruses, and 95% or greater amino acid identities to each other in all regions of their genome. Apart from the ORF1 NTPase region, their ORF1 regions had less than 90% identity to the genogroup III genotype 1 Bo/Jena/80/DE virus, confirming two genogroup III genotypes. A close antigenic relationship was demonstrated by ELISA between the genotype 2 viruses, which will allow their serological diagnosis.

Genomic characterization of the unclassified bovine enteric virus Newbury agent-1 (Newbury1) endorses a new genus in the family Caliciviridae.

The pathogenic bovine enteric virus, Newbury agent-1 (Bo//Newbury1/1976/UK), first identified in 1976, was characterized as a possible calicivirus by morphology, buoyant density in CsCl and the presence of a single capsid protein but genomic sequence could not be obtained. In the present study, the complete genome sequence of Newbury1 was determined and classified Newbury1 in a new genus of the Caliciviridae. The Newbury1 genome, of 7454 nucleotides, had two predicted open reading frames (ORFs). ORF1 encoded the non-structural and contiguous capsid proteins. ORF2 encoded a basic protein characteristic of the family Caliciviridae. Compared to the 4 recognized Caliciviridae genera, Norovirus, Sapovirus, Lagovirus and Vesivirus, Newbury1 had less than 39% amino acid (47% nucleotide) identity in the complete 2C-helicase, 3C-protease, 3D-polymerase and capsid regions but had 89% to 98% amino acid (78% to 92% nucleotide) identity to the recently characterized NB virus in these regions. By phylogenetic analyses, Newbury1 and NB viruses formed a distinct clade independent of the 4 recognized genera. However, amino acid identities showed that Newbury1 and the NB virus were distinct polymerase types (90% amino acid identity), but their complete capsid proteins were almost identical (98% amino acid identity). Analyses of contemporary viruses showed that the two polymerase genotypes, Newbury1 and NB, were circulating in UK cattle and antibody to Newbury1-like viruses was common in cattle sera. The present study defined the existence of a new genus in the Caliciviridae that we propose be named Becovirus or Nabovirus to distinguish the new clade from bovine noroviruses.

Genotype 1 and genotype 2 bovine noroviruses are antigenically distinct but share a cross-reactive epitope with human noroviruses.

The bovine enteric caliciviruses Bo/Jena/1980/DE and Bo/Newbury2/1976/UK represent two distinct genotypes within a new genogroup, genogroup III, in the genus Norovirus of the family Caliciviridae. In the present study, the antigenic relatedness of these two genotypes was determined for the first time to enable the development of tests to detect and differentiate between both genotypes. Two approaches were used. First, cross-reactivity was examined by enzyme-linked immunosorbent assay (ELISA) using recombinant virus-like particles (VLPs) and convalescent-phase sera from calves infected with either Jena (genotype 1) or Newbury2 (genotype 2). Second, cross-reactivity was examined between the two genotypes with a monoclonal antibody, CM39, derived using Jena VLPs. The two genotypes, Jena and Newbury2, were antigenically distinct with little or no cross-reactivity by ELISA to the heterologous VLPs using convalescent calf sera that had homologous immunoglobulin G titers of log10 3.1 to 3.3. CM39 reacted with both Jena and heterologous Newbury2 VLPs. The CM39 epitope was mapped to nine amino acids (31PTAGAQIAA39) in the Jena capsid protein, which was not fully conserved for Newbury2 (31PTAGAPVAA39). Molecular modeling showed that the CM39 epitope was located within the NH2-terminal arm inside the virus capsid. Surprisingly, CM39 also reacted with VLPs from two genogroup II/3 human noroviruses by ELISA and Western blotting. Thus, although the bovine noroviruses Jena and Newbury2 corresponded to two distinct antigenic types or serotypes, they shared at least one cross-reactive epitope. These findings have relevance for epidemiological studies to determine the prevalence of bovine norovirus serotypes and to develop vaccines to bovine noroviruses.

A chimeric bovine enteric calicivirus: evidence for genomic recombination in genogroup III of the Norovirus genus of the Caliciviridae.

The Norovirus genus of the Caliciviridae encompasses viruses that cause outbreaks of gastroenteritis in human and viruses that have been associated with diarrhea in cattle. The two bovine noroviruses, Bo/Newbury2/76/UK and Bo/Jena/80/DE, represent two distinct genetic clusters in the newly described genogroup III. In the present study, Jena-like polymerase sequences were identified for the first time in the UK, but one of these, Bo/Thirsk10/00/UK, was a chimeric virus. Bo/Thirsk10/00/UK had a Jena-like polymerase gene but Newbury2-like capsid and ORF3 genes by comparison of their genome organization, nucleotide, and amino acid identities and phylogenetic analyses. The present study is one of few studies to clearly demonstrate the existence of chimeric genomes in the Norovirus genus and the first, to our knowledge, to identify a chimeric genome in genogroup III. It provides additional support that genomic recombination is part of the natural evolution of noroviruses and is relevant to the diagnosis and immunological control of norovirus diarrhea outbreaks.

Molecular characterization of bovine enteric caliciviruses: a distinct third genogroup of noroviruses (Norwalk-like viruses) unlikely to be of risk to humans.

Bovine enteric caliciviruses (BoCVs) have been classified in the Norovirus (Norwalk-like virus) genus of the Caliciviridae, raising questions about zoonotic transmission and an animal reservoir for the human Norwalk-like viruses (NLVs), an important cause of nonbacterial gastroenteritis in humans. We examined the genetic relationship of human NLVs to BoCVs that were identified by using reverse transcription-PCR with primer pairs originally designed to detect human NLVs. Polymerase, capsid, and open reading frame 3 (ORF3) gene sequence analyses of BoCVs that were identified from 1976 to 2000 from throughout the United Kingdom showed that BoCVs formed a distinct third genogroup of closely related viruses distinct from the human genogroup I and II NLVs. Evidence was not obtained to support the concept that BoCVs are circulating in humans and pose a threat to human health.

Identification and molecular characterization of a bovine G3 rotavirus which causes age-independent diarrhea in cattle.

G3 rotaviruses have been reported rarely in cattle, and none have been characterized. We report the first genomic characterization of a bovine G3 rotavirus, CP-1, which had been biologically characterized in vivo and shown to cause age-independent diarrhea. CP-1 was a G3 rotavirus as its VP7 had 92 to 96% deduced amino acid identity to those of G3 rotaviruses. However, initially, CP-1 was identified as a G10 rotavirus by RT-PCR even though the CP-1 VP7 had only 81 to 85% deduced amino acid identity to those of G10 rotaviruses. Rotavirus CP-1 was of P[5] specificity, a type common in cattle, and had a bovine NSP1 and NSP4. These results added another animal species to those in which G3 rotaviruses have been found, characterized a bovine rotavirus which caused age-independent diarrhea in calves, and raised the possibility that bovine G3 rotaviruses may be misdiagnosed as G10 rotaviruses.

Rotavirus cross-species pathogenicity: molecular characterization of a bovine rotavirus pathogenic for pigs.

Rotaviruses which cause disease in heterologous animal species have been reported but the molecular basis of cross-species infectivity and disease is not established. We report the molecular characterization of a cloned rotavirus, PP-1, which was originally obtained from cattle and which had been biologically characterized in vivo in two target animal species, gnotobiotic pigs and calves. In pigs, PP-1 caused severe clinical disease but in experimental calves it replicated subclinically. PP-1 was characterized as a G3 reassortant with a porcine VP4 and NSP4 but a bovine NSP1. The PP-1 VP4 had 96 to 97% deduced amino acid identity to P[7] porcine rotaviruses and P[7] specificity was confirmed with VP4-specific monoclonal antibodies. Sequence analysis of the PP-1 NSP1 showed 94 to 99.6% deduced amino acid identity to bovine rotaviruses but the NSP4 protein had 94 to 98% identity to the NSP4 genotype B porcine rotaviruses. G-typing PCR initially classified PP-1 as a G10 rotavirus but sequence analysis revealed 92 to 96% identity of the PP-1 VP7 with porcine, simian, and human G3 rotaviruses. These results, combined with the in vivo properties of PP-1 in the two target species, supported the concept that species-specific VP4 and NSP4, but not NSP1, are required to induce rotavirus disease, at least in calves and pigs. The results illustrate experimentally that rotaviruses circulating in one animal species can pose a risk to another by the emergence of a pathogenic reassortant rotavirus under appropriate conditions.

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.

The bovine Newbury agent-2 is genetically more closely related to human SRSVs than to animal caliciviruses.

The hypothesis that the enteric bovine calici-like virus Newbury agent (NA-2) belongs to the family Caliciviridae was examined by genome sequence analysis. Use of solid-phase immune electron microscopy allowed samples with good levels of virus to be identified and amplification of the genome was achieved by reverse transcription-polymerase chain reaction. Examination of a 216-amino-acid sequence in the RNA-dependent RNA polymerase gene and a 116-amino-acid sequence in the capsid gene showed that NA-2 had the closest deduced amino acid identity (77 to 80% for the polymerase region and 67 to 73% for the capsid region) to the morphologically indistinguishable human SRSVs (small round structured viruses) of genogroup 1, which are classified as members of the Caliciviridae. It had a weak relationship (<34.5% deduced amino acid identity) in both the polymerase and the capsid regions to animal caliciviruses, all of which have classical morphology. This is the first genomic data from a nonhuman virus with SRSV morphology. It confirms the hypothesis that the bovine enteric calici-like virus NA-2 is a member of the family Caliciviridae and endorses the observation to date that viruses with SRSV morphology are genomically distinct.

Viral determinants of rotavirus pathogenicity in pigs: evidence that the fourth gene of a porcine rotavirus confers diarrhea in the homologous host.

A porcine rotavirus (prv) monoreassortant, S-F4, which carries RNA segment 4 of the pig-pathogenic variant prv 4F in the genetic background of the pig-apathogenic variant prv 4S (G. I. Tauscher and U. Desselberger, J. Virol. 71:853-857, 1997), was found to be pathogenic in gnotobiotic piglets. This indicates that RNA segment 4 of the pig-pathogenic variant prv 4F is a major determinant of pathogenicity in its homologous host.

Determinants of rotavirus host range restriction--a heterologous bovine NSP1 gene does not affect replication kinetics in the pig.

The genetic basis of rotavirus host range restriction (host species specificity) is unknown but the NSP1 (fifth) gene has been implicated in some studies. We studied the replication kinetics in vivo of a NSP1 gene monoreassortant, E11, to assess the influence of a heterologous NSP1 gene on the ability to replicate in pigs. The monoreassortant possessed 10 genes from the porcine parent rotavirus SW20/21, which replicated productively in pigs, and the NSP1 gene from the bovine rotavirus UK which produced an abortive infection in pigs. Groups of up to four pigs were inoculated orally with 10(5) to 10(6) TCID50 of the monoreassortant, the porcine parent rotavirus, or the bovine parent rotavirus or were sham inoculated. The monoreassortant replicated productively in pigs with replication kinetics almost identical to the porcine parent rotavirus. During a 9-day observation period after inoculation, the number of days with virus in the faeces, the onset and duration of virus excretion, and peak titres in faeces were similar for the monoreassortant and the parent porcine rotavirus. The genetic composition of the viruses excreted in the faeces was confirmed as that of the inocula by PAGE. Thus possession of a heterologous NSP1 gene from a bovine rotavirus which failed to replicate in pigs did not produce an abortive infection or affect the replication kinetics in vivo. The genetic basis of host range restriction between porcine and bovine rotaviruses remains to be established.

A study of nurses' views about the prevention of nosocomial urinary tract infections.

This study sought to discover the contribution of nursing practice to the prevention of hospital-acquired or nosocomial urinary tract infections (NUTIs), the most commonly occurring nosocomial infection. Seventy-five per cent of such infections are associated with urethral catheters. The practices of nurses who are caring for patients on a 24 h basis would appear to be fundamental to achieving any reduction in the incidence of NUTIs. This qualitative study utilized unstructured interviews to explore the views of 12 registered nurses about three key issues: first, what care do nurses give with the aim of preventing catheter-associated NUTIs; secondly, what improvements in practice would further prevent catheter-associated NUTIs; thirdly, what do nurses see as constraints to the prevention of catheter-associated NUTIs? The nurses identified many of the measures that were cited in the literature as effective for preventing NUTIs; however in reality, they stated that their practice differed because of a lack of time to give care and to update themselves. The consequences of under-staffing were that junior and temporary staff (whose competence in preventing NUTIs was questioned) worked unsupervised. Those interviewed identified feelings of powerlessness in effecting preventative measures, and identified not only the role of medical staff in influencing NUTIs but also their inconsistent approach to care. All these forces effectively limited the nurses' ability to prevent NUTIs. The study is concluded with recommendations for changes in practice and further research.

A community systems approach to substance abuse prevention in a rural setting.

The Community Health Demonstration Project developed a community systems model of program development and interventions which provides consistent, ongoing prevention messages and services across multiple delivery systems and service providers. This model has brought together various sectors of a rural disadvantaged Appalachian community in Pennsylvania to plan, implement, and evaluate a community-wide campaign addressing the prevention of alcohol, tobacco, and other drug (ATOD) problems. The model for this community prevention effort focused primarily on the development of community awareness, resources, and programming-capacity building-all of which provides a foundation for continuing attitude and behavior change. Preliminary evaluative data indicate that awareness of local ATOD problems has increased. Moreover, new programming has been implemented and interaction and cooperation among services providers have improved dramatically during the course of the project.

The lesions of rotavirus infection in 1- and 10-day-old gnotobiotic calves.

Age-related resistance to rotavirus disease has been described with some rotaviruses. In the present study, we investigated age-related resistance to rotavirus disease by defining extent of intestinal infection, virus replication, and severity of intestinal lesions in groups of three 1- and 10-day-old gnotobiotic calves of mixed breed inoculated orally with a cloned bovine rotavirus of low virulence for calves (strain C3-160) and in two groups of three uninoculated control calves of mixed breed. One-day-old calves inoculated with rotavirus developed diarrhea 26 hours after inoculation, and their feces contained 10(8.5)-10(9.2) TCID50/g feces; inoculated 10-day-old calves did not develop diarrhea, virus excretion commenced on the second or third day after inoculation, and peak concentrations of virus in feces were 10(5.7)-10(7.9) TCID50/g feces. Calves were euthanatized within 8-30 hours after the attainment of peak virus shedding while they were still shedding virus at peak levels. The mean percentage of small intestinal epithelium that was immunostained for rotavirus was three times greater in 1-day-old calves than in 10-day-old calves, and the large intestine was infected more extensively in 1-day-old calves. Immunostaining for rotavirus was maximal in the mid small intestine. Staining of mucin was substantially less in the epithelium of the small intestines and colon of rotavirus-inoculated 1-day-old calves than in age-matched controls. The mean height of villi was reduced to approximately half that of controls in the mid and distal small intestine of rotavirus-inoculated 1-day-old calves and was unchanged in 10-day-old calves. Mean crypt cell production rates were greater than that in controls in both groups of rotavirus-inoculated calves, indicating increased enterocyte loss. Age-related resistance to disease was not due to an inability of rotavirus to infect and replicate in enterocytes with lethal effects but appeared to be associated with a slowing of the pathogenic process, which occurred because insufficient enterocytes became infected and destroyed for lesions to develop.

A definition of bovine rotavirus virulence.

Rotaviruses are accepted as enteric pathogens of calves but many natural infections are subclinical. In the present paper, the outcome of inoculation of gnotobiotic calves of three ages (the second day of life, the second week of life and calves aged 6 weeks and over) with doses of 10(5.0) to 10(6.5) TCID50 was compared for three bovine rotavirus isolates (C3-160, 17/4 and 39/58). The clinical outcome of infection was dependent on both calf age and rotavirus isolate. Age-dependent resistance to infection was not found. By contrast, age-dependent resistance to disease was found with rotavirus isolates C3-160 and 17/4 but not with 39/58. All three isolates caused disease in calves inoculated on the second day of life but only one, 39/58, caused disease in the two older groups. Peak levels and duration of virus excretion were similar in clinically normal (10(6.7 +/- 1.08) TCID50 per g of faeces for 4.6 +/- 1.2 days) and diseased (10(7.45 +/- 0.94) TCID50 per g of faeces for 5.3 +/- 0.98 days) calves of all ages, but the onset of virus excretion occurred sooner in clinically affected calves (1.6 +/- 0.63 days in clinically affected compared with 3.7 +/- 1.5 days in clinically normal calves, P < 0.01). The present study confirmed the findings of an earlier study (Bridger & Pocock, 1986) which showed that bovine rotaviruses differ in virulence for calves in the second week of life and that older calves are susceptible to rotavirus infection and disease. In addition the present study demonstrated for the first time, that differences in rotavirus virulence are not apparent with calves inoculated on the second day of life, an age which has been used commonly to assess rotavirus virulence. It is suggested that rotaviruses that cause disease in calves only on the second day of life should be described as of low virulence whereas those that cause disease in all ages should be described as virulent.

Temporal correlation between a single amino acid change in the VP4 of a porcine rotavirus and a marked change in pathogenicity.

We previously described a marked increase in the pathogenicity of a cell culture grown porcine rotavirus, PRV 4F, during serial passage in gnotobiotic piglets (Bridger et al., 1992). Here we report close temporal correlation between this change in pathogenicity and an amino acid change within a highly conserved hydrophobic domain of VP4 at position 469. Cell culture grown PRV 4F is unique in having a hydrophilic residue, glutamine, at amino acid 469; all previously sequenced VP4s have hydrophobic leucine or phenylalanine residues at the corresponding position. The detection of a point mutation causing a deduced amino acid change from glutamine to leucine at amino acid 469 of PRV 4F VP4 in virus obtained from one piglet at the second serial passage correlated exactly with the emergence of viral pathogenicity. However, given the multifactorial nature of virus pathogenicity, genetic studies are required to ascertain the degree to which this mutation is responsible for the observed change in pathogenicity.

Number and distribution of T lymphocytes in the small intestinal mucosa of calves inoculated with rotavirus.

An understanding of the immune response to rotavirus is needed to develop effective prophylaxis. There is evidence that cell-mediated responses may be involved and to extend these observations, rotavirus antigen and the three major T cell subsets, BoCD4+, BoCD8+, and BoWC1+ gamma/delta lymphocytes were immunostained in tissue sections from calves killed at 2, 4, 6, 8 and 10 days post inoculation and quantified by image analysis. It was established that in control calves, BoCD4+ lymphocytes were predominantly in the lamina propria, while the majority of BoCD8+ and BoWC1+ gamma/delta lymphocytes were in the epithelium. Rotavirus infection was seen throughout the small intestine with the greatest amount of viral antigen detected at 4 days post inoculation in the mid and distal small intestine. Increased numbers of all subsets were detected; small increases in intraepithelial BoCD4+ and BoWC1+ gamma/delta T lymphocytes were observed especially in the distal small intestine, while larger increases in BoCD8+ cells were detected in the epithelium and lamina propria of the proximal, mid and distal small intestine. The timing and location of these increases in T lymphocyte subsets is indicative of a specific immune response involving BoCD8+ and BoWC1+ gamma/delta T lymphocytes.