Breda Virus Research Articles

Infectious Rotavirus and Coronavirus of Calves

Recently, some progress has been made in the study of bacterial and viral infectious diseases of calves. However, as effective systems and methods are put in place to control these diseases, other new emerging diseases will also become important. Among diseases of the neonatal period in calves, diarrhea, which causes significant economic damage, is one of the main problems in cattle breeding. Calves diarrhea is divided into two groups: infectious and non-infectious. Diarrhea of infectious origin is mainly caused by Escherichia coli, Salmonella sp., Campylobacter sp., Clostridium perfringens type C, Rotavirus, Coronavirus, Parvovirus, Astrovirus, Calicivirus, Breda virus, Cryptosporidium, Coccidia, Giardia and Neoascaris vitulorum. During field studies, it was found that 75-95% of infectious diarrhea in calves is caused by enterotoxic Escherichia coli, rotaviruses, coronaviruses, cryptosporidium, including 27-36% rotaviruses, 20-26% coronaviruses. These diseases are often registered in livestock farms of the republic of Azerbaijan and cause the death of newborn calves.

Molecular characterization and phylogenetic analysis of the genome of porcine torovirus

In this study, we amplified and sequenced the first genome of porcine torovirus (PToV SH1 strain). The genome was found to be 28,301 bp in length, sharing 79 % identity with Breda virus. It mainly consists of replicase (20,906 bp) and structural genes: spike (4,722 bp), membrane (702 bp), hemagglutinin-esterase (1,284 bp), and nucleocapsid (492 bp). Sequence alignments and structure prediction suggest genetic differences among toroviruses, mainly in NSP1 (papain-like cysteine proteinase domain). Rooted phylogenetic trees were constructed based on the 3C-like proteinase and RNA-dependent RNA polymerase genes. PToV, Berne virus and Breda virus were clustered together, forming a separate branch from white bream virus that was distant from that of the coronaviruses.

Breda and Breda-like viruses: diagnosis, pathology and epidemiology.

Breda virus serotype 1 (Iowa) was isolated from a severe outbreak of neonatal calf diarrhoea in which 15% of the diarrhoeic animals died. Two further isolates, one from Iowa and one from Ohio, are antigenically related to the first isolate but belong to the Breda virus serotype 2 group by haemagglutination inhibition and by immuno-electron microscopy (IEM). The majority (88.5%) of cattle are serologically positive for Breda virus by enzyme-linked immunosorbent assay (ELISA). The diagnostic methods of choice should include a combination of electron microscopy, IEM, haemagglutination inhibition or ELISA, in order to identify all serotypes of Breda virus. Serotypes 1 and 2 infect and replicate in crypt and epithelial cells of the small intestine, from the mid jejunum region posteriorly through to the descending colon. Virus replicates in and is released from cells before the appearance of microscopic lesions and clinical signs. Diarrhoea starts within 24-72 hours of infection and death can occur within 36-48 hours after the onset of diarrhoea. The morphology of Breda virus, determined by negative staining and from ultrastructural studies of infected cells, is unique and probably diagnostic. Little is known about the epidemiology, other than that transmission is faecal-orally and may require close contact. Large quantities of virus are released in the faeces.

Association of enteric shedding of bovine torovirus (Breda virus) and other enteropathogens with diarrhea in veal calves.

To determine the prevalence, fecal shedding pattern, and association of bovine torovirus (BoTV) with diarrhea in veal calves at time of arrival and periodically throughout the first 35 days after their arrival on a veal farm. 62 veal calves. Fecal samples collected on days 0, 4, 14, and 35 after arrival were tested for BoTV by use of ELISA and reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Paired serum samples obtained from blood collected on days 0 and 35 were analyzed for BoTV antibodies with a hemagglutination inhibition assay. Fecal samples were also screened for other enteric pathogens, including rotavirus, coronavirus, and Cryptosporidium spp. Fecal shedding of BoTV was detected in 15 of 62 (24%) calves by use of ELISA and RT-PCR assay, with peak shedding on day 4. A significant independent association between BoTV shedding and diarrhea was observed. In addition, calves shedding > or = 2 enteric pathogens were more likely to have diarrhea than calves shedding < or = 1 pathogen. Calves that were seronegative or had low antibody titers against BoTV (< or = 1:10 hemagglutination inhibition units) at arrival seroconverted to BoTV (> 4-fold increase in titer); these calves were more likely to shed virus than calves that were seropositive against BoTV at arrival. Shedding of BoTV was strongly associated with diarrhea in neonatal veal calves during the first week after arrival at the farm. These data provide evidence that BoTV is an important pathogen of neonatal veal calves.

Enteric and nasal shedding of bovine torovirus (Breda virus) in feedlot cattle.

To assess fecal and nasal shedding patterns of bovine torovirus (BoTV) in cattle at time of arrival and periodically throughout the first 21 days after arrival at a feedlot. 57 steers. Fecal and nasal-swab samples collected on days 0, 4, 14, and 21 after arrival were tested for BoTV, using ELISA. A subset of samples from calves testing positive and negative for BoTV was analyzed, using reverse transcriptase-polymerase chain reaction (RT-PCR). Paired serum samples were collected on days 0 and 21 and tested for BoTV antibodies, using a hemagglutination inhibition assay. Overall rate of fecal shedding of BoTV was 21 of 57 (37%) by ELISA and 40 of 42 (95%) by RT-PCR with peak shedding on day 4. Diarrhea was more common in calves shedding BoTV than those not shedding the virus (odds ratio, 1.72). Overall rate of nasal shedding of BoTV was 15 of 57 (26%) by ELISA and 42 of 42 (100%) by RT-PCR, with peak shedding on day 0. Specificity of the RT-PCR product was confirmed by sequence analysis. Approximately 93% of the calves seroconverted to BoTV (> 4-fold increase in titer). Differences were not detected between calves shedding BoTV and nonshedders in relation to disease and treatments, perhaps because of the low number of cattle in the study. This study confirmed BoTV infections in feedlot cattle, including BoTV antigen and viral RNA in nasal secretions, and the shedding pattern during the first 21 days after arrival in a feedlot.

The novel hemagglutinin-esterase genes of human torovirus and Breda virus

Human torovirus (HTV) and Breda virus (BRV), members of the genus torovirus in the family Coronaviridae, are established infectious agents of humans and cattle, respectively. The hemagglutinin-esterase (HE) gene of Breda virus serotype 2 (BRV-2) has been identified and the nucleotide sequence for BRV serotype 1 (BRV-1) genome which contains the open reading frames for the viral structural proteins has been reported revealing the presence of a 1.25 kb gene whose nucleotide sequence is identical to that of the BRV-2 HE gene. In this study, we amplified the 1.2kb HE gene from the HTV genome using long RT-PCR and sequenced the amplicon directly. At the nucleotide level, the HTV HE gene manifests 85% sequence identity to the HE genes of BRV-1 and BRV-2 and 89% identity with the X pseudogene sequence of BEV. The 1.25 kb amplicons which contained the HE genes of BRV-1 and HTV were cloned and expressed in a baculovirus system and the proteins purified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Hyperimmune sera prepared in guinea pigs against these proteins were reactive with both bovine torovirus (BTV) and human torovirus (HTV) antigens. By immunoblot, they reacted specifically with a 65 kDa protein corresponding in size to the torovirus HE protein. Furthermore, the hyperimmune sera but not the preimmune sera reacted with a series of BTV-positive and HTV-positive fecal specimens by immunoblot and dot blot analysis. By immunoelectron microscopy (IEM) torovirus particles from BTV-positive specimens from calves with diarrhea and HTV-positive specimens from patients were aggregated by the hyperimmune sera. Human convalescent sera and gnotobiotic calf post-infection sera reacted by immunoblot with the expressed 65 kDa protein. The expressed HE protein of HTV has important diagnostic potential.

Bovine torovirus: Sequencing of the structural genes and expression of the nucleocapsid protein of Breda virus

Breda virus (BRV), a member of the genus torovirus, is an established etiological agent of diarrhea of cattle, which is found as two separate serotypes, BRV-1 and BRV-2. In this study, a 7.5 kb fragment of the BRV-1 genome that bracketed the genes for the structural proteins of BRV was amplified by long RT-PCR and the amplicon purified and sequenced directly. Sequence analysis revealed the presence of four open reading frames (ORF) corresponding to the peplomer (S), envelope (M), and nucleocapsid (N) genes, and an ORF for a novel 1.2 kb gene located between the M and N genes. This new gene was identical in nucleotide sequence to the hemagglutinin-esterase (HE) gene of BRV-2. With the exception of this new ORF, BRV-1 manifests 80% nucleotide sequence identity with the torovirus prototype, Berne virus (BEV) in the 7.5 kb region from the 3′ end of the genome that contains the genes for the structural proteins. A 504 base segment containing the ORF for the BRV-1 N gene was amplified by RT-PCR, and cloned into an Escherichia coli expression system. The resulting protein was purified by SDS-PAGE and used to immunize guinea pigs. Hyperimmune serum was reactive with bovine torovirus (BTV) and human torovirus (HTV) antigens. By immunoelectron microscopy, it was shown to aggregate broken but not intact torovirus particles from BTV-positive fecal specimens. By immunoblot, the hyperimmune serum reacted specifically with the 20 kD N proteins of both BTV and HTV, as well as with the expressed N protein. BRV-1 and BRV-2 immune sera from gnotobiotic calves, but not human convalescent sera from HTV-infected patients, reacted with the expressed N protein by immunoblot. These findings were applied to the design of a dot blot assay that could specifically detect BTV and HTV from fecal specimens.

Characterization of Torovirus from Human Fecal Specimens

The toroviruses, Berne virus (BEV) and Breda virus (BRV), are recognized pathogens of horses and cattle, respectively. Torovirus-like particles (TVLPs) that are immunologically related to BRV have been reported as etiological agents of gastroenteritis in humans. Of the toroviruses, only BEV has been shown to replicate in cell culture. Hence, these agents can be routinely detected only by electron microscopy (EM), although serological testing has been used as well. Our studies have provided supporting evidence that the TVLPs detected in the stool specimens of pediatric patients with gastroenteritis are human toroviruses. By EM, these particles are morphologically similar to BEV and BRV. Thin-section electron microscopy revealed that TVLPs contain toroidal-shaped nucleocapsids. Viruses purified from human fecal specimens agglutinate rabbit erythrocytes. BRV antiserum as well as convalescent sera from patients with gastroenteritis whose stools contain TVLPs were shown to contain antibodies that react with purified TVLPs as demonstrated by hemagglutination inhibition, immunoelectron microscopy, and immunoblotting. RNA extracted from partially purified TVLP preparations is amplifiable by RT-PCR using primers bracketing a 219-base region at the 3′ end of the Berne virus genome. Sequence analysis of amplicons from five isolates showed a high degree of identity with the corresponding BEV sequence.

Experimental infection of swine with a sandfly (Lutzomyia shannoni) isolate of vesicular stomatitis virus, New Jersey serotype.

parisons of bovine coronaviruses derived from neonatal calf 31. Van Kruiningen HJ, Khairallah LH, Sassevelle VG, et al.: 1987, diarrhea and winter dysentery of adult cattle. Arch Virol 140: Calfhood coronavirus enterocolitis: a clue to the etiology of 1303-1311. winter dysentery. Vet Pathol 24:564-567. 29. Van Kruiningen HJ, Castellano VP, Koopmans M, Harris LL: 32. Welch SW, Saif LJ: 1988, Monoclonal antibodies to a virulent 1992, A serologic investigation for coronavirus and Breda virus strain of transmissible gastroenteritis virus: comparison of reacantibody in winter dysentery of dairy cattle in the northeastern tivity with virulent and attenuated virus. Arch Virol 101:221United States. J Vet Diagn Invest 4:450-452. 235. 30. Van Kruiningen HJ, Castellano VP, Torres A, Sharpee RL: 33. Yolken RH: 1982, Enzyme immunoassays for the detection of 1991, Serologic evidence of coronavirus infection in New York infectious antigens in body fluids: current limitations and future and New England dairy cattle with winter dysentery. J Vet Diagn prospects. Rev Infect Dis 4:35-68. Invest 3:293-296.

A serologic investigation for coronavirus and Breda virus antibody in winter dysentery of dairy cattle in the northeastern United States.

Since 1980, the amount of evidence implicating a bovine coronavirus as the cause of winter dysentery has been increasing. Japanese workers were the first to recover a coronavirus from the feces of a cow with “epizootic diarrhea”; this discovery was followed by similar reports from Belgium and the United States. 1 Because coronavirus-like particles and coronavirus antigen can be found in the feces of a high proportion of normal dairy cows in some herds during the winter stabling season, the significance of these isolations has been interpreted with some reservation. Serologic studies revealed hemagglutination-inhibition seroconversion to reference strains of bovine coronavirus in 59% of affected Japanese cattle; 15 workers from Ohio reported 4-fold or greater rises in serum neutralization (SN) titers in 19 of 26 animals (73%); and the British, using a latex agglutination inhibition test, found seroconversion in 3 of 5 affected cattle? We recently reported 63% seroconversion by an enzyme-linked immunosorbent assay (ELISA) method, in 35 sick animals from 8 herds with winter dysentery. Additional evidence, and perhaps the most convincing, that a coronavirus is responsible for winter dysentery, is the demonstration by immunoperoxidase and electron microscopy methods of coronavirus in damaged colonic epithelial cells and mucosal macrophages of both spontaneous and experimentally induced cases. 18 In the Netherlands, however, there has been a serologic association of Breda virus infection with the occurrence of winter dysentery. Sera from 149 cows from 19 farms were tested by a blocking ELISA method, and 4-fold or greater seroconversion was found in 7-60% of cattle tested from 10 of the farms; cattle from 9 farms showed no seroconversion. In view of questions raised about the respective roles of Breda virus and coronavirus, previously assembled and some newly acquired sera were tested (retested in the case of previously assembled sera) for the presence of antibodies to these 2 viruses. The procedure used to obtain and select the sera used in this study was described in the prior report. Two serotypes of Breda virus are recognized; Breda virus serotype 1 (BRVl) represents the original isolate from Breda, Iowa, and serotype 2 (BRV2) comprises an isolate from Ohio and the second Iowa isolate. Thus far, Breda virus has not been successfully propagated in tissue culture; therefore all studies conducted with this agent employ density-gradientpurified virus particles obtained from an experimentally in-

CDNA probes for the diagnosis of bovine torovirus (Breda virus) infection

A genomic cDNA library of RNA from Breda virus (BRV), a bovine torovirus, was prepared. The nucleotide sequence of the 3' end of the genome was found to be highly conserved (93% identical) between BRV and Berne virus, the torovirus prototype. Cross-hybridization experiments were performed to select Berne virus cDNA clones for use as probes in a dot hybridization assay; the objective was to detect heterologous torovirus RNA in fecal material. A rapid RNA extraction method was employed to make the test applicable for routine diagnosis. Samples from calves after experimental and natural infection with BRV were assayed to establish the sensitivity and specificity of the test and to compare the test with the enzyme-linked immunosorbent assay (ELISA) for antigen detection. For this purpose, 53 samples from seven infected calves were tested with both methods. In the ELISA, BRV was detected in six fecal samples from three inoculated calves. By use of the hybridization test, 16 samples from seven calves reacted positively. With one exception, only postinoculation samples were found positive in hybridization. No signal was seen in feces from uninoculated calves or from calves infected with rotavirus or coronavirus.

Breda virus (Toroviridae) infection and systemic antibody response in sentinel calves

SUMMARY Enzyme-linked immunosorbent assays were established to detect Breda virus antigen in feces and homologous antibodies of the IgGl, IgM, and IgA isotypes in serum. With the aid of solid-phase immune-electron microscopy, torovirions in fecal material were observed. The course of natural infection was studied in 10 sentinel calves that had been obtained from different farms, and housed together at 1 week of age. They were separated from other cattle until the age of 10 months. Up to the age of 4 months, all calves regularly excreted Breda virus in the feces. Irrespective of the existence of IgG1 isotype maternal antibodies, all calves had early IgM responses in serum, but lack of IgA seroconversion. In 7 calves, antibody titer decreased below detection, whereas 3 calves had an isotype switch, resulting in persistent IgG1 titer. After introduction into the dairy herd at 10 months of age, all calves had diarrhea, and shedding of Breda virus was observed in 8 of them. Seroconversion for all antibody isotypes was observed, indicating lack of mucosal memory. In contrast, coronavirus infection in the presence of maternal antibodies led to isotype switch in all calves but one, and a memory response was observed after introduction into the dairy herd.

Seroepidemiology of Breda virus in cattle using ELISA

Two direct blocking enzyme linked immunosorbent assays (ELISA) for the detection of antibodies to Breda virus in sera of cattle were compared. An ELISA with consecutive addition of antigen and test serum to an antibody-coated plate gave higher positive: negative absorbence ratios than an ELISA in which antigen and test serum were added simultaneously. Sera collected from breeding and fattening herds in The Netherlands ( n = 1313) and the F.R.G. ( n = 716) were tested, and antibodies to Breda virus were demonstrated in 94% of adult cattle. Ninety percent of newborn calves had high levels of maternal antibodies, which waned until the age of 3 months. Active seroconversion occurred between 7 and 24 months in most animals.

Studies on bovine breda virus

Diarrheic feces from 21 calves were examined by electron microscopy and 16 contained particles morphologically similar to those of Breda virus. The particles were spherical or elongated, 60–270 nm in greatest dimension and had surface spikes 9–13 nm long. Convalescent serum from a human patient with Breda virus-associated diarrhea reacted with one of the bovine viruses by immune electron microscopy, suggesting a serological resemblance between human and bovine Breda-like viruses. Immune electron-microscopy and immunofluorescence demonstrated that isolates of bovine Breda virus from the U.S.A. were related to the French virus. One of the viruses had a density in sucrose solution of 1.16, similar to the value for Berne virus.

Viral gastroenteritis

ResumeDe très nombreux virus peuvent être responsables de gastro-entérites, surtout sévères chez les jeunes enfants en proie à des anomalies nutritionnelles ou immunitaires. Les plus fréquents de ces virus — et les seuls qui se prêtent à un diagnostic immunologique de routine (ELISA) — sont les rotavirus. Les autres : agent de Norwalk et virus apparentés, coronavirus et virus Breda ne sont pour le moment accessibles qu'à la microscopie électronique. Le traitement est symptomatique et le pronostic est fonction de la précocité de la réhydratation.

Detection of Breda virus antigen and antibody in humans and animals by enzyme immunoassay.

Enzyme immunoassays were developed for the detection of Breda virus antibody and antigen. Cattle sera collected in the United Kingdom were found to have a high prevalence of antibody (55%) to Breda virus when examined in a competitive enzyme-linked immunosorbent assay. A low prevalence of antibody was found in pigs (2.2%), and no antibody was found in sheep or goat sera. No antibody to either Breda virus or Berne virus was detected in human sera collected from veterinarians and farm workers. Only 1 of 430 human fecal specimens (0.2%) contained Breda virus antigen detectable by enzyme-linked immunosorbent assay.

The Haemagglutinating Activity of Berne Virus

Berne virus possesses haemagglutinating activity which is inhibited by antisera that neutralize the infectivity of the virus. In decreasing order, human, rabbit and guinea-pig erythrocytes were agglutinated whereas agglutination was not observed with rat, goose, chicken or horse red blood cells. This pattern is different from that seen with the closely related Breda virus of cattle. Haemagglutinin was found to co-sediment with viral infectivity in sucrose density gradients. Transmission electron microscopy showed that intact virus particles form bridges between adjacent erythrocytes. The viral envelope was seen at a distance from the erythrocyte surface suggesting that the peplomers possess haemagglutinating activity. Haemagglutination was decreased in the presence of fetuin and gangliosides and also by pretreatment of the erythrocytes with periodate, suggesting that the virus binds to glycoproteins and/or glycolipids on the erythrocyte surface.

A morphological study of the replication of Breda virus (proposed family Toroviridae) in bovine intestinal cells.

The morphological aspects of Breda virus serotype 2 replication in intestinal cells of gnotobiotic calves were investigated by electron microscopy. Ultrastructural findings suggest a morphogenetic pathway involving cytoplasmic vesicles, the Golgi apparatus and the cell nucleus. Virus uptake probably occurs via a receptor-mediated endocytosis-like mechanism. Endocytotic vesicles then carry virus to an as yet undetermined site of uncoating. Masses of tubules having the same diameters as Breda virion cores are found in nuclei, suggesting a role for the cell nucleus in replication of nucleocapsids. Similar tubules, as well as complete virions, were found in the Golgi region, the apparent site of virus assembly. Virus-containing Golgi vesicles then presumably move to cell surfaces where they fuse with apical and baso-lateral cell membranes to release virions in a way that permits more than one viral replicative cycle to occur without damage to host cell integrity. Virions are elongated with rounded ends and measure 42 X 100.5 nm. The morphogenesis and replication of Breda virus most closely resembles that of Berne virus of the proposed family Toroviridae.

Cellular lesions in intestinal mucosa of gnotobiotic calves experimentally infected with a new unclassified bovine virus (Breda virus).

Four gnotobiotic calves were inoculated intranasally with Breda virus within two hours after cesarean section. Three calves developed diarrhea between 48 and 60 hours after inoculation; one calf was killed at 36 hours. Consistent microscopic findings in the intestinal mucosae of calves with diarrhea were cytopathologic changes in enterocytes of the lower small intestine, large intestine, and dome epithelial cells and an acute inflammatory response with cellular infiltration and subtle changes in capillaries. Virions with average dimensions of 35 x 80 nm were seen in rounded cells that had severe cell swelling, dilatation of the cytocavitary network, and hydropic degeneration. Virions were most often in multiple, large autophagolysosomes. There was a good correlation in detection of virus in cells with electron microscopy and indirect immunofluorescence. No lesions were detectable in two control calves killed five days after inoculation.

Astrovirus and Breda virus infections of dome cell epithelium of bovine ileum.

A bovine enteric virus antigenically related to the United Kingdom isolate of bovine astrovirus was isolated from diarrheic feces, also containing rotavirus, of a calf in Florida. The astrovirus infected cell cultures and the epithelial cells of domes in the ileum, and there was cross-immunofluorescence with antiserum to the United Kingdom astrovirus. Calves infected with astrovirus alone did not develop clinical disease, but when astrovirus was mixed with rotavirus or Breda virus 2, the calves developed severe diarrhea and more extensive astrovirus infection of the dome epithelium. The dome epithelial cells showed degeneration associated with astrovirus infection, and a few cells showed degeneration with Breda virus 2 infection. Virions with a 30-nm diameter were seen in astrovirus-infected dome cells, and Breda virus 2 virions were also observed either in separate cells or, on occasion, with both viruses in one cell.