BVD - The Virus:

Mucosal Disease:

Vilcek and Nettleton 2006

Deregt, D., S. R. Bolin, et al. (1998). Mapping of a type 1-specific and a type-common epitope on the E2 (gp53) protein of bovine viral diarrhea virus with neutralization escape mutants. Virus Res 53(1): 81-90.

The envelope protein E2 of BVDV is a major immunodominant protein important for neutralization of virus by antibodies, and therefore important for vaccine production and serotyping. This study mapped differences in the epitopes of E2 between BVDV 1 and BVDV 2, using monoclonal antibodies. Sequences of the E2 gene of several BVDV 1 and BVDV 2 isolates are included in the paper. Single nucleotide changes at amino acid positions 9, 32 and 72, preventing the BVDV 1-specific monoclonal antibody 157 from neutralizing BVDV 2 isolates, were identified. Four amino acids were identified within position 71-77 of E2 common to BVDV 1 and BVDV 2, which were critical for neutralization by monoclonal antibody 348. Monoclconal antibody 348 was reactive to all BVDV 2 strains, but to a lesser extent than BVDV 1.

Vilcek, Durkovic et al 2005

Vilcek, S. and P. F. Nettleton (2006). Pestiviruses in wild animals. Vet Microbiol 116(1-3): 1-12.

This paper discusses pestiviruses in wild animals. Most pestiviruses are not host-specific - CSF, BVDV 1 and giraffe pestivirus circulate in wild animals. Listed in the paper are the different isolates found in wild animal species. CSF antibodies are found in wild boars as well as domestic pigs, and viruses isolated from wild boars have been linked to outbreaks of CSF in pigs. BVDV antibodies have been found in deer, buffalo, bighorn sheep, chamois, eland, pronghorn antelope, alpacas and llamas. The possibility of wild animals acting as a reservoir of BVDV and CSF has implications for their eradication programs in European countries. There is no evidence at present for transmission of BVDV from wild to domestic animals. It is likely that wild animals are infected with more pestivirus species than have been identified at present.

Ridpath 2003

Vilcek, S., B. Durkovic, et al. (2005). Genetic diversity of BVDV: consequences for classification and molecular epidemiology. Prev Vet Med 72(1-2): 31-5; discussion 215-9.

The discovery of a 12th subgenotype of BVDV 1 in Swiss cattle, BVDV 1k, found by comparing genomic sequences from the 5’ untranslated region (UTR), Npro and E2 regions is discussed. Isolates of BVDV 1d in India and BVDV 1c in Australia were also identified, and it was concluded that distribution of subgenotypes is unrelated to geographic origin of viral isolates. The authors question the ability of primers based on BVDV 1a and 1b to detect different isolates in diagnosis of the virus and vaccines containing BVDV 1a and 1b to protect against different subgenotypes.

Buckwold, Beer et al 2003

Ridpath, J. F. (2003). BVDV genotypes and biotypes: practical implications for diagnosis and control. Biologicals 31(2): 127-31.

This paper discusses the implications of the genetic diversity of bovine viral diarrhea virus (BVDV) for clinical presentation, virus detection and vaccine protection. The paper illustrates how pestivirus species can be divided into three main branches, with BVDV 1 and BVDV 2 in one branch, classical swine fever (CSF), border disease virus (BDV) and unique viruses in the giraffe and reindeer in another, and a virus in a pronghorn antelope in a third branch. BVDV occurs in two genotypes, BVDV 1 and BVDV 2. Genetic drift, brought about by mutations of the RNA genome, is responsible for creating different genotypes and subgenotypes, causing variation in detection and vaccine protection of BVDV. Included in the paper are monoclonal antibody panels for BVDV 1 and BVDV 2 isolates, showing the variation in antibody-binding to different isolates. BVDV 1 and BVDV 2 can exist as two biotypes – non-cytopathic and cytopathic. BVDV causes a wide range of clinical diseases, with non-cytopathic isolates responsible for causing in utero persistent infections. Some non-cytopathic BVDV 2 strains cause severe clinical disease, known as severe acute BVD.

Kummerer, Tautz et al 2000

Buckwold, V. E., B. E. Beer, et al. (2003). Bovine viral diarrhea virus as a surrogate model of hepatitis C virus for the evaluation of antiviral agents. Antiviral Res 60(1): 1-15.

This paper explores the advantages and disadvantages of using BVDV as a model of the hepatitis C virus (HCV) for studies into antiviral agents. BVDV is easy to grow in tissue culture, has a complete replication cycle and there are molecular clones available. The paper describes the structure and replication cycle of Flaviviridae, and compares BVDV to HCV. Despite low levels of sequence identity between HCV and BVDV, the authors believe that BVDV is a useful model for replication of HCV in studies into antiviral drugs, due to similarity in replication cycles, biology and homology of many gene products that are possible targets for antiviral drugs for HCV. The paper is positive about the potential use of BVDV as a model for antiviral agents directed against elements known as IRES in the 5’ UTR of HCV and BVDV, which initiate translation, as well as other proteins involved in replication (amino acid sequences of NS3 and NS5B in HCV and BVDV are aligned in the paper). The use of BVDV-HCV chimeras may be helpful in the development of anti-HCV antiviral agents.

Deregt, Bolin et al 1998

Kummerer, B. M., N. Tautz, et al. (2000). The genetic basis for cytopathogenicity of pestiviruses. Vet Microbiol 77(1-2): 117-28.

This paper describes the first cytopathic BVDV isolate to have resulted from a method other than recombination, by point mutations in the NS2 gene, and describes these mutations. In cytopathic BVDV strains, the nonstructural protein NS2-3 is cleaved to produce NS2 and NS3. Mucosal disease occurs when an animal persistently infected with a non-cytopathic virus is either superinfected by an antigenically-related cytopathic virus, or when a non-cytopathic virus mutates. This paper identifies differences in the genome of cytopathic and non-cytopathic pairs. Insertions, duplications, rearrangements and deletions identified in cytopathic strains are illustrated in a series of diagrams. A cytopathic BVDV isolate was cloned and sequenced, and no genomic recombination was present. In this strain, the 3’ terminal third of the NS2 gene was required for cleavage of NS2-3. Cleavage of NS2-3 resulted from point mutations within the NS2 gene, and cleavage efficiency was mapped to residue 1555 of the polyprotein, as illustrated in the paper. Expression of NS3 in cytopathic strains is brought about by integration of ubiquitin-coding sequences into BVDV genomes, which create a protease cleavage site in the viral polyprotein. This is cut by a cellular protease, releasing the N-terminus of NS3. It is thought that the N-terminus of NS3 is required for the cytopathic effect of the virus.

Brownlie, Thompson & Curwen, 2000

Bovine virus diarrhoea virus - strategic decisions for diagnosis and control. In Pract., Apr 2000; 22: 176 - 187.

This extensive article covers many aspects of BVDV from the virus at a molecular level, to the practicalities of its control on a farm basis. Numerous pictures and tables clarify the information. After classifying the virus and its different strains, and describing its structure, the importance of the different biotypes, cytopathic and non-cytopathic, are explained. The various diseases that occur with BVDV are described, including acute infections, haemorrhagic disease, mucosal disease and in utero infections. The diagnostic tests for BVDV are compared, including blood and bulk milk samples for antibody testing, and virus detection using blood samples. A comprehensive discussion on the options for control of BVDV is included, and concludes that combining eradication with vaccination is optimal. For this to be successful, PI calves, identified by Scandinavian countries as being super-shedders of BVDV, must be detected and culled, and the breeding herd must be vaccinated. Despite this paper being written nine years ago, the diagnostic tests and methods of control discussed are still applicable, except that RT-PCR is now commonly used in diagnosis.  

Tautz, N et al, 1994

Tautz, N., et al., Pathogenesis of mucosal disease: a cytopathogenic pestivirus generated by an internal deletion. J Virol, 1994. 68(5): p. 3289-97.

The genome of a novel cytopathic BVDV strain, CP9 was characterized in this study. This study is the first to discover a second, smaller length of RNA (8kb) occurring in the cytopathic strain CP9 in addition to the 12.3kb genome. This 8kb length of RNA was shown to be a defective interfering particle (DI), by cell culture experiments. These are unable to replicate without a helper virus, whose replication they interfere with. In the absence of the DI, the cytopathic effect of the virus was abolished, suggesting that the DI is involved in cytopathogenicity of the virus and the production of fatal mucosal disease. They established in this paper that BVDV CP9 requires a DI as a cytopathogenic agent as well as a non-cytopathic helper virus. A 4.3kb deletion occurs in the DI which includes all of the BVDV structural proteins, and part of the nonstructural protein p125, identified by cDNA cloning and sequencing. The importance of p80 as a protein only found in cytopathic strains is highlighted. They showed in this study that the genome of DI9 can express p80.

Sopp, P et al, 1994

Sopp, P., et al., Detection of bovine viral diarrhoea virus p80 protein in subpopulations of bovine leukocytes. J Gen Virol, 1994. 75 ( Pt 5): p. 1189-94.

BVDV is able to infect leukocytes, and it is thought that this may account for the defective immune response and immunotolerance seen with infection. This paper describes the detection of p80 protein in bovine leukocytes of viraemic cattle by flow cytometry and two-colour immunofluorescence, and concludes that this could be important for tolerance. The paper describes the use of different monoclonal antibodies for the detection of the non-structural protein, p80 and the structural protein, gp53. 23% of peripheral blood mononuclear cells (PBMCs) collected from eight viraemic animals were positive for viral antigen. Monocytes were found to express the highest level of p80, followed by CD2+ cells, and then by WC3+ B cells and WC1+ γδ T cells. These cells represented cells in which viral protein synthesis had occurred, as p80 is a non-structural protein. The staining of the PBMCs from a PI animal is shown. The proportions of PBMCs infected were independent of animal age. The proportions of subpopulations of leukocytes (B cells, monocytes, CD4+, CD8+ and WC1+ T cells) were no different in the PI animals to control animals. The varying proportions of the different types of PMBC in animals of different ages found in previous studies in discussed.

Nakajima, N et al, 1993

Nakajima N, Fukuyama S, Hirahara T, Takamura K, Okada N, Kawazu K, Ui S, Kodama K.  1993.  Induction of mucosal disease in cattle persistently infected with noncytopathic bovine viral diarrhea-mucosal disease virus by superinfection with cytopathic bovine viral diarrhea-mucosal disease virus.  J Vet Med Sci. 1993 Feb;55(1):67-72.

In this study the clinical outcome of three PI animals superinfected with homologous and heterologous cytopathic BVDV strains to the non-cytopathic strains they were infected with was observed. This paper supports evidence for a requirement of viral homogeneity between non-cytopathic and cytopathic strains for mucosal disease to occur. Despite the small sample size, this study is valuable as it included natural and experimental infection, describes the clinical outcome, and the results of extensive serological and virological studies. Virus isolation from these animals identified the strains with which they were persistently infected. Two of the animals were naturally superinfected with cytopathic BVDV, one of which displayed typical signs of mucosal disease, and died; the other became anorectic and pyrexic, but recovered, having produced antibodies to the strain. This animal was PI with an antigenically-different strain. This animal and another were later experimentally infected with other cytopathic strains. One of the animals succumbed to mucosal disease after inoculation with an antigenically-related strain to its cytopathic virus. The other animal (which had earlier recovered from natural infection with cytopathic virus) did not suffer from mucosal disease, as this animal was PI with an antigenically different strain, confirmed by cross-neutralisation. It was concluded that antigenic relationship between cytopathic and non-cytopathic strains is important in the development of mucosal disease.

Brownlie, J & Clarke, M C, 1993

Brownlie, J & Clarke M C. (1993). "Experimental and spontaneous mucosal disease of cattle: a validation of Koch's postulates in the definition of pathogenesis". Intervirology 35(1-4): p51-59.

This paper describes the fulfillment of Koch’s Postulates with regards to mucosal disease in cattle. The usefulness of postulates is evaluated, and their shortcomings explained. The clinical signs of mucosal disease are briefly described, being profuse diarrhea, with severe erosions in the intestine, targeting the Peyer’s patches, and ultimate death. The paper explains the pathogenesis of mucosal disease, and the requirement of a calf persistently-infected (PI) with non-cytopathic BVDV being superinfected with a cytopathic strain. The aetiology of mucosal disease is refined, with the discovery that the superinfecting cytopathic strain must be anitgenically homologous to the non-cytopathic strain in order for mucosal disease to occur, as PI calves can mount an immune response to heterologous strains. Evidence for the de novo production of cytopathogenic virus from persisting virus was demonstrated by the spontaneous development of mucosal disease in a PI animal that had been kept in strict isolation. Possible mechanisms for the production of cytopathic from non-cytopathic virus including genomic mutations, duplications and insertions are discussed, as well as the fact that cytopathic viruses cannot establish infection in the foetus.

Moennig, V et al. 1990

Moennig, V., et al., Reproduction of mucosal disease with cytopathogenic bovine viral diarrhoea virus selected in vitro. Vet Rec, 1990. 127(8): p. 200-3.

This study confirms the concept that pairs of antigenically closely-related cytopathic and non-cytopathic strains of BVDV are required for the production of fatal mucosal disease in cattle. Nine PI calves were inoculated with closely-related cytopathic strains, two were inoculated with closely related non-cytopathic strains, and two were inoculated with antigenically different cytopathic strains. Cytopathic viruses were selected using monoclonal antibodies to the envelope glycoprotein gp53. Superinfection with the closely-related cytopathic strains caused all nine animals to succumb to mucosal disease, and no neutralizing antibodies were produced. No signs of disease were recorded in the other animals, and those infected with unrelated cytopathic strains produced antibodies to the virus. In this study the non-cytopathic isolates in PI calves displayed almost identical reaction patterns, suggesting the presence of a single, uniform virus in the herd. This has severe implications as a mutation event could occur in one animal, and cause the superinfection of in-contact animals.

Brownlie, Clarke et al. 1984

Brownlie, J., M. C. Clarke, et al. (1984). "Experimental production of fatal mucosal disease in cattle." Vet Rec 114(22): 535-6.

In the space of two pages this pivotal paper begins to elucidate the complex aetiology of mucosal disease in cattle. Mucosal disease has been recognized since 1953, and an association with bovine viral diarrhea virus had been suspected, but the mechanism behind its onset had remained unknown. This paper was the first to fulfill Koch’s Postulates with regards to the causation of mucosal disease by reproducing mucosal disease in cattle and hypothesizing about the trigger for its onset, including the essential role of virus biotype. Brownlie et al showed experimentally that mucosal disease occurs when a calf persistently infected in utero with a non-cytopathic strain of BVDV becomes superinfected with a cytopathic strain of BVDV. Clinical appearance and post mortem examination, as well as the consistent finding of both cytopathic and non-cytopathic viruses in blood samples from cattle affected by mucosal disease were evidence for this. This has serious implications for herds with many persistently-infected calves, where a devastating outbreak of mucosal disease could occur.

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