Bovine Viral Diarrhoea Virus

Description

Across the globe, cattle of all ages are affected by bovine viral diarrhoea virus (BVDV). The virus gives rise to productive and reproductive losses, causing significant economic impact

Bovine Viral Diarrhoea Virus

Classification

Over 60 years ago, the viral aetiology of the disease bovine viral diarrhoea was established. In the 1960s-70s, bovine viral diarrhoea virus (BVDV), together with related agents causing disease in sheep and swine, was assigned to the newly-penned “Pestivirus” genus. At this stage, Pestiviruses were considered to be non-arthropod-borne togaviruses, although it was later realised by sequencing of genomic RNA they are more taxonomically suited to the family Flaviviridae (Collett et al, 1988; Meyers et al, 1989), where they reside today.

The Flaviviridae are primarily spread via arthropod vectors, particularly mosquitoes and ticks. Genera within the family include the Flaviviruses, which cause disease in both man (such as yellow fever virus and West Nile virus) and animals (for example, louping ill), and Hepacivirus, which contains Hepatitis C virus only.

Pestiviruses, however, are not arthropod-borne, and include pathogens of cattle (BVDV), sheep and pigs. The porcine Pestivirus, classical swine fever virus (CSFV), was first documented in 1833 in Ohio (quoted by Hanson, 1957). Acute disease (classical swine fever) typically includes a raised body temperature, inco-ordination of movement and hyperaemia of the skin followed by petechial or extensive haemorrhage (Dahle and Leiss, 1992).

Border disease, caused by Border disease virus (BDV) is the ovine pestivirus and was described by Hughes et al. in 1959. Congenital infection results in the birth of “hairy shaker” lambs, which suffer tonic-clonic tremors and have hairy rather than woolly coats (Sawyer, 1992). Severity of clinical signs can vary within a flock and among litter-mates.

Virus Structure

The BVDV genome comprises a single strand of positive sense RNA. By analysing the sequence of BVDV cDNA cloned into plasmid vectors, the genome has been found to be around 12.3Kb long (Donis, 1995). In infected cells, this is the only viral RNA present- no molecules of a subgenomic size are found. Figure 1.1 shows a diagrammatic interpretation of the BVDV genome. Genomic information is read in a single, large open reading frame (ORF) that commences at nucleotide 386 of the RNA. The ORF is 3898 codons long, and contains no non-coding sequences. BVDV polyprotein is translated directly from the ORF, and mature viral proteins arise from this following endoproteinase cleavages by both viral and cellular mechanisms (Dubovi, 1990; Donis, 1995).

The 5’ and 3’ untranslated regions (UTRs) are located at either end of the ORF. Unlike eukaryotic mRNA, BVDV genomic RNA has neither the 5’ methylated cap nor the 3’ poly-A tail at these positions. Instead, the UTRs are significantly long, at approximately 385 and 226 nucleotides for the 5’ and 3’ UTRs respectively (Donis, 1995). This allows them to accommodate a variety of functions normally conferred by the 5’ cap and the poly-A region, including the control of translation initiation and RNA stability. Entry of replicases required to produce viral progeny from the RNA template is also guided by the UTRs.

Figure 1.1 also shows the proteins encoded by the BVDV genome. The polyprotein produced is 3898 amino acids long, and has a molecular weight of 438kD (Donis, 1995). The genomic RNA codes both structural and non-structural proteins.

The first structural protein coded is Npro, which has a protease action that generates the N-terminus of the next protein, C. C packages genomic RNA and provides interactions necessary for formation of the enveloped virion. Erns, E1 and E2 are all glycoproteins. Erns has an RNase activity that is probably involved in viral replication and pathogenesis (van Gennip, 2005). E1 is membrane-anchored and initiates the translocation of E2 to the virion envelope where it is highly antigenic, causing the production of neutralising antibodies following infection or vaccination (Donis, 1995).

The next protein is P7. Although P7 function is uncertain, studies have suggested that the C-terminal part acts as a signal sequence required for correct processing and membrane translocation of NS2 (Tautz et. al, 1999).

NS2-3 is the first non-structural protein translated. Sequence similarities are shown by NS2-3 to a region that in other flaviviruses is split into two distinct polypeptides, NS2 and NS3 (Donis, 1995). NS2-3’s N-terminus is homologous to NS2, and its C-terminus to NS3. After the first 6 hours of infection, NS3 is found exclusively in cytopathic biotypes expressed as a separate polypeptide, making it a marker of cytopathic BVDV (Donis, 1995). NS2 is also expressed as a discrete polypeptide in some, but not all, cytopathic isolates.

The non-structural proteins play important roles in genome replication. The action of a serine protease domain within NS2-3 releases NS4A, NS4B, NS5A and NS5B (Harada et. al, 2000). NS4A goes on to act as a cofactor for the serine protease (Harada et. al, 2000), but knowledge of the role of NS4B is limited. NS5B possesses an RNA-dependent RNA polymerase activity (Zhong et. al, 1998). In the related Hepatitis C virus NS5B is an essential part of the replication complex (Brass et. al, 2006), and the function may be similar in BVDV.

The structural proteins and the genomic material created by the actions of the non-stuctural proteins come together to form the 40-60nm BVDV virion. A central core of RNA packaged in the capsid protein (C), which is surrounded by a lipid membrane. The glycoproteins E1 and E2 are anchored within the membrane, and Erns is loosely associated. Since naked BVDV RNA is infectious (Dubovi, 1990; Donis, 1995), it is clear that virions do not contain RNA replication proteins. Instead, virion proteins and lipids “capture” the viral genome from the host cell cytoplasm and deliver it to that of uninfected cells. Enzymes for the production of new RNA from the genomic RNA template are provided by the infected host cell.

Virus Genotypes

There are two recognised genotypes of BVDV; type 1 and type 2. These differ antigenically (Paton et al., 1995), although classification based on sequence variation is more precise. Analysis of the 5’UTR has divided BVDV1 into 11 subtypes and BVDV2 into 3 subtypes (Vilcek et al., 2001). Despite large intra-genotype antigenic differences, type 1 vaccines give some degree of cross-protection against disease caused by type 2 viruses (Carman et al., 1998; Cortese et al., 1998; van Oirschot et al., 1999).

A split in virulence between genotypes is evident. BVDV1 species, including classical strains and common vaccine strains, tend to cause milder disease (Deregt, 2004) and are found worldwide. Alternatively, BVDV2 isolates typically cause more severe disease, characterised by fever, diarrhoea, thrombocytopaenia, haemorrhage, respiratory signs, and high abortion and mortality rates (Corapi et al., 1989; Carman et al, 1998). These viruses were first reported in Canada and the USA, although their distribution is widening. The first British case of type 2 BVDV was identified in 2002 (Drew et al., 2002).

However, genotype is not always an accurate indicator of virulence. Some type 2 strains cause only subclinical or mild disease (Ahn et. al, 2005), and the spectrum of type 1 disease is also broad.

Virus Biotypes

Within both BVDV genotypes, isolates can be classified as one of two biotypes. Noncytopathic (ncp) viruses produce no visible cytopathic effect in cell cultures, and infected cells appear normal (Figure 1.2a). Conversely, cytopathic (cp) viruses cause cell vacuolation and death (Figure 1.2b) within 24-48 hours post-infection. Cytopathogenicity gives no indication of disease-causing potential.Noncytopathic BVDV is responsible for the majority of acute and persistent BVDV infections worldwide. Cytopathic biotypes are usually found in cases of the fatal BVD-associated condition, mucosal disease, and are always isolated alongside noncytopathic strains.

Cytopathic viruses have been shown to originate from noncytopathic strains by several mechanisms of mutation. These include insertions of cellular origin, such as ubiquitin sequences, and viral gene rearrangements, duplications and deletions (Deregt and Loewen, 1995). Accumulation of point mutations in the NS2 region and various RNA recombination events are also important (Tautz et al., 1994).

Serologically, the two BVDV biotypes are indistinguishable, but on a molecular level cytopathic viruses produce an additional protein, NS3, not found in cells infected with noncytopathic virus (Donis and Dubovi 1987; Pocock et al., 1987; Magar et al. 1998). This marker of cytopathic viruses is a smaller version of the larger structural protein, NS2-3, expressed in noncytopathic isolates. The mutational generation of cytopathic strains gives a cleavage site in NS2-3, resulting in the independent expression of NS3 as well as the larger protein in these strains (Meyers and Thiel, 1996).

Figure 2 summarises the classification of BVDV down to the biotype level. For each biotype, there are many virus strains all with varying virulence and distribution.

Transmission and Epidemiology

Epidemiology

• A major concern is that it can be confused with FMD (especially as it often occurs with clinical signs of salivation and depression)
• Virus is widespread: 60-70% exposure by 4 years of age
  • Often may sweep through a whole colony of young stock causing profuse diarrhoea (perhaps febrile) for a few days and then recover
  • Due to primary exposure to cytopathic strain of virus
• PI cows:
  • 100% vertical transmission to offspring
  • Are infected with BVDV-1nc and NEVER BVDV-1c
  • Are often antibody-negative (though they can show low levels of Ab to heterologous virus)
  • Show a wide range of clinical signs:
    • Severe congenital damage (ataxia)
    • Poor body condition
    • Increased susceptibility to enteric and respiratory disease
  • Act as the herd reservoir of BVDV
  • Can ONLY be identified by blood testing
• Transfer via semen, direct contact with acutely infected animals, or vertical from dam to offspring
• Transfer can be iatrogenic: repeated use of needles and gloves, etc.

Pathogenesis

BVDV-1c

• Infects cattle regardless of age
• Usually mild: diarrhoea with recovery in 10 dyas
• Immunosuppression can lead to secondary infection

BVDV-2nc

• Transient thrombocytopenia and leukopenia over 2 weeks
• Hemorrhages
• Secondary infection
• Death

BVDV-1nc

• Transplacental infection of naive heifers
• Outcome depends on age of fetus at contraction
  • 0-110 days: abortion or persistently infected (PI) calves born
  • 110-220 days: congenital damage with noticeable CNS and musculoskeletal lesions
  • 220 days to term: active immunity developed

Mucosal Disease

• Mucosal disease is caused by a superinfection of PI animals with a second homologous cytopathic biotype (eg BVDV-1nc followed by BVDV-1c)
• Infection typically occurs between 6-18 months of age but is variable
• Superinfection will quickly spread horizontally among PI animals
• Invariable fatal
• Characterized by oral and enteric erosions, particularly overlying Peyer's patches, and ulceration of the feet
• Animals can show anorexia, depression and/or diarrhoea for 2-5 days before death
• Vaccination can lead to iatrogenic infection in undiagnosed PI calves

Diagnosis

Laboratory Tests

• Traditional test: virus isolation followed by serology on infected cells
• ELISA for virus antigen in animals with persistent viremia (will show up 3-8 days post-infection)
• PI calves often appear virus negative as a result of receiving neutralizing Ab in colostrum: can be countered by RT-PCR
• Paired serum samples from cows with acute BVDV
• Herd sampling by ELISA for antibody on bulk milk

Pathology

Small erosions of MDV/BVDV - vesicles are microscopic (Courtesy of Alun Williams (RVC))

Coalescing lesions of BVDV (Courtesy of Alun Williams (RVC))

• Mucosal Disease: erosive condition produces small multiple, cleanly punched out lesion in mouth
• Neutrophils invade the ulcer and if bacterial colonisation occurs, further excavation follows. Either:

1. This lesion develops a granular base and becomes diphtheritic.
2. If bacterial colonisation does not take place, healing occurs within fourteen days.

• Seen in most parts of mouth (or maybe on muzzle) e.g. dental pad, cheeks, sides of tongue
• Lesions extend throughout gut with particularly big ulcers in small intestine over Peyers patches. Necrosis occurs in lymph nodes and spleen

• No vesicular stage, prickle cells die off from surface resulting in layer of necrotic debris over epithelial layer
• Infection penetrates inward through stratum germinativum.
• Epithelium does not recover as animal does not recover

Treatment and Control

• No known treatment to reverse persistent infection or to cure mucosal disease
• BUT, without exposure to BVDV, the whole herd is at risk as there is no developed immunity
• Vaccination of dams before pregnancy will prevent PI calves being born
  • Beta-propiolactone inactivated vaccine
  • Combine with screening for antigen and removal of PI animals