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

Initially, BVDV replicates in the nasal mucosa and tonsil to high titres. After spreading to regional lymph nodes, the virus disseminates throughout the body reaching highest concentrations in the tonsil, thymus and ileum. Leucocytes are also infected (Bruschke et al., 1998). BVDV can infect cells of the bone marrow (Spagnuolo et al., 1997), and intestinal mucosa. Lymphoid tissue of the Peyer’s patches and thymus is often depleted.

BVDV infection can result in a range of clinical diseases, from subclinical infections to the highly fatal mucosal disease. While inter-genotype virulence differences are partially responsible for the variations in clinical manifestation, host factors are also important. Immuno-competence or immunotolerance, pregnancy status, gestational age of foetus, passively versus active immunity and levels of environmental stress may all contribute to the severity of disease.

1.2.1 Infection of the Immunocompetent, Non-Pregnant, Seronegative Animal

BVDV is generally considered a mild disease in immunocompetent cattle; it has been estimated that 70% to 90% of BVDV infections occur without clinical signs (Ames, 1986). If closely observed, sub-clinically infected cattle may show a small increase in body temperature, mild leucopaenia, and agalactia (Perdrizet et. al, 1987; Moerman et. al, 1994).

Clinical disease is known as BVD. This tends to affect animals 6-12 months of age with high morbidity, although fatality is uncommon (Baker, 1995). An incubation period of 5-7 days is followed by pyrexia and leucopaenia. Viraemia is apparent from 4-5 days post-infection, and may continue until day 15 (Duffell and Harkness, 1985). BVD no longer seems to present as herd outbreaks of diarrhoea (Brownlie, 1985). Although diarrhoea does sometimes occur, clinical findings more commonly include depression, anorexia, occulo-nasal discharge, decreased milk production and, occasionally, oral lesions (Baker, 1995). A rapid respiratory rate resembling pneumonia may also be observed (Perdrizet et. al, 1987).

Acutely infected non-pregnant animals shed low concentrations of virus compared to persistently infected cattle (Duffell and Harkness, 1985). Animals produce antibodies to BVDV 2 to 4 weeks after infection (Baker, 1995), which persist for life.

“Severe BVD” also exists, seen in the UK in 1992-1993 (Hibberd and Turkington, 1993), and in herd outbreaks between 1993 and 1995 in Ontario (Carman et. al, 1998). Infected animals showed acute onset of diarrhoea, fever and decreased milk production, sometimes proving fatal. Non-cytopathic, type 2 viruses were implicated in these cases, raising the issue of the degree of cross protection afforded by type 1 vaccines. However, severe disease was only seen in cattle where vaccine manufacturers’ instructions had not been followed, implying protection is usually given.

BVDV2 infection may also result in haemorrhagic syndrome (HS), reported in both North America (Perdrizet et. al, 1987; Rebhun et.al, 1989) and Europe. This is characterised by significant thrombocytopaenia, giving rise to bloody diarrhoea, petechial haemorrhages of mucous membranes and epistaxis (Rebhun et. al, 1989). Fever and leucopaenia are also seen.

1.2.2 Infection of the Immunocompetent, Pregnant, Seronegative Animal

Immunocompetent, pregnant cattle show the same responses to BVDV infection as non-pregnant animals. However, BVDV has a high potential to cross the placenta and infect the developing foetus, meaning additional outcomes of infection may occur in the calf. The main factor influencing the virus’s effects on the foetus is the gestational age at the time of transplacental infection.

Infection at the time of insemination may result in reduced conception rates, and that shortly after increases loss of embryos (Carlsson et. al, 1989; McGowan et. al, 1993). Foetal infection in the first trimester (50-100 days) can cause death, although expulsion of the foetus may not occur until several months later.

Figure 1.4: (from Brownlie et al., 2000) A calf stillborn due to transplacental BVDV infection.

Transplacental infection between days 100 and 150 may result in congenital defects. At this stage the immune system is in the final phase of development, and mounts an inappropriate inflammatory response to BVDV to cause these effects (Duffell and Harkness, 1985). Growth defects in organs such as the thymus, and central nervous system pathologies such as cerebellar hypoplasia, often arise (Brownlie, 1985). Calves with cerebellar hypoplasia are ataxic, reluctant to stand and may suffer tremors (Baker, 1995). Infection at this point may also cause visual problems, including blindness and cataracts. Virus may localise to the vascular endothelium, causing vasculitis and associated inflammation, oedema, hypoxia and cellular degeneration (Brownlie, 2000). Weak, stunted calves may also be produced.

Infection in the third trimester trimester (over 180-200 days) elicits a response from the fully-developed immune system, giving rise to normal but seropositive calves.

1.2.3 Persistent Infection- Immunotolerant Animals.

Infection of the foetus with non-cytopathic virus before 120 days gestation may result in the birth of immunotolerant and persistently infected (PI) calves. The immune system, although competent, recognises the antigen as “self” rather than “foreign” and no response is mounted. The calf therefore develops a tolerant state to the virus which persists into neonatal life. Although no antibodies are produced against the original, transplacental-infecting strain, heterologous BVDV strains can elicit a response in PI cattle. Therefore, these may prove seropositive if tested (Bolin, 1985).

While they may appear clinically healthy, PI animals continuously shed large amounts of virus throughout their lives, providing a major source of infectious virus for naïve cattle. (Houe, 1999). PI dams produce PI calves, resulting in PI family lines which maintain the virus in a herd (Baker, 1995). 1-2% of the cattle population are PI (Houe, 1999), rising to 13% in foetal calves.

PI cattle are predisposed to other diseases, and have a reduced survival rate (Houe, 1993) with 50% dying within their first year (Duffell and Harkness, 1985). This increased susceptibility may be due to BVDV-associated immunosupression, considered in section 1.2.5. Animals may be undersized and slow-growing, and persistent infection is the prerequisite for mucosal disease.

1.2.4 Mucosal Disease

Mucosal disease (MD) primarily affects 6-18 month-old cattle and is invariably fatal (Brownlie et. al, 2000). Baker (1995) summarises the characterising symptoms, which last several days to weeks. These include pyrexia, depression and weakness. Anorexia gives emaciation and dehydration. Foul-smelling, sometimes bloody, watery diarrhoea develops 2-3 days after the onset of disease. Animals are often euthanised for humane reasons. 

As suggested by the name, lesions develop on mucosal surfaces including the oral mucosa, tongue, external nares and the buccal and nasal cavities (Brownlie, 1985). Coalition of lesions gives larger areas of necrosis (Baker 1995), leading to excessive salivation, lacrimation, and ocular discharge. The coronet and interdigital surface are also affected, causing the animal to become disinclined to walk and eventually recumbent (Brownlie, 1985). Lesions of the abomasum and small intestine are seen on post-mortem examination, and congestion of the large intestine mucosa results in a stripy, thickened appearance (Brownlie, 1985). Figure 1.5 shows examples of tongue and small intestine lesions.

MD occurs when animals persistently infected with noncytopathic BVDV are superinfected with an antigenically similar cytopathic strain. Cytopathic virus arises from the persistent noncytopathic virus by mutation (see 1.1.4), and may then be transmitted to cause MD in animals PI with the same noncytopathic strain. Immunotolerance induced by the noncytopathic strain prevents superinfecting virus being recognised by the immune system; the biotypes are “homologous” to the immunotolerance (Brownlie, 1990). “Heterologous” superinfection with a non-related cytopathic biotype causes an antibody response and mucosal disease does not usually occur.

Figure 1.5: (From Brownlie, 1985) a) Tongue of a calf suffering mucosal disease. Complete loss of the epithelium has occurred at the apex. b) Lesion of the small intestine due to MD. These may appear chronic, and have food adhering to the surface.

1.2.5 Immunosupression in Mixed Infections

BVDV-induced leucopaenia reduces the defences available against invading pathogens, enhancing the pathogenicity of co-infecting organisms. BVDV can therefore be considered an immunosuppressive agent.

BVDV-associated immunosupression has a particularly important role in bovine respiratory disease, with an association between BVDV antibody titre and respiratory disease treatment being demonstrated (Martin and Bohac, 1986). BVDV is the virus most frequently isolated from pneumonic lungs, often found in association with Pasteurella haemolytica (described by Baker, 1995). This pathogen combination causes severe fibrino-purulent bronchopneumonia, with the area of pneumonic lesions increasing by 35-60% compared to that caused by Pasteurella infection alone (Brownlie, 1985). Synergism is also displayed with parainfluenza, bovine rhino-tracheitis and respiratory syncitial viruses.

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