Bovine Viral Diarrhoea Virus

Description

Bovine viral diarrhoea is a viral disease that affects cattle worldwide. Caused by a pestivirus, it gives rise to significant economic losses in both dairy and beef cattle through its effects on production and reproduction. Bovine viral diarrhoea virus can lead to a variety of clinical outcomes that ranging from subclinical infections to the more severe presentations including abortion, infertility, and the fatal mucosal disease. The condition is highly immuno-suppressive and secondary respiratory and enteric complications often occur.

Bovine Viral Diarrhoea Virus

Classification

The viral aetiology of BVD was first established over 60 years ago, but it was not until the 1960s that the agent was assigned to the newly penned "Pestivirus" genus. At this stage Pestiviruses were considered to be non-arthropod-borne togaviruses; later, sequencing of genomic RNA showed that they are taxonomically better suited to the Flaviviridae family^{1, 2}. Many members of the Flaviviridae family are indeed arthropod-borne, such as the Flaviviruses West Nile Virus and yellow fever virus. However, Pestiviruses are not transmitted by insects, and the genus includes pathogens of cattle (BVDV), sheep (Border Disease virus) and pigs (Classical Swine Fever Virus).

Virus Structure

The BVDV genome comprises a single strand of positive sense RNA which is around 12.3 kilobases in length³. The genome is read in one 3898-codon open reading frame that contains no non-coding sequences. BVDV polyprotein is translated directly from the ORF and is cleaved by viral and cellular proteinases to form mature viral proteins^{3, 4}. At either end of the ORF, 5’ and 3’ untranslated regions exist. These regions are long, allowing them to accomodate fuctions conferred in eukaryotic DNA by the 5’ cap and the 3' poly-A tail, such as controlling the initiation of translation, facilitating the entry of replicases, and contributing to RNA stability⁴. BVDV's RNA genome encodes both structural and non-structural proteins. These include Npro, whose protease action generates the N-terminus of the protein C. C is the capsid protein that packages genomic RNA and assists in the formation of the eventual enveloped virion. Erns, E1 and E2 are all glycoproteins, with Erns possessing RNase activity involved in viral replication and pathogenesis⁵. E1 is membrane-anchored and initiates the translocation of the antigenic protein E2 to the envelope³. P7 has an uncertain function⁶. NS2-3 is the first non-structural protein to be translated. Sequence similarities are shown by NS2-3 to a region that in other Flaviviridae is split into two distinct polypeptides, NS2 and NS3. In BVDV, NS2 and NS3 can be expressed as separate polypeptides: NS3 is found exclusively in cytopathic isolates from 6 hours post-infection, making it a marker of this biotype³. NS2 is also expressed as a discrete polypeptide in some cytopathic isolates. NS2-3, along with the other non-structural proteins, plays an important role in genome replication. A serine protease domain within NS2-3 functions to release NS4A, NS4B, NS5A and NS5B⁷. NS4A is a cofactor for the serine protease⁷, and NS5B possesses an RNA-dependent RNA polymerase activity⁸. Knowledge of the role of NS4B is limited.

Newly formed genomic material is packaged by structural proteins to create the BVDV virion which is 40-60nm in diameter. The capsid is surrounged by a membrous envelope, in which the glycoproteins E1 and E2 are anchored. Naked BVDV RNA is infectious^{3, 4}, and so it can be deduced that the virions do not contain enzymes necessary for RNA replication: these are provided by the host cell.

Virus Genotypes

There are two antigenically distinct⁹ genotypes of BVDV, type 1 and type 2, which are most accurately characterised based on sequence variation. BVDV-1 and BVDV-2 contain 11 and 3 subtypes respectively, which have been demonstrated by analysis of the 5’UTR¹⁰. Despite the large antigenic differences between the genotypes, cross-protection against type 2 viruses is afforded by type 1 vaccines^{11, 12, 13}.

In general, the genotypes differ in virulence. BVDV-1 species are found worldwide and tend to cause milder disease ¹⁴, whereas BVDV-2 isolates typically cause more severe disease which is often haemorrhagic and is associated with a high mortality rate^{11, 15}. Type 2 viruses were first reported in Canada and the USA and have a more limited distribution than type 1 isolates. The relationship between genotype and virulence is, however, not fixed: some type 2 strains cause mild or subclinical disease¹⁶, and the spectrum disease caused by type 1 viruses is broad.

Virus Biotypes

BVDV isolates from either genotype can be of a cytopathic or non-cytopathic biotype. Non-cytopathic (ncp) viruses produce no visible effects in cell culture, whereas infection with cytopathic (cp) viruses gives cell vacuolation and death. Although non-cytopathic isolates are responsible for the majority of BVDV infections worldwide, cytopathogenicity gives no indication of disease-causing potential. Cytopathic biotypes of bovine viral diarrhoea virus are always isolated alongside non-cytopathic strains, and are found in cases of mucosal disease, a fatal BVD-associated condition.

Cytopathic viruses originate from non-cytopathic strains by mutation, including viral gene rearrangements, duplications and deletions¹⁷, and insertions of cellular origin, such as ubiquitin sequences. Point mutations in the NS2 region and various RNA recombination events are also important¹⁸. 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 non-cytopathic virus^{19, 20, 21}. This marker molecule arises from when NS2-3, expressed in non-cytopathic isolates, is cleaved at a site created by the mutations above²².

Transmission and Epidemiology

In most countries, BVDV is endemic and studies detecting antibody have estimated that between 70 and 100% of herds are either currently infected or have recently been infected with bovine viral diarrhoea virus²³.

BVDV can be transmitted from infected to susceptible cattle in several ways. Firstly, direct contact with a virus-shedding animal can cause disease. Both acutely and persistently infected animals shed virus, but levels of shedding are much higher in persistently infected cattle and transmission is more efficient. Transmission to heifers and cows may also occur venereally or via artificial insemination as acutely and persistently unfected bulls sheed bovine viral diarrhoea virus in their semen²⁴. The testes is an immunoprivileged site, and the virus can persist in this location despite otherwise systemic clearance²⁵. Indirect spread is possible: BVDV has been shown to spread through the re-use of needles, nose tongs²⁶ and rectal gloves²⁷, and blood feeding flies also give transmission.

Cattle that are persistently infected with noncytopathic BVDV serve as a natural reservoir for virus. Persistent infection develops when noncytopathic BVDV is transmitted transplacentally during the first 4 mo of fetal development. The calf is born infected with virus, remains infected for life, and usually is immunotolerant to the resident noncytopathic virus. Transplacental infection that occurs later in gestation results in abortion, congenital malformations, or birth of normal calves that have antibody against BVDV. The prevalence of persistent infection varies among countries and between regions within a country. In some areas, the prevalence of persistent infection in calves may be as high as 1-2% of cattle <1 yr of age. On a given farm, persistently infected cattle are often found in cohorts of animals that are approximately the same age. Persistently infected cattle can shed large amounts of BVDV in their secretions and excretions and readily transmit virus to susceptible herdmates. Clinical disease and reproductive failure often are seen after healthy cattle come in contact with a persistently infected animal. Biting insects, fomites, semen, biologic products, and possibly wild ruminants also can spread BVDV.

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.

Diagnosis

Although the clinical appearance may suggestive of BVD, disease presentation can vary widely and so laboratory testing is usually necessary.

Clinical Signs

Hibberd and Turkington, 1993 Carman et. al, 1998 The disease caused by bovine viral diarrhoea virus is known as bovine viral diarrhoea. It might be expected from this nomenclature that diarrhoea is a key clinical feature in BVDV infection, but disease can actually manifest in a variety of ways, ranging from subclinical disease to muscosal disease. Virulence factors related to genotype and strain are partially responsible for these variations, but host factors are also important. Pregnancy status, stage of gestation, immunity and the level of develoment of the foetal immune system all contribute to the outcome of BVDV infection.

Acute Infections: Non-Preganant Cattle

In the naive, non-pregnant, immunocompetent animal, BVD is normally mild: it is estimated that 70 to 90% of BVDV infections cause no clinical signs²⁸. If these subclinically affected cattle are observed closely, body temperature may marginally rise and mild leukopenia and agalactia may be seen ^{29, 30}. When clinical disease does occur in these animals, morbidity is high amongst cattle of 6-12 months of age. Following a 5-7 day incubation period, pyrexia and leukopenia is seen. Viraemia arises on days 4-5 days post-infection, and continues until around day 15³¹. Although some cattle suffer diarrhoea in BVDV infection, the disease no longer seems to present as herd outbreaks of diarrhoea³². Clinical signs more commonly include depression, anorexia, occulo-nasal discharge, decreased milk production and oral lesions³³, with a rapid respiratory rate resembling pneumonia sometimes apparent²⁹. Acutely infected, non-pregnant animals shed low concentrations of virus compared to persistently infected cattle³¹, and antibodies are produced 2-4 weeks post-infection which persist for life³³.

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.

Although BVDV infections in naive, non-pregnant animals are usually mild, outbreaks of a severe form of BVD have been known^{11, 34}. These were characterised by acute onset of diarrhoea, pyrexia and milk drop 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.

Acute Infections: Pregnant Animals

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.

Persistent Infections

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.

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.

Laboratory Tests

BVDV is diagnosed by the detection of virus or antibody in blood and milk samples. Antibody detection shows exposure of the herd to disease, whereas tests for antigen identify PI animals which may be antibody negative (Brownlie et. al, 2000).

Laboratory techniques for diagnosis of BVD virus Detection of antibody Serum neutralisation Serum neutralisation depends on the ability of antibodies in the serum to neutralise BVD virus and thereby prevent infection of cell culture. The test usually takes four to seven days to obtain a result but is dependent on cell culture facilities and an experienced observer. 201 202 ELISA An enzyme-linked immunosorbent assay (ELISA) technique for BVD virus antibodies that depends on binding of antibody to specific BVD virus antigen has been developed. The test takes one day. It requires purified ingredients but is simple to operate and the results can, if necessary, be recorded by eye. Detection of virus Cell culture BVD virus can be cultivated in cell culture monolayers (eg, calf testis or calf kidney). The cytopathic virus is identified by changes in the monolayers such as vacuolation of cell cytoplasm, rounding of cells and their subsequent lysis. Non-cytopathic virus produces no such changes. Both viruses can be visualised by fluorescein-coupled antibody (Fig 10). Primary identification of these viruses may be made in about seven days. Enzyme staining Virus grown on cell culture monolayers in microtitre assay plates or small petri dishes can be identified by enzyme-linked antibody. This assay may take only three to four days.

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

Once a herd’s status has been established, BVDV control measures can be implemented. Previously, PI animals have been used as natural “vaccinators” to increase herd immunity, although naïve animals must endure acute infection before this is achieved. Eradication by identification and culling of PI animals is possible, having been successfully accomplished in Scandinavia. However, this gives many seronegative, susceptible animals which is an imperfect solution for those units not completely biosecure or highly committed to the scheme. Killed and live vaccines afford a good level of protection providing they are used correctly and boosted regularly. A combination of eradication and vaccination gives the best level of control (Brownlie et. al, 2000), and the development of “marker” vaccines that allow natural- and vaccine-induced immunity to be distinguished will help this cause in future.

• No known treatment to reverse persistent infection or to cure mucosal disease

• • Beta-propiolactone inactivated vaccine
  • Combine with screening for antigen and removal of PI animals

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