title / Bovine viral diarrhoea virus: Virulence factors and improved control
/ DEFRA
project code / OD0342
Department for Environment, Food and Rural Affairs CSG 15
Research and Development
Final Project Report
(Not to be used for LINK projects)
Two hard copies of this form should be returned to:Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
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Project title / Bovine viral diarrhoea virus: Virulence factors and improved control
DEFRA project code / OD0342
Contractor organisation and location / Veterinary Laboratories Agency,
New Haw, ADDLESTONE
Surrey KT15 3NB
Total DEFRA project costs / £ 272,640
Project start date / 01/04/00 / Project end date / 31/03/03
Executive summary (maximum 2 sides A4)
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CSG 15 (Rev. 6/02) 3
Projecttitle / Bovine viral diarrhoea virus: Virulence factors and improved control
/ DEFRA
project code / OD0342
This project addressed selected aspects of the pathogenesis of BVD, and of BVDV diversity, which are relevant to work aiming at control of BVD. During the period this project was running two major viral diseases of livestock occurred in the UK, Classical Swine Fever in 2000, and Foot and Mouth Disease in 2001. Due to inevitable delays, most of the objectives could be rescheduled and carried out as planned.
Four different objectives were covered by this research project. The first focused on development of an in-vitro assay for virulence of bovine viral diarrhoea virus (BVDV). To serve as a "viral backbone" in which candidate genes determining virulence can be studied, cDNA copies of the viral RNA genome was prepared. In vitro propagation stability problems precluded use of the C24V reference strain as a genomic cDNA copy, but an infectious clone of the NADL reference strain was obtained, and has been used to rescue native or in-vitro modified infectious BVDV virions. The clinical recognition of cases of acute BVD caused by virulent BVDV strains often include haemorrhages, which can be correlated to a marked drop in the number of circulating platelets. To investigate whether this is caused by a direct effect of BVDV on platelet precursor cells, studies were carried out on stem cells harvested from the bone marrow of new-born calves. These were propagated in vitro, and parallel mock- and BVDV-inoculated cells were then labelled with monoclonal antibodies. Little evidence of infection with BVDV was seen in the inoculated cell cultures. However, the stability of the bone marrow cell cultures was not optimal, and it cannot be excluded that this might have influenced the outcome of the propagation of and staining for BVDV. A refined cell culture model would be needed to conclude on the suitability of this approach.
The second objective covered genetic and antigenic monitoring of UK and foreign BVDV strains. Recent surveys of UK ruminant pestiviruses have shown that in cattle BVDV type 1a predominates (95%), with a genogroup 1b/1i minority. In sheep, both BVDV-1a as well as border disease virus have been found; recent genetic typing of viruses previously classified antigenically as BVDV-2 has shown they most likely were cell culture contaminants. In contrast, BVDV obtained from continental European cattle (central Europe) are much more heterogeneous, altogether 11 genetic subtypes of BVDV-1 have been found, of which subtype 1a is a minority group. Additionally BVDV-2 is found regularly in some continental European countries, e.g. Belgium and Germany. The genetic homogeneity of UK BVDVs is shared with Ireland, and a similar pattern of genetically similar BVDVs (subtype 1b) has also been found in other countries with historically very limited international trade in live cattle (Norway). Monoclonal antibody typing of selected European BVDV-1 subtypes isolates has not shown specific patterns of antigenic differences, indicating that immunity to one is protective against infection with other BVDV type 1 viruses. Continued screening of viruses isolated from clinically “severe” cases of BVD in the UK has not shown other BVDV subtypes than the most commonly found 1a. Conversely, a BVDV-2 isolate was detected in a persistently infected bull born on a farm where embryo transfer had been used extensively. Although this BVDV-2 virus differed antigenically from the type 1 isolates which are most common in UK cattle, it was readily detected by the routine diagnostic assays in use by the VLA. Follow-up studies indicated that the type-2 BVDV had infected many animals on the original farm, but tracing of in-contact animals did not show spread beyond those coming from the original farm.
Reactivation of infectious BVDV in animals recovered from infection with BVDV is a potential threat to all BVD control programmes. To check for recrudescence of BVDV after acute infection of immunocompetent animals, immunological stress was simulated by dexamethason injections in 7 calves recovered from BVD. Excretion of infectious BVDV was monitored by virus culture of clinical samples, and by co-mingling susceptible sentinels. Despite continuous detection of viral RNA by RT-PCR before, throughout and beyond the immunological stress period of the recovered animals, no evidence of horizontal shedding of BVD was found. This supports previous observations of acute infection with BVDV as an evolutionary dead end for this virus.
The last ROAME objective was to develop alternative approaches to BVDV diagnosis in two important categories of animals, which usually fail to be detected by conventional diagnostic tests. These are foetuses and neonatal calves persistently infected with BVDV – the former primarily by their inaccessibility for sampling, and the latter since uptake of colostrum containing antibodies to BVDV interfere with detection of the virus until the calves are 2-3 months old. To obtain suitable samples, persistent infection with BVDV was induced in four calves by infecting pregnant heifers with noncytopathogenic strains of BVDV during early pregnancy. The animals were sampled regularly, and tested for BVDV using both conventional and alternative diagnostic tests. As expected, BVDV antigen ELISAs which have proven reliable for testing of older animals failed to diagnose the viraemia in three of the persistently infected calves until they were 7, 11 and 13 weeks old, whilst one calf remained negative even when last sampled at 13 weeks old. Conventional virus isolation in cell cultures gave similar inconsistent results. In contrast, a TaqMan RT-PCR assay gave continuously positive results, as did skin biopsies examined by immunohistochemistry. An interesting finding was that an antigen ELISA targeting a structural viral protein gave positive test results from between days 1 and 22 onwards; suggesting a differing humoral immune response against different viral antigens presented to the immune system of the dams pregnant with persistently infected foetuses. These results enable us to restructure diagnostic approaches to detect persistent infection in neonatal calves, which have remained difficult to identify and thereby posing a threat to the success of many BVD control programmes.
CSG 15 (Rev. 6/02) 3
Projecttitle / Bovine viral diarrhoea virus: Virulence factors and improved control
/ DEFRA
project code / OD0342
Scientific report (maximum 20 sides A4)
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CSG 15 (Rev. 6/02) 3
Projecttitle / Bovine viral diarrhoea virus: Virulence factors and improved control
/ DEFRA
project code / OD0342
Bovine viral diarrhoea virus (BVDV) causes a diverse disease syndrome (BVD) in cattle, with notably variable clinical signs of infection. In most countries with intensive cattle rearing the BVD prevalence is high, and thus considered to be a major loss-inducing endemic viral disease of cattle. Based on antigenic and genetic criteria, the causative virus has been classified as two distinct species (types 1 and 2). Within both species two different biotypes occurs (noncytopathogenic and cytopathogenic); and strains of both low and high virulence have been identified in either previously mentioned groups. Despite detailed studies of several virulent BVDV-2 isolates from North America, markers of virulence in BVDV have yet to be identified.
Together, these findings have indicated that BVDVs, or ruminant pestiviruses may be more diverse than previously assumed. Since the BVD prevalence in the continental European countries is comparable to that of the UK (between 60-90%), continued mapping of European virus isolates will provide an improved picture of BVDVs most likely to be introduced to the UK cattle population by live animal trade.
In Scandinavian countries, BVD control programmes have during the last decade lowered the prevalence to between 5 and 25%. The success of these control programmes has primarily depended on efficient use of diagnostic assays for surveillance and identification of persistently infected (PI) animals, but also on assumptions regarding modes of natural transfer of the virus in affected herds. Studies in immunocompetent animals has shown that after acute infection a lasting immune response develops, but the efficacy of this immunity in preventing BVDV recrudescence has not been examined. In large herds where PI animals have been removed, viral reactivation and of shedding from previously acutely infected animals could be a pathway towards new cycles of transplacental infection and birth of new PI animals.
Despite development of accurate and cost-efficient diagnostic tests for identification of PI animals, some categories of animals have remained beyond diagnostic reach. These are PI foetuses, or more accurately dams carrying PI foetuses after acute infection with BVDV during the first trimester, and young PI calves born to acutely infected dams. In case of pregnant animals, the unavailability of the foetus for sampling is the main problem. For young calves PI with BVDV, maternal antibodies are known to interfere with the detection of the virus up to at least three months of age. Novel approaches to BVDV diagnosis of these categories of animals are needed to supplement the tests successfully used for older animals.
Four main objectives were specified for this project. Development of in-vitro methods to study the basis of virulence would allow more accurate and rapid verification of suspected virulent BVDV strains. To estimate the risk foreign BVDV strains pose to the UK national herd through trade in livestock, we would seek more comprehensive data on the diversity of BVDV strains in primarily other European countries. The potential continued spread of BVDV in herds being cleared for PI animals would be looked into by investigating whether BVDV may recrudesce in animals having recovered from acute infection. Lastly, we would investigate novel approaches to BVDV diagnosis in pregnant cattle and in neonatal calves, to minimise the risk of not identifying BVDV viraemic individuals in herds being cleared for BVDV.
The project was running from April 2000 until March 2003, a period during which the UK saw two outbreaks of exotic viral diseases of livestock, CSF in 2000, and FMD in 2001. During both epidemics, VLA Virology Department staff designated for this project was engaged in diagnostic laboratory work to regain the disease-free status of the UK. Additionally, large animal movement restrictions in force during the FMD epidemic prevented transportation of experimental animals to the VLA site, thereby delaying work involving use of experimental animals. Despite these obstacles, rescheduling of planned activities made it possible to continue with most of the work objectives, allowing us to conclude the majority of the planned work.
Objective 1 - BVDV infectious clone / cell culture system for in vitro virulence monitoring
In a previous project the genome of a reference strain of BVDV widely used at the VLA (C24V Oregon) was sequenced, and cloned DNA fragments of the genome were available for assembly into a plasmid-born DNA copy of the full genome, from which infectious viral RNA can be copied – i.e. an infectious clone. The assembly of the C24V fragments continued during this project. However it became clear that sequence errors in these fragments made them unsuitable for digestion with restriction enzymes needed to join them together. It also turned out that the in vivo-stability of the plasmids carrying the C24V DNA copy fragments was poor, resulting in loss rather than propagation of ligated full length DNA copies of the viral genome. Instead of repeating the sequencing of the available clones (or generating new ones which might be more stable), we decided to make use of a DNA clone made on basis of the BVDV NADL reference strain, and made available to us by an American group. Both these reference strains originated in the USA, are cytopathogenic, and of the 1a subtype – i.e. the same genotype as the majority of UK field virus strains. The basis of cytopathogenicity of the two BVDV strains is different, in that for the NADL there is a 270 nt genomic insertion in the NS2-3 gene, whilst in the C24V several point mutations in the same region confers cytopathogenicity. The deletion of the NADL insertion is a simpler way to reconstruct a noncytopathogenic viral backbone for subsequent cell culture studies than reverting several point mutations individually.
The proposed in vitro-system for monitoring of virulence factors was based on platelet precursor cells – megacaryocytes. Such cells were harvested from the bone marrow of newborn calves, and maintained as primary cell cultures. However, stable cell lines with characteristics seen for megacaryocytes could not be established. Thus, primary cell cultures were inoculated either with a virulent strain of BVDV (or mock inoculated), and were monitored for replication of BVDV using a BVDV-specific monoclonal antibody (Mab) in a flow-cytometric assay. No evidence of infection with BVDV was seen in any cell population in these primary cultures. Whilst further refinement of the outlined approaches was considered, along with the alternative strategy of monitoring for arrest of maturation of progenitor cells into platelets by infection with virulent BVDVs, it should not be forgotten that asymptomatic persistent infections (after early foetal infection) with virulent strains of BVDV are fairly common. The latter fact suggests that disease signs seen during acute infection with virulent BVDV virus strains are "side effects" of the immune system clearing the infection. Thus, with the exception of specialised immune system effector cells, primary or established cell cultures which are known to replicate BVDV may not be suitable for assaying virulence properties of specific virus isolates.