Download Bovine viral diarrhoea virus: virulence factors and improved control

DEPARTMENT for ENVIRONMENT, FOOD and RURAL AFFAIRS
Research and Development
CSG 15
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
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to [email protected]
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
Project start date
£ 272,640
01/04/00
Project end date
31/03/03
Executive summary (maximum 2 sides A4)
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 invitro 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 invitro 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
CSG 15 (Rev. 6/02)
1
Project
title
Bovine viral diarrhoea virus: Virulence factors and improved
control
DEFRA
project code
OD0342
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)
2
Project
title
Bovine viral diarrhoea virus: Virulence factors and improved
control
DEFRA
project code
OD0342
Scientific report (maximum 20 sides A4)
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 BVDV2 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 diseasefree 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,
CSG 15 (Rev. 6/02)
3
Project
title
Bovine viral diarrhoea virus: Virulence factors and improved
control
DEFRA
project code
OD0342
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.
Regarding BVDV and specific virulence factors, it is interesting to note that no other scientific reports beyond
the one by Topliff and Kelling (1998) have emerged since this project was commissioned, nor has the putative
virulence marker reported by these two authors been verified by other groups. The same applies to classical
swine fever virus, for which projects to locate virulence markers have been running since 1995-6, without any
significant publications emerging.
Objective 2 - Genetic and antigenic monitoring of UK and foreign BVDV strains
As regards UK BVDVs, this activity has continued according to demand. BVD viruses associated with cases of
acute (clinically recognizable) BVD have been of primary interest. Antigenic and genetic typing has not
revealed other than subtype 1a BVDVs from such cases. In a study to validate a TaqMan based RT-PCR for
detection of ruminant pestiviruses, a BVD type 2 virus was identified among a set of blood samples diagnosed
as BVDV positive by antigen ELISA (Drew et al, 2002). This was the first detection of BVDV-2 in cattle in the
UK (previously, some reports indicated BVDV-2 - then "atypical pestiviruses" - had been isolated from UK
sheep during the mid 80-ies; but more recent scrutiny of those virus isolates (Vilcek and Paton, 2000) have
shown they were in vitro-derived contaminants). Clinical records in the herd where the virus came from, as well
as observations in experimental animals inoculated with the isolate showed that it was a low-virulence isolate.
Despite being vaccinated against BVDV, differential neutralisation tests on sera from the animals on the farm
showed that the majority had undergone acute infection with the BVDV type-2 virus, indicating a significant
ability to spread in even vaccinated UK cattle. Follow-up investigations of contact animals resulted in detection
of a heifer persistently infected with the BVDV-2, but no further evidence of spread of the infection was found.
Continued surveillance of foreign BVDVs (and to a lesser extent ovine pestiviruses) have encompassed
approximately 150 isolates from Austria, France, Hungary, Italy, Norway, Slovenia, Slovakia, Spain, Turkey
(inactivated virus isolate), United Arab Emirates (inactivated virus isolates) and Yugoslavia. This has included
mostly collaborative studies with colleagues from overseas, some as visiting scientists to the VLA, and to a
minor extent the VLAs role as an OIE reference laboratory for BVD. The main result of this genetic typing
shows that continental European BVDVs are genetically more diverse than UK isolates. (Vilcek et al, 2001;
Toplak et al, 2002, Sandvik et al 2002, Petrovic et al, in preparation). Whilst more than 95% of BVDVs from
the UK and Ireland have been shown to be of genetic subtype 1a; genotypes 1b, 1d, 1e and 1f appear to
dominate in continental Europe, with 1a, 1c, 1g, 1h, 1i and type 2 as minority genotypes. A tendency towards
regional distribution of genotypes was seen, supporting previous observations that the major factor for spread
on BVDV is trade in live animals. The over-all genetic divergence between UK and continental European
BVDVs indicate that we by genetic typing have a fair chance of identifying BVDVs imported with cattle from
continental Europe.
CSG 15 (1/00)
4
Project
title
Bovine viral diarrhoea virus: Virulence factors and improved
control
DEFRA
project code
OD0342
Selected viruses from each genetic subtype were typed antigenically using a panel of Mabs raised against
several BVDV 1a’s. Although the relative reactivity against heterologous genetic subtypes was slightly lower
than for subtype 1a viruses, over-all a significant cross reactivity was seen for all BVDV type 1 subtypes, as
opposed to BVDV-2. This in-vitro typing suggests that immunity acquired against one BVDV type 1 strain
should cover against infection with other BVDV-1 genetic subtypes, but not against BVDV-2.
Objective 3 - Recrudescence of BVDV in animals recovered from acute infection
In an experiment where viral replication and shedding was monitored in calves undergoing experimental acute
infection with (a virulent) UK BVDV strain, seven calves which had recovered were injected with
dexamethasone (DMS) over a five-day period to mimic immunological stress. Reactivation of the virus was
monitored for by isolation from blood and nasal swabs, by RT-PCR as well as for viraemia/seroconversion of
susceptible sentinel animals. After the DMS treatment, no virus was isolated from any of the recovered calves
for periods up to 32 days, and similarly none of the commingling sentinel animals became infected with BVDV.
By RT-PCR, BVDV RNA could still be detected in whole blood of all seven calves up to and beyond the DMS
treatment period, but the frequency of detection did not increase after DMS treatment. The conclusion was that
no reactivation of BVDV could be seen in the experimental animals. These results were presented at the 5th
International Congress of Veterinary Virology (Sandvik et al., 2000), and a paper on the findings has been
submitted for publication (Paton et al.). The lack of experimental horizontal transfer from animals recovered
from acute BVDV even after immunological suppression verifies earlier field observations. Although it cannot
be extrapolated that viral reactivation and subsequent vertical transfer never occurs in pregnant animals either,
the findings suggest that it may not be a major risk associated with breeding antibody positive cows. This is
also in accordance with field observations.
Objective 4 – Development of methods for earlier detection of BVDV in foetuses and newborn calves
This Objective required induction of persistent infection in calves – i.e. infecting pregnant animals susceptible
to BVDV during the first trimester. Initial delays due to accommodation capacity limitations were subsequently
prolonged by animal movement restrictions imposed during the FMD outbreak. As soon as the latter were
lifted, four heifers were brought on site at Weybridge, and inoculated with BVDV when pregnancy could be
verified. They were bled regularly throughout the pregnancy, and gave in September 2002 birth to four calves
persistently infected with either of two strains of BVDV, of low and high virulence, respectively. Samples were
regularly taken from the calves until at three months of age, and tested for BVDV by virus isolation, antigen
ELISA, RT-PCR and by immunohistochemistry (IHC). The time when BVDV specific maternal antibodies
ceased to interfere with BVDV antigen (NS2-3) detection by ELISA varied significantly between the calves, the
first remained negative after ingestion of colostrum until 7 weeks old whilst the last still remained negative
when sampled 14 weeks old. A different antigen ELISA targeting the Erns viral protein was less affected by
maternal antibodies than the NS2-3 ELISA, in detecting viral antigen in all calves from 1-3 weeks of age
onwards. Virus isolation from serum and nasal secretions gave positive results on precolostral samples, but
subsequently negative for between 1-5 weeks before virus again could be isolated. The RT-PCR was not
affected by presence of maternal antibodies – all blood samples were positive for BVDV. This test is thus the
only test for BVDV that reliably can detect BVDV in blood samples from calves less than 3-4 months old.
No apparent correlation between the virulence of the infecting virus strain and detectability in the various
diagnostic tests could be seen. On the other hand, a significant calf-to calf variation in the duration of BVDV
specific maternal antibodies was seen. This probably reflects the design of the study, where the calves were
given 1.5 litres of colostrum and then allowed to suckle ad libitum, to mimic natural conditions as much as
possible. These results have been summarised and accepted for oral presentation at the 6'th ESVV
Conference of Veterinary Virology in France, August 2003. A manuscript is also under preparation.
Sequential skin samples were stained for BVDV by IHC; all samples were likewise positive for BVDV antigen
in dermal epithelial and hair follicle cells, although the pattern of staining was slightly different in samples taken
on the day of birth and a few weeks onwards. This should not prevent an experienced diagnostician in
recognising persistent infection with BVDV in skin biopsies.
References, presentations & submitted manuscripts
CSG 15 (1/00)
5
Project
title
Bovine viral diarrhoea virus: Virulence factors and improved
control
DEFRA
project code
OD0342
Collen, T., Douglas, A. J., Paton, D. J., Zhang, G. and Morrison, W. I. (2000) Single amino acid differences are
sufficient for CD4(+) T-cell recognition of a heterologous virus by cattle persistently infected with bovine
viral diarrhea virus. Virology 276; 70-82.
Drew, T. W. and Paton, D. 2000. Acute bovine viral diarrhoea in calves: Haematological parameters of calves
infected with a virulent strain of virus. Brocci & Lavazza (Eds.) Proceedings of the 5th International
Congress of Veterinary Virology, Brescia, 27-30 August 2000. ESVV/Istituto Zooprofilattico Sperimentale
della Lombardia e dell’Emilia Romagna, pp 381-2.
Drew TW, Sandvik T, Wakeley P, Jones T, Howard P. (2002) BVD virus genotype 2 detected in British cattle.
Vet Rec, 151; 551.
Fray, M. D., Paton, D. J. and Alenius, S. (2000) The effects of bovine viral diarrhoea virus on cattle
reproduction in relation to disease control. Anim Reprod Sci 60; 615-627.
Harasawa, R., Giangaspero, M., Ibata, G. and Paton, D. J. (2000) Giraffe strain of pestivirus: Its taxonomic
status based on the 5 '-untranslated region. Microbiol Immunol 44; 915-921.
Paton DJ, Bensaude E, Drew TW, Sandvik T, Davis L, Turner T, Brownlie B. Effect of viral dose and
immunosuppression on persistence and transmission of bovine viral diarrhoea virus. Manuscript, Vet
Microbiol.
Petrović Т, Duričić B, Тoplak I, Lazić S, Barlič Maganja D, Hostnik P, Grom Ј, Sandvik Т. Isolation and
comparative genotyping of recent Serbian and Slovenian bovine viral diarrhoea viruses. Manuscript, Acta
Veterinaria.
Rossmanith W, Vilcek S, Wenzl H, Rossmanith E, Loitsch A, Durkovic B, Strojny L, Paton DJ. 2001. Improved
antigen and nucleic acid detection in a bovine virus diarrhoea eradication program. Vet Microbiol 81; 207218.
Sandvik T, Drew TW, Bensaude E, Davis L, Turner J, Brownlie J, Paton DJ. (2000) Acute BVD in calves effect of viral dose and immunosuppression on clinical signs of disease, transmission to susceptible
animals and virus persistence in tissues. In: Brocchi E and Lavazza A (Eds in chief) Proceedings of the 5th
International Congress of Veterinary Virology, 27-30 Aug. 2000, Brescia. Published on behalf of the ESVV
by Istituto Zooprofilattico Sperimentale e Zootecniche, Brescia, Italy, 401-402.
Sandvik T, Larsen I-L, Nyberg O. 2001. Influence of milk from cows persistently infected with BVD virus on
bulk milk antibody levels. Vet Rec 148; 82-84.
Sandvik T, Davis L, Ibata G. (2002) Genetic typing of pestiviruses from Norwegian cattle and sheep. Poster,
5th ESVV Pestivirus Symposium, Cambridge, UK 26-29 Aug. 2002.
Sandvik T, Moore S, Turner J, Baker M, Ibata G, Drew T. 2003 Diagnosis of persistent infection with bovine
viral diarrhoea virus in the neonate or young calf. Abstract, Proceedings of the 6th ESVV International
Congress of Veterinary Virology, Saint Malo, 24-27 August 2003.
Toplak I, Sandvik T, Barlič-Maganja D, Grom J, Paton DJ. (2002) Genetic typing of bovine viral diarrhoea
virus: most Slovenian isolates are of genotypes 1d and 1f. Manuscript, Vet Microbiol.
Topliff CL and Kelling CL. (1998) Virulence Markers in the 5'-Untranslated Region of Genotype 2 Bovine Viral
Diarrhea Virus Isolates. Virology 250; 164-72.
Vilcek, S., Greiser-Wilke, I., Nettleton, P. and Paton, D. J. (2000) Cellular insertions in the NS2-3 genome
region of cytopathic bovine viral diarrhoea virus (BVDV) isolates. Vet Microbiol 77; 129-136.
Vilcek, S. and Paton, D. J. (2000) A RT-PCR assay for the rapid recognition of border disease virus. Vet Res
31; 437-445.
Vilček Š, Paton DJ, Durkovic B, Strojny L, Ibata G, Moussa A, Loitsch A, Rossmanith W, Vega S, Scicluna MT,
Palfi V, 2001. Bovine viral diarrhoea virus genotype 1 can be separated into at least eleven genetic groups.
Arch. Virol. 146; 99-115.
CSG 15 (1/00)
6