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United States Department of Agriculture

Agricultural Research Service

Research Project: COUNTERMEASURES TO CONTROL AND SUPPORT ERADICATION OF BOVINE VIRAL DIARRHEA VIRUS (BVDV) Title: Genetic and antigenic characterisation of Bungowannah virus, a novel pestivirus causing myocarditis in pigs

Authors
item Kirkland, Peter - MACARTHUR AGRICULTURAL
item Frost, M - MACARTHUR AGRICULTURAL
item Finlaison, D - MACARTHUR AGRICULTURAL
item Cook, R - MACARTHUR AGRICULTURAL
item Srivastava, M - MACARTHUR AGRICULTURAL
item King, K - MACARTHUR AGRICULTURAL
item Ridpath, Julia
item Gu, X - MACARTHUR AGRICULTURAL

Submitted to: European Society for Veterinary Virology
Publication Type: Proceedings
Publication Acceptance Date: September 16, 2008
Publication Date: September 16, 2008
Citation: Kirkland, P.D., Frost, M.J., Finlaison, D.S., Cook, R., Srivastava, M., King, K.R., Ridpath, J.F., Gu, X. 2008. Genetic and Antigenic Characterisation of Bungowannah Virus, a Novel Pestivirus Causing Myocarditis in Pigs. In: 7th International Pestivirus Symposium, European Society of Veterinary Virology, September 16-19, 2008, Uppsala, Sweden. p. 44.

Technical Abstract: In June 2003 a syndrome of sudden death in sucker pigs, an elevation in the proportion of stillborn foetuses, increased preweaning losses and to a lesser extent increased mummification rates was recognised on a property in NSW, Australia [1]. This disease has been described as the porcine myocarditis syndrome (PMC). Intensive studies involving the in utero inoculation of foetuses combined with novel molecular methods led to the discovery of a new pestivirus that has been proposed as new species within the pestivirus genus [2]. A range of studies in pigs that aim to reproduce the lesions of PMC, investigate the pathogenesis of the disease and accumulate evidence that this novel pestivirus is the causative agent of PMC are described in a companion presentation [3]. The aim of this paper is to describe a series of studies that have been undertaken to investigate the genetic and antigenic characteristics of Bungowannah virus. Methods: As attempts at isolating a virus had been unsuccessful, a modification of a technique used for detection of novel viruses in serum [4] was utilised. Sequence independent single primer amplification (SISPA) was undertaken on nucleic acid extracted from a pool of foetal serum. PCR primers matching terminal sequences of nucleic acid fragments detected by SISPA were used to amplify intervening RNA segments. The resulting products were cloned, sequenced and the results compared with data in Genbank. Subsequently, PCR using 'primer walking' was used to determine the entire viral genome. The characteristics of the genome of this new pestivirus were subsequently compared with published data for representative viruses from other pestivirus species. Virus isolation was attempted using PK-15A cell cultures and viral replication was detected initially by PCR. Later, after optimising cell fixation, viral antigens were detected in cell culture by immunoperoxidase (IPX) staining using convalescent pig serum. Antigenic studies, involving both peroxidase linked assays and virus neutralisation, were undertaken with a collection of polyclonal antisera raised against a range of representative pestiviruses. Pan-reactive monoclonal antibodies to pestiviruses were also used in IPX assays. Results: Initially RNA segments including parts of the 5'UTR, N**pro and E2 coding regions of a pestivirus were detected. There was also limited sequence from the p7, E**rns, NS5A and NS5B regions of the genome. Later, the intermediate sequences were identified to complete an entire pestivirus genome with a total length of 12.659 Kb. Genetic analyses have shown a low degree of similarity with the currently recognised pestiviruses. Further, the construction of dendrograms show that this novel virus is genetically distinct from all of the established pestiviruses species (BVDV-1, BVDV-2, BDV and CSFV) and with other viruses that have been proposed as possible new species ('pestivirus of giraffe', HoBi and the pronghorn antelope virus). A number of features of the genome have been identified, some of which are shared by other pestiviruses and some appear to be unique. Viral replication was demonstrated in PK-15A cells, initially by PCR and later by IPX using a convalescent pig serum. IPX staining revealed cytoplasmic accumulations of viral antigens, consistent with a pestivirus. No staining was observed with any of the pan-reactive monoclonal antibodies. There was no cross-neutralisation detected when PMC convalescent pig serum was tested in virus neutralisation assays using reference strains of BVDV, BDV and CSFV, nor was there any evidence of neutralisation of Bungowannah virus by antisera to strains of BVDV1, BVDV2, BDV, CSFV, HoBi or pronghorn viruses. Limited cross reactivity with Bungowanah virus antigens was observed in peroxidase linked assays with some very high titred antisera to BVD2 and CSF viruses. Discussion and Conclusions: The genetic data available convincingly identify this as a unique pestivirus which is antigenically very different from other pestiviruses. It does not react with pan-reactive anti-pestivirus monoclonal antibodies. Further, it cannot be amplified with current 'pan reactive' PCR primers. Together, these features allow this virus to escape detection by current diagnostic assays. Collectively the results of the genetic and antigenic studies would suggest that this virus is the most divergent of the pestiviruses that have been recognised and warrants classification as a new species in the genus. As well as identifying the origins and geographic distribution of this virus, it will be important to determine whether this novel pestivirus plays a role in other disease syndromes. References: [1] McOrist, et al. (2004). Aust. Vet. J. 82, 509-11. [2] Kirkland et al., (2007) Virus Res 129, 26-34. [3] Finlaison et al. (2008) This conference. [4] Allander et al. (2001) PNAS 98, 11609-11614.

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