Molecular characterisation of bovine viral diarrhoea virus with special regard to cytopathogenicity

Abstract

Bovine viral diarrhoea virus is a major pathogen of cattle that causes significant economic losses worldwide in the cattle industry. Based on the ability to cause cytopathic effect in cell cultures, BVDV strains can be classified as cytopathogenic or non-cytopathogenic biotypes. The cp biotypes are generated in the persistently infected cattle by genomic alterations of the ncp biotype. Cytopathogenicity of BVDV has been shown to correlate with the presence of insertions of cellular sequences, duplication of viral genomic regions with or without insertions, deletions and point mutations in the genomes of cp strains. These genetic alterations are termed cytopathogenicity markers. In most cp BVDV strains the role of these cp markers is well defined, but in some cases further genetic and functional analyses are needed to elucidate the role of these genomic alterations. Since the Hungarian cp BVDV isolates have not been characterised so far, in the first study the possible cytopathogenicity markers in the genomes of six “archive” cp BVDV strains isolated in the 1970s have been examined. At that time a live BVDV vaccine (termed here for ethical reasons BVDV-X) was introduced and widely used in Central Europe until the beginning of the 1990s. The viruses were selected as representatives of various forms of BVDV infections: enteritis and mucosal disease presumably associated with the use of the live attenuated vaccine, as well as respiratory syndrome. The complete NS2-3 coding region of the six isolates and the vaccine virus were amplified by RT-PCR and were sequenced. The results showed that new cp markers were found in all cp BVDV strains at nucleotide position 4355 in the NS2 gene. These cp markers resemble to a very rare cp marker that is present in only reference strain BVDV CP7. The NS2-3 region of four isolates originated from the vaccination accidents and of the BVDV-X vaccine virus was identical proving that the vaccine caused early onset of mucosal disease. The cp marker proved to be a 45-nucleotide viral insertion that encodes 15 amino acids of the NS4B/NS5A junction region in a normal BVDV genome. In respiratory isolate H3887, a 21-nucleotide insertion of non-viral origin was found, which also located at nucleotide position 4355. The insertion had high similarity with a gene coding for murine interferon-induced guanylate-binding protein 1, and represented the first non-viral insertion identified at this position of the NS2 coding region. Respiratory isolate H3142 contained a 42-nucleotide viral duplication at close proximity of nucleotide position 4355. The insertion was identical to a part of the NS5B gene. This isolate also had a deletion of three nucleotides approximately 90 nucleotides downstream of the insertion. The genome rearrangements found in these isolates occurred preferentially at position 4355, suggesting that this part of the genome could represent a potential hot spot for recombination events in ncp BVDV, and may be termed position C. In the second study, recently isolated cp BVDV isolates were characterised. The use of BVDV-X vaccine contributed to the “positive effect” of causing MD in PI animals, reducing the natural source of BVDV. This may explain the phenomenon that cp BVDV strains were not isolated for nearly 30 years. Two cp BVDV strains have been recently isolated from MD cases in Hungary. The strains were examined for cytopathogenicity markers to check whether the newly found genomic alterations show any common feature with those of the recent cp BVDV isolates. In the genome of strain H4956, a jiv-like insertion was found similar to those described in reference strain NADL and in other BVDV 1, BVDV 2 and BDV strains. The jiv-like nucleotide sequence coding 133 amino acids was inserted at nucleotide position 4984, nine nucleotides upstream of that of strain NADL. The insertion showed 96% amino acid sequence identity with the cellular Jiv protein. In the genome of cp BVDV strain H115/PCR, an ubiquitin-containing duplication was found. The duplicated sequence started at nucleotide position 7978 in the NS4B gene. The duplication contained a complete ubiquitin monomer of 76 amino acids and the complete NS3 gene. The duplication located further downstream of the known ubiquitin-containing genomic regions of cp BVDV strains. The insertions and duplication of the recently isolated two cp BVDV strains further confirmed that recombinations occurring at positions A and B are the most common mechanisms leading to the development of BVDV cytopathogenicity. In the third study, complete genomic analysis of the BVDV-X vasccine was carried out. The BVDV-X vaccine was marketed many years ago (and not any more) as a derivate of the Oregon C24V strain. However, sequencing the whole NS2-3 region of the BVDV-X vaccine and of the cp BVDV strains originated from MD cases suggested recombinations between the vaccine and wild type variants of BVDV during the vaccine production. The analysed nucleotide sequences seemed to be distinct from BVDV Oregon C24V, therefore the genome of a pre-registration (termed here BVDV-Xpre) and of a marketed (BVDV-X) batches of the vaccine was analysed. Results of the complete genome analysis of BVDV-Xpre confirmed that the original virus strain used at the start of the vaccine production was Oregon C24V. Surprisingly, analysis of the complete nucleotide sequence of the BVDV-X marketed vaccine revealed that this strain belongs to the BVDV 1b subgroup, with a 93.7% nucleotide sequence identity with BVDV reference strain Osloss. The identity to BVDV Oregon C24V was significantly lower (77.4%), and a thorough sequence scanning showed that the genome of BVDV-X had not derived from Oregon C24V. These data indicate the very likely scenario that a strain different from Oregon C24V was picked up during the in vitro or in vivo passages for vaccine development. Despite the virus-switch, the BVDV-X vaccine continuously maintained its innocuity and efficacy, as proven by the regular quality testing data, and the presence of the alien virus remained unnoticed over many years. The results of this work emphasize that the contamination of commercially available live vaccines with exogenous BVDV strains is a real risk factor, and an unequivocal analysis, including molecular methods is needed to verify their authenticity. In the further two studies, the role of the cytopathogenicity marker found in the genome of BVDV-X was examined. Since the complete molecular analysis showed that other possible factors contributing to the cytopathogenicity of BVDV-X are not present in the genome, in the first step the role of the 45-nucleotide insertion in the expression of NS3 was investigated in the case of the vaccine virus. The whole NS2-3 gene of this virus and a PCR-directed mutagenesis-generated insertion-negative variant were cloned in pCI mammalian expression vector, and were expressed in BT cells. Western blot analysis revealed that the insertion contributed to a partial cleavage of NS2-3 generating NS3, the marker protein of cytopathogenicity. In addition, it was also demonstrated that the NS4B/NS5A junction of the insertion that is cleaved in the BVDV polyprotein is not processed in this case. In order to further examine the possible role of the 45-nucleotide insertion in the cytopathogenicity of BVDV-X, in the final step, a full-length infectious cDNA clone of the BVDV-X vaccine was generated. The recovered virus, BVDV-XR showed slight retardation in growth in comparison with the wild-type BVDV-X, but was appropriate for further reverse genetic studies. Since the natural ncp counterpart of the vaccine virus was not available, an ncp mutant was generated by PCR-directed mutagenesis. The recovered virus, BVDV-XR-INS- also showed the same growth characteristics as its cp counterpart, and caused no CPE. This observation gave a final proof that the insertion is indispensable in the cytopathogenicity of BVDV-X. In summary, these studies provide novel information on the biology of BVDV from aspects of virus recombination, which has an important impact, both on basic and applied research of veterinary virology.