A 45-Nucleotide Insertion in the NS2 Gene is Responsible for the Cytopathogenicity of a Bovine Viral Diarrhoea Virus Strain

Abstract

Cytopathogenicity (cp) markers have recently been investigated in the genomes of field isolates of bovine viral diarrhoea virus (BVDV). Most of the isolates originated from mucosal disease (MD) cases observed after vaccination with a live attenuated vaccine, termed here BVDV-X. The NS2-3 genes of these isolates and of the vaccine proved to be identical, including a 45-nucleotide (nt) viral insertion at nt position 4355. The insertion originated from the NS4B/5A junction region of the BVDV genome. Interestingly, in BVDV strain CP7 a 27-nt insertion originating from the NS2 is located exactly at the same position. Complete genome analysis of BVDV-X did not reveal further potential cp markers. Furthermore, expression studies indicated that the insertion promotes NS2-3 cleavage. In order to examine the possible role of the 45-nt insertion in viral cytopathogenicity in details, a full-length infectious cDNA clone of BVDV-X was generated, and bovine turbinate (BT) cells were transfected with RNA transcribed from the clone. The recovered virus, termed BVDV-XR, showed slight retardation in growth in comparison with the original BVDV-X, and induced cytopathogenic effect (CPE). Since the natural non-cytopathogenic (ncp) counterpart of the vaccine virus was not available, an insertion-negative mutant cDNA clone was generated from BVDV-XR by PCR-directed mutagenesis. The recovered virus, termed BVDV-XR-INS-, showed the same growth characteristics as its cp counterpart BVDV-XR, but caused no CPE. These findings provide a direct proof that the 45-nt insertion at position 4355 has a basic role in the cytopathogenic character of this BVDV strain.

Complete Genome Sequences of Both Biotypes of a Virus Pair of Bovine Viral Diarrhea Virus Subgenotype 1k

“…Thus, an insertion of cellular origin (14), or 27-nt and 45-nt insertions of viral sequences of the NS2 and the NS4B-NS5B junction regions were found in the BVDV-1b strain Cp7 and in several vaccine-derived viruses, respectively (14, 15). In accordance with these reports (15, 16), replacing the entire NS2 region of SuwaNcp with the corresponding region of SuwaCp yields a cytopathic virus. However, whether the insertion alone or only in combination with the point mutations in NS2 is responsible for the cp phenotype remains to be determined.…”

A novel NS3/4A protease dependent cleavage site within pestiviral NS2

“…It is worth noting that this cleavage site is located right downstream of the nine aa insertion of the CP7 (). Interestingly, previous studies have identified insertions of a similar size in several other cp isolates at highly similar NS2 positions, one of which was shown to have comparable effects in terms of NS2-3 cleavage and cytopathogenicity as the CP7 insertion [45, 46]. Remarkably, we did not detect any obvious effect of the nine aa insertion on the efficiency of the internal NS2 cleavage in our experiments (), despite the fact that the CP7 insertion is located directly upstream of the cleavage site in NS2 recognized by the NS3/4A protease.…”

“…3a). Interestingly, previous studies have identified insertions of a similar size in several other cp isolates at highly similar NS2 positions, one of which was shown to have comparable effects in terms of NS2-3 cleavage and cytopathogenicity as the CP7 insertion [45,46]. Remarkably, we did not detect any obvious effect of the nine aa insertion on the efficiency of the internal NS2 cleavage in our experiments (Fig.…”

“…The cleavage site is conserved between pestivirus species A, B and D all infecting ruminant hosts. Further cp BVDV strains with insertions in close proximity to the internal NS2 cleavage site as in strain CP7 [46] and possibly others also maintain this cleavage. Thus, an attractive hypothesis derived from these results would be that internal processing of NS2 plays an important role in the complex biology of the infection of ruminant hosts.…”

Herpetiform Genital Lesions in a Heifer with Mucosal Disease

“…18 Another possibility is that the PI animal was superinfected, most likely with a homologous antigenically related CP BVDV strain 5, 6, 20 or even a heterologous strain. 2, 17 There is no possibility that the superinfecting virus could have been vaccine-derived, 1, 16 because the only vaccine used in this herd was inactivated.…”