Viral sequence insertions and a novel cellular insertion in the NS2 gene of cytopathic isolates of bovine viral diarrhea virus as potential cytopathogenicity markers.
Cytopathogenicity of bovine viral diarrhea virus (BVDV) has been shown to correlate with the presence of insertions of cellular sequences, duplication of viral sequences with or without insertions, deletions, and point mutations in the genomes of cytopathogenic (cp) strains. In the present study we have investigated cytopathogenicity markers in the genomes of six cp BVDV isolates. The viruses were selected as representatives of various forms of BVDV infection, in some cases presumably induced by vaccination with a live attenuated vaccine. The complete NS2-3 coding region of the six isolates and of the vaccine virus were amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced. In the genomes of four isolates (H6379, H6712, H8427 and H-BVD MD) and of the vaccine virus, a 45-nucleotide viral insertion was found at nucleotide position 4355, encompassing nucleotides 8402-8446, that encode 15 amino acids of the NS4B/NS5A junction region in a normal BVDV genome. Isolate H3887 had a 21-nucleotide insertion of non-viral origin, also located at nucleotide position 4355. This insertion has a high homology with a gene coding for murine interferon-induced guanylate-binding protein 1, and represents the first non-viral insertion identified at this position of the NS2 coding region. Isolate H3142 carries a 42-nucleotide insertion at position 4361, identical to a part of the NS5B gene mapping to position 11078-11119. Additionally, this isolate also has a deletion of three nucleotides (positions 4448-4450). The role of the 45-nucleotide insertion in expression of NS3 was investigated using the vaccine virus. The NS2-3 gene of this virus, and that of a generated insertion-negative variant were cloned in the mammalian expression vector pCI, and expressed in bovine turbinate cells. Western blot analysis revealed that the insertion contributed to a partial cleavage of NS2-3 generating NS3, the marker protein for cytopathogenicity in BVDV. 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. The molecular mechanism underlying this phenomenon, however, remains to be elucidated.
Bálint
Adám
A
Department of Virology, Central Veterinary Institute, Tábornok u. 2, H-1149, Budapest, Hungary.
Pálfi
Vilmos
V
Belák
Sándor
S
Baule
Claudia
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D003588:Cytopathogenic Effect, Viral / Q000235:genetics*; D018076:DNA, Complementary; D030241:Diarrhea Virus 1, Bovine Viral / Q000235:genetics* / Q000302:isolation & purification; D019204:GTP-Binding Proteins / Q000235:genetics; D015321:Gene Rearrangement; D005814:Genes, Viral; D005819:Genetic Markers; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics*; D014765:Viral Vaccines
D018076:DNA, Complementary; C490288:Gbp2b protein, mouse; D005819:Genetic Markers; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D014765:Viral Vaccines; C074956:p125 protein, bovine diarrhea virus; D019204:GTP-Binding Proteins
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