J Bacteriol Virol.  2002 Mar;32(1):63-72.

Isolation and Complete Nucleotide Analysis of a Noncytopathic BVDV Variant using a Dominant Selective Marker

• Lee YM

Abstract

Upon infection, bovine viral diarrhea virus (BVDV), one of the pestiviruses in the Flaviviridae family, is divided into two biotypes, cytopathic (cp) and noncytopathic (ncp). The mechanism of cytopathogenicity, however, is not elucidated yet. In this study, we have investigated viral genetic element affecting the cytopathogenicity of BVDV by using an infectious cDNA molecular clone containing a dominant selective marker, puromycin acetyltransferase gene (pNADL/pac). From the recombinant cDNA clone pNADL/pac, viral RNA was synthesized by T7 RNA polymerase in vitro. By selecting the MDBK cells transfected with in vitro transcribed cp NADL/pac viral RNA with puromycin, we obtained the selected MDBK cells harboring cp NADL/pac viral RNA, which did not show cytopathic effect and could be passaged. Little cytopathic effect was observed upon infection of naive MDBK cells with the viral particles released from the selected cp NADL/pac-transfected MDBK cells. Complete nucleotide analysis of viral particles released from cp NADL/pac-selected MDBK cells revealed 5 substitutions in the viral open reading frame, but not in the 5' and 3'NTRs. Interestingly, only two point mutations (G8147A and T11343C) changed their amino acid code (M2588V in NS4B and T2653M in NSSB), and the other 3 substitutions (C1627T, T3097C, and T3376C) was silent. Therefore, our results suggest that NS4B and NSSB proteins of BVDV may play a role in cytopathogenicity.

Keyword

Bovine viral diarrhea virus; Cytopathogenicity; Puromycin acetyltransferase; Pestiviruses

MeSH Terms

Clone Cells
Diarrhea
DNA, Complementary
DNA-Directed RNA Polymerases
Flaviviridae
Humans
Open Reading Frames
Pestivirus
Point Mutation
Puromycin
RNA, Viral
Virion
DNA, Complementary
DNA-Directed RNA Polymerases
Puromycin
RNA, Viral