Sequence A: gtgcaactgc aggtggcggc attattgtga tgggttcgtt Sequence B: gtgttgcacg gatgaaatat gtagctgtgg atgatttacc

Introduction

Bovine viral diarrhea virus (BVDV) is a small enveloped RNA virus, and a member of the genus Pestivirus belonging to the family Flaviviridae which also includes hog cholera virus of swine and Border disease virus (BDV) of sheep (Nettleton and Entrican, 1995; Moennig and Plagemann, 1992). The single-stranded positive sense RNA genome is about 12.5 kb in length and contains one large open reading frame preceded by about 380 base untranslated region (UTR) (Collett et al., 1988). The 5' UTR is highly conserved among pestivirus species and has been found useful for differentiating virus members (Boye et al., 1991; Harpin et al., 1995)

Sequence A. Materials and methods

1.1. Virus isolation and cells

Bovine fetal muscular (BFM) cell cultures were prepared as described by Shimizu and Satou (1987) and used for virus isolation and serum neutralization (SN) test. The cells were grown in Eagle's minimum essential medium containing 10% goat serum determined to be free of BVDV and neutralizing antibody to BVDV, 10% tryptose phosphate broth and 0.015% sodium bicarbonate. NCP BVDV was assessed based on the capacity to inhibit the propagation of the CP BVDV strain in cell cultures (Gillespie et al., 1962). The new field isolates and Japanese reference strains, Nose (CP) (Kodama et al., 1974), T-20 (CP) (Hashiguchi et al., 1978), No. 12 (NCP) (Omori et al., 1967), KS86-1-NCP (Shimizu and Satou, 1987) and KS86-1-CP (Shimizu et al., 1989a and Shimizu et al., 1989b), were propagated in BFM cells and cloned by limiting dilution prior to use.

1.2. Antisera

Antisera against KZ-91-CP and OY-89 were prepared with sheep by the intravenous injection of 2 ml of each virus suspension containing approximately 105.5 TCID50/ml of virus. Sera were obtained at 4 weeks after inoculation and stored at -20°C and heat-inactivated at 56°C for ...