Identification of the bovine atypical pestivirus in biological samples

Atypical cattle pestivirus (BVDV3; HoBi-like) is an unclassified candidate for the genus Pestivirus of the Flaviviridae family. The agent was isolated for the first time in 2004 from fetal bovine serum from Brazil, and shows a high degree of similarity with the BVDV1 and BVDV2. Its presence in the cattle populations can potentially reduce the effectiveness of control programs of bovine viral diarrhea. The article presents the results of developing a method for identifying of BVDV3 in the biological samples based on polymerase chain reaction. Synthetic oligonucleotide primers complementary to positions 9202-9218 and 9501-9521 of the reference strain D32/00_ 'HoBi' genome were selected. The basic parameters of the reaction have been worked out. The sensitivity of PCR was 7.4½10-1 copies/μl. It has high specificity and does not reveal RNA of the BVDV1, BVDV2 and classical swine fever virus. With the help of the developed PCR 18 samples of fetal bovine serum (FBS) from various sources, 11 types of continuous cell culture lines used for virus cultivation in several Russian research institutes, 10 attenuated live vaccines, 189 internal organs, 1383 blood sera from cattle, 168 blood sera from reindeer, and 63 blood samples from red deers were investigated. The virus was revealed only in seven lots of FBS obtained from two manufacturers and produced in South America. Phylogenetic analysis of amplicons showed all positive lots grouped with BVDV-3 strain D32/00 _'HoBi (Brazilian group). Given the potential risk of using contaminated fetal serum, further research of the spread of BVDV3 in Russia is needed. The virus was not found in continuous cell culture lines, vaccines used for human, cattle and small domestic animals. Additionally, no evidence has been found out of virus circulation among cattle of various breeds, including those imported from another countries, reindeers and red deers in the Krasnodar territory, Siberia and the Republic of Kazakhstan. The presence of the virus in the FBS used in the production of vaccines does not exclude its spread in Russia. The findings confirm the need for continuous updating and improvement of methods for diagnosing pestiviruses and tightening the rules for the international FBS trading.