Genetic Variability of Bovine Viral Diarrhea Virus in the 5 '-UTR in the Central Anatolia of Turkey

Abstract

Background: The genus Pestivirus in the family Flaviviridae comprises the members bovine viral diarrhea virus type 1 (BVDV-1), classical swine fever virus and border disease virus. The BVDV enveloped and the genome is a single-strand positive sense RNA molecule of approximately 12.3 kilobases in length. The genome is transcribed as a single open reading frame, flanked by 5' and 3' untranslated regions. Genetic typing of BVDV has usually been performed using sequences from the 5'-UTR, N-pro and E2 regions. BVDV is an RNA virus with a high genome variability having practical consequences on epidemiology, diagnosis and disease control. Genetic monitoring was suggested as the first step in BVDV control because genetic typing of BVDV shows evidence of an increasing number of variants. For this reason circulating genetic typing of BVDV is important update these data. Circulating BVDV in the field shows genetic and antigenic diversity. 5'-UTR nucleotide sequence analysis has been widely used for pestivirus genotype identification. To further characterize the BVDV, the nucleotide sequence of the 5'-UTR that represents a conserved region of the virus genome was analyzed in many studies. The purpose of the current study was to investigate genotypes of pestivirus were circulating in cattle populations in Central Anatolia Region of Turkey. Materials, Methods & Results: Blood samples from 160 animals in randomly selected seven cattle dairy farms that lives with more than 1100 cattle, were collected between November 2009 and March 2010 from Kirikkale (n = 57), Corum (n = 50), Ankara (n = 21), Yozgat (n = 17), Kirsehir (n = 15) cities where are located in Central Anatolia region of Turkey. To detect BVDV in cattle, viral RNA was extracted from whole blood samples using QIAamp Viral RNA Kit and the 5'-UTR were targeted using RT-nested PCR accomplished with first round primers pair panpestivirus and with second round BVDV-1a, BVDV-1b and, BVDV-2 pooled blood samples, respectively. It was detected in second round of RT-nested PCR that BVDV-1a and, BVDV-2 rate are 0.625%, 7.5% in the cattle respectively but not BVDV-1b. Positive PCR amplicons were purified from agarose gel by using commercial DNA purification kit GeneClean III. Two panpestivirus positive PCR amplicons were sequenced using 326 primer. To determine genetic typing of circulating BVDV in the cities, two panpestivirus positive PCR amplicons were sequenced to found genetic diversity and all data were deposited in GenBank under accession numbers; BVDV/Turkey/Kirikkale/01 (HQ393488.2) and BVDV/Turkey/Kirikkale/02 (HQ393489.2). Gene sequences were compared to Mega 4.1 and ClustalW analyzing software. Discussion: The BVDV has a world-wide distribution and causes significant economical losses especially on cattle farms. In this study, it was investigated genetic variability of BVDV subtypes by identifying the 5'-UTR nucleotide sequences of two panpestivirus amplicons from field samples. It was found that BVDV-1a and BVDV-2 in terms of BVDV epidemiology is genotyping, 0.625% and 7.5% using RT-nested PCR respectively. Genetic typing is important for the precise classification and molecular epidemiology of BVDV-1 and epidemiological information on currently epidemic viruses is also important for BVDV prevention and control. We suggest that vaccines should contain at least one strain of both species in Turkey. The study of genetic diversity of BVDV is useful for the understanding of pestivirus field locations as well as for epidemiological studies and planning future BVDV control and vaccination programs in Turkey.