Faculty of Veterinary Medicine University of Zagreb
Abstract
Between 2007 and 2011, a total of 1937 sera samples and five spleen samples from nine Croatian dairy herds were tested for the bovine viral diarrhea virus (BVDV) using virus isolation and the immunoperoxidase test. BVDV was detected in 13 persistently infected (PI) cattle with a non-cytopathogenic biotype, while in five animals with fatal mucosal disease, isolates from spleen samples were of the cytopathogenic biotype. To reveal the genetic typing of Croatian BVDV isolates, viral RNA was extracted from infected cell cultures and amplified by RT-PCR, with primers targeting the 5’-UTR and the Npro gene, followed by direct sequencing of purified PCR products. Sequence and phylogenetic analysis of the 5’-UTR genome region determined that all Croatian isolates belonged to BVDV genotype 1; 11 isolates were grouped with BVDV-1b and 7 with BVDV- 1f viruses. The phylogenetic tree inferred by the Bayesian approach, using combined 5’-UTR/Npro, supported clustering of Croatian isolates in two subgroups. The deduced aminoacid sequence of the Npro region revealed 5 sites unique for four domestic BVDV-1f isolates.Od 2007. do 2011. godine na virus virusnog proljeva goveda (VPG) pretraženi su uzorci pet slezena i 1937 uzoraka seruma podrijetlom iz 9 stada mliječnih goveda. Imunoperoksidaznim testom (IP) u 13 je uzoraka dokazan necitopatogeni biotip virusa VPG te je potvrđena perzistentna zaraza. Iz svih pet uzoraka slezena izdvojen je citopatogeni virus VPG. U svrhu genotipizacije vlastitih izolata virusa VPG, iz nadtaloga inficiranih staničnih kultura izdvojena je virusna RNK i umnožena lančanom reakcijom polimerazom uz prethodnu reverznu transkripciju (RT-PCR). Umnoženi odsječci 5’-UTR i gena Npro su potom sekvencirani. Filogenetska analiza hrvatskih izolata virusa VPG pokazala je da svi pretraženi izolati pripadaju genotipu 1; 11 izolata svrstano je u podtip 1b, a 7 u 1f. Filogenetska analiza na temelju spojene 5’-UTR/Npro genske sekvencije potvrdila je rezultate genotipizacije pretraženih izolata. Na Npro dijelu genoma utvrđeno je pet aminokiselinskih promjena osebujnih za hrvatske izolate iz podtipa 1f
