Isolation and molecular detection of bovine parainfluenza virus type 3 in cattle in Serbia

Abstract

The presence of bovine parainfluenza virus type 3 (BPIV3) was examined in 119 nasal swabs collected from cattle with severe respiratory infection. All samples were conducted for virus isolation on the MDBK cell line. The cytopathic effect was observed after 48h to 72h in cells inoculated with eight samples (8/119; 6.7%). The confirmation of isolated strains of BPIV3 was done by the virus-neutralization test. In addition, all samples of bovine nasal swabs were also examined for the presence of BPIV3 virus using RT-PCR with primers specific for the part of HN gene. The presence of BPIV3 was detected in eight samples (8/119; 6.7%) that were also positive upon virus isolation. The molecular characterization based on nucleotide sequencing of the part of the HN gene showed that all BPIV3 isolates belonged to genotype C of BPIV3. They branched in one distinct cluster with three different branches, but these branches were very similar to each other (98.1% to 99.8%). Serbian BPIV3c isolates were most similar to the Chinese BPIV3c isolates SD0805, SD0809 and SD0835 (from 97.92% to 99.7%), and to South Korean (12Q061), Japanese (HS9) and American (TVMDL16 and TVMDL20) BPIV3c strains (from 97.1% to 98.8%), and distinct from American (TVMDL15and TVMDL17) and Australian (Q5592) BPI3V genotype B strains (only 79.9% to 82.3% similarity), as well as from the genotype A BPIV3 strains from different countries published in GenBank.

Ukupno je ispitano 119 uzoraka nosnih briseva goveda na prisustvo parainfluenca 3 virusa goveda (bovine parainfluenza virus type, eng. - 3 BPIV3). Iz svih uzoraka nosnih briseva je vršena izolacija virusa na ćelijskoj liniji MDBK. Pojava citopatogenog efekta na kulturi ćelija, nakon 48h, odnosno 72h, utvrđena je kod osam uzoraka nosnih briseva (8/119; 6.7%). Identifikacija izolovanih sojeva BPIV3 je izvršena primenom virus neutralizacionog testa. Dodatno, svi uzorci nosnih briseva goveda su ispitani na prisustvo BPIV3 i primenom metode RT-PCR uz korišćenje prajmera specifičnih za deo HN gena virusa. Prisustvo virusne nukleinske kiseline je utvrđeno kod osam uzoraka nosnih briseva (8/119; 6.7%), koji su bili pozitivni i na izolaciji virusa. Molekularna karakterizacija zasnovana na sekvenciranju dela HN gena izolata BPIV3 iz Srbije je potvrdila da svi pripadaju genotipu C BPIV3 (BPIV3c). Oni su se u filogenetskom stablu granali u tri različite grane koje su međusobno veoma slične (98.1% do 99.8%). Izolati BPIV3 iz Srbije su pokazali visok stepen sličnosti nukleotidnih sekvenci sa BPIV3c sojevima SD0805, SD0809 i SD0835 iz Kine (97.92% do 99.7%), odnosno sa BPIV3c sojevima 12Q061 iz Južne Koreje, HS9 iz Japana i TVMDL16 i TVMDL20 iz Amerike (sličnost od 97.1% do 98.8%), kao i različitosti u odnosu na nukleotidne sekvence sojeva TVMDL15 i TVMDL17 izolovanih u Americi i soja Q5592 izolovanog u Australiji, a koji su pripadali genotipu B BPIV3 (sličnost od 79.9% do 82.3%). Slična razlika je utvrđena i sa nukleotidnim sekvencama sojeva virusa, poreklom iz različitih država, svrstanih u genotip A BPIV3, a objavljenih u genskoj bazi podataka (NCBI GenBank).