RESUMO
Molecular analysis of the polymorphic region of the bovine coronavirus (BCoV)-S gene using recent Japanese field isolates and reference strains revealed that the 148 isolates collected from 1999 to 2008 from 13 prefectures, covering all regions of Japan (Hokkaido, Tohoku, Kanto, Chubu, Kinki, Chugoku, Shikoku, and Kyusyu region) and divided into 3 clusters, show distinctive divergence from the prototype enteric BCoV strains. Almost all isolates after 2005 were clustered into group 4, and there was no regional specificity in these clusters. To differentiate the genotypes without sequencing, a simple technique-reverse transcriptase-polymerase chain reaction/restriction fragment length polymorphism analysis (RT-PCR/RFLP)-was developed. The availability of a simple and easy diagnostic assay will enable larger epidemiological studies of BCoV.
Assuntos
Bovinos/virologia , Coronavirus Bovino/genética , Filogenia , Animais , Proteínas do Capsídeo/genética , Análise por Conglomerados , Primers do DNA/genética , Genótipo , Japão , Polimorfismo de Fragmento de Restrição/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterináriaRESUMO
The difference between the attenuated live vaccine strain 758-43 and its parent virulent strain 758 was investigated genetically. These viruses were propagated in Madin-Darby bovine kidney cells, and viral DNA was obtained from the culture supernatants of the infected cells. Based on a previous report, a large deleted region would seem to exist in the Hind III J fragment located between nucleotide numbers 2439 and 11,270. Three pairs of primers were designed based on the complete BHV-1 DNA sequence. With one pair of primers used, the PCR products derived from strains 758 and LA resulted in fragment sizes of 1850 bp, whereas that from the vaccine strain was smaller than those from the virulent strains. The attenuated live vaccine strain, 758-43, lacked 652 bp in the PCR product region, accounting for approximately 84% of the coding region of the UL49 homolog gene of BHV-1. The present results provide a new and important information to distinguish the vaccine strain 758-43 clearly from wild-type BHV-1 isolates in Japan. The UL49 homolog gene seems to participate in pathogenicity in herpesvirus infections.
Assuntos
Sequência de Bases , Genoma Viral , Vacinas contra Herpesvirus/genética , Rinotraqueíte Infecciosa Bovina/virologia , Deleção de Sequência , Animais , Bovinos , Linhagem Celular , Herpesvirus Bovino 1/genética , Japão , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Vacinas Atenuadas/genéticaRESUMO
An outbreak of contagious mastitis occurred among cattle on a farm, and bovine herpesviruses were isolated from the affected mammary tissues, scabs and abscess discharge of the cattle. A bovine herpesvirus type 4 (BoHV-4)-specific fragment was amplified from the isolates by polymerase chain reaction (PCR). Restriction endonuclease analyses demonstrated that the isolates were related to Movar-like European type BoHV-4. To determine the ratio of BoHV-4 subclinical infection in the cattle, a genomic survey was performed by PCR for cattle that were moved to the animal hygiene service station in Ibaraki prefecture. The BoHV-4 genome was occasionally detected in peripheral blood leukocytes, lymph nodes and nervous tissues. The rate of BoHV-4 subclinical infection was relatively high in the cattle.