Genetic characterization of influenza virus circulating in Brazilian pigs during

Background Influenza A viruses circulating in pigs in Brazil are still not characterized, and only limited data are available about swine influenza epidemiology in the country. Therefore, we characterized the hemagglutinin (HA) and neuraminidase (NA) genes of influenza viruses isolated from Brazilian pigs. We also evaluated one case of probable swine-to-human transmission. Methods Twenty influenza viruses isolated from pigs during 2009-2010 in five Brazilian states (Minas Gerais, Sao Paulo, Parana, Rio Grande do Sul, and Mato Grosso) were used. One human isolate, from a technician who became ill after visiting a swineherd going through a respiratory disease outbreak, was also used in the study. Phylogenetic analysis for the HA and NA genes and hemagglutinin amino acid sequence alignment were performed. Results All isolates clustered with pandemic H1N1 2009 (pH1N1) viruses and appeared to have a common ancestor. Genetic diversity was higher in the HA than in the NA gene, and the amino acid substitution S203T in one of HA's antigenic sites was found in most of the samples. The human isolate was more related to swine isolates from the same herd visited by the technician than to other human isolates, suggesting swine-to-human transmission. Conclusion Our results show that pH1N1 was disseminated and the predominant subtype in Brazilian pigs in 2009-2010

Canine distemper virus induces apoptosis in cervical tumor derived cell lines

Apoptosis can be induced or inhibited by viral proteins, it can form part of the host defense against virus infection, or it can be a mechanism for viral spread to neighboring cells. Canine distemper virus (CDV) induces apoptotic cells in lymphoid tissues and in the cerebellum of dogs naturally infected. CDV also produces a cytopathologic effect, leading to apoptosis in Vero cells in tissue culture. We tested canine distemper virus, a member of the Paramyxoviridae family, for the ability to trigger apoptosis in HeLa cells, derived from cervical cancer cells resistant to apoptosis. To study the effect of CDV infection in HeLa cells, we examined apoptotic markers 24 h post infection (pi), by flow cytometry assay for DNA fragmentation, real-time PCR assay for caspase-3 and caspase-8 mRNA expression, and by caspase-3 and -8 immunocytochemistry. Flow cytometry showed that DNA fragmentation was induced in HeLa cells infected by CDV, and immunocytochemistry revealed a significant increase in the levels of the cleaved active form of caspase-3 protein, but did not show any difference in expression of caspase-8, indicating an intrinsic apoptotic pathway. Confirming this observation, expression of caspase-3 mRNA was higher in CDV infected HeLa cells than control cells; however, there was no statistically significant change in caspase-8 mRNA expression profile. Our data suggest that canine distemper virus induced apoptosis in HeLa cells, triggering apoptosis by the intrinsic pathway, with no participation of the initiator caspase -8 from the extrinsic pathway. In conclusion, the cellular stress caused by CDV infection of HeLa cells, leading to apoptosis, can be used as a tool in future research for cervical cancer treatment and control

Canine distemper virus detection in asymptomatic and non vaccinated dogs

A quantitative real time polymerase chain reaction (PCR) revealed canine distemper virus presence in peripheral blood samples from asymptomatic and non vaccinated dogs. Samples from eleven domestic dogs with no signs of canine distemper and not vaccinated at the month of collection were used. Canine distemper virus vaccine samples in VERO cells were used as positive controls. RNA was isolated with Trizol®, and treated with a TURBO DNA-free kit. Primers were designed for canine distemper virus nucleocapsid protein coding region fragment amplification (84 bp). Canine b-actin (93 bp) was utilized as the endogenous control for normalization. Quantitative results of real time PCR generated by ABI Prism 7000 SDS Software showed that 54.5% of dogs with asymptomatic canine distemper were positive for canine distemper virus. Dissociation curves confirmed the specificity of the real time PCR fragments. This technique could detect even a few copies of viral RNA and identificate subclinically infected dogs providing accurate diagnosis of this disease at an early stage.<br>A reação em cadeia da polimerase (PCR) em tempo real revelou a presença do vírus da cinomose canina em amostra de sangue de cães assintomáticos e não vacinados. Amostra de onze cães domésticos sem nenhum sinal clínico de cinomose e que não foram vacinados no mês da coleta de sangue foram utilizados para análise. Amostra vacinal do vírus da cinomose canina em células VERO foi utilizada como controle positivo. O RNA total foi isolado utilizando-se Trizol®, e tratadas com o Kit TURBO DNA-free. Os iniciadores foram desenhados para amplificar a região do nucleocapsídeo viral com 319pb e 84pb para a PCR convencional e PCR em tempo real, respectivamente. O fragmento alvo da b-actina canina com 93pb foi utilizado como controle endógeno e normalizador. Resultados quantitativos da PCR em tempo real gerados pelo programa ABI Prism 7000 SDS demonstraram que 54,5% dos cães assintomáticos foram positivos para o vírus da cinomose canina. As curvas de dissociação confirmaram a especificidade dos fragmentos da PCR em tempo real. A detecção precoce do RNA viral é importante para a identificação de cães subclinicamente infectados e limitar a difusão da doença

Distribution of caprine arthritis encephalitis virus provirus, RNA, and antigen in the reproductive tract of one naturally and seven experimentally infected bucks

AbstractCaprine arthritis encephalitis is a worldwide, multisystemic disease caused by a small ruminant lentivirus. Although the main route of transmission is oral, detection of proviral DNA of the caprine arthritis encephalitis virus (CAEV) in caprine semen has been previously described. However, the presence of viral antigens in the male reproductive tract has apparently never been reported. The objective was to study lesions in the buck reproductive system and to detect, in these tissues, the presence of proviral DNA, viral RNA and CAEV antigens. Tissues from eight CAEV-infected bucks (one naturally and seven experimentally infected) were analyzed by histopathology, nested polymerase chain reaction, reverse transcriptase-polymerase chain reaction, and immunohistochemistry. Interstitial pneumonia, synovitis, and lesions in the male reproductive tract were detected in some of the bucks. Proviral DNA was detected in the lungs and joints as well as in the reproductive systems of all animals, whereas viral RNA was detected only in the genital tract of the naturally infected buck. Viral antigens were immunostained in most of the organs of the male reproductive tract. This report was apparently the first to clearly demonstrate CAEV antigen expression in the male reproductive tract, which indicates the possibility of venereal transmission of CAEV