Gissandra Farias Braz

Genetic characterization of influenza virus circulating in Brazilian pigs during 2009 and 2010 reveals a high prevalence of the pandemic H1N1 subtype

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.

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.

Caspase-3/-8/-9, Bax and Bcl-2 expression in the cerebellum, lymph nodes and leukocytes of dogs naturally infected with canine distemper virus.

Canine distemper is an immunosuppressive disease caused by the canine distemper virus (CDV). Pathogenesis mainly involves the central nervous system and immunosuppression. Dogs naturally infected with CDV develop apoptotic cells in lymphoid tissues and the cerebellum, but this apoptotic mechanism is not well characterized. To better understand this process, we evaluated the expression of Bax, Bcl-2, and caspase-3, -8 and -9, by evaluating mRNA levels in the peripheral blood, lymph nodes and cerebellum of CDV-infected (CDV+) and uninfected (CDV-) dogs by real-time polymerase chain reaction (PCR). Blood samples from 12 CDV+ and 8 CDV- dogs, diagnosed by reverse transcription-PCR, were subjected to hematological analysis and apoptotic gene expression was evaluated using real-time-PCR. Tissues from the cerebellum and lymph nodes of four CDV+ and three CDV-dogs were also subjected to real time-PCR. No significant differences were found between CDV+ and CDV- dogs in the hemotological results or in the expression of caspase-3, -8, -9, Bax, and Bcl-2 in the peripheral blood. However, expression of Bax, caspase-3, -8 and -9 was significantly higher in the cerebellum of CDV+ compared to CDV- dogs. Expression of caspase-3 and -8 was significantly higher in the lymph nodes of CDV+ compared to CDV- dogs. We concluded that infection with CDV induces apoptosis in the cerebellum and lymph nodes in different ways. Lymph node apoptosis apparently occurs via caspase-3 activation, through the caspase-8 pathway, and cerebellum apoptosis apparently occurs via caspase-3 activation, through the caspase-8 and mitochondrial pathways.

Serological evidence of swine influenza in Brazil

Please cite this paper as: Rajão et al. (2013). Serological evidence of swine influenza in Brazil. Influenza and Other Respiratory Viruses 7(2), 109–112.

Detecção do vírus da cinomose canina em cães assintomáticos e não vacinados

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.

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

Caprine 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.

Vero cells infected with the Lederle strain of canine distemper virus have increased Fas receptor signaling expression at 15 h post-infection.

We evaluated the expression of the Fas receptor gene in Vero cells infected with the Lederle vaccine strain of canine distemper virus using RT-PCR. Vero cells were plated, and after being grown for 24 h in MEM with 5% FBS, 80-90% confluent monolayer cultures were infected with the virus. The cells were harvested at 3, 6, 9, and 15 h post-infection. Uninfected Vero cells were used as a control. Total RNA was isolated from Vero cells using 1 mL Trizol(®) LS, and RT was performed using 2 μg total RNA. Primer pairs for RT-PCR amplification for the canine distemper virus nucleocapsid gene, the S26 reference gene, and the Vero rFas gene were used to analyze expression in Vero cells. RT-PCR results revealed virus activity at 3, 6, 9, and 15 h in the virus-infected Vero cells. The S26 housekeeping gene was amplified in virus infected and control samples. However, expression of the cell death receptor Fas was detected in Vero cells only at 15 h post-infection. We suggest that the Lederle vaccine induces apoptosis by Fas receptor signaling, possibly through caspase-8 signaling rather than through mitochondrial signaling in the infected cells.