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Abstract 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.

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. [ABSTRACT FROM AUTHOR]

Canine distemper is a systemic viral disease characterized by immunosuppression followed by secondary infections. In most cases the clinical diagnosis of distemper is difficult to confirm. Considering the great infectious potential of the disease, the possibility of carrying out an antemortem diagnosis of distemper is important. Absolute quantitative real time PCR (AQPCR) was used for detection and diagnosis of canine distemper virus (CDV) in blood of 5 asymptomatic dogs and vaccine samples. CDV-specific oligonucleotide primers of 20 and 21 nt length were designed for the amplification of an 84 bp target CDV nucleocapsid protein coding region fragment. Another target fragment of 93 bp of canine â-actin was utilized as endogenous control. After conventional reverse transcription at 45°c, cDNA samples were used for AQPCR in ABI Prism 7000 SDS with the PE Biosystems SYBR®Green and protocol. Standard calibration curves were determined for both CDV and â-actin target amplicons. Quantitative results were generated by ABI-Prism Software. Dissociation curves for target fragments confirmed the AQPCR specificity. Slopes for calibration curves from both CDV and â-actin in the range of -3.2 demonstrated a reaction efficiency of 100% and a high linearity with r² = 0.989. Positive controls confirmed that the product detected was CDV, and negative (no template) controls showed no CDV PCR products. The AQPCR detected 3 positives in 5 dogs asymptomatic for CDV. The present protocol detected as little as 10-8 ng of CDV cDNA in asymptomatic dogs, and may be suitable for future viral quantification and epidemiologic studies. (Sponsored by CNPq-Brasil). [ABSTRACT FROM AUTHOR]

Canine distemper virus (CDV) causes canine distemper diseases in dogs and in other carnivores. CDV is characterized by fever, nasal discharge, encephalomyelitis, conjunctivitis, gastroenteritis and pneumonia. The cellular pathogenesis includes mainly complications in the CNS and immunosuppressive systems, which are not completely understood. Several viruses have been shown recently to cause cell death by means of apoptosis induction. Dogs naturally infected with CDV show apoptosis in lymphoid tissues and cerebellum. However, apoptotic pathway mechanisms for CDV are not well determined. Our goal was to develop a sensitive method for diagnosis of CDV. We used quantitative PCR to investigate expression of the pro-apoptotic caspases 3 and 8, and antiapoptotic Bcl-2, in leukocytes of infected dogs. CDV diagnosis was performed by gel-based RT-PCR, using previously characterized primers. Six CDV positives and 6 negative samples were selected for apoptotic gene expression comparison by a Real Time PCR SYBR®GREEN protocol. Caspase -3 cDNA expression (ng/IJg total RNA) was 4.7 times higher in the CDV positive (0.14) than in the CDV negatives (0.03); Caspase -8 was 6.2 times higher in the CDV positive (0.52) than in the CDV negatives (0.083); and Bcl-2 levels did not change in CDV positives (0.035) and negatives (0.037). Results demonstrated that CDV infection induced transcription of caspases -3 and -8, while Bcl-2 expression did not change. This suggests that CDV might cause immune suppression induced by apoptosis in leukocytes via caspase-3 activation, but the main cascade for this induction may occur due to activation of the caspase-8 pathway. [ABSTRACT FROM AUTHOR]