In vitro inhibition of Chikungunya and Semliki Forest viruses replication by antiviral compounds: synergistic effect of interferon-alpha and ribavirin combination.

Chikungunya virus (CHIKV) and Semliki Forest virus (SFV) were used in our laboratory to screen active antiviral compounds against viruses of the Alphavirus genus. Antiviral activity was estimated by the reduction of the cytopathic effect of each alphavirus on infected Vero cells and by virus titer reduction. Cytotoxicity was evaluated by determining the inhibition of Trypan blue exclusion in confluent cell cultures and by the evaluation of the inhibitory effect on cell growth. With CHIKV and SFV, the selectivity indices of human recombinant interferon-alpha and iota-carrageenan were much higher than that of ribavirin, which has been previously investigated for its inhibitory effect on alphavirus infections. Compared to ribavirin, 6-azauridine was more effective against CHIKV and showed a similar antiviral activity against SFV. IFN-alpha2b, glycyrrhizin, 6-azauridine, and ribavirin caused a concentration-dependent reduction in the virus yield with CHIKV and SFV. Moreover, the combination of IFN-alpha2b and ribavirin had a subsynergistic antiviral effect on these two alphaviruses and should be evaluated for the treatment of these infections.

Comparison of flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcription-PCR assay for detection of flaviviruses targeted to a conserved region of the NS5 gene sequences.

Arthropod-transmitted flaviviruses are responsible for considerable morbidity and mortality, causing severe encephalitic, hemorrhagic, and febrile illnesses in humans. Because there are no specific clinical symptoms for infection by a determined virus and because different arboviruses could be present in the same area, a genus diagnosis by PCR would be a useful first-line diagnostic method. The six published Flavivirus genus primer pairs localized in the NS1, NS3, NS5, and 3' NC regions were evaluated in terms of specificity and sensitivity with flaviviruses (including the main viruses pathogenic for humans) at a titer of 10(5) 50% tissue culture infectious doses (TCID(50)s) ml(-1) with a common identification step by agarose gel electrophoresis. Only one NS5 primer pair allowed the detection of all tested flaviviruses with the sensitivity limit of 10(5) TCID(50)s ml(-1). Using a heminested PCR with new primers designed in the same region after an alignment of 30 different flaviviruses, the sensitivity of reverse transcription-PCR was improved and allowed the detection of about 200 infectious doses ml(-1) with all of the tick- and mosquito-borne flaviviruses tested. It was confirmed that the sequenced amplified products in the NS5 region allowed predictability of flavivirus species by dendrogram, including the New York 99 West Nile strain. This technique was successfully performed with a cerebrospinal fluid sample from a patient hospitalized with West Nile virus encephalitis.