Construction of an infectious cDNA clone of Enterovirus 71: insights into the factors ensuring experimental success.

Enterovirus 71 (EV 71) is a causative agent of mild Hand Foot and Mouth Disease but is capable of causing severe complications in the CNS in young children. Reverse genetics technology is currently widely used to study the pathogenesis of the virus. The aim of this work was to determine and evaluate the factors which can contribute to infectivity of EV 71 RNA transcripts in vitro. Two strategies, overlapping RT-PCR and long distance RT-PCR, were employed to obtain the full-length genome cDNA clones of the virus. The length of the poly(A) tail and the presence of non-viral 3'-terminal sequences were studied in regard to their effects on infectivity of the in vitro RNA transcripts of EV 71 in cell culture. The data revealed that only cDNA clones obtained after long distance RT-PCR were infectious. No differences were observed in virus titres after transfection with in vitro RNA harbouring a poly(A) tail of 18 or 30 adenines in length, irrespective of the non-viral sequences at the 3'-terminus.

The HIV type 1 epidemic in Belarus: predominance of Eastern European subtype A strains and circulation of subtype B viruses.

To study the molecular epidemiology of HIV-1 in Belarus, where the rapid spread of HIV-1 has been registered since 1996, we obtained HIV-1 sequences from 30 individuals living in five cities in both the main geographic areas of the epidemic (Gomel and Minsk regions) and territories where spreading of the epidemic remains limited (Grodno region). Analysis of env V3 and gag p17/p24 sequences demonstrated that infections in all 12 injecting drug users and 14 of 18 individuals infected through sexual contacts were caused by subtype A viruses that are specific for the epidemic in the former Soviet Union (IDU-A viruses), while the remaining four infections were caused by phylogenetically unrelated to each other subtype B viruses. Extrapolation of these results to the total population of HIV-1-infected individuals in Belarus allowed us to estimate that IDU-A viruses account for nearly 95% of HIV-1 infections in Belarus.