Luba is one of the four historical foci of Human African Trypanosomiasis (HAT) on Bioko Island, in Equatorial Guinea. Although no human cases have been detected since 1995, T. b. gambiense was recently observed in the vector Glossina palpalis palpalis. The existence of cryptic species within this vector taxon has been previously suggested, although no data are available regarding the evolutionary history of tsetse flies populations in Bioko.
We analyzed RNA splice site usage in three HIV-1 subtype B primary isolates through reverse transcriptase polymerase chain reaction (RT-PCR) amplification of spliced RNAs using a fluorescently labeled primer, with computerized size determination and quantification of PCR products, which were also identified by clone sequencing. In one isolate, P2149-3, unusual and unreported spliced transcripts were detected. This isolate preferentially used for rev RNA generation a 3 splice site (3ss) located five nucleotides upstream of A4a, previously identified only in a T cell line-adapted virus and in a group O isolate, and designated A4d. P2149-3 also used an unreported 3ss for rev RNA generation, designated A4h, located 20 nucleotides upstream of 3ss A4c. Additionally, unusual nef RNAs using 3ss A5a and A7a and with exon composition 1.3.7 were identified. The identification of several unusual and unreported spliced transcripts in an HIV-1 primary isolate suggests a greater diversity of splice site usage in HIV-1 than previously appreciated.
The HIV-1 primary transcript undergoes a complex splicing process by which more than 40 different spliced RNAs are generated. One of the factors contributing to HIV-1 splicing complexity is the multiplicity of 3 splice sites (3ss) used for generation of rev RNAs, with two 3ss, A4a and A4b, being most commonly used, a third site, A4c, used less frequently, and two additional sites, A4d and A4e, reported in only two and one isolates, respectively. HIV-1 splicing has been analyzed mostly in subtype B isolates, and data on other group M clades are lacking. Here we examine splice site usage in three primary isolates of subtype C, the most prevalent clade in the HIV-1 pandemic, by using an in vitro infection assay of peripheral blood mononuclear cells. Viral spliced RNAs were identified by RT-PCR amplification using a fluorescently-labeled primer and software analyses and by cloning and sequencing the amplified products. The results revealed that splice site usage for generation of rev transcripts in subtype C differs from that reported for subtype B, with most rev RNAs using two previously unreported 3ss, one located 7 nucleotides upstream of 3ss A4a, designated A4f, preferentially used by two isolates, and another located 14 nucleotides upstream of 3ss A4c, designated A4g, preferentially used by the third isolate. A new 5 splice site, designated D2a, was also identified in one virus. Usage of the newly identified splice sites is consistent with sequence features commonly found in subtype C viruses. These results show that splice site usage may differ between HIV-1 subtypes.
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