We report the second human immunodeficiency virus (HIV) belonging to the new HIV type 1 (HIV-1) group P lineage that is closely related to the simian immunodeficiency virus found in gorillas. This virus was identified in an HIV-seropositive male hospital patient in Cameroon, confirming that the group P virus is circulating in humans. Results from screening 1,736 HIV-seropositive specimens collected in Cameroon indicate that HIV-1 group P infections are rare, accounting for only 0.06% of HIV infections. Despite its rarity, group P shows evidence of adaptation to humans.

HIV-1 strain diversity in Cameroon did not significantly change, suggesting a mature and relatively stable epidemic.

The simian virus 40 (SV40) origin region includes the viral replication origin and the early and late promoters and consists of a few palindromes, a 17-base-pair (bp) A + T-rich sequence, three copies of a G + C-rich 21-bp repeat, and two copies of a 72-bp repeat. We have made sequential deletions in the SV40 origin region and determined the early promoter efficiencies of these truncated DNA segments by connecting them in the correct orientation with the coding regions of selectable marker genes and assaying the expression of the chimeric marker genes in vivo in different host cell lines. A truncated SV40 early promoter segment containing only the TATA box and the major in vivo mRNA initiation sites has essentially no promoter efficiency. We have located the major component of the SV40 early promoter within the 21-bp repeated sequences, which consist of an alternating and mutually overlapping array of two C-rich oligonucleotides having the consensus sequences Y-Y-C-C-G-C-C-C (Y = pyrimidine nucleoside) and G-C-C-C-(C)-TA-AT-A(T)-C-T. Between one and two copies of the 21-bp repeat were adequate for gene expression under conditions in which the enhancement effect of the 72-bp repeat was minimal. We also find that the SV40 72-bp repeat exhibits a pronounced host range in its enhancement of gene expression; the enhancement is only 2-fold in the nonpermissive mouse cells but amounts to 10- or 20-fold in the permissive monkey cells or the semipermissive human cells, respectively.

bMolecular surveillance is essential to monitor HIV diversity and track emerging strains. We have developed a universal library preparation method (HIV-SMART [i.e., switching mechanism at 5= end of RNA transcript]) for next-generation sequencing that harnesses the specificity of HIV-directed priming to enable full genome characterization of all HIV-1 groups (M, N, O, and P) and HIV-2. Broad application of the HIV-SMART approach was demonstrated using a panel of diverse cell-cultured virus isolates. HIV-1 non-subtype B-infected clinical specimens from Cameroon were then used to optimize the protocol to sequence directly from plasma. When multiplexing 8 or more libraries per MiSeq run, full genome coverage at a median ϳ2,000؋ depth was routinely obtained for either sample type. The method reproducibly generated the same consensus sequence, consistently identified viral sequence heterogeneity present in specimens, and at viral loads of <4.5 log copies/ml yielded sufficient coverage to permit strain classification. HIV-SMART provides an unparalleled opportunity to identify diverse HIV strains in patient specimens and to determine phylogenetic classification based on the entire viral genome. Easily adapted to sequence any RNA virus, this technology illustrates the utility of next-generation sequencing (NGS) for viral characterization and surveillance.

Surveillance of emerging viral variants is critical to ensuring that blood screening and diagnostic tests detect all infections regardless of strain or geographic location. In this study, we conducted serological and molecular surveillance to monitor the prevalence and diversity of HIV, HBV, and HTLV in South Cameroon. The prevalence of HIV was 8.53%, HBV was 10.45%, and HTLV was 1.04% amongst study participants. Molecular characterization of 555 HIV-1 specimens identified incredible diversity, including 7 subtypes, 12 CRFs, 6 unclassified, 24 Group O and 2 Group N infections. Amongst 401 HBV sequences were found a rare HBV AE recombinant and two emerging sub-genotype A strains. In addition to HTLV-1 and HTLV-2 strains, sequencing confirmed the fifth known HTLV-3 infection to date. Continued HIV/HBV/HTLV surveillance and vigilance for newly emerging strains in South Cameroon will be essential to ensure diagnostic tests and research stay a step ahead of these rapidly evolving viruses.