Screening of human populations with high exposure to NHPs has resulted in the successful discovery of novel retroviruses, including HTLV-3, HTLV-4, and simian foamy virus (SFV) [1, 7, 8, 32]. We have previously demonstrated that hunter-collected DBS specimens from wild-caught NHPs are not only an effective collection strategy to demonstrate STLV diversity but also allow for monitoring of retroviral cross-species transmission events at the primate-hunter interface . Using these primate DBS specimens, we recently identified novel STLV-3s in wild-caught C. mona and C. nictitans monkeys by analysis of partial gene sequences . To characterize this new virus, we obtained its complete proviral genome using nucleic acids extracted entirely from two DBS, collected by two hunters in the field. To our knowledge, this is the first full-length genome of a simian retrovirus obtained entirely from DBS. The ability to generate a complete viral genome from the equivalent of about 0.25 ml whole blood demonstrates further the utility of this collection strategy for monitoring and characterizing viral diversity.
Robust phylogenetic analysis of both the conserved tax region and gag-pol-env-tax concatenated sequences inferred a novel lineage with high statistical support within the PTLV-3 clade that is highly divergent. The formation of a fourth lineage within the diversity of PTLV-3, containing STLV-3 sequences from two distinct primate species (C. mona and C. nictitans), strongly supports the proposed nomenclature and classification of this new virus as STLV-3 subtype D. The discovery of nearly identical STLV-3d(Cmo8699AB and Cni7867AB) viruses in two different primate species within the same region of Cameroon and the inferred ancient divergence of STLV-3d about 115,000 ya also suggests a higher prevalence and a more widespread distribution for this virus.
Our results also suggest that taxonomic subdivision within the current subtype B strains is warranted. PTLV-3 from Cameroon (STLV-3(Cto604), HTLV-3(Pyl43), and HTLV-3(Lobak18)) are distantly related to other PTLV-3 subtype B strains from West-Central Africa ((HTLV-3(Cam2026ND), STLV-3(NG409), STLV-3(PPAF3), and STLV-3(Lal9589NL) sharing < 90% nucleotide identity. Since both PTLV-3 lineages are presently known as B subtypes, we propose re-naming them as subtypes B1 (from Cameroon only) and B2 (from West-Central Africa). A similar nomenclature strategy has been previously adopted for subtyping HTLV-2 . This re-classification and the categorization of the new STLV-3 subtype D are based upon highly supported phylogenetic division of these subtypes and genetic distances of at least 5% across the genome, as recently proposed for PTLV classification .
PTLVs have an ancient evolutionary history with the ancestral HTLVs being inferred to have first occurred many thousands of years ago following zoonotic transmission from STLV-infected NHPs [14, 24, 29, 42, 61]. This finding contrasts with the relatively recent emergence of the human immunodeficiency virus (HIV) from simian immunodeficiency virus-infected NHPs in the last century [62, 63]. The recent discovery of HTLV-3 and HTLV-4 and novel STLV-1-like viruses among people who hunt and butcher NHPs suggests that these interspecies transmission events are not rare and are most likely contemporaneous [1, 7]. From phylogenetic analysis it has been inferred that STLV-1 may have crossed species boundaries to humans on at least seven separate occasions resulting in the multiple HTLV-1 subtypes . Given the inferred ancient origin of STLV-3d(Cmo8699AB) and PTLV-3, the wide geographic distribution of STLV-3 across Africa, the long history of human exposure to simians in Africa and the lack of screening for HTLV in blood banks in Africa, human infections with STLV-3d-like viruses might be expected to occur there. Thus, although HTLV-3 so far has only been identified in three persons from Cameroon and all three are subtype B viruses, it is tempting to speculate that like HTLV-1 diversity, HTLV-3 diversity will be driven by transmission of each of the four STLV-3 subtypes to humans. More surveillance studies at the NHP-human interface are needed to determine the prevalence, diversity, and epidemiology of STLV-3d(Cmo8699AB) and HTLV-3.
Molecular differences between HTLV-1 and HTLV-2 Tax proteins have been proposed to modulate function, transmissibility, and pathogenesis . We therefore examined the predicted protein sequences of STLV-3d(Cmo8699AB) to determine whether important functional and regulatory motifs were present to infer the replication-competency and pathogenic potential for this divergent viral subtype. All enzymatic, structural, and regulatory proteins were preserved in STLV-3d(Cmo8699AB), including the ubiquitous Tax binding domains CBP/P300, NES, and CR2, which are all important for viral transcription and transformation [64–66]. In addition, the presence of a PDZ-binding motif in the STLV-3d Tax, which has been shown to be critical in signal transduction and T-cell transformation of HTLV-1-infected cells [50, 51], suggests that the STLV-3d(Cmo8699AB) Tax is more similar to the Tax of PTLV-1 and other PTLV-3s, than it is to the Tax of PTLV-2 which lacks a PDZ motif [14, 42]. Furthermore, as has been demonstrated with all PTLVs, STLV-3d also possesses a conserved ASP basic leucine zipper motif in the antisense strand between the env and tax/rex gene regions. ASP has been shown to participate in regulation of viral replication and possibly oncogenesis [50, 51]. Combined, these findings show that the STLV-3d(Cmo8699AB) genome is intact, is likely to be replication competent, and may have a pathogenic potential similar to HTLV-1 which is also predicted for HTLV-3 subtype B; however, further studies are required to validate this hypothesis.
The LTR region of STLV-3d(Cmo8699AB) has two of the three 21-bp repeat Tax-responsive elements (TRE) typically found in the HTLV-1 and HTLV-2 LTRs. The three TREs (distal, central, and proximal) are involved in basal transcription and have been shown to confer Tax1, Tax2, and Tax3 responsiveness . Studies have also shown that mutations in the central TRE compared to the distal or proximal TRE-1 result in the greatest loss of basal transcription levels . As with all PTLV-3s, the STLV-3d(Cmo8699AB) LTR lacks only the distal TRE, which does not appear to have deleterious effects on gene expression and viral replication [30, 69]. Nonetheless, more studies are necessary to determine if these differences will affect the transcriptional activity of STLV-3d(Cmo8699AB).
Another notable difference of STLV-3d from other PTLV-3s was observed in the leucine-rich activation region of the putative NES domain of the Rex protein involved in regulation of viral expression. STLV-3d(Cmo8699AB) has a single aa mutation from aspartic acid or glycine to alanine at position 94 similar to that seen in the HTLV-2 Rex protein (Rex2). Mutagenesis studies substituting alanine for serine residues in this region have demonstrated a significant reduction in the phosphorylation activation required for efficient RNA binding of Rex-2 . These results suggest that the alanine mutation at aa position 94 of the STLV-3d(Cmo8699AB) Rex may also have a similar loss of biologic activity. The effects of these changes on the processing of viral transcripts and regulation of viral replication by the STLV-3d Rex will require further investigation.