In this study, we trimmed sequences from a partial pol gene which included the prot. gene of HIV-1 sequences from Ghana. The results of this study unlike others presented the opportunity to determine phylogenetic relationships as the sequences were shortened from longer fragments and not by sequencing partial pol genes of the HIV-1 strains [15, 17, 18]. Our results indicate that the T/S values are different for different lengths of sequences and should be considered when building trees with fragments of the pol gene. The similarity in topology between RTs and RT shows that the 661 bp can be confidently used for subtyping of HIV-1 strains from Ghana.
Similar length of sequences in the env as compared to the prot. have been used to establish phylogenetic relationship in HIV-1 strains from Ghana . This may mean that the variability in the prot gene, especially for CRFs, may not be sufficient to establish strain relationships. Our results for the prot. phylogeny are in contrast to that of others [12, 14, 15], but confirm the study by Pasquier et al . The differences obtained from these studies are likely to be mainly due to the number of reference subtypes included. It is therefore important that in determining the true relationships of sequences, at least the nine pure subtypes and circulating recombinants commonly found within the region under study, are used for tree building. The subtyping done by Kinomoto et al. using only the prot. gene may therefore not be reliable .
The repeat sequences introduced had bootstrap values of 100% for the pol, RT and RTs phylogenetic trees but not prot. It can therefore be inferred that using a bootstrap value of 70% for the RT and 57% for the RTs which accounted for the CRF 02_AG cluster will be sufficient to determine subtypes. Although other studies have used higher values, our results indicate that it may be necessary to include repeat reference sequences in order to ascertain the reliability of the length of sequences being used for bootstrapping analysis. Since the repeat sequences in the prot. gene had bootstrap values < 100%, which did not reflect in the others, this test can be used as a standard to test for the reliability for HIV-1 phylogeny rather than arbitrarily fixing bootstrap values that support the confidence of relationships.
Our results confirm those of other studies that the pol and RT genes are useful for subtyping [17, 18]. The loosely arranged pol gene sequences in the phylogenetic trees also reflected in the recombination analysis done, and confirm loosely arranged HIV-1 strains in previous studies . Fragments of a previously characterized Ghanaian 02-AG sequence [GeneBank: AB286862 (4 in Additional file 1)] were found in only two sequences, GHN36 and GHN81 [see Additional file 1]. Since GHN36 was the only pure 02_AG strain found, this may suggest that the pol genes may have evolved away from this prototype into other sequences. The pol gene of 02_AG sequences may be undergoing complex recombination processes that may further complicate its use for subtyping. Furthermore, since GHN90 was clearly an out-group when the 24 Ghana sequences were analyzed alone, it is likely that the evolution is towards that strain. This may explain why fragments of CRF 02_AG strains [GeneBank: AJ286956 (5 in Additional file 1)] and [GeneBank: AJ583728 (7 in Additional file 1)] which were common in GHN90 were frequently seen in other GHN sequences [see Additional file 1].
Although GHN21 and GHN117 did not cluster with significant reliability with the AG recombinant reference sequences DDJ362 [GeneBank: AY521632] and DDJ364 [GeneBank: AY521633] even in the pol gene (Figure 1b), this can be explained with the recombination analysis done [see Additional file 1]. GHN21 and GHN117 both had fragments of CRF 02_AG strains in their sequences, with GHN117 having 5 as compared to one in GHN21 [see Additional file 1]. It will be impossible to make these inferences about the purity of GHN21 and GHN117, and the other GHN strains [see Additional file 1], without the RIP analysis.
Thus, the polymerase genes of HIV-1 strains from Ghana are made up of recombinants of several CRF 02_AG strains from Ghana, Senegal and Cameroon, but the clinical implications are unknown. A continuous surveillance of pol gene sequences from Ghana is needed to understand this evolutionary pattern.