We also compared the consensus sequences from each subtype with the consensus sequence for the B′ subtype to try and identify mutations that were unique to B′. This is shown in Fig. 3. Eight and nine distinct signature mutation sites unique to B′ were found in the p17 and V3 regions, respectively. Notably, these differences existed in almost all of the B′ samples, regardless of when and where the samples were collected, and thus they appear to be characteristic of the B′ subtype (supplemental Table S1 and Table S2).
The emergence of B′ was sudden and forced people to recognize within-subtype variety of HIV-1 at the nucleonic  or even antigenic [40,41] level. Although phylogenetically distinguishable, the difference between B′ and typical B subtype has never been clearly determined and many of the B′ sequences have merely been classified as belonging to the B subtype; thus, it took us a great deal of effort to collect a reliable set of B′ sequences. After retrieving and verifying this set, we found that B′ was mainly prevalent in south-east Asia and China. The majority of B′ sequences were found in Thailand, Myanmar and China, and sporadic incidences of B′ were found in other countries such as Malaysia, Singapore, India, Indonesia and Cambodia. This finding is consistent with many previously published epidemiology reports [2–11,42–47].
Each of the different datasets used in this study indicate a similar origin of B′ around 1985. Given the documented explosive epidemic of B′ is in 1988, the epidemic of B′ seems to have been established in about 3 years. Compared with other epidemics (e.g. the HIV-1 C subtype , HIV-1 B subtype in Haiti and USA [21,25]), the transmission of B′ was very fast. This is consistent with findings from earlier studies of the Asian HIV-1 epidemic situation, which suggest that B′ is closely associated with initial phase of the blood-born HIV-1 transmission in Asia, which involved the epidemic among IDUs in south-east Asia and China [3–11,42,47–50] and the epidemic in paid blood donors (PBDs) in China [11,43,46]. HIV is more efficiently transmitted by virus-polluted injecting equipment than other forms of transmission, because it enables the virus to bypass the body tissues and HIV can pass directly into the blood. This is the reason why IDUs and PBDs are the most vulnerable populations under the threat of HIV. In addition, because they may also pass on HIV infection sexually , creating a ‘critical mass’ of infections within sexual networks; even wherever the numbers of people injecting drugs are relatively small, their contribution to the overall HIV epidemic in a country can be considerable. Modeling shows that in situations wherein HIV has remained low for years despite low condom use, a sharp rise in HIV infection among drug injectors could ‘kick-start’ an HIV epidemic that may otherwise have taken many decades to develop . Our result highlights that an epidemic of HIV in IDUs can be established in an astonishingly short period.
In an epidemic that spreads rapidly through a population, the evolutionary rate of HIV is relatively low  and, thus, the diversity of virus is limited, indicating that the molecular character of the founder virus of the epidemic can be preserved on a comparatively large scale. We observed more conserved amino acid regions in B′ and CRF01_AE, which have been transmitted rapidly in Asia than in other subtypes. The signature sites of B′ we identified exist widely among the sequences, regardless of their sampling date and location, which provides insight into the molecular characteristics of the ancestor of B′. These results in turn confirmed our identification of B′ sequences as almost all of these sequences exhibited a majority of these motifs, in sharp contrast to the B subtype sequences (supplemental Table S1 and Table S2). Although only the p17 and V3 regions were analyzed, it is likely that additional signatures exist in other regions, and the amino acid difference between B′ and typical B subtype should be taken into consideration in epidemic investigation and vaccine design.
The complex drug traffic provides routes for various HIV strains to co-circulate in IDUs. As the drug-use and sexual networks intermix in the IDUs, the molecular epidemic situation in this group is changing. After less than a decade, the IDUs in Thailand, Myanmar, Malaysia and China experienced an increase of recombinant forms of HIV, and the dominant position of B′ in those populations had been replaced. The recombinant forms originate from two sources: the CRF01_AE that used to be prevalent in infections via sexual transmission expanded into the IDUs [5,40,41,47,54–56]; new recombinant forms that had been emerging in this group because of the co-circulation of B′ and different subtypes [12,15–17,45,54,57–61]. These new recombinant forms are playing increasingly important roles in the epidemic in Asia. In addition to the role played by B′ in the IDUs–FSWs (female sex workers) driven HIV-1 epidemic in Asia, the B′ remains a dominant subtype of HIV-1 prevalence in rural China as a consequence of illegal blood collection [11,43,46]. However, its impact is no longer restricted to former PBDs, as B′ was transmitted further into the general population through the established pattern of sexual and vertical transmission.
Our finding is the first in-depth investigation of the HIV-1 B′ subtype at a global level, which investigates both the genetic and transmission aspects, providing a valuable reference for HIV scientists, public health officials and HIV vaccine designers.
Explanation: Although our results indicate that B′ represents a variant of subtype B, for consistency with previously published research, and for clarity we refer to this variant as subtype B′ or B′ throughout the article.
The authors would like to thank Xiao Tong for helpful suggestions regarding data interpretation.
Authors contribution: R.Y., X.D., and S.R. conceived the original idea. X.D. and S.R. designed the experiment. X.D. collected the data and performed the genealogical and demographic analysis. S.R. developed the software and performed amino acid analysis. H.L. set up the analysis platform. X.D., S.R., R.Y., and Y.S. contributed to the writing of the article.
Conflicts of interest: The authors have declared that there is no conflict of interest.
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B' is always reported to be so distant from other B subtype sequences that its relation with B subtype and other HIV-1 subtypes seems obscure and it triggers the wildest guess about its origin. To illustrate the relationship between B' and other HIV-1 subtypes, we downloaded two reference sequences suggested by the Human Retroviruses and AIDS compendium for each HIV-1 subtype from the Los Alamos HIV database. Adding four B' sequences to the selected reference sequences, a full-length alignment was created. MrBayes, V3.1.2 was then used to build a reference tree. We ran two independent chains, sampled every 100th generation, and 250 samples of each run were discarded as burn-in. Our phylogenetic analysis of the full-length B'+ reference sequences alignment showed that although formed a monophyletic group supported by highest posterior value (P = 1), B' still has a closer association with B subtype than with other subtypes (Fig. 1).
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