Identification of CRF65_cpx sequences from the Los Alamos HIV Sequence Database
As shown in the neighbor-joining phylogenetic tree (Figure 1a), we identified a CRF65_cpx cluster located inside subtype C clade with high bootstrap confidence (99%). Figure 1b shows that some subtype C and 1 B/C recombinant form sequences were intermingled with CRF65_cpx sequences in this cluster, indicating they should be classified as CRF65_cpx sequences. After the removal of duplicated sequences, a total of 32 CRF65_cpx pol sequences were obtained. Of note, almost half of the 32 CRF65_cpx sequences (n = 32 [46.9%]) were misclassified—14 sequences were misclassified as subtype C and 1 sequence was misclassified as B/C recombinant form (Table 1).
Expansion of CRF65_cpx in China
Figure 2 illustrates the geographic distribution of CRF65_cpx in China. In the present study, we found that the CRF65_cpx strains were detected in seven provinces that are geographically far from each other: southern provinces (Yunnan and Guangdong), northern provinces (Jilin and Heilongjiang), eastern province (Anhui), and central provinces (Beijing and Hebei). Yunnan Province and Beijing were the two areas where the majority (71.9%, 23/32) of CRF65_cpx strains were detected. Hebei, Anhui, Guangdong, Jilin, and Heilongjiang provinces collectively harbored one-quarter (25%, 8/32) of CRF65_cpx strains, and the geographic origin of one strain (accession number: KP698506) was not reported. Although the number of CRF65_cpx strains detected in these five provinces was small, the strains were all detected in recent years, which provided evidence for onward transmission of CRF65_cpx. The detection of CRF65_cpx strains in an increasing number of provinces suggested the rapid expansion of this variant in China.
As shown in the ML tree (Figure 3), all CRF65_cpx sequences formed a monophyletic cluster with a high bootstrap value of 1000. CRF65_cpx sequences from Yunnan Province were located near the root of this cluster, and the remainder of CRF65_cpx sequences from the other six provinces formed an MSM group and positioned inside the CRF65_cpx cluster. To our surprise, Yunnan CRF65_cpx sequences were mainly derived from a heterosexual risk group (84.6%, 11/13), whereas the CRF65_cpx sequences in other provinces were almost exclusively derived from an MSM population (94.4%, 17/18). These results suggested that CRF65_cpx initially originated among heterosexuals in Yunnan Province and subsequently spread to MSM in other provinces, which was in line with our previous study observations . The ML phylogenetic tree also showed that all MSM group sequences (n = 19) were classified into three different clusters (Figure 3). Cluster I included two Hebei sequences and one Heilongjiang sequence. Cluster II involved Hebei, Beijing, and Guangdong sequences and one sequence whose geographic origin was not available. We found one larger cluster (cluster III) that grouped 12 sequences from four provinces: nine from Beijing, one from Hebei, one from Jilin, and one from Anhui. The inclusion of sequences from different provinces in all three MSM clusters illustrated the active transmission of CRF65_cpx among MSM, regardless of a large geographic distance.
Natural presence of the V179D and K103R/V179D mutations in CRF65_cpx strains
Among the 32 patients harboring CRF65_cpx strains, the great majority (96.9%, 31/32) were treatment naive and the remaining one patient was treatment experienced. We surprisingly found NNRTI-resistance mutations were present in all of the 32 patients. With the exception of one patient who had NNRTI-resistance mutation V179E, the other 31 patients harbored V179D mutation, which resulted in the natural presence of V179D in CRF65_cpx strains (Table 1). All V179D mutations were encoded by codon GAT, whereas the V179E mutation was encoded by codon GAA. A single T-to-A transversion at the third position of codon 179 in CRF65_cpx may be responsible for the emergence of V179E mutation in one patient. Obviously, V179D mutation was a signature mutation in CRF65_cpx patients due to a strong founder effect.
Variants emerging at RT position 179 were highly heterogeneous and most often accompanied by other DRMs. V179D occurred alone in 22 patients, accounting for nearly 70% of all patients with CRF65_cpx. Surprisingly, the combination of V179D and K103R, conferring intermediate resistance to EFV and NVP, was detected in seven treatment-naive patients, also indicating the natural presence of K103R/V179D in CRF65_cpx strains (Table 1). We also found that two polymorphic accessory mutations, K103R and V179E, occurred simultaneously in one patient. In addition, V179D mutation was accompanied by E138A mutation in one patient, and these two mutations resulted in low-level resistance to ETR and RPV. However, the effects of combinations of K103R plus V179E and E138A plus V179D on NNRTI susceptibility are still not clear, which suggested that these two mutation patterns deserve further investigation. V179D, Y181C, and H221Y were detected in the one treatment-experienced patient.
We next sought to investigate the distribution of the K103R/V179D mutation in different risk groups and geographic areas. As shown in Figure 3, none of Yunnan sequences harbored the K103R/V179D mutation, regardless of the risk group. Of note, all seven CRF65_cpx sequences harboring K103R/V179D mutation were exclusively present in MSM cluster III. Moreover, sequences harboring K103R/V179D mutation accounted for more than half (58.3%, 7/12) of all cluster III sequences, demonstrating the natural presence of the K103R/V179D mutation in this larger MSM cluster. With respect to geographic distribution, K103R/V179D mutation was detected in four provinces: four patients from Beijing, one patient from Hebei, one patient from Jilin, and one patient from Anhui.