Identification of a Novel HIV Type 1 CRF01_AE/B′/C Recombinant Isolate in Sichuan,China

Recombination between HIV-1 subtypes is one of the main mechanisms which contribute to the genetic diversity and complexity of HIV-1 genomes. To date more than 75 circulating recombinant forms (CRFs) and innumerable unique recombinant forms (URFs) have been reported worldwide (Los Alamos National Laboratory www.hiv.lanl.gov/content/sequence/HIV/CRFs/CRFs.html). Recombination is frequently found in many places in the world where different subtypes and CRFs co-circulate, so-called “recombination hotspots,” continuously giving rise to URFs. ¹ According to the national cross-sectional study of HIV molecular epidemiology in 2006, HIV-1 genotypes A, B, B′, C, G, and CRF01_AE, CRF02_AG, CRF06_cpx, CRF07_BC, CRF08_BC, and other unique circulating forms (URFs) have been detected in China. ² Therefore, the opportunities for the emergence of various novel recombinant forms are greatly created by co-circulation and dual infection of multiple HIV-1 genotypes (subtypes/sub-subtypes, CRFs, and URFs) in HIV-1 high-risk groups of the same region in China, such as JL.RF01, ³ CRF65_cpx, ⁴ and CRF78_cpx. ⁵

In this study, we separated a recombinant strain of HIV-1 (XC2014EU01) from a 52-year-old male, who was infected through heterosexual sex in Sichuan, China, and presented with CD4⁺ T cell count 366 cells/μl and viral load 93,500 copies/ml on March 13, 2014. The informed consent was signed before the sample collection. This study was reviewed by the Institutional Research Ethics Community of the Chinese Center for Disease Control and Preventive (No. 201334).

The viral RNA was extracted from the virus isolated from the peripheral blood mononuclear cells using QIAamp^® Viral RNA Mini Kit (QIAGEN) and then transcribed into cDNA. Through gradient dilution of cDNA, the near full-length genome (NFLG) was amplified by nested polymerase chain reactions (nest-PCRs). ⁶ The following PCR conditions for both of the two rounds were used, including an initial denaturation 94°C for 2 min, followed by 35 cycles of 94°C for 30 s, 62°C for 30 s, and 68°C for 4 min, followed by a final extension at 68°C for 10 min. The positive PCR products were purified and sequenced. The sequences were spliced and assembled by Sequencher v4.9.

The NFLG sequence of XC2014EU01 was aligned against all known HIV-1 group M reference sequences representing subtypes/sub-subtypes and CRF relative to our study in China, obtained from the Los Alamos HIV Sequence Database (www.hiv.lanl.gov/content/sequence.html). Nucleotide sequences were first aligned by using Clustal W and then adjusted manually using BioEdit. ⁷ Phylogenetic and subregion tree analyses were implemented in MEGA 5.0, using the neighbor-joining method based on the Kimura two-parameter model with 1,000 bootstrap replication test. ⁸ Recombination breakpoints were determined using RIP, jpHMM (www.hiv.lanl.gov), and Simplot software v3.5.1. ⁹

The NFLG sequence of XC2014EU01 was obtained with the length of 8,891 bp (relative to the HXB2 nucleotide numbering system: positions 635 to 9613). Phylogenetic analyses revealed that the NFLG sequence of XC2014EU01 clustered with CRF01_AE and CRF78_cpx with 0.94 bootstrap values, but formed a monophyletic branch distinct from them (Fig. 1). Recombination analyses show that XC2014EU01 was composed of CRF01_AE, C, and B′, with two C segments and one B′ segment inserted in CRF01_AE backbone (Fig. 2A). The breakpoints relative to HXB2 nucleotide positions were defined by HIV Sequence Locator (www.hiv.lanl.gov/content/sequence/LOCATE/locate.html). The unique mosaic structure of the recombinant virus XC2014EU01 is as follows: I_{CRF01_AE} (790–1,176 nt), II_C (1,197–2,743 nt), III_B (2,827–3,067 nt), IV_C (3,139–4,697 nt), and V_{CRF01_AE} (4,913–9,613 nt) (Fig. 2B). To determine the parental origins of each segment, subregion tree analyses of XC2014EU01 were constructed with MEGA 5.0 (Fig. 3). Subregion tree analyses demonstrated that two subtype C segments clustered with India C lineages (95IN21068) with 0.705 and 0.977 bootstrap values, respectively (Fig. 3II, IV), which was also the parental origin of CRF07_BC and CRF08_BC. The subtype B segment clustered with China subtype B lineage (RL42.U71182), with 0.887 bootstrap values (Fig. 3III), so the parental origin of the B segment was most likely from China subtype B′ lineage. Although the two CRF01_AE segments originated from Thailand CRF01_AE (Fig. 3I, V), they were not related to the seven distinct phylogenetic clusters of CRF01_AE epidemic in China. ¹⁰

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FIG. 1.

Phylogenetic tree analyses of the NFLG sequence of XC2014EU01. It was constructed with MEGA 5.0 using the neighbor-joining method with the Kimura two-parameter model and 1,000 bootstrap replication test. XC2014EU01 clustered with the CRF01_AE and CRF78_cpx reference sequences, but formed a distinct monophyletic branch distantly related to the reference sequences. A solid triangle (▲) marks XC2014EU01, which was also displayed as a bold line throughout the article. Bootstrap values (>0.7) were showed at the corresponding nodes. NFLG, near full-length genome.

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FIG. 2.

Recombinant analysis of XC2014EU01. (A) Posterior probabilities analysis of the recombinant calculated by jpHMM. Inset: the different symbols represent different references. (B) Genome map of the NFLG sequence of XC2014EU01 (based on HXB2 numbering). The mosaic map was generated using the Recombinant HIV-1 Drawing Tool (www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html). Four unique recombinant breakpoints divided the NFLG into five segments.

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FIG. 3.

Subregion tree analysis of the novel identified XC2014EU01. Phylogenetic trees for five segments (I–V) defined by bootscanning plot analysis were constructed with MEGA 5.0 using the neighbor-joining method with the Kimura two-parameter model and 1,000 bootstrap replication test. A solid triangle (▲) marks each segment of XC2014EU01, which was displayed as a bold line in each phylogenetic tree. The subtypes of all sequences were pointed in the right side of the trees. The scale bars were shown at the bottom of each tree. Bootstrap values (>0.7) were showed at the corresponding nodes.

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The viral NFLG of XC2014EU01 kept the CRF01_AE parental backbone with three subtype B′ and C segments inserted into gag and pol genes, respectively. Compared with XC2014EU01, the recombinant form of JL.RF01 identified that there were six subtype B′ and C segments inserted into gag and pol genes of CRF01_AE parental backbone. ³ The recombinant form of XC2014EU01 was also different from CRF65_cpx and CRF78_cpx. ^4,5 The mosaic structure of CRF65_cpx recombinant genome was composed of fourteen subtype C, B′, and CRF01_AE segments. ⁴ The recombinant form XC2014EU01 was similar to CRF78_cpx, both of which were three subtype C and B segments inserted in the CRF01_AE parental backbone. ⁵ The majority of gag and pol genes of XC2014EU01 were subtype C segments with a small subtype B′ segment (322 bp) inserted in p51gene, whereas there were three small subtype C and B segments inserted in gag and pol gene of the CRF01_AE parental backbone in CRF78_cpx. ⁵

In this study, XC2014EU01 was identified as CCR5-tropic on Ghost cells consistent with partly CRF01_AE found in China. It was largely due to whole env sequence of XC2014EU01 belonged to CRF01_AE. ¹¹ Moreover, the neutralizing activity depended critically on HIV-1 envelope protein. ¹² So the plasma of XC2014EU01 infected person was detected for neutralizing activities against 15 HIV pseudotyped viruses in TZM-bl cells (not shown). These pseudoviruses include 12 pseudovirus of global Panel, 2 Tier 1 pseudoviruses of MW965 and SF162, and 1 murine retrovirus of SVA-MLV (murine retrovirus Env-pseudovirus) as unrelated negative control. The 50% inhibitory dose (ID₅₀) to pseudovirus infection was calculated in times of dilution, and ID₅₀ < 20 was counted as negative. VRC01 at 200 μg/ml was detected as the control.

For SVA-MLV control pseudovirus, VRC01 and the plasma of the patient (XC2014EU01) showed negative neutralizing results (<20). These results verified patient free of antiretrovirus drug therapy. For the other 14 pseudoviruses detected, VRC01 showed broad neutralizing activity, which neutralized 13 to 14 of the 14 pseudoviruses (neutralizing breadth >90%), consistent with other report. ¹³ The plasma of XC2014EU01 showed media neutralizing activity, which neutralized 10 to 12 of the 14 pseudoviruses (neutralizing breadth 70%–90%). The neutralizing potency of the plasma (XC2014EU01) was 69.78 lower than 195.59 of VRC01, without producing the broad-neutralizing antibodies.

The emergence of the novel CRF01_AE/B′/C recombinant suggested the continued generation of new recombinant strains in high-risk populations in Sichuan. XC2014EU01 was still identified as CCR5-tropic, and the plasma of XC2014EU01 infected person did not have the broad neutralizing activity. The emergence of XC2014EU01 may indicate the increasing complexity of the HIV-1 epidemic among high-risk populations in Sichuan. And whether the recombination may confer selective advantages forming circulating clusters over parental viruses remains to be investigated.

Sequence Data

The NFLG sequence of XC2014EU01 has been submitted to GenBank with the accession number KX353919.