Identification and Characterization of Two New HIV Type 1 Unique (B/C) Recombinant Forms in China

Abstract

Recombination was most important in the generation of new viral strains and in the increase of HIV diversity. There were more and more new HIV-1 strains. Not only circulating recombinant forms (CRFs) but also unique recombinant forms (URFs) have been reported around the world. CRF07_BC and CRF08_BC were the two predominant CRFs circulated in Yunnan Province, southwest China. In the present study, we identified two new HIV Type 1 unique (B/C) recombinant gorms in Yunnan Province by nucleotide sequencing in two halves of HIV genome. Although the genomic structures of the two B/C recombinants were different from previously identified CRFs (CRF07_BC and CRF08_BC) and URFs in Yunnan Pprovince, they have several common recombination sites with CRF07_BC and CRF08_BC.

Introduction

O ne of the most significant properties of the human immunodeficiency viruses is their high genetic diversity. HIV evolves at an extremely high rate because of its high mutation rate^1,2 and short generation time^3,4 in conjunction with a large population of productively infected cells within the host.⁵ According to phylogenetic analysis of circulating viral strains, HIV-1 is classified into three distinct groups (M, N, and O) and nine subtypes (A to D, F to H, J, and K) within group M, which is the major form responsible for the AIDS pandemic.

Genetic recombination was considered as one of the major mechanisms for HIV to acquire sequence diversity. It was most important in the generation of new viral strains and in the increase of HIV diversity. Now, at the Los Alamos HIV database web site (http://hiv-web.lanl.gov/CRFs/CRFs.html), more and more circulating recombinant forms (CRFs) have been recognized. Meanwhile multiple unique recombinant forms (URFs) have been reported in many regions around the world.

Yunnan Province was thought to be an epicenter of HIV-1 epidemic in China where subtype B and subtype C were co-circulating in the early 1990s.^6,7 The co-circulation of subtype B and subtype C had resulted in the emergence of two circulating recombinant forms, CRF07_BC and CRF08_BC.^8,9.Meanwhile, multiple unique B/C recombinant forms have been detected in Yunnan Province. In the present study, we have found two new HIV Type 1 unique (B/C) recombinant forms in Yunnan Province, China.

Materials and Methods

Plasma samples were obtained from two HIV-1 infected individuals living in Yunnan Province. They gave written consent for the use of their blood samples for research and publication. The detailed information of the two patients is listed in Table 1.

Table 1.

Information of Samples Used in this Study

Patient	Age	Gender	Date sample collected	Transmission	Viral load	CD4+T cell count	Antiretroviral treatment
309	34	male	2007/09/03	heterosexual behavior	2,504	NA^a	D4T+3TC+NVP
341	35	male	2007/11/17	injecting drug using	17,963	319	D4T+3TC+NVP

NA, not available.

Viral RNA was extracted from the stored plasma samples using high pure viral RNA kit (Roche) according to the manufacturer's protocol. For plasma sample 341 containing 17,963 RNA copies/ml, RNA was directly extracted. Sample 309 with low viral loads (2504 copies/ml) was concentrated by centrifugation at 23,000 g for 1 h at 4°C prior to the same extraction procedure.¹⁰ RNA was collected from the spin columns and dissolved with Elution Buffer in a final volume of 50 μl. The extracted RNA was immediately reverse transcribed.

Reverse transcription of RNA to single-strand cDNA was performed using the SuperScript III according to the manufacture's instruction (Invitrogen, Carlsbad, CA). We used reverse primers 07Rev8 (5’-CCTARTGGGATGTGTACTTCT GAACTT-3’; nt 5,193–5219; HXB2 numbering) and 1.R3.B3R (5’-ACTACTTGAAGCACTCAAGGCAAGCTTTATTG-3’; nt 9,611–9,642;HXB2 numbering) to synthesize cDNA that served as templates for amplification of 4.4 kb 5’-half viral genome fragment and 4.7 kb 3’-half viral genome fragment. The RT reaction volume was 20 μl; 8 μl RNA, 1 μl dNTP(10 mM),0.25 μl Primer(20 pmol/μl), 3.75 μl DNase/RNase Free Water were mixed and incubated for 5 min at 65°C to denature the secondary structure of the RNA template. The tube was transferred to an ice cooler and incubated for at least 1 min. The contents of the tube were collected by brief centrifugation. Then 4 μl 5X First-Strand buffer, 1 μl DTT (100 mM), 1 μl RNaseOUT(40 U/μl), 1 μl Superscript III(200 U/μl) were added to the above reaction mixture. Tubes were then incubated at 50°C for 1 h. Then 1 μl SuperScript III was added to the mixture and the tubes were incubated for 1 h at 55°C for increasing the cDNA yield and transcription of difficult template. Finally, the reaction was inactivated and the RNA template digested. The cDNA was frozen at −80°C until further analysis or used immediately for PCR amplification.

Near-endpoint PCR was performed to generate 5’- and 3’-half viral genome fragments using specific primers. In a 20 μl reaction volume, PCR amplifications were performed in the presence of 1X High Fidelity Platinum PCR buffer, 2 mM MgSO4, 0.2 mM of each dNTP, 0.2 μM of each primer, and 0.025 U/μl Platinum Taq High Fidelity polymerase (Invitrogen). First round PCR primers for amplifying the 5’-half genome were 1.U5.B1F and 07Rev8. PCR reactions were performed in MicroAmp 96-well reaction plates as the following: one cycle of 94°C for 2 min, and three cycles of a denaturing step of 94°C for 30 s, an annealing step of 55°C for 1 min, an extension step of 68°C for 5 min, followed by 32 cycles of a denaturing step of 94°C for 15 s, an annealing step of 55°C for 30 s, an extension step of 68°C for 4.5 min, and a final extension of 10 min at 68°C. 1 μl first-round PCR product was added to the second-round PCR to generate the 5’-half genome with the primers Upper1A and Rev11. The second-round PCR conditions were the same as the first round. For amplification of the 3’-half genome, we used primers 07For7 and 2.R3.B6R as the first-round primers, and then used primers VIF1 and Low2c as the second-round primers. PCR conditions were the same as for amplification of the 5’-half genome. Second-round PCR products were run on a 1% TAE agarose gel to check for positive amplification. For one sample, two positive 5’-half genomes and two positive 3’-half genomes were chosen and subjected to direct sequencing. The nearly full-length genome sequence was assembled by overlapping the sequences of the two half genome fragments and merging them into one sequence. The primers for amplification of 3’- and 5’-half genome were available upon request.

The two nearly full-length sequences were aligned with the HIV-1 reference strains (http://www.hiv.lanl.gov/content/hivdb/SUBTYPE_REF/align.html) using Clustal W. Phylogenetic tree was constructed by the neighbor-joining method based on Kimura's two-parameter distance matrix with 1000 bootstrap replicates using Mega 4.1. Bootscanning analysis was performed by the neighbor-joining method to assess the structures of the two nearly full-length HIV-1 sequences and recombination breakpoints, using subtype B strain RL42, subtype C strain 95IN21068, and subtype J strain J_97DC_KTB147 as references. Analysis was set by a sliding window of 200 nucleotides (nt) advanced in 20 nt increments. To further define the precise boundaries of recombination, an informative site analysis was performed with reference strains as previously described.¹¹ The insertion segments were used to build phylogenetic trees via the neighbor-joining method with bootstrapping to confirm the origin of the different segments. Recombinant HIV-1 Drawing Tool (http://www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html) was used to elucidate the vivid structures of the newly HIV Type 1 unique (B/C) recombinant forms.

Results

The assembled nearly full-length genome sequences of samples 309 and 341 were analyzed using the HIV sequence Locator (http://www.hiv.lanl.gov/content/sequence/LOCATE/locate.html). Both the sequences encoded intact canonical genes (gag-pol-vif-vpr-tat-rev-vpu-env-nef) except for a stop codon in rev2 which was very common in subtype C viruses.

Phylogenetic analysis of the nearly full-length genomes demonstrated that the two patients 309 and 341 clustered with CRF07_BC, CRF08_BC, and subtype C reference strain C.IN.95IN21068 (Fig. 1). Meanwhile, they presented the minimum sequence distances with subtype C reference strain C.IN.95IN21068 that was the backbone of CRF07_BC and CRF08_BC. Interestingly, they were distantly related to the other three HIV-1 subtype C reference strains distributed in African countries and Brazil.

FIG. 1.

Neighbor-joining phylogenetic tree of nearly full-length genomes of samples 309 and 341. The neighbor-joining tree is constructed with the reference strains for HIV group M (subtypes A–D, F–H, J, and K) and circulating recombinant forms (CRFs) using MEGA 4.1. Values on the branches represent the percentage of 1000 bootstrap replicates and bootstrap values over 70% are shown in the tree. The two URFs_BC are marked with a solid circle. The scale bar indicates 2% nucleotide sequence divergence.

Bootscanning analysis was performed to elucidate the structures of the two nearly full-length HIV-1 sequences and recombination breakpoints. Bootscanning plots of the two nearly full-length genomes using the representative reference strains revealed that evidence of possible recombination events were detected (Fig. 2). The two genomes were composed of subtype C and several small segments of subtype B. It was very similar to both CRF07_BC and CRF08_BC.

FIG. 2.

Bootscanning analysis of nearly full-length sequences of two samples (A: 309, B: 341). The bootstrap values are based on 500 replicates using neighbor-joining methods. The reference strains are shown at the bottom.

Informative site analyses were performed to further scrutinize and define the recombination breakpoints. According to informative site analysis, there were two small subtype B segments within sample 309 and one small subtype B segments within sample 341 respectively. Both sample 309 and sample 341 used subtype C as a backbone that was identical to that of bootscanning analyses. The genomic maps were constructed using the Recombinant HIV-1 Drawing Tool according to informative sites (Fig. 3). Recombinant breakpoints were indicated in relation to HXB2. Sample 309 displayed two small subtype B segments as follow: one in pol (2882–3109), and one in vpr-tat1-rev1-vpu (5827–6190). It was similar that one small subtype B segment, in gag (1204–1840), was detected in sample 341. One common subtype B segment (nt 1204–1840) located in gag p24 was shared between sample 341 and CRF08_BC. Additionally, one common subtype B segment (nt 2882–3109) located in pol p51 appeared in both sample 309 and CRF08_BC. Moreover, one segment (nt 5827–6190) located in vpr-tat1-rev1-vpu was very similar to one segment of CRF07_BC. Interestingly, one subtype B segment appeared in both CRF07_BC and CRF08_BC at the right end of genomes within nef gene while it did not occur in samples 309 and 341.

FIG. 3.

Schematic representation of the mosaic structures of the nearly full-length genomes of two B/C recombinants (A: 309, B: 341). The genomic maps were constructed using the Recombinant HIV-1 Drawing Tool available at the HIV Database. Subtype C segments and subtype B segments that compose HIV-1 genome appear as differently colored regions in the map. Recombinant breakpoints were identified and positions were indicated in relation to HXB2.

The phylogenetic trees of the subtype C insertion segments within sample 309 and 341 indicated that all the subtype C segments were closely related to subtype C reference strain from India with high bootstrap values (data not shown). All the subtype B segments within sample 309 and 341 clustered with subtype B reference strains (Fig. 4). But only the segment within 341 showed that it originated from subtype B-Thai reference RL42 from China. The other subtype B segments within sample 309 had uncertain origins because their lengths were too short to get high bootstrap values.

FIG. 4.

Phylogenetic relationship of subtype B segments within samples 309 and 341 to reference strains of subtype A, B, C, and J. The neighbor-joining tree is constructed using MEGA 4.1. Values at the nodes indicate the percent bootstraps in which the cluster to the right was supported. Bootstraps of 70% and higher only are shown. The scale bars are shown at bottom. (A) and (B) Phylogenetic trees of the first and second subtype B segments within sample 309. (C) Phylogenetic tree of subtype B segment within sample 341.

Discussion

In the present study, we identified two new HIV Type 1 unique (B/C) recombinant forms in Yunnan Province, China. The genomic structures of the two B/C recombinants were different from previously identified CRFs (CRF07_BC and CRF08_BC) and URFs in Yunnan province. McCutchan et al. ¹² reported that CRF07_BC and CRF08_BC shared five precise B/C boundaries based on the precise mapping of recombinant breakpoints. The two B/C URFs also have several similar recombination sites with CRF07_BC and CRF08_BC. Moreover, the subtype C segments used as backbone of the two URFs were closely related to those of Indian origin and distinct from clade C viruses isolated in South America and Africa which was identical to that of CRF07_BC and CRF08_BC.

Up to now, several papers have reported that multiple forms of URFs are present in Yunnan Province, especially in Dehong Prefecture.^13
–15 The patients whose plasma samples were used in this study were also living in Dehong Prefecture. The two new UFRs identified in this study reconfirmed the high prevalence of multiple forms of UFRs in Dehong Prefecture near the Myanmar border.^14,16 Most of previously identified UFRs were detected in IDUs, whereas in our study, one URFs was detected in patient 309 who was infected by heterosexual behavior. It suggested that the distribution of multiple forms of UFRs was not constrained to particular people with special high risk. In other words, not only IDUs but also people with other high risks were exposed to various UFRs to some extent.

HIV recombinants are an important and dynamic component of the global epidemic. The emergence of multiple forms URFs will undoubtedly baffle the development of effective vaccine. In Yang's article, he claimed that multiple forms of HIV-1 BC recombinants emerged in Yunnan Province with an on-going trend.¹⁴ The two new BC URFs identified in this study had different genomic structures from all URFs reported in other papers. This obviously added the variety of HIV genome in Yunnan Province and may complicate the design of HIV vaccine. Although multiple forms of URFs were present in Yunnan Province, there was a lack of sufficient sequence data on the HIV-1 nearly full-length genome in this region. Most sequences obtained in this area were partial regions of the HIV-1 genome. It was necessary to increase the number of HIV-1 nearly full-length genomes to better elucidate the genotyping, prevalence, and evolution of HIV-1 in this region.

Sequence Data

Sequences have been submitted to GenBank under accession numbers: HM776938 and HM776939.

Footnotes

Acknowledgments

This work was supported by the National Key S&T Special Projects on Major Infectious Diseases (Grant Nos.2008ZX10001-004, 2008ZX10001-012). We also acknowledge Dr. Feng Gao at the Duke Human Vaccine Institute, Duke University Medical Center, for providing the technical support in achieving nearly full-length HIV genome.

Author Disclosure Statement

No competing financial interests exist.

References

Preston

, Poiesz

, Loeb

. Fidelity of HIV-1 reverse transcriptase. Science, 1988; 242:1168–1171.

Mansky

, Temin

. Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase. J Virol, 1995; 69:5087–5094.

, Neumann

, Perelson

, Chen

, Leonard

, Markowitz

. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature, 1995; 373:123–126.

Wei

, Ghosh

, Taylor

et al. Viral dynamics in human immunodeficiency virus type 1 infection. Nature, 1995; 373:117–122.

Chun

, Carruth

, Finzi

et al. Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature, 1997; 387:183–188.

Graf

, Shao

, Zhao

, Seidl

, Kostler

, Wolf

, Wagner

. Cloning and characterization of a virtually full-length HIV type 1 genome from a subtype B -Thai strain representing the most prevalent B-clade isolate in China. AIDS Res Hum Retroviruses, 1998; 14:285–288.

Luo

, Tian

, Hu

, Kai

, Dondero

, Zheng

. HIV-1 subtype C in China. Lancet, 1995; 345:1051–1052.

Piyasirisilp

, McCutchan

, Carr

et al. A recent outbreak of human immunodeficiency virus type 1 infection in southern China was initiated by two highly homogeneous, geographically separated strains, circulating recombinant form AE and a novel BC recombinant. J Virol, 2000; 74:11286–11295.

, Graf

, Zhang

et al. Characterization of a virtually full-length human immunodeficiency virus type 1 genome of a prevalent intersubtype (C/B’) recombinant strain in China. J Virol, 2000; 74:11367–11376.

10.

Salazar–Gonzalez

, Bailes

, Pham

et al. Deciphering human immunodeficiency virus type 1 transmission and early envelope diversification by single-genome amplification and sequencing. J Virol, 2008; 82:3952–3970.

11.

Robertson

, Hahn

, Sharp

. Recombination in AIDS viruses. J Mol Evol, 1995; 40:249–259.

12.

McCutchan

, Carr

, Murphy

et al. Precise mapping of recombination breakpoints suggests a common parent of two BC recombinant HIV type 1 strains circulating in China. AIDS Res Hum Retroviruses, 2002; 18:1135–1140.

13.

Yang

, Kusagawa

, Zhang

, Xia

, Ben

, Takebe

. Identification and characterization of a new class of human immunodeficiency virus type 1 recombinants comprised of two circulating recombinant forms, CRF07_BC and CRF08_BC, in China. J Virol, 2003; 77:685–695.

14.

Yang

, Xia

, Kusagawa

, Zhang

, Ben

, Takebe

. On-going generation of multiple forms of HIV-1 intersubtype recombinants in the Yunnan province of China. AIDS, 2002; 16:1401–1407.

15.

Qiu

, Xing

, Wei

et al. Characterization of five nearly full-length genomes of early HIV Type 1 Sstrains in Ruili City: Implications for the genesis of CRF07_BC and CRF08_BC circulating in China. AIDS Res Hum Retroviruses, 2005; 21:1051–1056.

16.

Takebe

, Motomura

, Tatsumi

, Lwin

, Zaw

, Kusagawa

. High prevalence of diverse forms of HIV-1intersubtype recombinants in Central Myanmar: Geographical hot spot of extensive recombination. AIDS, 2003; 17:2077–2087.