Near Full-Length Genomic Characterization of a Novel HIV-1 Unique Recombinant (CRF55_01B/CRF07_BC) from a Malaysian Immigrant Worker in Zhejiang,China

Human immunodeficiency virus type 1 (HIV-1) shows extremely high level of genetic variation. It was reported that circulating HIV-1 strains differed from one another by 20% or more in relatively conserved regions and by up to 35% in envelope regions. ^1,2 This variation is growing over time. The extensive genetic variation of HIV-1 is due to high replication rate, error-prone reverse transcriptase, and, more importantly, recombination, which is considered to be a vital mechanism to generate novel HIV-1 variants. ^{3 –5} To date, HIV-1 has been divided into four groups, including M, N, O, and P. Among them, M group is further divided into 9 subtypes and 76 circulating recombinants forms (CRFs) (www.hiv.lanl.gov/components/sequence/HIV/search/search.html Accessed on July 23, 2016). Moreover, increasing number of unique recombinant forms have also been reported recently. ^6,7

As China is continuously open to the world, people from all around the world can come to China freely for business, travel, study, etc. This can facilitate diverse social and cultural exchange on one hand, but on another hand, it may potentially increase the transmission and spread of various diseases, including HIV-1/AIDS. Therefore, it is important and necessary to monitor HIV-1 infection status and viral dynamics among cross-border populations.

In the present study, plasma was collected from an HIV-1-positive patient (ZJCIQ15005) in March 2015 with informed consent. Epidemiological survey data showed that the patient was a 35-year-old male and a Malaysian citizen who came to Zhejiang, China, as an immigrant worker. Unfortunately, he refused to disclose background information related to infection time, route, and high-risk behaviors involved. The near full-length genome (NFLG) of ZJCIQ15005 (8974 bp) was amplified using primer sets and parameters described previously. ^8,9 Polymerase chain reaction products were purified and sequenced by an ABI 3730XL sequencer using BigDye terminators (Applied Biosystems, Foster City, CA). A BLAST search was performed to exclude the possibility of cross-contamination.

NFLG sequence of ZJCIQ15005 was aligned together with reference sequences obtained from the HIV databases (www.hiv.lanl.gov) using the Clustalx 1.81 program. ¹⁰ The genetic distances between HIV-1 sequences were calculated using Kimura's two-parameter model. ¹¹ Phylogenetic tree was generated using the neighbor-joining method implemented in MEGA 6 and then plotted using the TreeView software. As shown in Figure 1, the ZJCIQ15005 sequence clustered with CRF55_01B reference sequences but set up a distinct branch (Fig. 1).

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FIG. 1.

Phylogenetic analysis of the NFLG nucleotide sequence of the ZJCIQ15005 isolate. The neighbor-joining phylogenetic tree was constructed based on the NFLG nucleotide sequences of HIV-1 isolates using the MEGA 6 software package. All the reference strains were retrieved from the Los Alamos National Laboratory HIV Sequence Database. The ZJCIQ15005 isolate was labeled with a black solid circle in the highlighted branch. Bootstrap analysis was performed with 1,000 replications, and only bootstrap values >70% are shown. The scale bar represents 5% genetic distance. HIV-1, human immunodeficiency virus type 1; NFLG, near full-length genome.

To analyze the recombination form of ZJCIQ15005, the sequence was submitted to RIP (www.hiv.lanl.gov/content/sequence/RIP/RIP.html) using all default settings except the window size of 300 as described previously. ⁸ The result revealed a recombination form consisting of CRF55_01B and CRF07_BC, with an obvious break point between positions 5,500 and 7,500 approximately (Fig. 2).

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FIG. 2.

RIP analysis for ZJCIQ15005. Similarity distance analysis was performed using RIP from the Los Alamos National Laboratory HIV Database with default setting except for the window size of 300. RIP, Recombinant Identification Program.

Bootscan analysis was subsequently carried out using SimPlot (version 3.5.1) to position the recombination break points. The query sequence ZJCIQ15005 was bootscanned with reference sequences (CRF55_01B and CRF07_BC as parents, subtype G as outgroup) using all default settings. Bootscan results showed that ZJCIQ15005 was composed of 12 interlaced mosaic segments corresponding to CRF55_01B (I, III, V, VII, IX, and XI) and CRF07_BC (II, IV, VI, VIII, X, and XII), respectively (Fig. 3). All CRF55_01B segments were well confirmed by phylogenetic trees with bootstrap values between 88 and 100 (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/aid). For CRF07_BC segments, all of them were clustered with CRF07_BC reference strains, although the bootstrap value of phylogenetic clades was not high for segments II, IV, X, and XII, which seemed to be due to the short length of these segments (less than 200 bp). However, all larger segments were clustered with CRF07_BC reference strains with very high bootstrap values (Supplementary Fig. S1).

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FIG. 3.

Bootscan plots of ZJCIQ15005 using CRF55_01B, CRF07_BC, and subtype G as subtype references.

The break point positions were referred to the HXB2 coordinates and located by the HIV Sequence Locator (www.hiv.lanl.gov/content/sequence/LOCATE/locate.html). Therefore, the genome of the new recombinant ZJCIQ15005 was as follows: CRF55_01B (613–4,662 nt), CRF07_BC (4,663–4,787 nt), CRF55_01B (4,788–5,225 nt), CRF07_BC (5,226–5,295 nt), CRF55_01B (5,296–5,580 nt), CRF07_BC (5,581–5,672 nt), CRF55_01B (5,673–6,321 nt), CRF07_BC (6,322–8,414 nt), CRF55_01B (8,415–8,874 nt), CRF07_BC (8,875–9,030 nt), CRF55_01B (9,031–9,389 nt), and CRF07_BC (9,390–9,594 nt). The mosaic map of ZJCIQ15005 was generated using the Recombinant HIV-1 Drawing Tool (www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html) (Fig. 4).

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FIG. 4.

Genomic map of ZJCIQ15005. The break point positions referred to HXB2 coordinates located by the HIV Sequence Locator (www.hiv.lanl.gov/content/sequence/LOCATE/locate.html) and the mosaic map were generated using the Recombinant HIV-1 Drawing Tool (www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html).

The recombinant ZJCIQ15005 was discovered from a comprehensive HIV-1 molecular epidemiologic study among cross-border populations in Zhejiang province of China. Besides this CRF55_01B/CRF07_BC recombinant, we also detected CRF02_AG, CRF01_AE/07_BC, CRF55_01B, CRF03_AB, CRF13_cpx, CRF64_BC, and CRF65_cpx recombinant viruses, all of which were relatively new and rare for the Chinese populations (data not shown). Therefore, a continuing HIV-1 molecular epidemiologic investigation that focuses on cross-border populations is necessary to characterize their potential recombination and transmission, which may help to understand the dynamics of the HIV-1 epidemic.

In summary, we characterized an NFLG sequence from an immigrant worker and identified a novel HIV-1 recombinant, consisting of different recombinant break points between CRF55_01B and CRF07_BC strains. Understanding the properties of these newly emerging inter-CRF recombinants may provide a better comprehensive understanding to incorporate in the design of vaccines and other effective prevention measures to control and limit the HIV/AIDS epidemic in China.

Sequence Data

The NFLG sequences of ZJCIQ15005 have been deposited in GenBank with the accession number KX010453.