Recombination Form and Epidemiology of HIV-1 Unique Recombinant Strains Identified in Yunnan, China
China. PLoS ONE 7(10): e46777. doi:10.1371/journal.pone.0046777
Recombination Form and Epidemiology of HIV-1 Unique Recombinant Strains Identified in Yunnan, China
Lin Li 0
Lili Chen 0
Shaomin Yang 0
Tianyi Li 0
Jianjian Li 0
Yongjian Liu 0
Lei Jia 0
Bihui Yang 0
Zuoyi Bao 0
Hanping Li 0
Xiaolin Wang 0
Daomin Zhuang 0
Siyang Liu 0
Jingyun Li 0
Darren P. Martin, Institute of Infectious Disease and Molecular Medicine, South Africa
0 1 Department of AIDS Research, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology , Beijing , China , 2 Yunnan Provincial Hospital Infectious Disease, AIDS Care Center (YNACC) , Kunming, Yunnan , China , 3 Urumqi General Hospital of Lanzhou Military Area Command , Urumqi, Xinjiang , China
Several studies identified HIV-1 recombination in some distinct areas in Yunnan, China. However, no comprehensive studies had been fulfilled in the whole province up to now. To illustrate the epidemiology and recombination form of Unique Recombinant Forms (URFs) circulating in Yunnan, 788 HIV-1 positive individuals residing in 15 prefectures of Yunnan were randomly enrolled into the study. Full-length gag and pol genes were amplified and sequenced. Maximum likelihood tree was constructed for phylogenetic analysis. Recombinant breakpoints and genomic schematics were identified with online software jpHMM. 63 (10.2%) unique recombinant strains were identified from 617 strains with subtypes. The URFs distributed significantly differently among prefectures (Pearson chi-square test, P,0.05). IDUs contained more URFs than sexual transmitted population (Pearson chi-square test, P,0.05). Two main recombinant forms were identified by considering the presence of CRF01_AE segments in full length gag-pol genes, which were B9/C and B9/C/CRF01-AE recombinants. Three clusters were identified in the ML tree which contained more than three sequences and supported by high bootstrap values. One CRF was identified. Many of URFs contained identical breakpoints. The results will contribute to our understanding on HIV recombination and provide clues to the identification of potential CRFs in China.
Funding: This work was supported by the National Key Science and Technology Special Projects on Major Infectious Diseases (Grant No. 2008ZX10001-004) and
the National Natural Science Foundation of China (No. 81072348). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Human immunodeficiency viruses (HIV) were characterized
with high level of genetic variation. Recombination is one of main
mechanisms of HIV diversity. When a cell that was dually infected
by two different viruses produced progeny virions with genomic
RNAs from each virus, strand-switching would take place during
the next round of reverse transcription [1,2,3]. As the results,
recombination would happen and unique variants with genome
from two distinct parental viruses would be produced. Substantial
studies showed that recombination contributed to viral escaping
mutations under immune pressure, viral fitness and emergence of
viral drug-resistance . Up to now, 52 circulating recombinant
(http://www.hiv.lanl.gov\content\hivdb\CRFs\CRFs.html) have been reported, which is responsible
for more than 20% HIV infections in the global AIDS epidemic
. Many studies demonstrated that mosaic strains were arising
frequently, especially in populations with multiple subtypes
circulating , surveillance on the emergence and epidemic of
HIV-1 unique recombinant forms will be helpful for the prediction
of CRFs and HIV epidemic in different area.
Yunnan, a southwestern province of China that shares a border
with the famous heroin-producing area of Myanmar (Burma), was
considered as the epicenter of China. The first HIV-1 epidemic
that occurred among injecting drug users (IDUs) in 1989 in China
was reported inYunnan [7,8,9]. Now it is still the area with the
most severely HIV epidemic in China with highest number of new
reported cases. Multiple HIV-1 genotypes, including B, C,
CRF01_AE, CRF07_BC, and CRF08_BC, are all currently
circulating in the area , supposing the possible emergence of
a new recombination. CRF07_BC and CRF08_BC, the dominant
CRF strains spreading in China now, were believed to arose in
Yunnan in 1990s and spread to other areas [11,12]. In recent
years, the transmitting routes and prevalence of different HIV
subtypes or CRFs have changed dramatically in Yunnan .
Accordingly, URFs originated from prevalent subtypes in same
population would change. Recently, extensive recombination of
HIV was identified in Myanmar, which is close to Yunnan
province . All of these reasons urgent the surveillance of HIV
recombination in Yunnan, China. In this study, we characterized
the genomic schematic and illustrated the epidemic of HIV URFs
in whole Yunnan province basing on full length gag-pol gene
Materials and Methods
Study subjects and specimens
HIV-positive individuals were randomly recruited from the list
of Yunnan AIDS care center, with written informed consents. The
ratios of enrolled subjects from different prefectures were
determined according to the reported cases of prefectures to make
them representatively. The epidemiologic background was
collected through specific epidemiological investigation by trained
interviewers. Peripherial blood was sampled from Jan. 2008 to
Dec. 2009, and plasma was separated and stored in 280uC
freezer. The Ethical Review Board, Science and Technology
Supervisory Committee at the Beijing Institute of Microbiology
and Epidemiology approved the study.
HIV-1 RNA extraction, amplification and sequencing
Viral RNA was extracted from 500 ml HIV-1 positive plasma
specimens with high pure viral RNA kit (Roche, USA). Viral full
length gag (from 763 to 2400 according to the HXB2 calibrator)
and pol (from 2068 to 5221 according to the HXB2 calibrator)
genes were amplified separately with more than 300 bp
overlapping region using reverse transcriptional nest PCR as described
before . Positive PCR products were sequenced by Huada
genomics company (China) with a variety of internal specific
primers (available on request) after being purified. For variant
containing breakpoint in the overlapping regions of gag and pol
genes, 59 half genome was amplified and sequenced again using
methods provide in previous paper . The nearly full-length
genomes were reverse-transcripted and amplified in two halves
with 1 kb overlapping regions as described before .
Edit, Assemble, Genotyping and Phylogenetic Analysis of
All of the sequenced fragments were edited and assembled as
described before . To check for potential contamination, the
sequences obtained were compared to all known sequences in the
HIV database by a Basic Local Alignment Search Tool (BLAST)
search (http://hiv-web.lanl.gov/content/index). HIV genotype
was determined using the national center for biotechnology
information viral genotyping tool (http://www.ncbi.nih.gov/
projects/genotyping/formpage.cgi) by combining both gene
regions from same isolate and further confirmed by phylogenetic
analysis with reference sequences using the Neighbor-Joining
method in MEGA5.0 software and Maximum Likelihood method
in PhyML software . To fulfill the phylogenetic analysis, full
length gag and pol genes derived from same variant were assembled
together and aligned with reference sequences representing
subtypes AD, FH, J, K, CRF01_AE, CRF07_BC, CRF08_BC
and CRF31_BC (http://www.hiv.lanl.gov). ML tree was
constructed using GTR plus gamma model of nucleotide substitution
which was selected with jModelTest software [17,18]. SPR was
used for tree searching. 100 bootstrap replicates were fulfilled to
decide the branch support value. The possible intertype mosaicism
was screened with online Recombination Identification Program
(version 3.0; http://hiv-web.lanl.gov) and further confirmed by the
online software jpHMM-HIV (http://jphmm.gobics.de/). The
breakpoints were determined by a probabilistic approach using
jpHMM-HIV online software . The approach combines the
idea of a profile HMM with the jumping alignment (JALI)
approach proposed by Spang et al. in 2002 as a strategy to
Statistical analyses were performed using SPSS V.10.0 software.
Pearson chi-square test was used to analyze the differences of
demographic distributions of newly identified URFs and difference
s of composition of URFs genome. All statistical analyses were two
sided, and p,0.05 was considered statistically significant.
Demographic distributions of identified URFs
A total of 788 participants residing in Yunnan province were
enrolled into the study. Among of them, 617 subjects were
determined with infected viral subtypes. 63 strains (accession
number: JQ898162- JQ898277, JX679207), which were 10.2% of
subtype-determined isolates, showed different genomic schematic
structure to reported CRFs and were identified as URFs. The
prevalence of URFs was unequal in different prefectures in
Yunnan province (Table 1, Pearson chi-square test, P,0.05).
Prevalence of URFs in Dehong and Dali prefectures were higher
than 20%, which strongly suggested that there was high
prevalence of more than one subtype in the same population.
Relative low prevalence of URFs in Xishuangbanna and Lincang
prefectures were observed, which suggested relatively sole subtype
of HIV prevalent in the area. Epidemic of URFs in peoples
acquired HIV-1 through different transmitting routes also largely
varied (Pearson chi-square test, P,0.05). Among 129 strains from
IDUs, 20 URFs were identified (15.5%). In heterosexual
transmitted population, the prevalence of URFs was only 7.9%.
Genomic schematic structure analysis
Since CRF01_AE, CRF07_BC, CRF08_BC and subtype B9
were the main prevalent strains in Yunnan province, it is not
surprising for us to find that all genomes of the URFs were
comprised with them (Table 2). Strains composed of subtype B9
and C in the gag-pol regions (47 cases) were dominant in the URFs,
which were responsible for 74.6% of total URFs. Subtype B9 and
C were believed to be introduced into Yunnan province and
responsible for early HIV epidemic, especially in the IDUs
[20,21]. CRF01_AE was found to lead HIV epidemic in
heterosexual transmitted population in recent years . That
may be the reason why most of URFs with CRF01_AE as genomic
backbones were found in heterosexual transmitted population
(75%). The distribution of URFs with different backbones was
significantly different in IDUs and heterosexual transmitted
population. Comparing to the data published by Yang et al in
2002, the composition of gag-pol genes also changed. The ratios of
HIV-1 variants containing CRF01_AE segment in gag-pol genes
increased from 8.3% in 2002 in Yangs paper to 25.4% in 2009 in
this manuscript, which strongly suggested that the recombination
form of URFs might be changing.
Phylogenetic analysis of URFs
In the maximum likelihood tree constructed with full-length
gagpol genes sequenced in this study, all of unique recombinant strains
placed among the subtype reference sequences, with three clusters
containing more than 3 sequences with high branch supports in
the ML tree. (Figure 1) With detailed URF sequence data
presented by Yang et al in 2002, phylogenetic analysis was further
fulfilled to explore possible evolution relationship. To do this, all
gag-pol genes from URFs reported in this study were aligned with
12 URF sequences identified in Yangs paper and ML tree was
constructed basing on 2.6 Kb gag-RT genes as described in the
materials and methods. Similar topology was observed as the ML
tree constructed with only our sequences. DH003, DH008 and
DH012 from Yangs lab were found locating in Cluster III. To
identify more HIV-1 variants with similar recombinant form, all of
Asian URFs (57 strains) containing subtype B, C and CRF01_AE
gene segments in gag-pol regions were downloaded from Los
Alomas database and analyzed together with our sequences.
Phylogenetic tree showed that YNRL9607, YNRL9613 grouped
into cluster II. Strain 341 grouped into cluster III.
Prefectures and transmitting routes
Cases with subtypes
Analysis of recombinant breakpoint and identification of
a novel CRF
Recombinant breakpoints analysis basing on full length gag-pol
sequences showed that 56 of 63 URFs contained same breakpoints
with at least one of other strains.(Figure 2) Similar genomic
schematic structures among sequences in the same cluster were
found. In cluster I, 813 and 814 contained same breakpoints, while
816, 822 and 823 shared same breakpoint. All of those 5 strains
were from same area, it is hard to exclude epidemiological linkage.
In cluster II, 515 and 523 contained similar breakpoints in pol
gene, while 523 had a short 230 bp length subtype B9 gene
segment inserting in gag region. The other sequences locating in
cluster II contained longer subtype B9 sequences in pol gene. 1444
and 2347 contained same genomic schematic and might be
potential CRF. All of sequences locating in cluster III contained
same recombinant breakpoints (1229 and 1837 according to
HXB2 calculator) with subtype B9 gene segments inserted in gag
gene. Three strains (DH003, DH012 and DH015) in professor
Yangs paper contained same breakpoints as strains in cluster III
identified in this study. Epidemiological background investigations
excluded linkage among those participants (Table 3) and suggested
that those strains might represent a new CRF. All full length gag-pol
sequences of URFs containing B9, C or CRF01_AE segment from
Asian were obtained from database and submitted to breakpoints
analysis. The results showed that strain 341 had similar breakpoint
as 1434, 1439, 2315, 2327 strains, and also the strain DH003 from
Yangs paper. To determine the full length genomic schematic, 39
half genomic sequences of 1439 strain identified in this study were
obtained as described in the materials and methods. The jpHMM
analysis showed that 1439 and 341 strains displayed same genomic
structure (Figure 2). In the chimera, a subtype B9 segment was
inserted into the backbone of subtype C, with breakpoints
corresponding to HXB2 nucleotide positions 1229 and 1837
approximately. According to the criteria for designation of a new
CRF, those 6 strains constitute a new CRF identified in the
worldwide HIV-1 pandemic.
High heterogeneity of HIV was always observed in infected
individuals, which made it difficult for the amplification and
sequencing of long gene segments. In most of previous studies, the
determination of HIV subtypes prevalent in Yunnan province was
always based on short gene segment, which could not conclude
HIV-1 subtypes and identify recombination exactly. Long gene
segment, even full length genomic sequence, is now considered
more suitable for determining HIV subtypes. Many studies
obtained long gene segment through assembling many short
segments, however, it was hard to get gene segment originated
from same variants due to the existence of quasispecies. In this
study, we analyzed HIV genomic schematic structure basing on
full length gag (1584 bp according to HXBII calculator) and pol
(3147 bp according to HXBII calculator) genes, which make it
more exactly for identification of recombinant strains.
HIV epidemic in Yunnan province was characterized with
multiple strains prevalent simultaneously. The subtype constitution
in Yunnan has always been changing. Accordingly, epidemiology
of URFs would change. In 2002, Yang R et al. found that URFs
accounted for 70% of HIV prevalent in IDUs in Dehong
prefecture . In this more comprehensive study basing on long
gene segments, highest prevalence of URFs in IDU population
was also observed. However, we found that the prevalence of
URFs in IDUs in Dehong prefecture was 35% (14/40). Due to
different genomic regions were adopted in determining viral
subtypes, it is kind of hard to compare both results, but our data
supposed the possible change of URFs prevalence in Yunnan. The
recombinant forms of URFs were also in changing. In previous
studies, URFs containing genomes composed of subtype B9 and C
were dominant [22,23]. In this study, although recombinant
strains originated from subtype B and C segments were still
dominant in URFs, more URFs with CRF01_AE as the backbone
were identified. CRF08_BC strains were dominating HIV
epidemic in Yunnan province, which was responsible for more
than 50% HIV cases (data not shown). Many URFs containing
CRF08_BC breakpoints were identified in this study. So it was
Year of collectionRisk factor Viral Load
#: 341 strain was downloaded from HIV Los Alomas database which was also published by our lab. .
*: DH003 strain was identified by Yang et al. in 2002. .
necessary to pay attention to URFs originated from CRF08_BC
which may become a CRF. CRF01_AE was believed to be
introduced into Yunnan heterosexual transmitted population in
few years  and leading a new epidemic in Yunnan.
Identifications of URFs originated from it strongly suggested that
the subtype is spreading quickly and need to be put more attention
in the surveillance of HIV in the area.
In this study, at least three clusters of sequences with similar
recombinant sites were identified. One CRF was identified with
sequences obtained from HIV Los Alomas database and Yangs
paper . Initially, subtype B9 was believed to be main subtypes
in IDUs in Yunnan [20,21,24,25]. However, CRF07_BC and
CRF08_BC strains spread quickly after their formation and
became dominant in Yunnan and many other areas in China .
Comparing to their parental strains, CRF07_BC and CRF08_BC
showed more superiority of spreading in Chinese population.
Since many new URFs originated from now prevalent strains were
observed, there is the possibility of new CRFs displaced them and
become dominant. Further studies were necessary to explore the
biological characterizations of URFs and social factors related to
their epidemic, which will provide predictive information on HIV
in Yunnan and even China.
We also want to show our thanks to Dr. Feng Gao in Duke Human
Vaccine Institute, Duke University Medical Center for providing the
technical support in reverse transcription and nest-PCR.
Conceived and designed the experiments: LL Jingyun Li. Performed the
experiments: LL LC YL ZB HL XW DZ SL. Analyzed the data: LL TL
LJ. Wrote the paper: LL Jingyun Li LJ. Participated in the collections and
epidemiological investigation of patients: SY Jianjian Li BY.
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