Xueshan Xia

On-going generation of multiple forms of HIV-1 intersubtype recombinants in the Yunnan Province of China

ObjectivesTo investigate the molecular epidemiology of HIV in Chinas Yunnan Province, where the initial HIV-1 outbreak among injecting drug users (IDU) occurred in 1989, and to analyse the genesis and interrelationship of the epidemic with that in surrounding areas. DesignA molecular epidemiological investigation was conducted among IDU in three prefectures in Yunnan Province, including Wenshan (east), Honghe (southeast) and Dehong (west). MethodsThirty-nine specimens were collected from consenting IDU in 2000–2001. The nucleotide sequences of 2.6 kb gag-RT and 340 base pair (bp) env (C2/V3) regions were determined. Phylogenetic tree and recombination breakpoint analyses were performed. ResultsThe circulating recombinant form (CRF), CRF08_BC, predominated in east Yunnan near Guangxi Province (89% in Wenshan and 81% in Honghe), whereas it was not detected in Dehong (0/14) in the west. In contrast, 71% (10/14) of the Dehong isolates were unique recombinant forms (URF), mostly between subtypes B′ (Thailand variant of subtype B) and C, with distinct profiles of recombination breakpoints. The subtype B′ accounts for the remaining 29% (4/14) of Dehong isolates. Interestingly, two Honghe isolates (2/16) shared some of the precise B′/C recombination breakpoints with CRF07_BC. ConclusionNew recombinant strains are arising continually in west Yunnan near the Myanmar border. Some appeared to be secondary recombinants derived from CRF07_BC that had further recombined with other strains. The uneven distribution of subtypes, CRF and URF, suggests the presence of independent transmission networks and clusters among IDU in Yunnan.

High prevalence of HIV-1 and hepatitis C virus coinfection among injection drug users in the southeastern region of Yunnan, China.

&NA; The southeastern region of Yunnan province is a key site for drug trafficking and HIV‐1 infection spread from the west of Yunnan and Laos to southeastern China. To investigate the prevalence of HIV‐1 infection and hepatitis C virus (HCV) coinfection among injection drug users (IDUs) in southeastern Yunnan, three cohorts of 285 addicts, including 242 IDUs and 43 oral drug users, living in the cities of Gejiu and Kaiyuan and the county of Yanshan were studied. HIV‐1 and HCV infections were detected by enzyme‐linked immunosorbent assay and/or polymerase chain reaction. Data on the age, sex, risk behavior, drug use history, employment, ethnic background, and marriage status were obtained by interview. The overall prevalence of HIV‐1 infection was 71.9%. The rate of HCV coinfection among 138 HIV‐1‐infected IDUs was 99.3%. Most HIV‐infected IDUs were 20 to 35 years old (86.7%) and were ethnic Han (75.9%), suggesting that the epidemic in Yunnan is no longer confined to non‐Han ethnic minorities. HIV prevalence in female IDUs (81.2%) was significantly higher than in male IDUs (68.2%) (p < .05). The prevalence of HIV infection reached 68.4% after 1 year of injection drug use. Needle/syringe sharing is the major high risk factor for the spread of HIV‐1 and HCV infections. Large‐scale educational campaigns are urgently needed to reduce the spread of HIV and HCV infection in these regions.

The unique HCV genotype distribution and the discovery of a novel subtype 6u among IDUs co-infected with HIV-1 in Yunnan, China†‡

The Yunnan province is the epicenter of HIV‐1 epidemics in China and a center for drug trafficking to the other parts of the world. In six prefectures of this province, a total of 132 IDUs were recruited to determine the sero‐prevalence of HCV and HIV‐1 and the positive rates were 93.94% and 68.18%, respectively (P < 0.001). Co‐infection with HCV and HIV‐1 was found among 89 IDUs, of whom several HCV fragments were amplified and sequenced. Sequences of the HCV 5′NCR‐C and NS5B region were determined from 82 IDUs. Phylogenetic analyses showed consistent genotyping among 80 IDUs. Among them HCV genotypes 1a, 1b, 3a, 3b, 6a, 6n, and a tentatively assigned novel 6u subtype were found in 1 (1.25%), 16 (20%), 19 (23.75%), 24 (30%), 4 (5%), 9 (11.25%) and 7 (8.75%) individuals, respectively. In two IDUs, genotyping results were discordant, suggesting mixed HCV infections or recombination. The proportion of patients with HCV 1b tended to decrease from the north to south and from the east to west in this province. Genotype 3 and 6 strains were more frequent in the southern prefectures. The novel subtype 6u strains were only detected in Dehong which borders Myanmar. Our findings showed a unique pattern of HCV genotype distribution, which is similar to that in the southeastern Asian countries but distinct from that among the general population in China. Routes of drug trafficking and the resulting high prevalence of HIV‐1 infection may have contributed to this pattern of HCV genotype distribution. J. Med. Virol. 80: 1142–1152, 2008.

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

ABSTRACT We identified a new class of human immunodeficiency virus type 1 (HIV-1) recombinants (00CN-HH069 and 00CN-HH086) in which further recombination occurred between two established circulating recombinant forms (CRFs). These two isolates were found among 57 HIV-1 samples from a cohort of injecting drug users in eastern Yunnan Province of China. Informative-site analysis in conjunction with bootscanning plots and exploratory tree analysis revealed that these two strains were closely related mosaics comprised of CRF07_BC and CRF08_BC, which are found in China. The genotype screening based on gag-reverse transcriptase sequences of 57 samples from eastern Yunnan identified 47 CRF08_BC specimens (82.5%), 5 CRF07_BC specimens (8.8%), and 3 additional specimens with the novel recombinant structure. These new “second-generation” recombinants thus constitute a substantial proportion (5 of 57; 8.8%) of HIV-1 strains in this population and may belong to a new but yet-undefined class of CRF. This might be the first example of CRFs recombining with each other, leading to the evolution of second-generation inter-CRF recombinants.

A Novel Genotype of GB Virus C: Its Identification and Predominance among Injecting Drug Users in Yunnan, China

GB virus C (GBV-C) is prevalent globally and particularly among individuals at risk of parental exposures. Based on genetic diversity, this virus is now classified into six genotypes and many subtypes with distinct geographical distribution. In this study, 120 Injecting Drug Users (IDUs) were recruited from Yunnan province, China. Among them, 43 (35.8%) were positive for GBV-C RNA, 70 (58.3%) and 103 (85.8%) sero-positive for HIV-1 and HCV respectively. This revealed 18.3% of IDUs having GBV-C/HIV/HCV triple infection, which is significantly higher than 7.5% of GBV-C/HIV-1 and 10% of GBV-C/HCV dual infection rates (P<0.05). Based on 5′UTR sequences, the identified 43 viral isolates can be classified into three phylogenetic groups: one (2.3%) and two (4.7%) belonged to genotype 3 and 4, respectively, and the remaining 40 (93%) formed a new group with 97% of bootstrap support. This new GBV-C group was further confirmed by characterizing the E2 region and full-length genome sequences. Analysis of 187 nt 5′UTR sequence showed three previous reported isolates from Southeast Asia were re-classified into this new group. It implies they have the same origin with strains from Yunnan. Although we provisionally assigned this new group as GBV-C genotype 7, a simpler five groups of GBV-C nomenclature is recommended. Genotype 4, 6 and the newly designated genotype 7 could be reclassified as one group, which may represent a single GBV-C genotype. The classification of the other four groups was corresponding to that of previous reported genotype 1, 2, 3 and 5. Furthermore, the diversity of amino acid sequence in the E2 region was analyzed. The inhibitory effect of GBV-C genotype 7 on HIV-1 cell entry could be deduced. Since GBV-C may have a beneficial effect on AIDS disease progression and interact with HCV during co-infection, this finding may raise interests in future studies on this virus that was previously thought to be a “non-pathogenic virus”.

A new HCV genotype 6 subtype designated 6v was confirmed with three complete genome sequences

BACKGROUND Although hepatitis C virus (HCV) genotype 6 is classified into 21 subtypes, 6a-6u, new variants continue to be identified. OBJECTIVE To characterize the full-length genomes of three novel HCV genotype 6 variants: KMN02, KM046 and KM181. STUDY DESIGN From sera of patients with HCV infection, the entire HCV genome was amplified by RT-PCR followed by direct DNA sequencing and phylogenetic analysis. RESULTS The sera contained HCV genomes of 9461, 9429, and 9461nt in length, and each harboured a single ORF of 9051nt. The genomes showed 95.3-98.1% nucleotide similarity to each other and 72.2-75.4% similarity to 23 genotype 6 reference sequences, which represent subtypes 6a-6u and unassigned variants km41 and gz52557. Phylogenetic analyses demonstrated that they were genotype 6, but were subtypically distinct. Based on the current criteria of HCV classification, they were designed to represent a new subtype, 6v. Analysis of E1 and NS5B region partial sequences revealed two additional related variants, CMBD-14 and CMBD-86 that had been previously reported in northern Thailand and sequences dropped into Genbank. CONCLUSION Three novel HCV genotype 6 variants were entirely sequenced and designated subtype 6v.

Complete genome sequencing and phylogenetic analysis of HCV isolates from China reveals a new subtype, designated 6u

Full length genome sequences were characterized for three novel hepatitis C virus (HCV) isolates (here named DH012, DH014, and DH028). The complete genomes were all isolated from injecting drug users (IDUs) who were co‐infected with HIV‐1 and lived in Dehong prefecture, Yunnan Province, China, which neighbors Myanmar. The three genomes are 9,443–9,470 nt in length and each contains a single open reading frame (ORF) of 9,069 nt long. Pairwise comparisons indicated nucleotide similarities of 97.9–98.6% among the three isolates, and similarities of 72.4–75.0% between the three isolates and 20 reference strains (representing HCV subtypes 6a–6q and 6t plus two unassigned genotype 6 isolates km41 and gz52557). Phylogenetic analyses demostrated that the three isolates formed a tight and well‐supported monophyletic cluster in the genotype 6 clade. No evidence of viral recombination was found using similarity plots and bootscanning analyses. Based on the current HCV classification criteria, we have assigned the three isolates to a new subtype, 6u. Although another “6u” isolate D83 has been reported very recently, it is subtypically distinct from the three isolates we described here. Because its designation does not meet the criteria described in the updated HCV nomenclature proposal, this “6u” isolate should be reclassified. J. Med. Virol. 80:1740–1746, 2008.

Targeted next-generation sequencing of candidate genes reveals novel mutations in patients with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a major cause of sudden cardiac death and heart failure, and it is characterized by genetic and clinical heterogeneity, even for some patients with a very poor clinical prognosis; in the majority of cases, DCM necessitates a heart transplant. Genetic mutations have long been considered to be associated with this disease. At present, mutations in over 50 genes related to DCM have been documented. This study was carried out to elucidate the characteristics of gene mutations in patients with DCM. The candidate genes that may cause DCM include MYBPC3, MYH6, MYH7, LMNA, TNNT2, TNNI3, MYPN, MYL3, TPM1, SCN5A, DES, ACTC1 and RBM20. Using next-generation sequencing (NGS) and subsequent mutation confirmation with traditional capillary Sanger sequencing analysis, possible causative non-synonymous mutations were identified in ~57% (12/21) of patients with DCM. As a result, 7 novel mutations (MYPN, p.E630K; TNNT2, p.G180A; MYH6, p.R1047C; TNNC1, p.D3V; DES, p.R386H; MYBPC3, p.C1124F; and MYL3, p.D126G), 3 variants of uncertain significance (RBM20, p.R1182H; MYH6, p.T1253M; and VCL, p.M209L), and 2 known mutations (MYH7, p.A26V and MYBPC3, p.R160W) were revealed to be associated with DCM. The mutations were most frequently found in the sarcomere (MYH6, MYBPC3, MYH7, TNNC1, TNNT2 and MYL3) and cytoskeletal (MYPN, DES and VCL) genes. As genetic testing is a useful tool in the clinical management of disease, testing for pathogenic mutations is beneficial to the treatment of patients with DCM and may assist in predicting disease risk for their family members before the onset of symptoms.

Phylogenetic analysis of dengue virus reveals the high relatedness between imported and local strains during the 2013 dengue outbreak in Yunnan, China: a retrospective analysis

BackgroundAn outbreak of dengue virus (DENV) occurred in Yunnan province. More than 2,000 individuals were infected from August to November 2013. In this study, we aimed to characterize the origin and prevalence of DENV in Dehong prefecture of Yunnan province using phylogenetic and evolutionary analyses of DENV strains collected from local patients and foreign travelers.MethodsA total of 41 DENV-positive serum samples were randomly collected from travelers who entered China at Ruili port or local patients with dengue fever in Dehong prefecture of Yunnan province, China. The envelope (E) gene of DENV was amplified and sequenced. The distributions and evolutionary characteristics of different genotypes were elucidated by phylogenetic and Bayesian analyses.ResultsPhylogenetically, all of the 41 DENV-positive samples could be classified into genotype I (43.9%) of serotype DENV-1 and the Asian I genotype (56.1%) of serotype DENV-2. DENV strains derived from local patients and foreign travelers were scattered equally within these two clusters. Furthermore, the DENV strains from the two populations exhibited high relatedness based on evolutionary characteristics.ConclusionsThese results suggested that imported and local DENV strains occurring during the dengue outbreak in 2013 were highly related. Additionally, these data may suggest that this dengue outbreak was caused by a newly imported infection from the neighboring country of Myanmar.

HIV‐1 prevalence and subtype/recombinant distribution among travelers entering China from Vietnam at the HeKou port in the Yunnan province, China, between 2003 and 2012

The aim of this study was to assess HIV‐1 prevalence and the distribution of HIV‐1 subtypes among travelers crossing the border at the HeKou land port. Between 2003 and 2012, 22,799 persons were randomly recruited from people entering China from Vietnam. In this crossing border population, a total of 161 (0.71%) travelers were determined as HIV‐1‐positive. From them, 140 HIV‐1‐positive serum samples were collected for RNA extraction and subsequent RT‐nested PCR amplification of the group‐specific antigen (gag)‐RT with a length of 2.6 kb. The DNA sequences were analyzed to determine the HIV‐1 subtypes/recombinants. We found that the circulating recombinant form 01_AE (CRF01_AE) was the most common HIV‐1 subtype, accounting for 49.4% (41/83) of the subtyped 83 samples, followed by CRF08_BC (26.5%, 22/83) and CRF07_BC (7.2%, 6/83). Only 1 sample was classified as subtype C. Thirteen cases could not be clustered into any known subtypes or CRFs and presented as unique recombinant forms (URFs). Of them, 6 recombination patterns were identified. They had distinct structures consisting of fragments of subtypes B, C, CRF01_AE, CRF07_BC and CRF08_BC. Between 2003 and 2012, CRF01_AE and CRE08_BC were shown to be the most prevalent recombinant forms identified each year. But yearly change of each subtype is uncertain regular among in these travelers during the past decade. Understanding the distribution of HIV‐1 subtypes/recombinants and how it changes across time among individuals entering China from Vietnam through this land port is crucial to establish strategies for the prevention of HIV cross‐border transmission. J. Med. Virol. 87:1500–1509, 2015.