Rongge Yang

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.

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.

HIV protease inhibitor nelfinavir inhibits replication of SARS-associated coronavirus

n Abstractn n A novel coronavirus has been identified as an etiological agent of severe acute respiratory syndrome (SARS). To rapidly identify anti-SARS drugs available for clinical use, we screened a set of compounds that included antiviral drugs already in wide use. Here we report that the HIV-1 protease inhibitor, nelfinavir, strongly inhibited replication of the SARS coronavirus (SARS-CoV). Nelfinavir inhibited the cytopathic effect induced by SARS-CoV infection. Expression of viral antigens was much lower in infected cells treated with nelfinavir than in untreated infected cells. Quantitative RT-PCR analysis showed that nelfinavir could decrease the production of virions from Vero cells. Experiments with various timings of drug addition revealed that nelfinavir exerted its effect not at the entry step, but at the post-entry step of SARS-CoV infection. Our results suggest that nelfinavir should be examined clinically for the treatment of SARS and has potential as a good lead compound for designing anti-SARS drugs.n n

Closely related HIV-1 CRF01_AE variant among injecting drug users in northern Vietnam: evidence of HIV spread across the Vietnam-China border.

To investigate the nature of recent HIV outbreaks among injecting drug users (IDUs) near the Vietnam-China border, we genetically analyzed 24 HIV-positive blood specimens from 2 northern provinces of Vietnam (Lang Son and quang Ninh) adjacent to the China border, where HIV outbreaks among IDUs were first detected in late 1996. Genetic subtyping based on gag (p17) and env (C2/V3) sequences revealed that CRF01_AE is a principal strain circulating throughout Vietnam, including the provinces near the China border. The majority of CRF01_AE sequences among IDUs in Quang Ninh and Lang Son showed significant clustering with those found in nearby Pingxiang City of Chinas Guangxi Province, sharing a unique valine substitution 12 amino acids downstream of the V3 loop. This particular subtype E variant, uniquely found among IDUs in northern Vietnam and southeastern China, is designated E(v). The genetic diversity of CRF01_AE distributed in Quang Ninh (1.5 +/- 0.6%) and Pingxiang City (1.9 +/- 1.2%) was remarkably low, indicating the emerging nature of HIV spread in these areas. It is also noted that the genetic diversity of CRF01_AE among IDUs was consistently lower than that in persons infected sexually, suggesting that fewer closely related CRF01_AE variants were introduced into IDUs and, conversely, that multiple strains of CRF01_AE had been introduced via the sexual route. The data in the present study provide additional evidence that HIV outbreaks among IDUs in northern Vietnam were caused by the recent introduction of a highly homogeneous CRF01_AE variant (E(v)) closely related to that prevailing in nearby southern China.

Identification of a novel second-generation circulating recombinant form (CRF48_01B) in Malaysia: a descendant of the previously identified CRF33_01B.

A molecular epidemiological investigation conducted among injecting drug users in eastern Peninsular Malaysia in 2007 identified a cluster of sequences (n = 3) located outside any known HIV-1 genotype. Analyses of near full-length nucleotide sequences of these strains from individuals with no recognizable linkage revealed that they have an identical subtype structure comprised of CRF01_AE and subtype B′, distinct from any known circulating recombinant forms (CRFs). This novel CRF, designated CRF48_01B, is closely related to CRF33_01B, previously identified in Kuala Lumpur. Phylogenetic analysis of multiple CRF48_01B genome regions showed that CRF48_01B forms a monophyletic cluster within CRF33_01B, suggesting that this new recombinant is very likely a descendant of CRF33_01B. CRF48_01B thus represents one of the first examples of a “second-generation” CRF, generated by additional crossover with pre-existing CRFs. Corroborating these results, Bayesian molecular clock analyses indicated that CRF48_01B emerged in ∼2001, approximately ∼8 years after the emergence of CRF33_01B.

Augmentation of Human Immunodeficiency Virus Type 1 Subtype E (CRF01_AE) Multiple-Drug Resistance by Insertion of a Foreign 11-Amino-Acid Fragment into the Reverse Transcriptase

ABSTRACT A human immunodeficiency virus type 1 (HIV-1) subtype E (CRF01_AE) variant (99JP-NH3-II) possessing an in-frame 33-nucleotide insertion mutation in the β3-β4 loop coding region of the reverse transcriptase (RT) gene was isolated from a patient who had not responded to nucleoside analogue RT inhibitors. This virus exhibited an extremely high level of multiple nucleoside analog resistance (MNR). Neighbor-joining tree analysis of thepol sequences indicated that the 99JP-NH3-II variant had originated from the swarm of drug-sensitive predecessors in the patient. Population-based sequence analyses of 82 independently cloned RT segments from the patient suggested that the variants with the insertion, three or four 3′-azido-3′-deoxythymidine resistance mutations, and a T69I mutation in combination had strong selective advantages during chemotherapy. Consistently, in vitro mutagenesis of a drug-sensitive predecessor virus clone demonstrated that this mutation set functions cooperatively to confer a high level of MNR without deleterious effects on viral replication capability. Homology modeling of the parental RT and its MNR mutant showed that extension of the β3-β4 loop by an insertion caused reductions in the distances between the loop and the other subdomains, narrowing the template-primer binding cleft and deoxynucleoside triphosphate-binding pocket in a highly flexible manner. The origin of the insert is elusive, as every effort to find a homologue has been unsuccessful. Taken together, these data suggest that (i) HIV-1 tolerates in vivo insertions as long as 33 nucleotides into the highly conserved enzyme gene to survive multiple anti-HIV-1 inhibitors and (ii) the insertion mutation augments multiple-drug resistance, possibly by reducing the biochemical inaccuracy of substrate-enzyme interactions in the active center.

Molecular Epidemiology of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus in Former Blood Donors in Central China

The prevalence of hepatitis C virus (HCV) among human immunodeficiency virus (HIV-1)-positive former blood donors (FBDs) in Hubei province, central China, and the subtypes of these two viruses are identified. HIV-1-positive specimens were collected from FBDs, transfusion recipients, and their sexual partners in Hubei province, central China. The prevalence of HCV in HIV-1-positive FBDs was 78.6%. The dominant circulating HIV-1 subtype of FBDs was subtype B (Thai-B); one belonged to U.S.-European subtype B. HCV genotypes 2a (78.6%) and 1b (21.4%) were detected. No recombinant form of HIV-1 was identified. Non-B subtypes occurring among FBDs indicate the complexity of the HIV-1 prevalence in central China, where HIV is beginning to spread into the general population.

Short Communication: Isolation and Characterization of Replication-Competent Molecular DNA Clones of HIV Type 1 CRF01_AE with Different Coreceptor Usages

We have isolated replication-competent molecular clones of HIV-1 circulating recombinant form CRF01_AE with different coreceptor usages. After lambda phage cloning of unintegrated circular proviral DNAs derived from a CRF01_AE strain (HIV-1NH1), isolated in Japan, the infectious molecular clone, designated p93JP-NH1, was reconstituted. 93JP-NH1 showed an X4 and R5 phenotype in NP2 cell-based coreceptor utilization assays and exerted robust replication in human T cell lines, including MT2, M8166, and PM1 cells, whereas it propagated modestly in peripheral blood mononuclear cells. The CRF01_AE molecular clone with R5 phenotype (p93JP-NH2env) was then constructed by replacing the env gene of p93JP-NH1 with that of a nearly isogenic CRF01_AE R5 strain isolated from an epidemiologically linked case. The phylogeny and recombination break-point analysis confirmed that these clones shared an A/E recombinant structure similar to that of the prototype CRF01_AE strain, CM240. These replication-competent CRF01_AE molecular clones with different coreceptor usages would be useful tools for the study of CRF01_AE, one of the most prevalent strains in Asia.

Isolation and Characterization of a Full-Length Molecular DNA Clone of Ghanaian HIV Type 1 Intersubtype A/G Recombinant CRF02_AG, Which Is Replication Competent in a Restricted Host Range

We have isolated a replication-competent, full-length molecular clone of HIV-1 CRF02_AG, designated p97GH-AG1, by reconstituting two separately amplified genomic regions of an HIV-1 provirus of a 1997 Ghanaian isolate. The phylogenetic and recombination breakpoint analyses revealed that 97GH-AG1 had an A/G recombinant structure similar to that of prototype Nigerian isolate IbNG. The 17-nucleotide insertion downstream of the primer-binding site appeared to be a common sequence signature specific to most CRF02_AG strains, including 97GH-AG1. 97GH-AG1 showed an R5 phenotype and exerted productive infection in both HOS and NP2 cell infectivity assays, whereas it failed to show a detectable level of progeny production in peripheral blood mononuclear cells (PBMCs). The data may suggest the presence of unknown determinant(s) that dictate efficient replication in PBMCs, but that are not required for replication in immortalized cell lines.