To investigate more closely the determinants of transmission and escape in HIV-1 internal proteins, we analyzed the characterization of putative N-linked glycosylation sites (PNGSs) and the variable loop of CRF07_BC recombinant human immunodeficiency virus type 1 (HIV-1), isolated from intravenous drug users (IDUs). We studied the characterization of PNGSs and the variable loop in the C1-C5 and V1-V4 regions of the HIV-1 env gene in 12 intravenous drug users (IDUs) who were divided into two groups according to the length of infection time. In addition, two IDUs were longitudinally monitored from the time of seroconversion for 1.5 and 2.5 years. The longitudinal characterization within the individuals on PNGSs and the variable loop in the C1-C5 and V1-V4 region were also observed. Based on the above analysis, we found that PNGSs and the variable loop appeared to increase over time within IDU transmission of CRF07_BC recombinant HIV-1. We argue that limited PNGSs and the length of variable loops may be involved in selective transmission and escape.

To explore the temporal genetic variation of HIV-1 CRF07_BC and reconstruct its epidemic in Xinjiang, China, we studied 216 C2-V4 fragments of env genes sampled from 1996 to 2008. Phylogenetic analysis indicates that the viruses prevailing in Xinjiang form a large monophyletic cluster and may have originated from a common ancestor. The epidemic in Xinjiang was probably established around 1995 (95% confidence interval, 1994-1996). We noted an increased diversity of CRF07_BC over time, with a rapid evolutionary rate we estimated to be 8.3x10(-3) substitutions per site per year in the env gene. After 5-6 years of the epidemic (1997-2002), the transmission rate of CRF07_BC in Xinjiang slowed down, although CRF07_BC infection remained at a high prevalence.

HIV-1 CRF07_BC constitutes a large fraction of HIV-1 strains circulating in China and is responsible for the rapidly expanding epidemic across the country. Little is known about the biological characteristics of the CRF07_BC viruses, particularly the propensity by which this circulating recombinant form of HIV replicates within an infected individual. In this study, a near-full-length genome of a CRF07_BC virus from an injecting drug user (IDU) from the Xingjiang region in China was cloned and rescued by the functional 5' LTR (long terminal repeat region) and 3' LTR from pNL4-3 to produce a chimeric infectious molecular clone (NLXJDC6441X2). When cultured in interleukin-2-stimulated peripheral blood mononuclear cells (PBMCs) and the Ghost.R5 cell line, NLXJDC6441X2 produced a lower replication yield than expected. More study is needed to explore the key role that leads to the poor infectious activity of NLXJDC6441X2. This study has produced the first isolation of the HIV-1 CRF07_BC DNA clone and has provided a versatile molecular model for research focusing on the biological properties of this subtype.

OBJECTIVES: To investigate the expression of NKG2A on cytotoxic natural killer (NK) cells in HIV-1-infected patients. DESIGN: A cross-sectional study was conducted to investigate the NKG2A expression on NK cell subsets among HIV-infected individuals at different clinical stages and HIV negative controls. METHODS: 113 HIV-1 infected and 43 uninfected individuals were enrolled in this study. The HIV-1 infected patients were further categorized into three groups, CD4 cell count > 500 cells/microl (n = 44), CD4 cell count of 200-500 cells/microl (n = 49) and CD4 cell count < 200 cells/microl (n = 20). Flow cytometry was used to determine the expression of NKG2A on NK cell subsets. A flow-based assay was employed to quantify the NK cytotoxicity. RESULTS: Fewer cytotoxic NK cells and more dysfunctional NK cells were observed in patients with advanced clinical conditions. Higher NKG2A expression level in cytotoxic NK subset were found in later stages HIV infection, 25.6% in groups with CD4 cell count > 500 cells/microl, 40.9% in groups with CD4 cell count 200-500 cells/microl and 48.3% in groups with CD4 cell count < 200 cells/microl. Lower NK mediated cytotoxicity, which was associated with the decrease in cytotoxic NK cell number and higher NKG2A expression on cytotoxic NK subsets, was found in AIDS patients compared with patients at early stage of infection. A reverse association between the percentage of NKG2A positive cells in cytotoxic NK subset and CD4 cell count was observed in all HIV-1 infected groups. CONCLUSION: Fewer cytotoxic NK cells and higher NKG2A expression in cytotoxic NK subset was found in patients in late stage HIV infection. Such a phenomenon may relate to the escape of HIV-1-infected CD4+ T cells from being attacked by NK cells.

ABSTRACT: BACKGROUND: The characterization of HIV-1-specific T cell responses in people infected with locally circulating HIV-1 strain will facilitate the development of HIV-1 vaccine. Sixty intravenous drug users infected with HIV-1 circulating recombinant form 07_BC (CRF07_BC), which has been spreading rapidly in western China from north to south, were recruited from Xinjiang, China to assess the HIV-1-specific T cell responses at single peptide level with oligopeptides (OLP) covering the whole concensus clades B and C proteome. RESULTS: The median of the total magnitude and total number of OLPs recognized by CTL responses were 10925 SFC/million PBMC and 25 OLPs, respectively, when tested by clade C peptides, which was significantly higher than when tested by clade B peptides. The immunodominant regions, which cover 14%(58/413) of the HIV-1 proteome, are widely distributed throughout the HIV-1 proteome except in Tat, Vpu and Pol-PR, with Gag, Pol-RT, Pol-Int and Nef being most frequently targeted. The subdominant epitopes are mostly located in p24, Nef, integrase, Vpr and Vif. Of the responses directed to clade C OLPs, 61.75%(972/1574) can be observed when tested with corresponding clade B OLPs. However, Pol-PR and Vpu tend to be targeted in the clade B sequence rather than the clade C sequence, which is in line with the recombinant pattern of CRF07_BC. Stronger and broader CTL responses in subjects with CD4 cell counts ranging from 200 to 400/mm3 were observed when compared to those with less than 200/mm3 or more than 400/mm3, though there have been no significant correlations identified between the accumulative CTL responses or overall breadth and CD4 cell count or plasma viral load. CONCLUSIONS: This is the first study conducted to comprehensively address T cell responses in Chinese subjects infected with HIV-1 CRF07_BC in which subtle differences in cross-reactivity were observed, though similar patterns of overall immune responses were demonstrated with clade B infected populations. The immunodominant regions identified in this population can facilitate future HIV-1 vaccine development in China.

The partial pol gene was analyzed to determine circulating HIV-1 subtypes and the prevalence of drug resistance among recently infected injecting drug users in the west of China. Phylogenetic and bootscanning analyses showed that all 25 sequences belonged to CRF07_BC. No major drug resistance mutations were found, while the CRF07_BC sequences harbored a few highly prevalent polymorphic positions in both the protease and reverse transcriptase regions. It is notable that D60E, L63P and I93L substitutions, which were more common in HIV-1 isolates from PI-treated than untreated patients, were present in most of the isolates. Compared with subtype B, the CRF07_BC strains had a decreased genetic barrier for the V106M mutation, which is selected by efavirenz and leads to high-level resistance to all present nonnucleoside RT inhibitors. Further studies are needed to determine the impact of genetic features of CRF07-BC on antitretroviral susceptibility and evolution of drug resistance mutations.

Though HIV-1 CRF07_BC rapidly spread in China, there have been few reports about this subtype since its first genetic characterization nearly 10 years ago. It was urgent and necessary to know the current gene variation of circulating CRF07_BC strains. Xinjiang was the main region for the CRF07_BC epidemic and also an ideal region for research on the viral gene evolution. The strains of Ulumuqi and Yili in Xinjiang were isolated, cloned, and sequenced in this study. Analyses of phylogenetic, potential CTL epitopes and N-glycosites were preformed simultaneously. New CRF07_BC isolates showed higher genetic diversity and more potential N-glycosites than old isolates. It was interesting that although the env and nef genes are highly variable, highly conserved potential CTL epitopes and N-glycosites were found in deduced gp120 V3 and Nef product of all CRF07_BC isolates. The analysis of the sequences provides some valuable information on the investigation of the epidemiology and on vaccine development.

BACKGROUND: Transmission of HIV drug resistance (TDR) gives rise to reduced efficacy of initial antiretroviral treatment and has become a public health concern. METHODS: A nationwide survey on TDR was conducted in antiretroviral therapy-naive HIV-1-infected individuals from September 2004 to October 2005 in China. Drug resistance genotyping was performed on subjects' plasma samples. Drug resistance mutations were determined and scored by Stanford HIV Drug Resistance algorithm. RESULTS: Sequences were obtained from 676 individuals, of whom 61.2% were former plasma and blood donors, 17.3% were infected sexually, and 17.2% were intravenous drug users. Subtype B' HIV-1 strains were found in 73.5%, CRF01_AE in 13.9%, CRF07_BC in 6.2%, CRF08_BC in 2.7%, subtype C in 1.04%, subtype B in 0.9%, CRF02_AG in 0.4%, and B'/C intersubtype recombinant strains in 1.3% of the subjects. Twenty-six (3.8%) were found to harbor drug resistance strains. The rates of resistance to protease inhibitors, nucleoside reverse transcriptase inhibitors, and nonnucleoside reverse transcriptase inhibitors were 0.4%, 1.6%, and 2.1%, respectively. Though there was no significant difference in TDR rates between 2004 and 2005 (2.9% vs. 4.4%), an increased trend was observed in the rate of high-level drug resistance (0.8% in 2004 vs. 3.0% in 2005, P = 0.0634). CONCLUSIONS: The rate of TDR was relatively low in China, as compared with those in developed countries. Surveys among recently HIV-infected subjects should be performed continually to ensure the success of the scale-up antiretroviral treatment.

OBJECTIVE: To analyze the genetic characteristics of HIV-1 CRF01_AE strains prevailing in the four provinces of southern China. METHODS: Plasma samples were collected from the newly diagnosed HIV-1 individuals reported in 2006 in Guangdong, Guangxi, Jiangxi and Hunan province. The gag and env gene fragments were amplified from RNA template extracted from plasma using RT and nested PCR methods. CRF01_AE sequences were analyzed by phylogenetic methods and characterized by calculating the genetic distance and Entropy analysis. RESULTS: Two main epidemic clusters were found to exist in the CRF01_AE strains from 210 HIV-1 CRF01_ AE infected individuals collected in the 4 provinces, southern China. It was found that no international reference strain was closely correlated with cluster I, which including 123 samples. The strains in cluster II, consisting 57 cases of samples, were closely related with the strains identified in Vietnam. Genetic distance analysis of gag and env genes showed that the diversity of cluster I was obviously less than that of cluster II. Data on nucleotide polymorphism showed that nucleotides compositions of 42 sites in gag and 40 sites in env were significantly different between the two clusters. When compared with cluster II, the polymorphism decreased at 61 nucleotide sites but increased at 21 sites in cluster I. CONCLUSION: This was the first report describing that two main epidemic clusters were existed in CRF01_AE strains prevailing in the 4 provinces, Southern China. The virus in cluster I was the dominant strain in this region, with shorter period of circulation and higher proportion seen in the HIV-infected population, which might belong to CRF01_AE strain with certain features facilitating the spread of the virus. The virus in cluster II was highly homology with the CRF01_AE strains from Vietnam, and seemed to have had several events of epidemics in populations in border regions of China and Vietnam.

Serotonin transporter (SERT, 5-HTT) is a key element in the serotonergic system which is probably involved in the psychiatric disorders commonly observed in people living with HIV/AIDS. However no information is available about the effects of HIV infection on SERT expression. In this study, a TaqMan(R) real-time RT-PCR method was established, levels of SERT mRNA in the peripheral blood mononuclear cells (PBMCs) and various tissues from normal Chinese rhesus macaques, in PBMCs from 32 SHIV-sf162p4 infected rhesus macaques and from 8 rhesus macaques before and 7, 14, 21, 28 and 196 days after SHIV-sf162p4 infection, and in PBMCs before and after in vitro phytohemagglutinin (PHA) stimulation were examined. It was found that SERT mRNA was widely distributed in lymphoid tissues; the level of SERT mRNA was significantly reduced in PBMCs from SHIV infected rhesus macaques and in PBMCs stimulated with PHA. The most evident decrease (to about one-tenth) in SERT mRNA level was observed at day 7 after SHIV infection. Difference in PBMC SERT mRNA level between 5-HTTLPR genotypes was not statistically significant. These data indicated that, in addition to previously observed abnormality in serotonin metabolism, SERT expression might be affected in HIV/AIDS, which might be associated with depression and other psychiatric disorders in HIV/AIDS. Besides, this study provided a basis for quantitative analysis of SERT gene expression under effects of host and environmental factors, such as 5-HTTLPR genotypes, SERT targeting drugs or other infectious agents.