[The study of an in-house method for drug resistance genotyping testing on HIV-1 strains prevailing in China].

OBJECTIVE: To evaluate the amplification rate and the lowestlower detection limit of an in-house HIV-1 Drug resistant (HIVDR) genotyping test. METHODS: A total of 30 plasma samples were selected, which covered all major HIV-1 subtypes predominating prevailing in China (B', CRF07_BC, CRF01 _AE). The viral loads of the 30 selected samples were detected in triplicate by Easy Q method and the average values were taken as the viral loads of the samples. Each sample was diluted to the concentration of > 1000 copies/ml, 401-1000 copies/ml, 101-400 copies/ml, 50-100 copies/ml and < 50 copies/ml with HIV-negative plasma. After extraction of nucleic acids, RT-PCR and nested PCR amplification were performed, the efficiency of amplification of each subtype and the minimum detection limit were determined statistically based on the PCR results. RESULTS: The viral loads of the selected samples ranged from 2.03 x 10(2)-5.92 x 10(4) copies/ml. The sample of 50-1000 copies/ml have a high amplification rate (86%). CONCLUSION: The In-house method for HIV-1 drug resistance genotyping has a high sensitivity with a high successful amplification rate, especially in the samples with low viral load. This method can be used to the detection of drug-resistant virus and to provide scientific data to treatment options for patients.

[Feasibility of using dried blood spots to detect HIV drug resistance genotyping].

OBJECTIVE: This study aimed at exploring the feasibility of using dried blood spots (DBS) to detect HIV drug resistance genotyping in China by comparing the results of drug resistance from DBS, plasma and whole blood samples. METHODS: Blood samples were collected from 39 AIDS patients from Anhui (10), Yunnan (13), Hunan (6) and Xinjiang (10) provinces and autonomous regions. The HIV strains that infected these patients covered all the major HIV-1 subtypes prevailing in China (B, CRF01_AE, CRF07_BC). HIV drug resistance genotyping assay was performed on DBS as well as on the whole blood and plasma samples from the same patients simultaneously by using an in-house nest RT-PCR method. Drug resistance levels were determined based on Stanford University HIV drug resistance database, and the results from these three types of samples were compared. RESULTS: The percentages of successful amplification of protease and reverse transcriptase regions in the pol gene were 95% (37/39) from DBS, 92% (36/39) from whole blood and 100% (39/39) from plasma samples. The sequences from the three types of samples showed more than 99% identity.86% (31/36) of the DBS samples had the same set of drug resistance mutations as those which were detected from plasma samples. The differences probably resulted from mixed bases. CONCLUSIONS: There was no major difference in detecting HIV drug resistance genotyping among DBS, plasma and whole blood samples. Therefore, DBS is useful for detection of HIV drug resistance genotyping and is particularly valuable in developing countries like China, especially in remote rural regions.

[CD8+ cell noncytotoxic antiviral response (CNAR) to HIV in nosocomial HIV-infected individuals].

OBJECTIVE: To study the CD8+ cell noncytotoxic antiviral response (CNAR) to HIV in nosocomial HIV infected individuals, and reveal the relationship between the CNAR and CD4+ cell count. METHOD: CD8+ cells from HIV-1 sero-positive individuals were separated by immunomagnetic beads and mixed with CD4+ cells at different CD8 CD4 cell input ratios (2:1, 1:1, 0.5:1 and 0.25:1). Reverse transcriptase (RT) activity of cocultured supernatant was tested and compared with negative control and the suppression rate of HIV-1 replication was measured. RESULT: The average CD8:CD4 cell input ratios to reach 80% suppression of HIV replication in the group with CD4 < 300/microl and CD4 > 300/microl were 2.4:1 and 1.3:1, respectively (P < 0.05). CONCLUSION: CNAR activity in HIV infected individuals is associated with CD4+ cell count. The ability to suppress HIV replication in subjects with CD4 > 300 is stronger than those with CD4 < 300.

[Sequence analysis of gag and env genes of HIV type 1 circulating in former blood donors of Fuyang city, Anhui province].

OBJECTIVE: To determine the subtype and analyze the genetic characteristics of the HIV-1 predominantly circulating in the former blood donors of Fuyang city, Anhui province. METHODS: Whole blood samples were collected from 294 HIV-positive former blood donors of Fuyang city, 157 males and 137 females, aged 42 +/- 8. The fragments of HIV-1 env and gag genes were amplified by nested-PCR from the whole blood samples and thereafter sequenced. The env and gag sequences derived from 244 and 245 HIV infected individuals respectively were analyzed by using MEGA software, and related researches were also done according to the disease progression of the HIV infected individuals. RESULTS: Phylogenetic trees showed that both the env and gag strains were clustered with the Thailand B reference strains. The internal nucleotide distances of the env and gag genes were 9.11% and 3.59% respectively. The nucleotide distances of both env and gag genes significantly increased as the CD4 T-cell counts decreased or as the viral load rose (both P < 0.001). The V3 loop tip motifs were dramatically dominated by GPGQ in the long-time non-progressors, and by GPGR in the slow progressors (P = 0.038). CONCLUSION: The predominant strains circulating in the HIV-1 infected former blood donors of Fuyang city are of the Thailand B clade. Low CD4 T-cell count and high viral load are associated with the increase of genetic distances among viral isolates. The V3 loop tip motif changes from GPGQ to GPGR along with the progression of disease.

Epidemiology, clinical and laboratory characteristics of currently alive HIV-1 infected former blood donors naive to antiretroviral therapy in Anhui Province, China.

BACKGROUND: Unregulated commercial blood/plasma collection among farmers occurred between 1992 and 1995 in central China and caused the second major epidemic of human immunodeficiency virus type 1 (HIV-1) infection in China. It is important to characterize HIV-1-infected former blood donors and to study characteristics associated with disease progression for future clinical intervention and vaccine development. METHODS: A cross-sectional study was performed on HIV-1-infected former blood donors (FBDs) and age-matched HIV-seronegative local residents. Demographic, epidemiologic, clinical and key laboratory data were collected from all study participants. Both unadjusted and adjusted multivariate linear regressions were employed to analyze the association of the decrease of CD4(+) T-cell counts with other characteristics. RESULTS: Two hundred and ninety-four HIV-1-infected FBDs and 59 age-matched HIV-seronegative local residents were enrolled in this study. The unregulated blood/plasma collection occurred more than a decade (10.8 - 12.8 years) ago, which caused the rapid spread of HIV-1 infection and the high prevalence of co-infection with hepatitis C virus (HCV, 89.5%); hepatitis B virus (HBV) co-infection was observed in only 11 HIV(+)participants (3.7%). Deterioration in both clinical manifestation and laboratory parameters and increase of viral loads were observed in parallel with the decrease of CD4(+) T-cell counts. The decrease of total lymphocyte counts (P < 0.001) and hemoglobin levels (P < 0.001) and the appearance of dermatosis (P = 0.03) were observed in parallel with the decrease of CD4(+) T-cell counts whereas viral loads (P < 0.001) and CD8(+) T-cell counts (P = 0.01) were inversely associated with CD4(+) T-cell counts. CONCLUSIONS: Co-infection with HCV but not HBV is highly prevalent among HIV-1-infected FBDs. CD4(+) T-cell counts is a reliable indicator for disease progression among FBDs. Total lymphocyte counts, hemoglobin level and appearance of dermatosis were positively associated with CD8(+) T-cell counts and viral loads were inversely associated with the decreased CD4(+) T-cell counts.

[Identification of signature amino acids in the env V3-V4 and flanking regions of human immunodeficiency virus type 1 predominant strains in China].

OBJECTIVE: To identify signature amino acids in the V3-V4 and flanking regions of the env gene from human immunodeficiency virus type 1 (HIV-1) predominant strains in China and to elucidate the role of these signature amino acids on epidemiologic tracking and the development of vaccine. METHODS: Fragments of the HIV-1 env gene were amplified by nested-PCR from the whole blood of HIV-1 infected individuals from 12 provinces in China. Then, the PCR products were directly sequenced by using ABI 377 DNA SEQUENCER. The sequences covering the env V3-V4 region of the strains were used for the analyses described here. Envelope sequence subtypes were assigned using BLAST (http://www.HIV-Web.lanl.gov). Phylogenetic analyses were performed using GCG and MEGA as well as signature amino acids were identified using VESPA. RESULTS: Subtype B' strains and two recombinants (B'/C and CRF01-AE) were discovered among 157 currently circulating strains in China. The most prevalent subtypes were B'/C (38.85%), followed by B' (34.40%), and CRF01-AE (26.75%). Phylogenetic tree analysis of env V3-V4 region showed that subtype B' strains were closely related to B.CN.RL42, while most of B'/C strains clustered with 97CN54A and 97CNGX6F, CRF01-AE strains clustered into two distinct subgroups, which were closely related to THCM240 and 97CNGX2F. Analysis of signature amino acids revealed that eight of positions were identified as conserved signature amino acid sites in the env V3-V4 and flanking regions of the subtype B' and B'/C strains and almost all signature amino acids were found in their reference strains. Interestingly, eleven signature amino acids were demonstrated in the same regions of the CRF01-AE strains, but nine out of 11 signature amino acid sites were distinct from the same positions of the reference strains 97CNGX2F and TH.CM240. It is noteworthy that these 9 signature amino acids were found in the strains from all of the selected provinces except those from Yunnan province. CONCLUSION: Analysis of the signature amino acids suggest that much of the current Chinese epidemics of subtype B' and B'/C strains are descended from a single introduction into China, while the epidemic caused by the CRF01-AE strain is caused by multiple introductions into China from Thailand. These results will contribute to the policy of AIDS prevention and control as well as the ongoing development of AIDS vaccine.

[Sequence variation in the env V3-V4 region of HIV type 1 predominant subtype B and C strains circulating in China].

OBJECTIVE: To identify genetic variation of HIV-1 predominant subtype B and C strains in China during rapid horizontal transmission and to elucidate the potential relationship between genetic variation and selective pressure. METHODS: After the fragments of HIV-1 env gene were amplified by nested-PCR from the whole blood of 258 HIV-1 infected individuals, PCR products were directly sequenced using ABI 377 DNA sequencer. The sequences covering env V3-V4 region of 72 HIV-1 subtype B(n=37) and C(n=35) strains were selected for phylogenetic analysis. In addition, the ratios of synonymous (Ks) substitutions per nonsynonymous (Ka) substitutions were calculated using DIVERGE. RESULTS: The genetic distances of the V3-V4 region of subtype B strains were higher than that of subtype C strains. Furthermore, sequence analysis revealed that the V4 region was more variable than the V3 region for both subtype B and C strains. What's more, the V3 loop was less variable compared with the V3 upstream region and C3 region for subtype C Ks/Ka ratios of the entire aligned sequence of the two subtypes were below 1 0, with the lowest values found in the V3 region of subtype B strain and the V4 region of subtype C strain. CONCLUSIONS: The majority of variation in both subtypes B and C strains occurred in the V4 rather than the V3 region. It is important that our study found for the first time the V3 loop was more conservative than the V3 upstream region and C3 region for subtype C. Calculations of the Ks/Ka ratios throughout the V3-V4 region demonstrate that significant selective pressures experienced during the rapid horizontal spread of the virus in the Chinese HIV-1 infected population may have directed change in the V3 loop for the subtype B strain and the V4 loop for the subtype C strain. These results will contribute to the policy of AIDS prevention and control and the ongoing development vaccine.