Similar neutralizing activity in the HIV-1 infected long term non-progressors(LTNPs) and typical progressors(TPs).

Neutralizing antibodies are considered to be an important protective parameter used in HIV-1 vaccine evaluation. However, the exact role that neutralizing antibodies plays in controlling the disease progression of HIV-1 infected peoples is still undetermined. In this paper, we compared the protective function of the neutralizing antibody response in the plasma from LTNP and TP against clade B and clade C pseudoviruses. No difference in the neutralizing activities between the plasma from LTNP and TP was found, which was consistent with the most recent reports. In addition, no correlations between the titer or breadth and CD(4+) or viral load in HIV-1 infected individuals were found. The protective roles played by neutralizing antibodies in controlling disease progression of HIV-1 infected people need to be considered in a new viewpoint.

[Prevalence status and mutation pattern of H221Y in subtype B' of HIV-1].

OBJECTIVE: To explore the prevalence and mutation pattern of H221Y at reverse transcriptase (RT) among the subtype B' of human immunodeficiency virus1 (HIV-1) in antiviral therapy-failure patients. METHODS: A total of 1363 sequences, comprising of 1205 therapy-failure individuals and 158 therapy-naive individuals, were submitted to the Stanford HIV drug resistance database (SHDB) to analyze the frequency and mutation pattern of H221Y. RESULTS: The prevalence of mutation H221Y in the therapy-failure population was significantly higher than that of the therapy-naive (6.59% vs 0.60%) (χ(2) = 6.59, P = 0.027). The emergence of H221Y usually accompanied the position mutations of T215, M184, K103 and Y181 of RT, and the pattern of TAMs/H221Y/Y181C/I was common. Frequency of H221Y in the regimen of AZT/ddI/NVP was more popular than the other 4 regimens (14.6% vs 3.5%, 4.9%, 2.3%, 2.6%, all P < 0.01). CONCLUSION: With a unique mutation pattern, H221Y has a low prevalence in the individuals of first-line therapy-failure patients.

[Occurrence of human immunodeficiency virus-1 resistance through a six-year surveillance in rural areas of Henan].

OBJECTIVE: To analyze the occurring rules of human immunodeficiency virus (HIV) drug resistance under an unique therapy model among HIV-1 infected individuals on antiretroviral therapy (ART) in rural areas of Henan, China. METHODS: A cohort of 75 individuals on an ART regimen of zidovudine (ZDV), dideoxyinosine (ddI) and nevirapine (NVP) was established in March 2003. A total of 12 surveillance were conducted and 788 person-times were studied until 2010. The parameters of CD4 cell count and viral load (VL) were tested in each survey. And genotypic resistance testing was performed in patients with a failure of viral suppression. Survival analysis was used to estimate the occurrence time of resistance. RESULTS: The cumulative mortality rate was 16% (12/75) in the cohort. And the cumulative resistance rate was 88% (66/75) from 2004 to 2010. The rate of resistance reached 54.7% and the probability from susceptibility to drugs developing resistance decreased drastically from 100% to 45.3% within the first 1 year of initiation. The occurrence time of resistance for half of individuals in the cohort was at 12.0 months (95%CI 8.6 - 17.0) after initiation, 25.1 months (95%CI 19.0 - 33.3) in those whose VL was less than 4.0 lgU/ml and 4.8 months (95%CI 4.1 - 5.6) at VL > 4.0 lgU/ml during the first investigation. The individuals with an early occurrence of resistance within 12 months carried high risks for a failure of viral suppression and a decrease of CD4 counts. CONCLUSION: The occurrence of resistance rises with the course of therapy. And the greatest probability for resistance is within the first 1 year of initial therapy. A high level of VL has a significant impact on the development of resistance. Preventing the occurrence of resistance during the initial therapy remains a key goal.

[Screening program on novel drug resistance mutations of subtype B' in human immunodeficiency virus type 1 in China].

OBJECTIVE: To screen the level of novel drug resistance mutations in subtype B' in China. METHODS: 451 pol sequences collected from the previous study, which including 354 AIDS patients who had received antiretroviral treatment (ART) and 97 the untreated patients. Entire protease gene (codons 1 - 99) and full-length reverse transcriptase gene (codons 1 - 560) were included. Variation of mutations between the treated and the untreated patients with consensus/ancestral sequences were compared and the mutations with higher frequencies in the treated patients than in the untreated patients were screened before submitting the mutations to the Stanford HIV Drug Resistance Database (SHDB) (http: //hivdb.stanford.edu/). Relation between the mutations and resistance preliminarily was then analyzed, according to the information including SHDB. RESULTS: Frequencies of 7 mutations at 6 positions, D123E, V292I, K366R, T369A, T369V, A371V and I375V, 2 at DNA polymerase domain and 5 at connection domain of reverse transcriptase (RT) were higher in the treated patients than in the untreated patients. The information of 7 mutations including the SHDB showed that 7 mutations were major variants at corresponding positions, and theirs frequencies were higher in the treated patients using some drugs, than in the untreated patients. CONCLUSION: 7 mutations being screened from the China subtype B were possibly associated with the resistance, which called for the construction of mutated viruses by site-directed mutagenesis to identify their effects on the susceptivity of different drugs.

[Studying on the prevalence and mutation pattern of N348I which related to the resistance of HIV-1].

OBJECTIVE: To elucidate the prevalence and the mutation pattern of N348I that related to the resistance seen in the AIDS patients, in China. METHODS: Partial pol gene of HIV-1 comprising of full protease (PR) and reverse transcriptase (RT) was obtained from plasma samples of therapy-failure individuals (n = 614) and therapy-naive individuals (n = 619) by using reverse transcription polymerase chain reaction (RT-PCR). 1233 sequences were then submitted to the HIV-1 drug resistance database of the Stanford University to analyze the prevalence and the emergence pattern of N348I. RESULTS: The prevalence of N348I was 6.5% in the therapy-failure patients and 0.8% in the naive individuals, respectively. The prevalence of N348I was more popular among those patients whose ART regimens containing zidovudine (AZT or ZDV) than those without AZT in regimens (14.1% vs. 4.7%, χ² = 10.21, P < 0.01). N348I always emerged, and combined with others mutations among patients of ART, whose frequencies were: 85.0% in combination with thymidine analog mutations (TAMs) and 52.5% with M184V/I, respectively. CONCLUSION: N348I was somehow prevalent in the therapy-failure patients when using the first-line antiretroviral drugs, and it emerged as unique patterns. This study laid the ground in improving the technology of drug resistance genotypes detection and providing theoretical basis to study the mechanism of resistance and the law of molecular evolution.

[Evaluation on HIV antibody testing strategy currently used in China].

OBJECTIVE: To evaluate the yield of HIV antibody testing strategy currently used on different populations, in China. METHODS: (1) The following samples were collected and tested according to the currently used HIV antibody testing strategy in China. 103 133 samples from the general populations (outpatients, new recruits and blood donors), 1276 people under high risk (spouses of the HIV infected individuals, intravenous drug users) and 2323 biochemical or immunological abnormal samples. (2) Retrospective analysis was done on data from the HIV testing among outpatients in General Hospital of People's Armed Police Forces, from Jan., 2002 to Dec., 2008 and in three provincial central HIV test and confirmatory laboratories. RESULTS: (1) The yields of HIV antibody screening were significantly different in different populations. The probability of screening reactive to be true positive was 50% in high risk population, significantly higher than in the general population. The probability of screening reactive to be true positive was 19.58% in the confirmatory laboratory mainly towards the general population, but significantly lower than results from the confirmatory laboratories done on the high risk population. (2) From 2002 to 2008, in the General Hospital of People's Armed Police Forces, the probability of screening reactive to be true positive in the clinical HIV test was increasing from 3.7% to 16.0%, where as the efficiency of the repeat screening testing decreased from 92.6% to 61.5%. CONCLUSION: The predictive value of HIV antibody screening reactive was significantly greater in high risk population than in general population. The precision of HIV antibody initial screening was substantially increased with the improvement of HIV antibody test kits and of quality control in the HIV test laboratories in recent years. It is suggested that different HIV test strategies to be implemented in different populations.

Indinavir resistance evolution in one human immunodeficiency virus type 1 infected patient revealed by single-genome amplification.

UNLABELLED: Human Immunodeficiency Virus Type 1 exists in vivo as quasispecies, and one of the genome's characteristics is its diversity. During the antiretroviral therapy, drug resistance is the main obstacle to effective viral prevention. Understanding the molecular evolution process is fundamental to analyze the mechanism of drug resistance and develop a strategy to minimize resistance. OBJECTIVE: The molecular evolution of drug resistance of one patient who had received reverse transcriptase inhibitors for a long time and had treatment which replaced Nevirapine with Indinavir was analyzed, with the aim of observing the drug resistance evolution pathway. METHODS: The patient, XLF, was followed-up for six successive times. The viral populations were amplified and sequenced by single-genome amplification. All the sequences were submitted to the Stanford HIV Drug Resistance Database for the analysis of genotypic drug resistance. RESULTS: 149 entire protease and 171 entire reverse transcriptase sequences were obtained from these samples, and all sequences were identified as subtype B. Before the patient received Indinavir, the viral population only had some polymorphisms in the protease sequences. After the patient began Indinavir treatment, the variants carrying polymorphisms declined while variants carrying the secondary mutation G73S gained the advantage. As therapy was prolonged, G73S was combined with M46I/L90M to form a resistance pattern M46I/G73S/L90M, which then became the dominant population. 97.9% of variants had the M46I/G73S/L90M pattern at XLF6. During the emergence of protease inhibitors resistance, reverse transcriptase inhibitors resistance maintained high levels. CONCLUSION: Indinavir-resistance evolution was observed by single-genome amplification. During the course of changing the regimen to incorporate Indinavir, the G73S mutation occurred and was combined with M46I/L90M.

Allele-specific real-time PCR testing for minor HIV-1 drug resistance mutations: assay preparation and application to reveal dynamic of mutations in vivo.

BACKGROUND: It is very important for the clinical management to test for minor HIV-1 resistance mutations accurately and sensitively. The conventional genotypic assays of HIV drug resistance detection based on sequencing can only discriminate the mutations which present in more than 20% - 30%. The aim of this study was to evaluate allele-specific real-time PCR (ASPCR) to detect the resistance-related mutations located at positions 103, 184 and 215. METHODS: We developed the allele-specific PCR assay, using the most common drug resistance mutations in Chinese AIDS patients, K103N, M184V/I, T215F/Y as a model system. The standards were constructed by cloning the wild-type and mutant DNA fragments into the T-vector. We designed specific primers to discriminate mutant templates in the real-time PCR using SYBR green as a fluorescence reporter. And then we evaluated the ASPCR assay and tested 140 clinical samples using this method. RESULTS: The sensitivities of ASPCR assay were 0.04% for K103N, 0.30% for M184I, 0.40% for M184V, 0.03% for T215F and 0.02% for T215Y. The intra-assay and inter-assay coefficients of variation were less than 0.42. One hundred and forty plasma samples were tested by ASPCR and dynamic resistance curves of ten patients were obtained. CONCLUSIONS: Drug resistance emerged half a year after the start of antiretroviral therapy. The mutation of T215Y emerged 1 to 1.5 years after starting treatment and then increased rapidly. The ASPCR assay we developed was a sensitive, accurate and rapid method to detect the minor HIV-1 variants and it can provide earlier and more drug-resistance information for HIV research and AIDS antiretroviral therapy.

Competitive capacity of HIV-1 strains carrying M184I or Y181I drug-resistant mutations.

BACKGROUND: Virus with nucleoside reverse transcriptase inhibitors (NRTIs) or nonnucleoside reverse transcriptase inhibitors (NNRTIs) resistant mutations show different evolution tendencies when the anti-viral therapies are interrupted. Understanding the replication fitness of drug-resistant virus is important for the study of the prevalence of drug-resistance. For this purpose, we characterized the replication capacity of HIV-1 virus carrying lamivudine (3TC) or nevirapine (NVP) resistant mutations. METHODS: 3TC and NVP resistant variants were induced in vitro by selecting wild type virus in the presence of drugs. For the competitive replication assay, drug-resistant variants were cocultured with wild-type virus in the presence or absence of drugs. The ratios of the viral species were determined over time by using a real-time RT-PCR-based assay. RESULTS: 3TC-resistant (M184I mutation) and NVP-resistant (Y181I mutation) virus should be selected in vitro in two different ways. The competitive replication assay showed that the ratio of virus carrying a M184I mutation increased from 98.8%, while the wild type virus decreased to 1.2% after 4 passages in the presence of 3TC; the percentage of virus carrying the Y181I mutation increased to 90.5%, while wild type virus decreased to 9.5% in the presence of NVP. In the absence of drugs, the ratio of virus carrying the M184I mutation decreased to 5.3%, while wild type virus increased to 94.7%; the ratio of virus carrying Y181I increased to 75%, while wild type virus decreased to 25% after 4 passages. CONCLUSIONS: The NVP-resistant virus is fitter than wild type virus even in the absence of NVP that may be the reason that NNRTIs-resistant virus is spreading quickly.

Immunogenicities of Env glycoproteins from circulating HIV-1 isolates in China focusing on the strategy of "DNA prime plus protein boost".

BACKGROUND: The adenovirus-based HIV-1 vaccine developed by Merck Company suffered from an unexpected failure in September 2007. This generated a big shift in the strategy of HIV vaccine development with renewed focus on the induction of neutralizing antibodies. A major challenge in developing an HIV-1 vaccine is to identify immunogens and adopt delivery methods that can elicit broadly neutralizing antibodies against primary isolates of different genetic subtypes. METHODS: Most circulating HIV-1 isolates in China are composed of clades Thai-B, CRF_BC and CRF01_AE. In order to construct DNA vaccines against these 3 HIV-1 subtypes, DNA vaccines carrying the gp120 regions from HIV-1 isolates of GX48(AE), GX79(AE), NX22(BC), GS22(BC), HN24(Thai-B) were constructed. Expression of gp120 from these DNA vaccines was detected by Western blotting in transiently transfected 293T cells. Pilot immunizations of New Zealand white rabbits were performed using the strategy of "DNA prime plus protein boost" and the neutralizing antibody response was detected in a Tzm-bl cell based assay against different HIV-1 strains. RESULTS: Response of gp120-specific antibody was relatively low after DNA primes (mean titer = 10(4.72)); however, the titer of gp120-specific antibody went up with 2 protein boosts (mean titer = 10(6.81)). Above all, neutralizing antibody (Nab) titers induced by this combined approach were much better than those elicited by DNA or protein used alone (P < 0.01). Neutralizing activities of immunized rabbit sera against several pseudoviruses and laboratorial strains were evaluated, most rabbit sera primed with monovalent vaccine were capable of neutralizing only 1 of 5 viruses, however, sera primed with the polyvalent DNA vaccines were able to neutralize at least 2 of 5 viruses. CONCLUSION: Polyvalent DNA prime plus protein boost is an effective immunization strategy to broaden the neutralization breadth and further research should be performed on the basis of this pilot study.

[Research on the selective kinetics of HIV-1 nucleoside reverse transcriptase inhibitor drug resistance-associated mutations among 4 AIDS patients receiving highly active antiretroviral therapy].

OBJECTIVE: To elucidate the molecular evolutional characteristics of HIV-1 nucleoside reverse transcriptase inhibitor (NRTI) drug resistance-associated mutations in patients with AIDS receiving highly active antiretroviral therapy. METHODS: We selected 4 AIDS patients receiving highly active antiretroviral therapy (HAART) with good adherence under a HIV-1 drug resistance cohort from a rural region in central China. Those people carried susceptible virus at the beginning of treatment and gradually came to produce virus resistant to NRTIs during the process of antiretroviral therapy (ART). Reverse transcriptase (RT) genes from each patient's peripheral blood samples (from 3 to 33 months after withdrawal) were cloned and sequenced in succession. RESULTS: We sequenced a total number of 855 clones and obtained the HIV-1 NRTI drug resistance-associated mutations patterns of the 4 patients. Typical resistance mutations of thymidine analogue mutations (TAMs) pattern 1, such as L210W, T215Y and M41L, were generated in patient 'A'. TAMs pattern 2, including D67N, K70R and K219Q mutations, was discovered in patient 'B'. Interestingly, in patient 'C', some clones comprising not only TAMs pattern 1 mutations (T215Y) but also TAMs pattern 2 mutations (K70R, D67N). CONCLUSION: The four patients show different pathways on HIV-1 NRTI drug resistance-associated mutations, including TAMs pattern 1, TAMs pattern 2 and the fusion pattern of TAMs-1 & TAMs-2. We also noticed that the tendency of gradual accumulation was obvious and those mutations detected earlier tended to be the predominant strains.

[Study on the difference of the APOBEC3G mRNA levels among HIV long-term nonprogressors, slow progressors, people at high risks and normal people].

OBJECTIVE: To examine the APOBEC3G (hA3G) mRNA levels of four different groups in the human immunodeficiency virus (HIV) prevalent areas in central China and to analyze the relationship between hA3G mRNA levels and HIV disease progression. METHODS: We collected peripheral blood and isolated the peripheral boold monouuclear cells (PBMCs), and then cryo-preserved the PBMCs in liquid nitrogen. Prior to the total extraction of RNA, PBMCs were resuscitated and mRNA were reverse Transcripted to cDNA in vitro. Real-time polymerase chain reaction (PCR) was used to test hA3G mRNA levels of different groups. RESULTS: There were 13 HIV long term non-progressors with the mean CD4+ T lymphocyte count as (716 +/- 169) per microl and the mean affection time as (12.5 +/- 2.3) years. There were 48 HIV slow progressors with the mean CD4+ T lymphocyte count as (233 +/- 144) per microl and the mean affection time as (10.7 +/- 2.2) years. The hA3G mRNA level of HIV long term nonprogressors was higher than that of normal people while the hA3G mRNA level of HIV slow progressors was higher than that of normal people and high risk people. There were no correlations between CD4+ T lymphocyte count and hA3G mRNA levels of HIV long-term nonprogressors as well as in HIV slow progressors. CONCLUSION: There was difference found in the hA3G mRNA levels of four groups in the HIV popular area in central China while no correlation between CD4+ T lymphocyte count and hA3G mRNA levels of HIV long term nonprogressors as well as in HIV slow progressors were found.

[Study on the indeterminate results of characterization and verification of HIV antibody from Western blot test].

OBJECTIVE: To study the serological characterization of indeterminate Western blot (WB) results of HIV antibody and to find a new way to verify the HIV antibody indeterminate results and provide references for editing "National Guideline for Detection of HIV/AIDS". METHODS: All of the 42 subjects who were confirmed as indeterminate HIV antibody in People' Liberation Army HIV Confirmation Laboratory from 2005 to 2006, were collected. Line immunoassay, HIV viral load test and HIV-1 p24 were tested and followed up for 3-6 months' to compare the changes of WB bands patterns. RESULTS: (1) For the 42 individuals with indeterminate HIV antibody, a total of 8 different patterns of bands were found in WB test including 45.2% of them were p24 mono band, 30.9% were gp160 mono band, 11.9% were gp160 with p24, 2.4% (only one case) were gp160gp120 +/-, gp41p24, p24p17, gp41 or gp120 respectively. It was noticed that the most patterns of common bands with indeterminate results were p24 mono band, gp160 mono band and gp160 with p24, which composed 88.0% of the whole indeterminate WB band patterns. (2) Twenty three cases had been followed up for more than 3 months with 22 giving no WB band image change and were confirmed as HIV sero-negative. The other one with case gp160 and p24 had developed to more bands in the period of 77 days follow-up with more bands, including gp160, gp120, p66, p31, p24 and p17,showed up and was confirmed as HIV primary infection. (3) Line immunoassay was applied to all of those 23 cases who had been followed up and the results showed that only one serological change was found and the case was confirmed to be HIV-positive. Among the other 22 cases without serological changes, 16 cases were proved to be HIV-negative, 6 cases were still indeterminate. The specificity was 72.7%. P24 antigen test showed negative in all the 23 cases, including the case which later was confirmed as HIV-positive. Of all the 23 originally indeterminate cases, viral loads were tested in 7 cases. Positive result was found in the case which was proved later to be HIV-positive. No viral loads were detected in the other 6 cases (< LDL). CONCLUSION: The most common band patterns of indeterminate HIV antibody were mainly p24 monoband, gp160 monoband or with p24. Most of them (95.6%) were not infected by HIV, the bands showed up in WB test and demonstrated as non-specific reactions. Line immunoassay could determine about 70% of the indeterminate reactions. Results from viral load test also suggested that it was an efficient method to discriminate indeterminate results. With these two techniques, HIV serology could be diagnosed without 3 months' follow-up in primary infection which gave indeterminate WB results.

[Study on the antiviral therapy program among people with human immunodeficiency virus in Guangxi Zhuang Authority].

OBJECTIVE: To explore the reconstitution of immune function and viral suppression condition and to analyze the occurrence of drug resistance HIV-1 variants and its prevalence after using HAART in Guangxi Autonomy Region. METHODS: From July 2004 to October 2005, 133 HIV infected individuals who had received HAART for more than three months were recruited, and 58 infected persons with no antiviral therapy were selected as controls. Questionnaire was used to collect information about the adherence of HAART therapy. Immune reconstruction and viral suppress conditions were obtained by CD4+ counts and viral load and RT-PCR were used to amplify the PR and RT regions of HIV-1 genome while HIV-1 drug resistance rates were analyzed to show the occurrence and prevalence in both treated and naive patients. RESULTS: In terms of CD4+ T cell counts: 70.69% of the treated patients showed obvious increase and 23.28% had no apparent change but 6.03% of them went down. 70.48% of the patients who had received antiviral therapy more than 3 months had their viral load lower than the low detectable limitation. When comparing the log of viral load between treated and untreated cohort, the mean value of the treated was obviously less than the untreated (P < 0.05). However,the result of drug resistance showed no obvious difference between the treated and untreated groups. CONCLUSION: The antiviral therapy being used in Guangxi region, had achieved obvious effect on the reconstruction of immune system and the suppression of viral replication in vivo under good adherence while the occurrence of drug-resistant HIV strain did not show obvious difference between treated and naive patient groups.

[Cohort study on human immunodeficiency virus discordant couples in the countryside of central China].

OBJECTIVE: To establish a cohort of human immunodeficiency virus (HIV) discordant couples for follow-up studies and to collect data on frequency of HIV heterosexual transmission and related factors. METHODS: A total of 52 HIV discordant couples were identified by face to face interview and serological testing, in which the HIV negative individuals had no HIV infection behaviors including injecting drug use, blood transfusion or having sexual partners other than his/her own wife/husband. Three times of follows-up studies were carried out in 0.5 year, 1 year and 2.5 years to collect information on their sexual practices and condom use through face to face interview together with 20 ml whole blood collected to test HIV antibody, CD4+ T cell count and viral load. RESULTS: (1) In the period of 2.5 years follow-up, no HIV seroconversion and HIV transmission was found. (2) The frequencies of sexual intercourse between once per month to once per week were 65.4%, 72.9%, 71.7% and 80.0% at the time of cohort setup: 0.5 year, 1 year and 2.5 years of follow-up respectively. The rates of "occasional use" to "never use" condoms were 76.9%, 66.6%, 69.1% and 60.0% at the time of cohort setup as: 0.5 year, 1 year and 2.5 years of follow-up, respectively. No significant difference between different times of follow-up for sexual intercourse or condom use. (3) 85.4%, 66.6% and 60.0% of the HIV positive individuals kept their CD4+ T cell count stabilized or raised during the 0.5 year, 1 year and 2.5 years follow-up period, respectively. However, 66.7% of them showed stable or declined viral load in the period of 2.5 years follow-up. It appeared that stable or raised CD4+ T cell and the stable/declined viral load happened simultaneously. CONCLUSION: No transmission was identified in this study. The stabilized CD4+ T cell count and viral load might be account for the reason of no transmission while the biological factors from host and virus related with transmission need to be further studied.

Phenotypic resistance of resistant strains of HIV type-1 subtype B in China.

BACKGROUND: This study was aim to explore the characteristics of phenotypic resistance of resistant strains of HIV type-1 (HIV-1) subtype B and to compare the concordance between the phenotypic resistance and genotypic resistance. METHODS: The genotypic resistance assay for the HIV-1 clinical isolates was performed. One isolate without resistance mutation was chosen as a drug-sensitive reference strain and seven subtype B isolates with resistance mutations were phenotypically tested. Fifty percent inhibitory concentrations (IC50) between resistant and sensitive viruses were compared. The resistance extent was determined by the folds of the increased IC50. The concordance between the phenotypic resistance and genotypic resistance was also analyzed. RESULTS: IC50 of resistant isolates were 0.0006 - 0.1300 micromol/L for zidovudine (AZT), 0.0016 - 0.0390 micromol/L for lamivudine (3TC), 0.0104 - 0.4234 micromol/L for nevirapine (NVP), and 0.0163 - 0.1142 micromol/L for indinavir (IDV), respectively. Genotypic and phenotypic resistance assays indicated that the resistant strains were intermediately and highly resistant to nucleotide analog reverse transcriptase inhibitors and non-nucleotide analog reverse transcriptase inhibitors. The phenotypic assay was consistent with the genotypic assay. For measuring the potential resistance, the genotypic assay was more sensitive than the phenotypic. In evaluating the resistance to protease inhibitors, these two assays were discrepant. CONCLUSIONS: Both the phenotypic and genotypic assays indicate that the resistant viruses exist in HIV-infected patients in China who have received treatment. Phenotypic and genotypic assays have high concordance, and the genotypic assay could replace the phenotypic assay to predict the HIV-1 resistance.

Prevalence and evolution of drug resistance HIV-1 variants in Henan, China.

To understand the prevalence and evolution of drug resistant HIV strains in Henan China after the implementation of free antiretroviral therapy for AIDS patients. 45 drug naïve AIDS patients, 118 AIDS patients who received three months antiretroviral therapy and 124 AIDS patients who received six months antiretroviral treatment were recruited in the southern part of Henan province. Information on general condition, antiretroviral medicines, adherence and clinical syndromes were collected by face to face interview. Meanwhile, 14 ml EDTA anticoagulant blood was drawn. CD4/CD8 T cell count, viral load and genotypic drug resistance were tested. The rates of clinical improvement were 55.1% and 50.8% respectively three months and six months after antiretroviral therapy. The mean CD4 cell count after antiretroviral therapy was significantly higher than in drug naïve patients. The prevalence rate of drug resistant HIV strains were 13.9%, 45.4% and 62.7% in drug naïve patients, three month treatment patients and six month treatment patients, respectively. The number of resistance mutation codons and the frequency of mutations increased significantly with continued antiretroviral therapy. The mutation sites were primarily at the 103, 106 and 215 codons in the three-month treatment group and they increased to 15 codon mutations in the six-month treatment group. From this result, the evolution of drug resistant strains was inferred to begin with the high level NNRTI resistant strain, and then develop low level resistant strains to NRTIs. The HIV strains with high level resistance to NVP and low level resistance to AZT and DDI were highly prevalent because of the AZT+DDI+NVP combination therapy. These HIV strains were also cross resistant to DLV, EFV, DDC and D4T. Poor adherence to therapy was believed to be the main reason for the emergence and prevalence of drug resistant HIV strains. The prevalence of drug resistant HIV strains was increased with the continuation of antiretroviral therapy in the southern part of Henan province. Measures, that could promote high level adherence, provide new drugs and change ART regimens in failing patients, should be implemented as soon as possible.

[Genotypic antiretroviral resistance testing and phylogenetic analysis of protease and reverse transcriptase in antiretroviral drug-naïve AIDS patients in Henan province].

OBJECTIVE: Frequency, type and clinical implications on protease and reverse transcriptase drug resistance mutations were investigated and phylogenetic analysis in antiretroviral drug-naïve AIDS patients was carried out in Henan province. METHODS: 45 plasma samples were separated from the anticoagulatory whole blood, from which reverse transcription-polymerase chain reaction technique was used to amplify the partial pol gene. The sequences were analysed for genotypic antiretroviral resistance and phylogenetic relation through landing the websites http://hivdb.stanford.edu and http://hiv-web.lanl.gov, under BioEdit and DNAClub software. RESULTS: Partial pol sequences of 36 samples were successfully amplified. The major mutation rate of resistance to protease was 8.3% (3/36), including types D30A, V32A, G73C and V82A. Minor mutation rate of resistance was 100%, including types of L63PS (36/36), I93L (35/36), V77IL (34/36), A71IVT (10/36) and D60E (2/36). The mutation rate of resistance to reverse transcriptase was 38.9% (14/36). Mutation-scoring and clinical implication clewed drug resistance rates were 5.6% (2/36) and 22.2% (8/36) to protease inhibitors and reverse transcriptase inhibitors respectively, while 1 sample was potentially low-level resistant to all of the protease inhibitors and 3 samples to part of the reverse transcriptase inhibitors. Phylogenetic analysis revealed that the pol gene of 36 samples were highly homologous and having a near relative to B.US.83.RF ACC M17451. 36 samples seemed to have the same infection source while their resistance mutations were not due to drug-resistant virus infection but to the evolving of virus in vivo. CONCLUSION: Most of the antiretroviral drug-naïve AIDS patients in Henan province were sensitive to the currently available antiviral medicine, but antiviral treatment must be in accordance with the strict procedure and to keep better adherence, to avoid the epidemics caused by drug-resistant virus.

[Cloning and characterization of a full-length HIV-1 genome of a prevalent subtype B-Thai strain in Henan Province].

OBJECTIVE: To clone, identify and phylogenetically characterize a clade B-Thai HIV isolate representing the most prevalent virus in Henan province. METHODS: Peripheral blood mononuclear cells (PBMCs) from an HIV-1 infected patient in Henan Province were separated, and co-cultivated with phytohemagglutinin-stimulated healthy donor PBMCs. Proviral DNA was extracted from productively infected PBMCs. The full-length HIV-1 genome was amplified by using the LA Tag long template PCR system. Primers were positioned in conserved regions within the HIV-1 long terminal repeats. Purified PCR products were T-A ligated into a pWSK29-T vector(CNHN 24 clone). Three recombinant clones containing virtually full-length HIV-1 genome were identified by PCR. The full-length genome was sequenced by using the primer-walking approach. Nucleotide sequence similarities were calculated by the local-homology algorithm. Phylogenetic trees of gag, pol and env reading frames were constructed using the Phylip software. RESULTS: HIV-1 C3V4 sequences indicate that the epidemic in this area was B-Thai subtype. V3 loop multiple amino acid sequence alignments showed amino acid alterations at nine positions. The 9,010 bp genomic sequence derived from isolate CNHN 24 contained all known structural and regulatory genes of an HIV-1 genome. No major deletions, insertions, or rearrangements were found. The highest homologies of the gag, pol, vpr, and vif reading frames to the corresponding clade B-Thai RL 42 sequences were 95.42%-97.08%. Phylogenetic trees showed the closest relationship of CNHN 24 and RL 42. CONCLUSION: The cloning and characterization of a virtually full-length HIV-1 B-Thai subtype in central China was completed in our laboratory. The data should be helpful to future studies on the genetic diversity of HIV-1.