Features of Ebola Virus Disease at the Late Outbreak Stage in Sierra Leone: Clinical, Virological, Immunological, and Evolutionary Analyses.

We performed Ebola virus disease diagnosis and viral load estimation for Ebola cases in Sierra Leone during the late stage of the 2014-2015 outbreak (January-March 2015) and analyzed antibody and cytokine levels and the viral genome sequences. Ebola virus disease was confirmed in 86 of 1001 (9.7%) patients, with an overall case fatality rate of 46.8%. Fatal cases exhibited significantly higher levels of viral loads, cytokines, and chemokines at late stages of infection versus early stage compared with survivors. The viruses converged in a new clade within sublineage 3.2.4, which had a significantly lower case fatality rate.

Design, synthesis, and biological evaluation of novel trifluoromethyl indoles as potent HIV-1 NNRTIs with an improved drug resistance profile.

A novel series of trifluoromethyl indole derivatives have been designed, synthesized and evaluated for anti-HIV-1 activities in MT-2 cells. The hydrophobic constant, acute toxicity, carcinogenicity and mutagenicity were predicted. Trifluoromethyl indoles 10i and 10k showed extremely promising activities against WT HIV-1 with IC50 values at the low nanomolar level, similar to efavirenz, better than nevirapine, and also possessed higher potency towards the drug-resistant mutant strain Y181C than nevirapine. Preliminary SAR and docking studies of detailed binding mode provided some insights for discovery of more potent NNRTIs.

Diverse reactivity in microwave-promoted catalyst-free coupling of substituted anilines with ethyl trifluoropyruvate and biological evaluation.

Diverse reactivity by coupling of substituted anilines with ethyl trifluoropyruvate was developed under microwave irradiation without catalysts to generate 3-trifluoromethyl-3-hydroxy oxindoles, aromatic hydroxy trifluoromethyl esters, and 1,2-dicarbonyl compounds in a fast and efficient manner. The plausible mechanism for obtaining different products was proposed. Furthermore, the anti-HIV activity of aromatic hydroxy trifluoromethyl esters was first reported. The best inhibitory activity against wild-type HIV-1 IIIB was exemplified by trifluoromethyloxindole 3q with an IC50 = 5.8 µM, which also displayed potential activity against Y181C mutant virus with an IC50 = 7.5 µM. More significantly, the activities of oxindoles 3q and 3r to inhibit K103N/Y181C double mutant HIV-1 reverse transcriptase (RT) are probably similar to that of the second-generation nonnucleoside inhibitor HBY 097 by docking calculation.

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.

[Antiviral effect of lamivudine on HIV-1 targeting MT2 cells influenced by morphine].

OBJECTIVE: To determine whether morphine having the ability to influence the antiviral effect of lamivudine (3TC) in vitro study. METHODS: MT2 cells were randomly assigned into morphine + 3TC treatment group, morphine + naloxone + 3TC treatment group, naloxone + 3TC treatment group. Both 3TC and virus control groups were set up. The corresponding MT2 cells were treated with opiates antagonist (naloxone) for 0.5 hours before the 24-hours morphine treatment program was implemented while all of the groups were then infected with equal amounts of cell-free HIV-1 IIIB strain and 3TC. HIV-1 p24 antigen in culture supernatants collected at days 3, 4, 5 and 6 after infection status was tested and the inhibition of 3TC anti-HIV-1 p24 antigen of various treatment groups calculated. RESULTS: Inhibition of 3TC anti-HIV-1 p24 antigen of Morphine + 3TC treatment group was the lowest when HIV-1 infected cells at 3(rd)and 4(th) day and showed significant difference (P < 0.05) when compared to the 3TC control. However, there was no statistically significant difference among them (P > 0.05), when virus was infected the cells at 5(th)and 6(th) day. The difference of 3TC anti-HIV-1 p24 antigen inhibition between the morphine + naloxone + 3TC treatment group and the naloxone + 3TC treatment group was not significant (P > 0.05). Similar results were obtained when these two groups were compared to the 3TC control group (P > 0.05), respectively. The 3TC anti-HIV-1 p24 antigen inhibition of each treatment group reduced as the time of infection prolonged, showing a significant and time-course effect. CONCLUSION: The 3TC antiviral effect was reduced by morphine in the early stage of infection, and could be blocked by naloxone.

[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.

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.

A few specialized issues that should be focused on anti-HIV drug evaluation in vitro.

Since the introduction of antiretroviral therapy (ART), the lifespan and quality of life of patients infected with HIV have been significantly improved. But treatment efficacy was compromised eventually by the development of resistance to antiretroviral drugs, and more new anti-HIV drugs with lower toxicity and higher activity were needed. Based on the experience and lessons learned from the treatment in the developed countries, US FDA suggested that more pharmacodynamical researches should be considered ahead of the clinical trials. To facilitate the anti-HIV drug research and development, we reviewed a few specialized issues that should be focused on drug evaluations in vitro, including: 1) Mechanism of action studies, demonstrating the candidate drug's efficacy to specifically inhibit viral replication or a virus-specific function and confirm the drug target. 2) Drug resistance studies, selecting the drug-resistant variants in vitro and determining the activities inhibiting HIV isolates resistant to approved antiretroviral drugs of the same class. 3) Antiviral activity in vitro in the presence of serum proteins, ascertaining whether an investigational product is significantly bound by serum proteins. 4) Combination activity analysis, evaluating in vitro antiviral activity of an investigational product in two-drug combinations with other drugs approved.

[In vitro selection and identification of HIV strain which is resistance to two new HIV-1 nonnucleoside reverse transcriptase inhibitors].

JB25 and JB26 are new HIV-1 nonnucleoside reverse transcriptase inhibitors, and show potent anti-HIV activities. Sequential passage experiments with wild-type virus were performed to select and identify mutations induced by these two compounds in vitro. For the initial passage, compounds were present at approximately 2-fold IC50 in MT-2 cells. When cytopathic effect (CPE) was observed in more than 75% of the cells, the culture supernatants were collected. For the subsequent passages, fresh MT-2 cells were infected with 1 mL supernatants from the previous passage (regardless of the virus titer) and cultured in the presence of the compounds at concentrations that were increased 2-fold compared with that in the previous passage. This procedure was repeated with increasing concentrations for 12 passages. JB25 had amino acid substitution L100I (TTA-->ATA) at passage 6, and then changed into 100 M (ATA-->ATG) at passage 12, which was rare mutation form and had not been reported. At the same time, Y188C (TAT-->TGT) mutation appeared at passage 10. For JB26, there was a L100I (TTA-->ATA) mutation at passage 10. In a word, JB25 and JB26 showed a low genetic barrier to the development of resistance, and the resistance to JB26 developed slower than JB25. The mutations selected by JB25 and JB26 were mainly associated with codons 188 and 100 of HIV-1 reverse transcriptase.

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 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.