High prevalence and viremia of human pegivirus 2 in the HIV-infected population in Honghe Prefecture, Yunnan Province.

Human pegivirus 2 (HPgV-2) is a recently recognized pegivirus of the family Flaviviridae. To investigate the epidemic features of HPgV-2 circulating in the human immunodeficiency virus (HIV)-infected population, we tested for antibodies and viral RNA of HPgV-2 and hepatitis C virus (HCV) with retrospective plasma samples collected from 771 HIV infections with multiple risk behaviors in Honghe Prefecture of Yunnan Province. A total of 195 subjects (25.29%) were seroreactive to HPgV-2, and 41 (5.32%) were RNA positive. Although the positive rate of HPgV-2 antibodies in HIV/HCV-coinfected individuals (27.69%) was significantly higher than that of HIV monoinfections (20.82%) (p = 0.036), this is the first report of HPgV-2 viremia in HIV-infected individuals without HCV infection and the presence of two HPgV-2 lineages in China. Our data indicate that HPgV-2 can also be transmitted sexually, which might be facilitated when combined with HCV infection, injecting drug use, and risky sexual behavior, which appear to have a synergistic effect on HPgV-2 infection. Phylogenetic analysis of 26 near-full-length genome sequences showed that the HPgV-2 strains in China are divided into two clusters.

The prevalence of drug resistance among treatment-naïve HIV-1-infected individuals in China during pre- and post- 2004.

BACKGROUND: The widespread use of antiretroviral therapies has led to considerable concerns about the prevalence of drug-resistant, as transmission of drug-resistant (TDR) strains poses a challenge for the control of the HIV-1 epidemic. METHODS: We conducted an epidemiological study enrolling treatment-naïve HIV-1-positive subjects at the Peking Union Medical College Hospital since 1991. Drug resistance was determined by submitting the sequences to the Stanford University Network HIV-1 database. RESULTS: Of 521 participants, 478 samples were amplified and sequenced successfully. HIV Transmitted drug resistance prevalence in China was determined to be 6.7 %. We did not find significant differences in the TDR rate by demographic characteristics. No significant time trend in the prevalence of overall TDR was observed (p > 0.05). CONCLUSIONS: We identified an intermediate prevalence of transmitted drug resistance (TDR), exhibiting a stable time trend. These findings enhance our understanding of HIV-1 drug resistance prevalence and time trend, and provide some guidelines for the comprehensive public health strategy of TDR prevention.

Characterization of two HIV-1 infectors during initial antiretroviral treatment, and the emergence of phenotypic resistance in reverse transcriptase-associated mutation patterns.

BACKGROUND: Highly active antiretroviral therapy (HAART) is recommended to control the infection of HIV-1. HIV-1 drug resistance becomes an obstacle to HAART due to the accumulation of specific mutations in the RT coding region. The development of resistance mutations may be more complex than previously thought. METHODS: We followed two HIV-1 infectors from a HIV-1 drug resistance surveillance cohort in Henan province and evaluated CD4+ T-cell number and viral load thereafter at ten time-periods and characterized their reverse transcriptase-associated mutation patterns at each time point. Then we constructed the recombinant virus strains with these mutation patterns to mimick the viruses and test the phenotypic resistance caused by the mutation patterns on TZM-b1 cells. RESULTS: CD4+ T-cell number initially increased and then decreased rapidly, while viral load decreased and then dropped sharply during initial antiretroviral treatment. The number of mutations and the combination patterns of mutations increased over time. According to the phenotypic resistance performed by recombinant virus strains, VirusT215Y/V179E/Y181C/H221Y exhibited high levels of resistance to EFV (5.57-fold), and T215Y/V179E-containing virus increased 20.20-fold in AZT resistance (p < 0.01). VirusT215Y/V179E/Y181C increased markedly in EFV resistance (p < 0.01). The IC50 for VirusT215Y/V179E/H221Y was similar to that for VirusT215Y/V179E/Y181C. VirusT215Y/K103N/Y181C/H221Y induced a dramatic IC50 increase of all the four agents (Efavirenz EFV, Zidovudine AZT, Lamivudine 3TC, and Stavudine d4T) (p < 0.01). As for VirusT215Y/K103N/Y181C, only the IC50 of EFV was significantly increased. T215Y/K103N resulted in a 26.36-fold increase in EFV (p < 0.01). T215Y/K103N/H221Y significantly increased the resistance to AZT and 3TC. The IC50 of EFV with T215Y/V179E was lower than with T215Y/K103N (F = 93.10, P < 0.0001). With T215Y/V179E, Y181C significantly increase in EFV resistance, while the interaction between 181 and 221 in EFV was not statistically significant (F = 1.20, P = 0.3052). With T215Y/K103N, neither H221Y nor Y181C showed a significant increase in EFV resistance, but the interaction between 181 and 221 was statistically significant (F = 38.12, P = 0.0003). CONCLUSIONS: Data in this study suggests that pathways of viral evolution toward drug resistance appear to proceed through distinct steps and at different rates. Phenotypic resistance using recombinant virus strains with different combination of mutation patterns reveals that interactions among mutations may provide information on the impact of these mutations on drug resistance. All the result provides reference to optimize clinical treatment schedule.

A considerable proportion of CRF01_AE strains in China originated from circulating intrasubtype recombinant forms (CIRF).

BACKGROUND: In this study, the prevalence of HIV-1 CRF01_AE intrasubtype recombinants in China is estimated and their contributions to the epidemic are explored. METHODS: Available HIV-1 complete genomes of CRF01_AE were retrieved from the HIV database. The two alignments were evaluated with RDP3. Recombinants were defined as cases in which the recombination signal was supported by at least 3 methods with P-values of ≤0.05 after Bonferroni correction for multiple comparisons implemented in RDP3. Phylogenetic analysis was performed to further investigate the role of intrasubtype recombinants in epidemics. RESULTS: Here, 124 out of the 339 sequences from around the world (36.6 %) showed significant evidence of recombination. Here, 84 of these recombinants were from China, accounting for 54.9 % of local total sequences (84 out of 153). The results indicated non-negligible levels of intrasubtype recombination. Subsequent phylogenetic analysis indicated that a considerable proportion of CRF01_AE strains in China originated from circulating intrasubtype recombinant forms. Three large, well-supported intrasubtype recombinants clusters were identified here. Through a survey of risk factors and sampling cities and provinces, cluster I and cluster II were found to be prevalent primarily among men who have sex with men in major northern cities. Cluster III was prevalent among heterosexuals and intravenous drug users in southern and southwestern provinces. CONCLUSIONS: The current work highlighted the remarkable prevalence of intrasubtype recombination within the CRF01_AE epidemic and emphasized the value of intrasubtype recombinants, which came to circulate in the same manner as intersubtype recombinants.

Subtype CRF01_AE dominate the sexually transmitted human immunodeficiency virus type 1 epidemic in Guangxi, China.

Human immunodeficiency virus type 1 (HIV-1) infection by sexual transmission in Guangxi, China had increased dramatically. However, limited information is available on the genetic characterization of the HIV-1 epidemic. In this study, HIV-1 seropositive drug-naïve patients infected by heterosexual transmission were enrolled. The full length gag and pol genes were sequenced followed by phylogenetic analysis, recombinant analysis and drug resistant analysis. Multiple subtypes were identified, including CRF01_AE (80.1%), CRF07_BC (6.4%), CRF08_BC (10.2%), subtype B (1.7%), and URFs (1.7%). In the phylogenetic tree, two large CRF01_AE clusters were identified. One cluster is originating from Vietnam strains as being reported previously in intravenous drug users. One novel cluster was identified and showed close relationship to strains from Fujian province. Inter-subtype recombination among CRF01_AE, subtype B and C was identified. Low level drug-resistance in drug-naïve heterosexually transmitted infections was found. The results suggested that multiple originating CRF01_AE strains dominated the HIV-1 epidemic in heterosexual transmission in Guangxi province.

[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.

High level soluble expression, one-step purification and characterization of HIV-1 p24 protein.

BACKGROUND: P24 protein is the major core protein of HIV virus particle and has been suggested as a specific target for antiviral strategies. Recombinant p24 protein with natural antigenic activity would be useful for various studies, such as diagnostic reagents and multi-component HIV vaccine development. The aim of this study was to express and purify the p24 protein in soluble form in E.coli. RESULTS: According to the sequence of the p24 gene, a pair of primers was designed, and the target sequence of 700 bp was amplified using PCR. The PCR product was cloned into pQE30 vector, generating the recombinant plasmid pQE30-p24. SDS-PAGE analysis showed that the His-tagged recombinant p24 protein was highly expressed in soluble form after induction in E. coli strain BL21. The recombinant protein was purified by nickel affinity chromatography and used to react with HIV infected sera. The results showed that the recombinant p24 protein could specifically react with the HIV infected sera. To study the immunogenicity of this soluble recombinant p24 protein, it was used to immunize mice for the preparation of polyclonal antibody. Subsequent ELISA and Western-Blot analysis demonstrated that the p24 protein had proper immunogenicity in inducing mice to produce HIV p24 specific antibodies. CONCLUSION: In this work, we report the high level soluble expression of HIV-1 p24 protein in E. coli. This soluble recombinant p24 protein specifically react with HIV infected sera and elicit HIV p24 specific antibodies in mice, indicating this soluble recombinant p24 protein could be a promising reagent for HIV diagnosis.

[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.

Different Profiles of Antibodies and Cytokines Were Found Between Severe and Moderate COVID-19 Patients.

Objectives: Our objective was to determine the antibody and cytokine profiles in different COVID-19 patients. Methods: COVID-19 patients with different clinical classifications were enrolled in this study. The level of IgG antibodies, IgA, IgM, IgE, and IgG subclasses targeting N and S proteins were tested using ELISA. Neutralizing antibody titers were determined by using a toxin neutralization assay (TNA) with live SARS-CoV-2. The concentrations of 8 cytokines, including IL-2, IL-4, IL-6, IL-10, CCL2, CXCL10, IFN-γ, and TNF-α, were measured using the Protein Sample Ella-Simple ELISA system. The differences in antibodies and cytokines between severe and moderate patients were compared by t-tests or Mann-Whitney tests. Results: A total of 79 COVID-19 patients, including 49 moderate patients and 30 severe patients, were enrolled. Compared with those in moderate patients, neutralizing antibody and IgG-S antibody titers in severe patients were significantly higher. The concentration of IgG-N antibody was significantly higher than that of IgG-S antibody in COVID-19 patients. There was a significant difference in the distribution of IgG subclass antibodies between moderate patients and severe patients. The positive ratio of anti-S protein IgG3 is significantly more than anti-N protein IgG3, while the anti-S protein IgG4 positive rate is significantly less than the anti-N protein IgG4 positive rate. IL-2 was lower in COVID-19 patients than in healthy individuals, while IL-4, IL-6, CCL2, IFN-γ, and TNF-α were higher in COVID-19 patients than in healthy individuals. IL-6 was significantly higher in severe patients than in moderate patients. The antibody level of anti-S protein was positively correlated with the titer of neutralizing antibody, but there was no relationship between cytokines and neutralizing antibody. Conclusions: Our findings show the severe COVID-19 patients' antibody levels were stronger than those of moderate patients, and a cytokine storm is associated with COVID-19 severity. There was a difference in immunoglobulin type between anti-S protein antibodies and anti-N protein antibodies in COVID-19 patients. And clarified the value of the profile in critical prevention.

Infection by Diverse HIV-1 Subtypes Leads to Different Elevations in HERV-K Transcriptional Levels in Human T Cell Lines.

Human endogenous retroviruses (HERVs) make up ~8% of the human genome, and for millions of years, they have been subject to strict biological regulation. Many HERVs do not participate in normal physiological activities in the body. However, in some pathological conditions, they can be abnormally activated. For example, HIV infection can cause abnormal activation of HERVs, and under different infection conditions, HERV expression may be different. We observed significant differences in HERV-K transcription levels among HIV-1 subtype-infected individuals. The transcriptional levels in the HERV-K gag region were significantly increased in HIV-1 B subtype-infected patients, while the transcriptional levels in the HERV-K pol region were significantly increased in CRF01_AE and CRF07_BC subtype-infected patients. In vitro, the transcriptional levels of HEVR-K were increased 5-fold and 15-fold in MT2 cells transfected with two different HIV-1 strains (B and CRF01_AE, respectively). However, there was no significant difference in transcriptional levels among regions of HERV-K. When MT2 cells were infected with different subtypes of HIV-1 Tat proteins (B, CRF01_AE), which is constructed by lentiviruses, and the transcription levels of HERV-K were increased 4-fold and 2-fold, respectively. Thus, different subtypes of HIV-1 have different effects on HERV-K transcription levels, which may be caused by many factors, not only Tat protein.

Multiple HIV-1 Subtypes Were Found Circulating in Suqian District of Jiangsu Province, China.

Jiangsu province has severe HIV-1 epidemic in China. Suqian which is located in north of the province has limited HIV epidemic information. Therefore, this study aimed to characterize the epidemic details in the area. A total of 196 plasma samples were collected from treated HIV-1-positive cases and viral RNA was extracted. Then HIV partial pol genes (nucleotide 2147-3462 by using HXB2 as calibrator) were amplified and sequenced. Finally, 84 partial pol genes were successfully obtained. The subtyping results indicate that multiple HIV-1 subtypes are circulating in Suqian district. Thereinto, CRF01_AE has been the dominant stains here and belonged to multiple lineages of CRF01_AE identified in China previously. Moreover, there is a high level of HIV drug resistance. All these results suggest HIV-1 epidemic in Suqian is rather complex and more measures must be performed for prevention and intervention in the area.

Construction and characterization of a full-length infectious molecular clone from the HIV type 1 subtype Thai-B isolated in Henan province, China.

Among the various subtypes of the M group of human immunodeficiency virus type 1 (HIV-1), subtype Thai-B is the most prevalent in China, particularly in the country's central region. Here we report on the construction of an infectious molecular clone (CNHN24) of this HIV-1 subtype. We show that the viral stock obtained after transfection of CHNH24 could replicate efficiently in PBMC and MT4 cells. Unlike other previously reported HIV infectious clones, CNHN24 was constructed with the low copy plasmid pLG338, allowing for the HIV genome to be very stable during the process of molecular manipulation. Given the prevalence of subtype Thai-B in China's HIV epidemic, the availability of pCNHN24 as the first infectious molecular clone of this subtype provides a useful tool for a wide range of studies including antiviral drug and vaccine research as related to this subtype of viruses.

Performance of the Abbott RealTime HIV-1 assay for quantification of HIV-1 clades prevalent in China.

OBJECTIVE: To evaluate the performance of the Abbott RealTime HIV-1 assay for measuring viral loads of the prevalent HIV-1 clades in China. STUDY DESIGN: Serially diluted samples, as well as 521 clinical samples from 213 untreated HIV-1-infected individuals, 56 HIV-1-infected patients receiving ART, 60 HCV- and 57 HBV-infected patients and 135 healthy blood donors, were tested with RealTime and EasyQ. RESULTS: Both assays exhibited linearity coefficients of >0.98. RealTime and EasyQt detected HIV-1 RNA in 87.36% and 86.99% of 269 HIV-1-seropositive samples, respectively. The correlation coefficients between the two assays for quantifying HIV-1 clades B', BC and AE were 0.884, 0.813 and 0.881, respectively, and the mean differences between the two assays for clades B', BC and AE were -0.087, 0.314 and 0.559 log(10)IU/mL, respectively. The correlation coefficient between the two assays for measuring samples from patients receiving ART was 0.945 (mean difference, 0.085 log(10)IU/mL). No false-positive samples were found among the 60 HCV-infected patients, 57 HBV-infected patients and 135 healthy blood donors. CONCLUSIONS: The Abbott RealTime HIV-1 assay shows good linearity and specificity. ART drugs and HIV-1 clades B' and BC do not affect the performance of the assay. Based on the comparison data, [corrected] clade AE may be more [corrected] readily detected by using this method [corrected]

Phylodynamic Analysis Revealed That Epidemic of CRF07_BC Strain in Men Who Have Sex with Men Drove Its Second Spreading Wave in China.

CRF07_BC was originally formed in Yunnan province of China in 1980s and spread quickly in injecting drug users (IDUs). In recent years, it has been introduced into men who have sex with men (MSM) and become the most dominant strain in China. In this study, we performed a comprehensively phylodynamic analysis of CRF07_BC sequences from China. All CRF07_BC sequences identified in China were retrieved from database. More sequences obtained in our laboratory were added to make the dataset more representative. A maximum-likelihood (ML) tree was constructed with PhyML3.0. Maximum clade credibility (MCC) tree and effective population size were predicted by using Markov Chains Monte Carlo sampling method with Beast software. A total of 610 CRF07_BC sequences coving 1,473 bp of the gag gene (from 817 to 2,289 according to HXB2 calculator) were included into the dataset. Three epidemic clusters were identified; two clusters comprised sequences from IDUs, while one cluster mainly contained sequences from MSMs. The time of the most recent common ancestor of clusters that composed of sequences from MSMs was estimated to be in 2000. Two rapid spreading waves of effective population size of CRF07_BC infections were identified in the skyline plot. The second wave coincided with the expanding of MSM cluster. The results indicated that the control of CRF07_BC infections in MSMs would help to decrease its epidemic in China.

Genetic Characterization of a Unique Recombinant Strain Identified in Yunnan with Genome Comprising B and C.

Since the first identification of HIV-1 outbreak in Dehong, Yunnan province has been the epidemic center of HIV in China. Owing to the special geographic location and the frequent population mobility, Yunnan province contained complex HIV subtype distribution. Many new circulating recombinant forms (CRFs) and unique recombinant forms (URFs) have been found in recent years. In this study, a unique HIV-1 recombinant strain genome (YN10134) was characterized from an HIV-positive female in Yunnan, China. This virus genome had a complex intersubtype recombinant structure with eight breakpoints, composed of subtypes B and C. Although the sequence had a similar breakpoint with CRF07_BC in the start position in Env, the phylogenetic analysis showed that the segment was not originated from CRF07_BC. The identification of the URF indicated the severity of the HIV epidemic in Yunnan province and the urgent need for epidemiological surveillance of the new recombination.

Near Full-Length Genomic Sequences of Two Novel HIV-1 Recombinant Forms Identified in Shenzhen, China.

Most HIV subtypes prevalent in China can be found in Shenzhen, including CRF07_BC, CRF01_AE, CRF08_BC, CRF55_01B, and subtype B. Multiple subtypes spreading in the same population always lead to the emergence of unique recombinant strains. Here, we report two unique recombinant forms (SZ44LS7251 and SZ95LS8027) of HIV-1 identified in a heterosexual population. Recombinant analyses were fulfilled based on the near full-length genomes. Both strains comprise subtypes B, C, and CRF01_AE. Phylogenetic analysis reveals that SZ44LS7251 is the second generation recombination originated from CRF55_01B andCRF07_BC, whereas SZ95LS8027 comprises CRF01_AE and CRF07_BC.The emergence of second generation recombination of HIV with complicated genomic structures supposed that high ratio of super infections or coinfections might happen in the Shenzhen area.

Phylodynamics of major CRF01_AE epidemic clusters circulating in mainland of China.

As the most dominant HIV-1 strain in China, CRF01_AE needs to have its evolutionary and demographic history documented. In this study, we provide phylogenetic analysis of all CRF01_AE pol sequences identified in mainland China. CRF01_AE sequences were collected from the Los Alamos HIV Sequence Database and the local Chinese provincial centers of disease control and prevention. Phylogenetic trees were constructed to identify major epidemic clusters. Bayesian coalescent-based method was used to reconstruct the time scale and demographic history. There were 2965 CRF01_AE sequences from 24 Chinese provinces that were collected, and 5 major epidemic clusters containing 85% of the total CRF01_AE sequences were identified. Every cluster contains sequences from more than 10 provinces with 1 or 2 dominant transmission routes. One cluster arose in the 1990s and 4 clusters arose in the 2000s. Cluster I is in the decline stage, while the other clusters are in the stable stage. Obvious lineage can be observed among sequences from the same transmission route but not the same area. Two large clusters in high-level prevalence were found in MSM (Men who have sex with men), which highlighted that more emphasis should be placed on MSM for HIV control in mainland China.

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.

HIV-1 is spreading out of former high-risk population through heterosexual transmission in Hebei, China.

INTRODUCTION: Multiple specific populations, including MSMs, IDUs, and FPDs, are involved in HIV epidemic in China. In the recent years, HIV transmission due to heterosexual transmission also contributed greatly to HIV epidemic in China. Very few studies have been fulfilled to characterize relationships of HIV-1 strains prevalent in different populations. In this study, the phylogenetic relationships of HIV-1 spreading in different populations were investigated. MATERIALS AND METHODS: HIV-1 sero-positive patients infected through different routes were enrolled into the study. Nested RT-PCR was used to amplify HIV gag and pol genes followed by sequencing. RESULTS: Multiple subtypes, including subtype B (52.1%), CRF01_AE (34.4%), CRF07_BC (6.3%), subtype C (4.2%), CRF02_AG (1.0%), CRF08_BC (1.0%) and unique recombination forms (1.0%) were identified. Phylogenetic analysis showed that strains from MSM, IDU, and FPDs grouped into clusters separately. However, strains identified in heterosexual transmitted population intermixed with all of other high risk populations. DISCUSSION AND CONCLUSION: The genetic data supposed that HIV-1 was spreading out of MSMs, IDUs, and FPDs through heterosexual transmission in Hebei, China. Urgent prevention and behavior intervention in the population will be necessary. Furthermore, the detailed sequence data will help the design of HIV-1 vaccines in China. Sequence Data: All of sequences have been deposited into the GenBank with the accession number: KJ820007-KJ820144.

The Antiviral Activity of Approved and Novel Drugs against HIV-1 Mutations Evaluated under the Consideration of Dose-Response Curve Slope.

OBJECTIVES: This study was designed to identify common HIV-1 mutation complexes affecting the slope of inhibition curve, and to propose a new parameter incorporating both the IC50 and the slope to evaluate phenotypic resistance. METHODS: Utilizing site-directed mutagenesis, we constructed 22 HIV-1 common mutation complexes. IC50 and slope of 10 representative approved drugs and a novel agent against these mutations were measured to determine the resistance phenotypes. The values of new parameter incorporating both the IC50 and the slope of the inhibition curve were calculated, and the correlations between parameters were assessed. RESULTS: Depending on the class of drug, there were intrinsic differences in how the resistance mutations affected the drug parameters. All of the mutations resulted in large increases in the IC50s of nucleoside reverse transcriptase inhibitors. The effects of the mutations on the slope were the most apparent when examining their effects on the inhibition of non-nucleoside reverse transcriptase inhibitors and protease inhibitors. For example, some mutations, such as V82A, had no effect on IC50, but reduced the slope. We proposed a new concept, termed IIPatoxic, on the basis of IC50, slope and the maximum limiting concentrations of the drug. The IIPatoxic values of 10 approved drugs and 1 novel agent were calculated, and were closely related to the IIPmax values (r > 0.95, p < 0.001). CONCLUSIONS: This study confirms that resistance mutations cannot be accurately assessed by IC50 alone, because it tends to underestimate the degree of resistance. The slope parameter is of very importance in the measurement of drug resistance and the effect can be applied to more complex patterns of resistance. This is the most apparent when testing the effects of the mutations on protease inhibitors activity. We also propose a new index, IIPatoxic, which incorporates both the IC50 and the slope. This new index could complement current IIP indices, thereby enabling predict the efficacy of pre-clinical drugs for which human pharmacokinetic is not available.