Construction of HIV-1 B subtype pseudovirus system in Guangxi / 实用医学杂志

Objective To establish a pseudovirus system for phenotypic drug-resistance detection and provide a relatively cheap and easy method for drug-resistance testing. Methods EGFP gene was amplified from plasmid pSV-EGFP and then cloned to backbone plasmid pNL4-3.Luc. E-R-by double enzyme digestion;env gene was amplified from RNA isolated from HIV-1-infected persons and cloned to eukaryotic expression plasmid cells and EGFP or ENV expression. Pseudovirus was produced by co-transfection of two recombinant plasmids to 293t cells. Infection of pseudovirus was determined by co-cultured with TZM-b1 cells and immunofluorescent test. Results Two recombinant plasmids(mass ratio,pcDNA3.1-env:pNL4-3.EGFP.E-R-.=2:1)were co-transfected to 293t cells. Cultured supernatants containing pseudovirus were harvested at 48 h post-transfection. Fluorescence was observed in TZM-b1 cells after TZM-b1 cells were infected with pseudovirus at 48 h post-infection. Conclusion The recombinant pseudovirus carrying EGFP gene is constructed successfully and it could be used for phenotypic drug-resistance detection.

Analysis on influencing factors of late detection for newly diagnosed HIV/AIDS positive patients in Guangxi in 2015 / 中华疾病控制杂志

Objective To explore the influencing factors of late diagnosis for newly diagnosed HIV/AIDS positive patients in Guangxi in 2015.Methods The CD4 + T lymphocytes count which was first detection for newly diagnosed HIV/AIDS positive patients in Guangxi during 2015 was collected.Data were statistically analyzed.Results We collected 8 586 newly diagnosed HIV/AIDS whose median CD4+ T lymphocytes counts was 237.5 cells/μl,and 43.12％ of them had less than 200 cells/μl.Gender,age,occupation,marriage,nation,education,route of transmission,types of testing and region had effects on late HIV diagnosis(all P ＜ 0.05).Logistic analysis found that risk factors associated with the late diagnosis of HIV were male(OR =1.851,95％ CI:1.673-2.048),migrant worker (OR =1.387,95％ CI:1.242-1.549),education below middle and secondary school(OR =1.619,95％ CI:1.400-1.873),currently married(OR =1.207,95％ CI:1.075-1.354),divorced or widowed(OR =1.508,95％ CI:1.309-1.738).Voluntary testing was a protective factor.Conclusions The prevalence the late diagnosis of HIV was high in Guangxi in 2015,it is crucial for related departments to enhance the testing and screening effort for HIV/AIDS.

Distribution of subtypes of pol gene in HIV-1 epidemic strains in Guangxi Zhuang Autonomous Region, 2010- 2012 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the distribution and proportion of subtypes of pol gene in HIV-1 epidemic strains in Guangxi Autonomous Region.</p><p><b>METHODS</b>152 HIV-1 patients were enrolled from 11 cities in Guangxi Autonomous Region from 2010 to 2012 by convenient sampling. Inclusion criterias were listed as the fdlowing: HIV-1 infection was confirmed by Western blot, HIV-1 viral load >1 000 copies/ml, > 18 year-old, and without any serious illnesses. 5 ml of peripheral blood samples were obtained from each patient. The viral RNA was isolated from plasma and used for amplification of full-length pol gene by nested RT-PCR. The amplified products were sequenced. After editing and modification, all sequences were characterized for preliminary subtyping by genotyping and confirmed with phylogenetic tree constructed by MEGA 5.03 software. The recombinant identification of 2 unknown recombinant strains was determined by RIP and jpHMM at GOBICS.</p><p><b>RESULTS</b>Among 152 patients, 137 full-length pol genes were successfully amplified and 127 HIV-1 subtypes were identified. The distribution and proportion of subtypes was summarized as the following 71 cases of CRF01_AE, accounting for 55.9% (71/127), 38 CRF08_BC, 29.9% (38/127), 13 CRF07_BC, 10.2% (13/127), and 3 B (B'), 2.4% (3/127), 2 unknown recombinant strains, 1.6% (2/127). In 11 cites of Guangxi Autonomous Region, subtype CRF01_AE was the dominant strain. Among heterosexual transmitted patients and drug abusers, the proportions of subtype CRF01_AE were 67.4% (58/86) and 34.1% (14/41), respectively. There was a significance different in the distribution of CRF01_AE in different routes of transmission (χ(2)=15.07, P<0.001). In age 21- 35, age 36- 60 and age>60 groups, the proportions of CRF01_AE was 43.6% (17/39), 57.6% (38/66), 77.3% (17/22), and CRF08_BC was 43.6% (17/39), 28.8% (19/66), 9.1% (2/22), respectively, the difference in proportions was significant(χ(2)=8.48, P= 0.014). The patterns of two unknown recombinant strains were found to be CRF01_AE/B (B') and CRF01_AE/C/B(B'), respectively.</p><p><b>CONCLUSION</b>CRF01_AE was the dominant HIV-1 subtype in Guangxi Autonomous Region from 2010 to 2012, with heterosexual transmission as its main spreading route. The two unknown recombinant strains in Guangxi Autonomous Region were reconstructed by subtype CRF01_AE and CRF_BC.</p>

Investigation of the HIV-1 drug resistance in Guangxi during 2009 to 2012 / 中华微生物学和免疫学杂志

Objective To investigate the HIV-1 drug resistance in Guangxi during 2009 to 2012 and to analyze the correlations between drug resistance and HIV-1 subtypes.Methods Patients with human immunodeficiency virus infection or acquired immune deficiency syndrome ( HIV/AIDS) were randomly re-cruited from different areas in Guangxi.HIV-1 RNA was extracted from blood samples of the subjects and converted into complementary DNA ( cDNA) by using reverse transcription.The pol gene was amplified and sequenced.Subtyping analysis was performed by using the online analysis tool of Genotyping in combination with the MEGA 5.03 software.The HIV resistance mutations were determined and scored with the use of Stanford HIV Drug Resistance Database.Results A total of 196 pol gene sequences were obtained from 103 antiretroviral therapy (ART)-treated subjects (52.55%) and 93 ART-na?ve subjects (47.45%).The 196 pol gene sequences were classified into four subtypes including CRF01_AE, CRF08_BC, CRF07_BC and B, accounting for 48.47%, 44.90%, 6.12%and 0.51%, respectively.The HIV drug resistance rates in sub-jects with and without ART were 10.68% and 7.53%, respectively.Among the 196 subjects, 14 cases showed low level of drug resistance, 3 cases showed moderate level of drug resistance and 4 cases showed high level of resistance.Only one case was resistant to both nucleoside reverse transcriptase inhibitors ( NR-TIs) and non-nucleoside reverse transcriptase inhibitors ( NNRTIs) .The resistance rates of the 196 cases to protease inhibitor (PIs), NRTIs, NNRTIs, and integrase inhibitors (INs) were 6.63%, 3.06%, 11.22%and 8.67%, respectively.The frequencies of PIs-related mutations in subtypes CRF01_AE, CRF07_BC and CRF08_BC were 6.32%, 41.67% and 2.27%, respectively.Most of the PI-related A71V/T mutations were identified in strains belonging to subtype CRF07_BC, accounting for 75% of all A71V/T mutations found in the 196 strains.The NNRTI-related E138A mutations only appeared in strains belonging to subtype CRF08_BC.Conclusion The drug resistance rate among patients with HIV-1/AIDS in Guangxi was higher than the average level in China.The drug resistance rates varied with the subtypes of HIV-1 strains.

Influence of Opiate Abuse on Expression of Toll-like Receptor 9 in Peripheral Blood Mononuclear Cells of HIV-1-Infected Individuals / 病毒学报

The aim of this study was to investigate the influence of opiate abuse on the expression of Toll-like receptor 9 (TLR9) in the peripheral blood mononuclear cells (PBMCs) of HIV-1-infected patients and to elucidate possible mechanisms involved in the enhancement of HIV-1 replication by opiate abuse. A total of 200 participants were enrolled in the study by random selection from methadone treatment centers and voluntary HIV counseling and testing centers in the cities of Nanning, Liuzhou, and Qinzhou. These participants included 50 HIV-positive opiate abusers (Opiates HIV(+) group), 50 HIV-negative opiate abusers (Opiates HIV(-) group), 50 HIV-positive subjects who were not opiate abusers (Non-opiates HIV (+) group), and 50 HIV-negative subjects who were not opiate abusers (Control group). PBMCs were isolated from the peripheral blood samples from the subjects and the expression levels of TLR9 mRNA and protein were determined by q-PCR and western blot respectively. There was no significant difference among the four groups in age, gender, nationality, domicile, marital status, educational background or duration of drug abuse (P > 0.05). The median viral loads of the Opiates HIV(+) were significantly higher than those of the Non-Opiates HIV(+) groups (4.450 x 10(3) and 3.977 x 10(3) copies/mL respectively, P < 0.05). The relative expression levels of TLR9 mRNA in the Opiates HIV(+), Non-Opiates HIV(+), Opiates HIV(-) and Control groups were (2.13 +/- 1.59) x 10(-3), (3.66 +/- 2.22) x 10(-3), (1.96 +/- 1.42) x 10(-3) and (7.66 +/- 4.87) x 10(-3), respectively. The expression of TLR9 mRNA was significantly lower in both HIV-1-infected and -uninfected groups of opiate abusers compared with groups of non-abusers (P < 0.05). There was no significant difference in TLR9 mRNA expression levels between the Opiates HIV(+) group and the Opiates HIV(-) group (P > 0.05). However, in the non-opiate groups, the expression levels of TLR9 mRNA in the HIV(+) group were significantly lower than that of the control group (P< 0.05). Western blot results confirmed that the expression of TLR9 protein was lower in the Opiates HIV(+), Non-Opiates HIV(+), and Opiates HIV(-) groups compared to the control group. These results suggest that opiate abuse can decrease the expression of TLR9 in PBMCs, which may result in the enhancement of HIV-1 infection and replication due to a decline in immune response mediated by the TLR9 pathway.

Expressions of Toll-like receptor 2 and the downstream proteins in human immunodeficiency virus/Mycobacterium tuberculosis co-infection patients / 中华传染病杂志

Objective To explore the expressions of Toll-like receptor 2 (TLR2 ) and the downstream proteins in patients with human immunodeficiency virus /Mycobacterium tuberculosis (HIV /M TB) co-infection .Methods A total of 119 subjects were randomly enrolled .The subjects were divided into four groups :HIV group (n = 32) ,HIV /M TB group (n = 30) ,M TB group (n = 28) and healthy control group (n= 29) .Peripheral venous blood was collected and the HIV-1 viral load was determined by standard method .The expression levels of TLR2 mRNA in peripheral blood mononuclear cells (PBMC) were determined by real-time quantitative PCR (qPCR) and mean fluorescent intensity (MFI) of TLR2 protein was detected by flow cytometry .The plasma interleukin (IL)-6 and tumor necrosis factor (TNF)-α levels were measured with enzyme-linked immunosorbent assay kits .The data were statistically analyzed by chi-square test ,students t test ,analysis of variance and rank sum test when appropriate .Results The viral load in HIV /M TB group ([5 .113 ± 1 .018] lg copy/mL ) was significantly higher than that in HIV group ([4 .416 ± 1 .020] lg copy/mL ; t = 3 .449 , P among HIV ,HIV/M TB ,M TB and healthy control groups were 1 .397 ± 0 .601 ,1 .463 ± 0 .702 ,1 .429 ± 0 .630 ,and 0 .970 ± 0 .488 ,respectively ,which was significantly different among the 4 groups (F =4 .197 , P= 0 .007) .The MFI of TLR2 protein expressions on PBMC among HIV ,HIV /M TB ,M TB and healthy control groups were 28 .12 ± 4 .55 ,38 .11 ± 11 .77 ,31 .13 ± 12 .10 and 23 .33 ± 5 .14 ,respectively . The TLR2 protein expression levels were significantly different among 4 groups (F= 13 .976 ,P< 0 .01) . The plasma IL-6 and TNF-α concentrations were significantly different among 4 groups (Z = 19 .088 , 15 .475 ,both P< 0 .01) .The IL-6 concentrations in three patient groups were higher than that in healthy control group ,but the TNF-α concentrations were lower than healthy control group .Conclusions The co-infection of HIV-1 and M TB may enhance the activation of TLR2 signaling pathway ,which leads to the increased expression of IL-6 .

The impact of methamphetamine on the expressions of macrophage inflammatory proteins in human immunodeficiency virus-infected patients / 中华传染病杂志

Objective To investigate the impact of methamphetamine (Meth) on the expressions of macrophage inflammatory protein (MIP)-1α ,MIP-1β ,interleukin (IL)-6 among human immunodeficiency virus(HIV)-infected patients .Methods The investigation was performed among 15 Meth-abuse and HIV-infected subjects (Meth + HIV ) ,15 non-Meth-abuse and HIV-infected subjects (non-Meth + HIV ) ,15 Meth-abuse and HIV-uninfected subjects (Meth) ,and 15 healthy subjects (HC) .CD4 + T lymphocyte counts in peripheral blood were detected by flow cytometry .The HIV viral loads in HIV-infected patients were detected by standard detection method .The levels of plasma MIP-1α ,MIP-1β and IL-6 from four groups were determined by enzyme-linked immunosorbent assay (ELISA ) .Intergroup difference was compared using t-test and interactive analysis was conducted using analysis of variance .Results In HIV-infected patients ,CD4 + T lymphocyte counts in Meth + HIV group was significant lower than non-Meth +HIV group (t= 5 .431 , P 0 .05) ,neither between HIV infection and the levels of cytokines (P> 0 .05) .Conclusion Meth abuse results in elevated expressions of MIP-1αand MIP-1β ,which indicates that Meth abuse may play a regulating role on promoting HIV infection .

The efficacy of antiviral therapy and drug resistance analysis among HIV/AIDS patients with heroin addiction in Guangxi Zhuang Autonomous Region / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the impact of heroin for antiviral treatment, drug resistance, mutation types and frequency in HIV/AIDS patients in Guangxi Zhuang Autonomous Region.</p><p><b>METHODS</b>HIV/AIDS patients were recruited in Methadone Maintenance Treatment Clinics, HIV/AIDS Clinic and HIV Voluntary Counseling and Testing Center Liuzhou and Baise city from April 2008 to October 2009. The patients were grouped by the situation of antiviral treatment and use of heroin. A total of 435 HIV/AIDS patients were recruited, among which 108 cases in antiviral treatment and heroin group, 93 cases in antiviral treatment and never using drug group, 105 cases in no antiviral treatment and using heroin group, 129 cases in no antiviral treatment and never using drug group. The effect of antiviral treatment was evaluated by questionnaire survey, viral load measurement and CD4(+) T lymphocyte count. HIV-1 RNA from plasma was extracted, and then the pol genes were amplified and sequenced. The sequences were analyzed for HIV-1 genotype drug-resistance.</p><p><b>RESULTS</b>For the patients who received antiviral treatment, the viral load in heroin group was higher than that in never using drug group (lg (2.61 ± 1.24) vs lg (2.08 ± 0.80), t = 3.54, P < 0.05) , and the percentage of viral load lower than 1 000 copies/ml in heroin group was significantly less than that in never using drug group (63.9% vs 86.0%,χ(2) = 12.76, P < 0.05). For the patients who received antiviral treatment, the difference has no significance in CD4(+) T lymphocyte count between heroin group and never using drug group ((337.92 ± 181.66) vs (326.14 ± 254.98), t = 0.38, P = 0.703). For the patients who didn't receive antiviral treatment, the difference also has no significance in CD4(+) T lymphocyte count between heroin group and never using drug group ((373.73 ± 155.97) vs (337.53 ± 209.26), t = 1.47, P = 0.143). For the patients who received antiviral treatment, there was no difference in the percentage of the CD4(+) T lymphocyte count more than 350/ml between heroin group and never using drug group (48.1% vs 43.0%, χ(2) = 0.53, P = 0.466). 319 HIV-1 pol gene sequences were obtained. Among the patients who received antiviral treatment, the mutation frequency of M184V/I, T215Y/F, L210W and T69N/S in heroin abuser group were significantly higher than that in never using drug group (14.9% (11/74) vs 4.4% (3/68), 12.2% (9/74) vs 1.5% (1/68), 12.2% (9/74) vs 1.5% (1/68) and 10.8% (8/74) vs 1.5% (1/68) respectively) (P < 0.05).</p><p><b>CONCLUSION</b>Using heroin may promote HIV replication, reducing the virological response to antiviral treatment and increasing the frequencies of drug resistance loci among HIV/AIDS patients.Heroin rehabilitation may benefit from the antiviral treatment and obtain better antiviral effect.</p>

Expression of TLR4 in peripheral blood mononuclear cells and plasma concentrations of TNF-αin patients with HIV/MTB co-infection / 中华微生物学和免疫学杂志

Objective To investigate the expression of TLR 4 and its downstream factor TNF-αin the patients with human immunodeficiency virus and Mycobacterium tuberculosis ( HIV/MTB) co-infection. Methods A total of 119 subjects including 32 patients with HIV infection (HIV group), 30 patients with HIV/MTB co-infection (HIV/MTB group), 28 patients with MTB infection (MTB group) and 29 healthy subjects ( control group ) were recruited continuously from the Fourth People′s Hospital of Nanning City , Guangxi.The expression of TLR4 in peripheral blood mononuclear cells (PBMCs) from the patients was de-termined by flow cytometry .ELISA was performed to detect TNF-αin plasma samples .The HIV-1 viral load was determined by standard method .Results The mean fluorescence intensity ( MFI) for TLR4 expression in PBMCs from HIV, HIV/MTB, MTB and control groups were 21.62±4.67, 18.29±3.87, 16.79±4.45, and 22.85±5.80, respectively, showing significant differences among four groups (F=8.105, P<0.01). The TLR4 levels in MTB and HIV/MTB groups were significantly lower than those in control group ( both P<0.01) and HIV group (P<0.01, P=0.014).The plasma concentrations of TNF-αin HIV, HIV/MTB, MTB and control groups were 15.892 (10.494-21.646) pg/ml, 13.142 (8.014-22.038) pg/ml, 16.284 (11.916-24.005) pg/ml, and 26.657 (16.321-34.541) pg/ml, respectively, that were significantly dif-ferent from each other (F=4.350, P=0.006).The levels of TNF-αin plasma from patients with HIV and HIV/MTB infection were significantly lower than those of healthy subjects (P=0.009 and P=0.001).The viral load in patients from HIV/MTB group (5.113 ±1.018 copies/ml) was significantly higher than that from HIV group (4.416±1.020 copies/ml) (t=3.449, P=0.001).Conclusion MTB infection might promote HIV replication by inhibiting the expression of TLR 4.HIV infection might increase host′s suscepti-bility to MTB infection by reducing the production of TNF-α.Suppressed expression of TLR and TNF-αpro-duction could contribute to the occurrence of HIV /MTB co-infection .

Inhibition of Toll-like receptor 2 expression by siRNA in human monocyte-derived macrophages / 中国免疫学杂志

To screen the siRNAs (small interference RNA sequences ) which specifically inhibit the gene expression of TLR2 in human monocyte-derived macrophage , and discuss their prospects on the treatment of HIV at the level of molecular immunology.Methods:We obtained the mRNA sequences of human TLR 2 gene from NCBI gene bank ,then designed three siRNAs by siDESIGNTM software.The siRNA targeting human housekeeping gene GAPDH was used as positive control.The fluorescent labeling missense siRNA sequences (NC-FAM ) was used as negative control.We collected fresh peripheral blood from healthy volunteers and isolated mononuclear cells from the blood samples.The human mononuclear macrophages were then purified from mononuclear cells by utilizing adherence method.Cationic liposome reagent Lipofectamine 2000 was used to mediate siRNAs into the human mononuclear macrophages.The levels of TLR2 mRNA expression of siRNA-transfected monocyte-derived macrophage were determined by q-PCR.Expression of TLR2 protein was determined by Western blot.Results: At 72 h after transfection ,we found that the expression of GAPDH mRNA and protein in positive control group decreased significantly.Also found there existed significant differences between each siRNA group (F=41.957,P<0.001).Compared with negative control ,the relative expression of TLR2 mRNA in all siRNAs groups decreased significantly (P<0.05 ) , and the inhibition rates were 46%, 43%, 43% by three miRNAs respectively.Western blot showed that the expression of TLR 2 protein in siRNAs groups decreased significantly compared with that of control (P<0.05 ).Conclusion: The designed siRNAs in this study could inhibit the expression of TLR 2 gene in human monocyte-derived macrophage ,indicating that mediation of TLR-2 expression by siRNA might be a novel strategy for HIV treatment from the per-spective of molecular immunology.

Observation on Polytene Chromosomes of the Ovarian Nurse Cell of Anopheles minimus from Yuanjiang / 中国寄生虫学与寄生虫病杂志

Anopheles minimus collected from Yuanjiang, Yunnan Province, were bred with standard methods in lab. The ovarian nurse cells of A.minimus were separated and stained, and the whole polytene chromosomes were photographed under light microscope and compared with A.minimus from Guangxi. 365 samples of ovarian nurse cells were observed. The chromosomes included one telocentric sex-chromosome X, two submetacentric autosomes II(autosome II right arm, 2R and autosome II left arm, 2L) and two metacentric autosomes III(autosome III right arm, 3R, and autosome III left arm, 3L). The X is the shortest chromosome and the 2R is the longest one. In comparison with the pattern of polytene chromosomes of A. minimus from Guangxi, difference at 12 positions has been found at the parts of arms in banding sequences.