Construction and expression of an optimized, novel human immunodeficiency virus type-1 lentiviral vector containing green fluorescent protein

The human immunodeficiency virus （HIV） lentiviral vector is an ideal vector for gene therapy. In the present study, the wild-type HIV-1 genome was segregated into four plasmids, and an optimized novel HIV-1 lentiviral vector containing green fluorescent protein and vesicular stomatitis virus G pseudo-capsule was constructed. The plasmids were pHR-CMV-EGFP, pCMVΔ8.9, pRSV-Rev, pCMV-VSV-G. The four plasmid system was co-transfected into 293T cells, and green fluorescent protein expression was observed. The present study obtained lentiviral particles by high-speed centrifugation, and the lentiviral particle titer was 4 × 108 TU/mL after centrifugation. Thus, an optimized novel HIV-1 lentiviral vector was successfully constructed.

INTRACELLULAR EXPRESSION OF MULTIMERIZED ANTISENSE TAR-CORE RNAS INHIBIT THE REPLICATION OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 IN HUMAN CD_4+T LYMPHOCYTES

Gene therapy is one of several approaches that are being tested in the search for an effective anti HIV treatment. In this strategy, a “resistant” gene would be introduced into target cells, rendering them resistance to the infection of HIV. The HIV 1 Tat protein transactivate HIV 1 gene expression at the transcriptional level by interacting with its response element(TAR) in the long terminal repeat(LTR). Previously, we have shown that antisense polyTAR Core RNAs can inhibit the transactivation of HIV 1 Tat protein in transiently transfected Jurkat cells. To determine whether this antisense polyTAR Core RNAs could inhibit HIV 1 replication in CD 4+ T cells, we transfected the antisense polyTAR Core gene to MT4 cells and challenged them with HIV 1 SF33 strain. Levels of HIV 1 p24gag antigen were reduced more than 4 fold in cultures of the transduced MT4/LR cells infected with HIV 1SF33 strain. In contrast, cultures of nontransduced MT4 cells and control LX vector transduced MT4/LX cells infected with the same viruses had high levels of HIV 1 p24gag. Our work showed that antisense polyTAR Core RNAs were able to inhibit HIV 1 replication in CD 4+ T cells, and could be used as resistance gene in further studying for gene therapy against HIV 1.

Human mpox co-infection with advanced HIV-1 and XDR-TB in a MSM patient previously vaccinated against smallpox:A case report

Mpox is a zoonotic infectious disease caused by the mpox virus(MPXV).Historically,the majority of mpox cases have been documented in Central Africa.However,since May 2022,there has been a notable rise in reported cases from regions beyond Africa.Currently,over 110 countries spanning Europe,North America,South America,Asia,and other territories have reported mpox infections.This report details a case involving a patient who identifies as a man who has sex with men(MSM)and is concurrently infected with MPXV,human immunodeficiency virus type 1(HIV-1),Pneumocystis jiroveci,as well as extensively drug-resistant tuberculosis(XDR-TB).This patient had also received a vaccination for smallpox in the past.Additionally,we provide photographic documentation charting the progression of dermatological manifestations associated with mpox.This case highlights the significance of sexual intercourse as a crucial mode of transmission for mpox.The rapid and widespread dissemination of the MPXV across various regions,especially among MSM communities,underscores the importance of enhancing preventive education efforts targeted at high-risk populations.

Differential selection in HIV-1 gp120 between subtype B and East Asian variant B'

HIV-1 evolves strongly and undergoes geographic differentiation as it spreads in diverse host populations around the world.For instance,distinct genomic backgrounds can be observed between the pandemic subtype B,prevalent in Europe and North-America,and its offspring clade B＇ in East Asia.Here we ask whether this differentiation affects the selection pressure experienced by the virus.To answer this question we evaluate selection pressure on the HIV-1 envelope protein gp120 at the level of individual codons using a simple and fast estimation method based on the ratio ka/ks of amino acid changes to synonymous changes.To validate the approach we compare results to those from a state-of-the-art mixed-effect method.The agreement is acceptable,but the analysis also demonstrates some limitations of the simpler approach.Further,we find similar distributions of codons under stabilizing and directional selection pressure in gp120 for subtypes B and B＇ with more directional selection pressure in variable loops and more stabilizing selection in the constant regions.Focusing on codons with increased ka/ks values in B＇,we show that these codons are scattered over the whole of gp120,with remarkable clusters of higher density in regions flanking the variable loops.We identify a significant statistical association of glycosylation sites and codons with increased ka/ks values.

Full-length clone and characterization of a human immunodeficiency virus type 1 subtype B' isolated from Hubei Province,China

There are two types of Human Immunodeficiency Virus （HIV）： HIV-1 and HIV-2. HIV-1 dominates epidemics in many different parts of the world, and HIV-2 is principally responsible for the epidemic in westem Africa. In China, Zeng et al have reported the first individual infected with HIV-1 in 1985. And in the 1990s, there was a severe epidemic involving the HIV-1 B＇ strain among people who sold blood and plasma in Henan, Hubei and adjacent provinces. To further study in HIV/AIDS vaccines and HIV-1 drug resistance for people in these regions, we need to construct an infectious HIV-1 B＇ molecular clone which is representative of the virus in these areas. To this end, we have isolated a HIV-1 B＇ virus from a child who was infected with HIV-1 from his mother in Hubei province, and have constructed a full-length clone from this genome.

Association of Neutralization Sensitivity of HIV-1 Primary Isolates With Biological Properties of Isolates From HIV-1 Infected Chinese Individuals

Objective Although HIV-1 infection is prevalent in many regions in China, it remains largely unknown on the biological characteristics of dominant circulating isolates. This study was designed to isolate the circulating viral strains from different prevalent regions and to characterize their biological properties and neutralization sensitivity. Methods Primary viruses were isolated from fresh PBMCs using the traditional co-culture method and their capacity of inducing syncytium was tested in MT-2 cells. Meanwhile, their coreceptor usage was determined with two cell lines: Magi and GHOST (3) stably expressing CD4 and the chemokine receptor CCR5 or CXCR4. Furthermore, the sensitivity of these viruses to neutralization by HIV-1-infected patients’ plasma which were highly active to neutralize SF33 strain, was quantified in GHOST cell-based neutralization assay. Results Six primary viral strains were isolated from 4 separated regions. Isolates LTG0213,LTG0214 and XVS032691 induced syncytia in MT-2 cells, and used CXCR4 as coreceptor. Isolates XJN0021, XJN0091, or SHXDC0041 did not induce syncytia, and used CCR5 as coreceptor. Overall neutralization sensitivity differed among four representative strains: HIV-1 XVS032691>LTG0214>XJN0091≈SHXDC0041. Conclusion The neutralization sensitivity of HIV isolates is linked with the phenotype of isolates, in which syncytium-inducing (SI) or CXCR4-tropic (X4) viruses are more easily neutralized than non-syncytium-inducing (NSI) or CCR5-tropic (R5) viruses. The genetic subtypes based on the phylogeny of env sequences are not classical neutralization serotypes.

Prokaryotic Expression and Purification of HIV-1 Vif and hAPOBEC3G, Preparation of Polyclonal Antibodies

To prepare HIV-1 Vif and hAPOBEC3G and to produce their antibodies, the full length gene fragment of HIV-1 vif was amplified by PCR from a plasmid of HIV-1 NL4.3 cDNA, and the APOBEC3G gene was obtained by RT-PCR from the total RNA of H9 cells. The resulting DNA construct was cloned into a prokaryotic expression vector (pET-32a). Recombinant pET-vif and pET-APOBEC3G were expressed respectively in Eserichia coli BL21 (DE3) as an insoluble protein. The vector also contained a six-histidine tag at the C-terminus for convenient purification and detection. To express and purify the HIV-1 Vif and hAPOBEC3G in E.coli cells, the accuracy of inserted gene and specificity of proteins were detected by the two enzyme digestion method, SDS-PAGE, and Western blotting. Rabbits were then immunized by Vif or APOBEC3G protein and serum samples were tested by indirect ELISA to determine the level of antibodies. Immunoenzyme and immunofluorescence assays were performed to identify the specificity of polyclonal antibodies. The titer of the anti-Vif antibodies was 1:204800, and that of the anti-APOBEC3G antibodies was 1:102400. Thus the antibodies could detect the antigen expression in the cells, demonstrating that fusion proteins with high purity and their corresponding polyclonal antibodies with high titer and specificity were achieved.

Identification of Aristolactam Derivatives That Act as Inhibitors of Human Immunodeficiency Virus Type 1 Infection and Replication by Targeting Tat-Mediated Viral Transcription

Despite the success of antiretroviral therapy(ART),efforts to develop new classes of antiviral agents have been hampered by the emergence of drug resistance.Dibenzo-indole-bearing aristolactams are compounds that have been isolated from various plants species and which show several clinically relevant effects,including anti-inflammatory,antiplatelet,and antimycobacterial actions.However,the effect of these compounds on human immunodeficiency virus type 1(HIV-1)infection has not yet been studied.In this study,we discovered an aristolactam derivative bearing dibenzo[cd,f]indol-4(5 H)-one that had a potent anti-HIV-1 effect.A structure-activity relationship(SAR)study using nine synthetic derivatives of aristolactam identified the differing effects of residue substitutions on the inhibition of HIV-1 infection and cell viability.Among the compounds tested,1,2,8,9-tetramethoxy-5-(2-(piperidin-1-yl)ethyl)-dibenzo[cd,f]indol-4(5 H)-one(Compound 2)exhibited the most potent activity by inhibiting HIV-1 infection with a half-maximal inhibitory concentration(IC50)of 1.03 lmol/L and a half-maximal cytotoxic concentration(CC50)of 16.91 lmol/L(selectivity index,16.45).The inhibitory effect of the compounds on HIV-1 infection was linked to inhibition of the viral replication cycle.Mode-of-action studies showed that the aristolactam derivatives did not affect reverse transcription or integration;instead,they specifically inhibited Tat-mediated viral transcription.Taken together,these findings show that several aristolactam derivatives impaired HIV-1 infection by inhibiting the activity of Tat-mediated viral transcription,and suggest that these derivatives could be antiviral drug candidates.

Cloning and analysis of the envelope protein clone of HIV-1, CHNHLJ03009c34 from an infected individual in Heilongjiang province

To analyze the variability and phenotype of envelope glycoprotein （Env） of human immunodeficiency virus type 1 （HIV-1） prevalent in Heilongjiang province, cloning of the full-length env gene from the peripheral blood mononuclear cells （PBMCs） of an HIV-1 positive individual in Heilongjiang province in China was performed by using conserved region primers. The amplified PCR products were cloned into a plasmid vector and sequenced. Phylogenetic analysis was done upon the full-length Env amino acid sequence. Subsequently, an HIV-1 pseudotyped virus bearing the envelope protein was constructed and the infectivity was examined using U87 cell lines expressing CD4 with either CCR5 or CXCR4. As the result, two functional env clones named as CHNHLJ03009c34 （GenBank Accession No： AY905493 ） and CHNHLJ03009c33 were obtained. It was found that the homology between CHNHLJ03009c34 and an HIV-1 subtype B＇ strain, RIA-2, isolated from Yunnan province, was 91.52% through comparing and analyzing full-length Env amino acid sequence of HIV-1 isolated from either China or abroad. Phylogenetic analysis indicated that CHNHLJ03009c34 has the closest molecular relation with strain RIA2 based on analyzing the full-length of the Env, while it became an independent branch upon analyzing the sequences of C2-V3 region of the Env. The secondary structure analysis of the envelope protein showed that the antigenicity and hydrophobicity of the strain demonstrated have no definite difference from that of RL42. Examination of infectivity showed that pseudovirus CHNHLI03009c34 could only infect U87. CD4. CCR5 cells, indicating that it was a RS-tropic HIV-1. In the conclusion, two HIV-1 env clones from an infected individual in Heilongjiang province have been identified as subtype B＇ and RS-tropic HIV-1. This is the first report on the analysis of primary isolates in Heilongjiang province.

Construction and characterization of a new simian/human immunodeficiency viruses clone carrying an env gene derived from a CRF07_BC strain

Background The CRF07_BC recombinant strain has been one of the most predominantly circulated HIV-1 strains in China, it is therefore necessary and urgent to develop a relevant animal model to evaluate candidate vaccines targeting HIV-1 CRF07 BC. A highly replication-competent simian/human immunodeficiency viruses （SHIV） construct containing the Chinese CRF07_BC HIV-1 env gene with the ability to infect Chinese rhesus monkeys would serve as an important tool in the development of HIV vaccines. The aim of this study was to examine whether SHIV XJDC6431 with the env fragment from a Chinese HIV-1 isolate virus could infect the human and monkey peripheral blood mononuclear cell （PBMC）, establish infection in Chinese rhesus macaque. Methods A SHIV strain was constructed by replacing the rev/env genes of SHIV KB9 with the corresponding fragment derived from the HIV-1 CRF07_BC strain. The infectious activity of the SHIV clones was determined in vitro in PBMCs from both non-human primate animals and humans. Finally, one Chinese rhesus macaques （Macaca mulatta） was infected with one SHIV via intravenous infusion. Results One SHIV clone designated as SHIV XJDC6431, was generated that could infect macaque and human PBMC. The virus produced from this clone also efficiently infected the CCR5-expressing GHOST cell lines, indicating that it uses CCR5 as its coreceptor. Finally, the virus was intravenously inoculated into one Chinese rhesus macaque. Eventually, the animal became infected as shown by the occurrence of viremia within 3 of infection. The viral load reached 105 copies of viral RNA per ml of plasma during the acute phase of infection and lasted for 10 weeks post infection. Conclusions We conclude that SHIV XJDC6431 is an R5-tropic chimeric virus, which can establish infection not only in vitro but also in vivo in the Chinese rhesus macaque. Although the animal inoculated with SHIV XJDC6431 became infected without developing a pathologic phenotype, the virus efficiently replicated with a persistent level of viral load in the plasma. This suggested that the SHIV could be used as a tool to test candidate AIDS vaccines targeting the Chinese HIV-1 CRF 07BC recombinant strain.

siRNA, miRNA and HIV: promises and challenges

Small interfering RNA (siRNA) and microRNA (miRNA) are small RNAs of 18-25 nucleotides (nt) in length that play important roles in regulating gene expression. They are incorporated into an RNA-induced silencing complex (RISC) and serve as guides for silencing their corresponding target mRNAs based on complementary base-pairing. The promise of gene silencing has led many researchers to consider siRNA as an anti-viral tool. However, in long-term settings, many viruses appear to escape from this therapeutical strategy. An example of this may be seen in the case of human immunodeficiency virus type-1 (HIV-1) which is able to evade RNA silencing by either mutating the siRNA- targeted sequence or by encoding for a partial suppressor of RNAi (RNA interference). On the other hand, because miRNA targeting does not require absolute complementarity of base-pairing, mutational escape by viruses from miRNA- specified silencing may be more difficult to achieve. In this review, we discuss stratagems used by various viruses to avoid the cells’ antiviral si/mi-RNA defenses and notions of how viruses might control and regulate host cell genes by encoding viral miRNAs (vmiRNAs).

Protective effect of curcumin on tumor necrosis factor-alpha-induced neuronal damage in the rat hippocampus A relationship to the inhibition of neuronal Ca^(2+) influx

BACKGROUND： Previous studies of curcumin have focused mainly on its cytotoxic properties for antitumor therapy. There are few studies addressing the application of curcumin in the prevention and treatment of nervous system diseases. OBJECTIVE： To observe the protective effect of curcumin against tumor necrosis factor-alpha （TNF-α）-induced neuronal damage in the rat hippocampus and to explore the intervention effect of curcumin on Ca^2＋ influx following neuronal damage. DESIGN, TIME AND SETTING： A cell morphological and physiological study was performed at the Institute of Brain Research, Medical College of Jinan University, China, from December 2006 to June 2007. MATERIALS： Curcumin （Sigma, USA） and TNF-α （Sigma, USA） were used in this study. METHODS： Hippocampal neurons were isolated from one-day neonatal rats and primarily cultured for 5 days. Following this they received 1 pmol/L curcumin and 100 ng/mL TNF-a pre-treatment. Dynamic morphological changes were observed for 1 hour by inverted microscopy. At 48 hours post-treatment, static morphological characteristics of the neurons were observed using inverted microscopy. Subsequently, hippocampal neurons were primarily cultured for 7 days, after receiving 1 pmol/L curcumJn and 4.5 ng/mL TNF-a pre-treatment. Intracellular free Ca^2＋ was measured using Fluo 3/acetoxymethyl ester. MAIN OUTCOME MEASURES： Effects of curcumin on TNF-a-induced neuronal damage and Ca^2＋ influx in the rat hippocampus were measured. RESULTS： Following curcumin treatment, TNF-a-induced neurons grew as normal. TNF-a induced a rapid Ca^2＋ influx into the neuronal cytoplasm; however, Ca2＋ fluorescence intensity only slightly increased when neurons were co-perfused with curcumin and TNF-α. CONCLUSION： Curcumin has a protective effect on rat hippocampal neurons possibly by reducing the TNF-α-induced rapid Ca^2＋ influx into neuronal cytoplasm and by maintaining the Ca^2＋ homeostasis.

Exploration of a Sequential Gp140-Gp145 Immunization Regimen with Heterologous Envs to Induce a Protective Cross-Reactive HIV Neutralizing Antibody Response In Non-human Primates

Raising a heterologous tier 2 neutralizing antibody(nAb)response remains a daunting task for HIV vaccine development.In this study,we explored the utility of diverse HIV-1 envelope(Env)immunogens in a sequential immunization scheme as a solution to this task.This exploration stemmed from the rationale that gp145,a membrane-bound truncation form of HIV Env,may facilitate the focusing of induced antibody response on neutralizing epitopes when sequentially combined with the soluble gp140 form as immunogens in a prime-boost mode.We first showed that gp140 DNA prime-gp145 Tiantan vaccinia(TV)boost likely represents a general format for inducing potent nAb response in mice.However,when examined in rhesus macaque,this modality showed little effectiveness.To improve the efficacy,we extended the original modality by adding a strong protein boost,namely native-like SOSIP.664 trimer displayed on ferritin-based nanoparticle(NP),which was generated by a newly developed click approach.The resulting three-immunization regimen succeeded in eliciting tier-2 nAb response with substantial breadth when implemented in rhesus macaque over a short 8-week schedule.Importantly,the elicited nAb response was able to effectively contain viremia upon a heterologous SHIV challenge.Collectively,our studies highlighted that diversification of Env immunogens,in both types and formulations,under the framework of a sequential immunization scheme might open new opportunity toward HIV vaccine development.

The Establishment of an In Vivo HIV-1 Infection Model in Humanized B-NSG Mice

Suitable animal models for human immunodeficiency virus type 1(HIV-1)infection are important for elucidating viral pathogenesis and evaluating antiviral strategies in vivo.The B-NSG(NOD-PrkdcscidIl2rgtm1/Bcge)mice that have severe immune defect phenotype are examined for the suitability of such a model in this study.Human peripheral blood mononuclear cells(PBMCs)were engrafted into B-NSG mice via mouse tail vein injection,and the repopulated human T-lymphocytes were observed at as early as 3-weeks post-transplantation in mouse peripheral blood and several tissues.The humanized mice could be infected by HIV-1,and the infection recapitulated features of T-lymphocyte dynamic observed in HIV-1 infected humans,meanwhile the administration of combination antiretroviral therapy(cART)suppressed viral replication and restored T lymphocyte abnormalities.The establishment of HIV-1 infected humanized B-NSG mice not only provides a model to study virus and T cell interplays,but also can be a useful tool to evaluate antiviral strategies.

Activity of superior interferon α against HIV-1 in severe combined immunodeficient mice reconstituted with human peripheral blood leukocytes

Background Interferon （IFN） can inhibit human immunodeficiency virus type 1 （HIV-1） replication in vitro and in clinic.However, IFN therapy for HIV infection was limited by its moderate antiviral efficacy and its frequent adverse effects. In the present study we evaluated the anti-HIV efficacy of a novel synthesized superior interferon α （slFNα）.Methods We performed in vitro experiments with HIV-1 IIB infected MT4 cells, and evaluated in vivo anti-HIV efficacy of slFNα in severe combined immunodeficient （SClD） mice reconstituted with human peripheral blood leukocytes （hu-PBL-SClD mice）.Results We found that the 50% effective concentrations （EC5o） of slFNα against the replication of HIV-1 in MT4 cells was 0.06 ng/ml, representing stronger antiviral activity than interferon-α in vitro. In the hu-PBL-SCID mice, a dose-dependent protection pattern was observed： with 0.45 μg and 1.35 μg slFNα daily treatments, parts of SCID mice were protected from HIV infection, whereas 2.25 μg sIFNα daily treatments resulted in a terminally complete protection.Conclusions slFNα shows good anti-HIV activity both in vitro and in SCID mice, may be a promising anti-HIV agent deserving clinical investigation, especially considering the potential of IFN-α to inhibit HIV replication in patients infected with drug-resistant variants or co-infected with hepatitis C virus （HCV）.

The V1 region of gp120 is preferentially selected during SIV/HIV transmission and is indispensable for envelope function and virus infection

A transmission bottleneck occurs during each human immunodeficiency virus(HIV) transmission event, which allows only a few viruses to establish new infection. However, the genetic characteristics of the transmitted viruses that are preferentially selected have not been fully elucidated. Here, we analyzed amino acids changes in the envelope protein during simian immunodeficiency virus(SIV)/HIV deep transmission history and current HIV evolution within the last 15–20 years. Our results confirmed that the V1V2 region of gp120 protein, particularly V1, was preferentially selected. A shorter V1 region was preferred during transmission history, while during epidemic, HIV may evolve to an expanded V1 region gradually and thus escape immune recognition. We then constructed different HIV-1 V1 mutants using different HIV-1 subtypes to elucidate the role of the V1 region in envelope function. We found that the V1 region, although highly variable, was indispensable for virus entry and infection, probably because V1 deletion mutants exhibited impaired processing of gp160 into mature gp120 and gp41. Additionally, the V1 region affected Env incorporation. These results indicated that the V1 region played a critical role in HIV transmission and infection.

Association of IL-7 with disease progression in Chinese HIV-1 seropositive individuals

Background Elevated levels of interleukin-7 （IL-7） have been correlated with CD4^＋ T cell depletion and the emergence of syncytium-inducing （SI） variants in human immunodeficiency virus type-1 （HIV-1） infection, and suggested as an indicator of acquired immunodeficiency syndrome （AIDS） disease progression. Therefore, we investigated the effects of IL-7 on disease progression and virus phenotype in Chinese HIV/AIDS patients. Methods In a cross-sectional study of 71 untreated HIV-1 seropositive individuals and 12 healthy donors, plasma IL-7 levels were determined by an ultra sensitive enzyme-linked immunosorbent assay （ELISA）, and its relations to CD4^＋ T cells, CD8^＋ T cells, plasma viral loads and HIV phenotypes were analyzed. Results Significant higher IL-7 levels were found in Chinese HIV/AIDS patients [（3.33±3.60） pg/ml] than those of health controls [（1.2±0.81） pg/ml] （P〈0.05）, and IL-7 levels were inversely associated with CD4^＋ T cell counts （r=-0.497, P〈0.01）. Furthermore, IL-7 levels were significant higher in patients with SI variants [（9.12±4.55） pg/ml] than those with non-syncytium-inducing variants [（1.50±2.69） pg/ml] （P〈0.01）. Conclusions Increased IL-7 levels were found in Chinese HIV/AIDS patients and significantly associated with disease progression, thus increased IL-7 plasma levels may indicate disease progression.

A new approach for sequencing virion genome of Chinese HIV-1 strains subtype B and BC from plasma

Background Although it was widely accepted that full-length HIV genome sequences is important in studying virus genetic evolution and variation as well as developing vaccine candidate,to directly sequencing HIV-1 genome of virion RNA remains as a challenge worldwide.Up to date,no published genomic sequences from virion RNA are available for Chinese prevalent HIV-1 strains due to the absence of specialized protocol and appropriate lab equipments.In this study we developed a straightforward approach for amplifying and sequencing HIV virion RNA from plasma by modifying published protocols and further confirmed it is suitable to process Chinese samples.Methods The methods for viral RNA extraction and gene amplification was modified and optimized as could be widely used in most Chinese labs.Gene alignment of Chinese HIV-1 strains was employed for designing specialized primer sets for Thai-B and BC recombinant strains.Based on comprehensively consideration of high variable gene region and recombinant breakpoints in BC recombinant strains,a three-amplicon strategy （including 4.3-kb gag-pol,2.9-kb pol-env and 2.7-kb env-ne） was developed.In addition,one amplicon （9 kb near full-length genome） was also used in 32 samples with varied viral loads.All amplicons were directly sequenced by DNA automated sequencer.Results Twenty-five percent（8/32） amplification efficiency was achieved by the one-amplicon strategy and 65.6%（21/32） by three-amplicon strategy.For one amplicon strategy,none of complete near full-length genome sequences was obtained by DNA sequencing.For three-amplicon strategy,75% sequences were achieved in DNA sequencing.Amplification efficiency but not sequencing efficiency was closely associated with viral loads.Conclusion Three-amplicon strategy covering all encoding regions of HIV-1 is suitable for Thai-B and BC recombinant strains and could be potentially employed in less-well equipped Chinese labs.

DYNAMICS BEHAVIOR OF MUTATION DURING REPRODUCTION ON HIV-1 DRUG RESISTANCE

To study the transition dynamics of resistant-type human immunodeficiency virus 1 （HIV-1） in highly active antiretroviral therapy （HAART） and the affect of neutral mutation in the evolution of HIV-1, a mathematical model is proposed when mutation occurs mainly during reproduction. The derived results show that the resistant-type will certainly colonize in patients once mutation occurs. Furthermore, a neutral mutation is closely related to the colonized pattern of resistant-type HIV-1 quasispecies and there are some changes in the pattern of transmission dynamics when mutation occurs mainly during reproduction or in the absence of reproduction, which may lead to significant strategies for predicting or checking HIV-1 drug resistance in HAART.

HLA-DR expression on regulatory T cells is closely associated with the global immune activation in HIV-1 infected subjects naive to antiretroviral therapy

Background The frequencies of regulatory T cells （Tregs） increased over the HIV infection but its counts actually decreased. We proposed that the decrease of Treg counts may cause the reduction of inhibitory effect and thereby account for the over-activation of Tregs during HIV infection. However, it remains unknown whether Tregs are also over-activated and thereafter the activation induced death may lead to the decrease of Tregs. Methods Tregs were defined as CD4＋CD25＋CD127lo/-T cells. Eighty-one HIV-1 infected patients were enrolled in our study, and twenty-two HIV-1 seronegative donors were recruited as the control. The levels of HLA-DR on Tregs were determined by FACSAria flow cytometer. Results Compared to HIV-1 seronegative donors, the levels of HLA-DR on CD4＋CD25＋CD127lo/- Tregs were significantly increased in HIV-1 infected patients, and its increase was positively associated with viral loads （r=0.3163, P=-0.004） and negatively with CD4 T-cell counts （r=-0.4153, P 〈0.0001）. In addition, significant associations between HLA-DR expression on CD4＋CD25＋CD127lo/- Tregs and the percentages of HLA-DR, CD38, Ki67 expressing CD4＋ and CD8＋ T cells were also identified. Conclusion HLA-DR on Tregs is a good marker for viral replication and disease progression. The over-activation of Tregs might result in the decrease of Tregs.