Identification and characterization of a novel bipartite nuclear localization signal in the hepatitis B virus polymerase

AIM:To characterize the nuclear import of hepatitis B virus(HBV)polymerase(P)and its relevance for the viral life cycle.METHODS:Sequence analysis was performed to predict functional motives within P.Phosphorylation of P was analyzed by in vitro phosphorylation.Phosphorylation site and nuclear localization signal(NLS)were destroyed by site directed mutagenesis.Functionality of the identified NLS was analyzed by confocal fluorescence microscopy and characterizing the karyopherin binding.Relevance of the structural motives for viral life cycle was studied by infection of primary Tupaia hepatocytes with HBV.RESULTS:We identified by sequence alignment and functional experiments a conserved bipartite NLS containing a casein kinaseⅡ(CKⅡ)phosphorylation site located within the terminal protein domain(TP)of the HBV polymerase.Inhibition of CKⅡimpairs the functionality of this NLS and thereby prevents the nuclear import of the polymerase.Binding of the import factor karyopherin-α2 to the polymerase depends on its CKⅡ-mediated phosphorylation of the bipartite NLS.In HBV-infected primary Tupaia hepatocytes CKⅡinhibition in the early phase(post entry phase)of the infection process prevents the establishment of the infection.CONCLUSION:Based on these data it is suggested that during HBV infection the final import of the genome complex into the nucleus is mediated by a novel bipartite NLS localized in the TP domain of HBV polymerase.

Differentiation of Sheeppox and Goatpox Viruses by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism

In the present study,the partial gene sequences of P32 protein,an immunogenic envelope protein of Capripoxviruses (CaPV),were analyzed to assess the genetic relationship among sheeppox and goatpox virus isolates,and restriction enzyme specific PCR-RFLP was developed to differentiate CaPV strains.A total of six goatpox virus (GTPV) and nine sheeppox virus (SPPV) isolates of Indian origin were included in the sequence analysis of the attachment gene.The sequence analysis revealed a high degree of sequence identity among all the Indian SPPV and GTPV isolates at both nucleotide and amino acid levels.Phylogenetic analysis showed three distinct clusters of SPPV,GTPV and Lumpy skin disease virus (LSDV) isolates.Further,multiple sequence alignment revealed a unique change at G120A in all GTPV isolates resulting in the formation of Dra I restriction site in lieu of EcoR I,which is present in SPPV isolates studied.This change was unique and exploited to develop restriction enzyme specific PCR-RFLP for detection and differentiation of SPPV and GTPV strains.The optimized PCR-RFLP was validated using a total of fourteen (n=14) cell culture isolates and twenty two (n=22) known clinical samples of CaPV.The Restriction Enzyme specific PCR-RFLP to differentiate both species will allow a rapid differential diagnosis during CaPV outbreaks particularly in mixed flocks of sheep and goats and could be an adjunct/supportive tool for complete gene or virus genome sequencing methods.

A Rapid and Sensitive One Step-SYBR Green Based Semi Quantitative Real Time RT-PCR for the Detection of peste des petits ruminants Virus in the Clinical Samples

敏感、快速的单个步实时(rt ) RT-PCR 用一步舞被标准化出众的格林·西布尔工具包吗？为 peste des petitis ruminants 病毒(PPRV ) 的察觉和 semi-quantitation 使用病毒 RNA 和矩阵(M) 蛋白质基因特定的教材并且与确定的常规 RT-PCR 和 TaqMan RT-PCR 相比。试金与 78.28 ～ 78.50 的 Tm 放大 PPRV M 基因的 124 bp 碎片。试金在到有 0.5 fg 的察觉限制(敏感) 的 0.5 fg 总数病毒 RNA 的 50 ng 的一个范围以内是线性的。基于现场稀释的 PPR 疫苗的病毒的连续冲淡，察觉限制是 0.0001 房间文化传染剂量 50% 单位(TCID50 ) 。另外，与疫苗的病毒的已知的 titre 刺的拖把材料同等地好在试金检测了。标准化 rt RT-PCR 容易在这块地和试验性的临床的样品里直接为 PPRV nucleic 酸的察觉被采用。试金从试验性的样品在拖把材料象 3 天柱子感染(dpi ) 和多达 20 dpi 一样早检测了 PPRV nucleic 酸。试金比在从 PPR 的 PPRV nucleic 酸的察觉的 TaqMan 和常规 RT-PCR 怀疑临床的样品绵羊和山羊的快速、更敏感。因此，确定的、简化 SYBR 绿 rt RT-PCR 是一种选择测试各种各样的诊断试金到已经存在并且能为有在减少污染的风险的优点的快速的临床的诊断有用。

Detection of Bluetongue Virus Group-specific Antigen Using Monoclonal Antibody Based Sandwich ELISA

A monoclonal antibody (MAb) specific for the bluetongue virus (BTV) group specific antigen (VP7) was characterized for its reactivity with purified virus and recombinant BTV VP7 (rVP7) protein and its suitability for use in the sandwich ELISA.The MAb,designated as 5B5 was specific to VP7 and belongs to IgG2a subclass and was selected for the development of the sELISA in this study.The MAb had a titer of 1:25 with BTV and 1:2 with the rVP7 protein.The sELISA is based on capturing of BTV antigen with VP7 specific MAb followed by detection using BTV polyclonal antiserum raised in rabbits.The assay was evaluated with six cell culture adapted serotypes of BTV that have been isolated from India,1,2,15,17,18 and 23.The assay could detect BTV antigen as early as day 8 in blood.It was also successfully applied for the detection of BTV group specific antigen in clinical samples of blood,washed RBCs,buffy coat and plasma.A total of 102 field samples from animals,suspected of being infected with BTV,were tested and 29.42% were positive.The blood samples were also amplified in cell culture which improved the sensitivity of the assay.Results confirmed that the sELISA is rapid and specific.

Study on Passive Immunity:time of Vaccination in Kids Born to Goats Vaccinated Against Peste des petits ruminants

In this study,the decay of maternal peste des petits ruminants virus(PPRV) antibodies in kids born to goats vaccinated with Asian lineage IV PPR vaccine and the efficacy of passive immunity against PPRV was assessed to determine the appropriate period for vaccination in kids.Serum samples collected from kids born to vaccinated,unvaccinated and infected goats at different time intervals were tested by PPR competitive ELISA and serum neutralization test(SNT).Maternal antibodies in kids were detectable up to 6 months with a decline trend from the third month onwards and receded below the protective level by the fourth month.The kid with an SN titre of 1:8 at the time of immunization showed significant PPRV specific antibody response(percentage inhibition of 76;SN titers >1:16),when tested on 21 day post-vaccination and was completely protected from infection upon virulent PPRV challenge.Similarly,the kid with 1:8 SN titers was completely protected from PPR infection on active challenge.Therefore,PPR vaccination is recommended in kids,aged 4 months and born to immunized or exposed goats.This could be a suitable period to avoid window of susceptibility in kids to PPRV and the effort to eliminate PPR infection from susceptible populations.

Determination of 50% endpoint titer using a simple formula

Two commonly used methods for calculating 50% endpoint using serial dilutions are Spearman-Karber method and Reed and Muench method. To understand/apply the above formulas, moderate statistical/mathematical skills are necessary. In this paper, a simple formula/method for calculating 50% endpoints has been proposed. The formula yields essentially similar results as those of the SpearmanKarber method. The formula has been rigorously evaluated with several samples.

Differential reactivity of mouse monoclonal anti-HBs antibodies with recombinant mutant HBs antigens

AIM: To investigate the reactivity of a panel of 8 mouse anti-hepatitis B surface antigen (HBsAg) monoclonal antibodies (mAbs) using a collection of 9 recombinant HBsAg mutants with a variety of amino acid substitutions mostly located within the “a” region.METHODS: The entire HBs genes previously cloned into a mammalian expression vector were transiently transfected into COS7 cells. Two standard unmutated sequences of the ayw and adw subtypes served as controls. Secreted mutant proteins were collected and measured by three commercial diagnostic immunoassays to assess transfection efficiency. Reactivity of anti-HBs mAbs with mutated HBsAgs was determined by sandwich enzyme-linked immunosorbent assay (ELISA).RESULTS: Reactivity of anti-HBs mAbs with mutated HBsAgs revealed different patterns. While three mutants reacted strongly with all mAbs, two mutants reacted weakly with only two mAbs and the remaining proteins displayed variable degrees of reactivity towards different mAbs. Accordingly, four groups of mAbs with different but overlapping reactivity patterns could be envisaged. One group consisting of two mAbs (37C5-S7 and 35C6-S11) was found to recognize stable linear epitopes conserved in all mutants. Mutations outside the “a” determinant at positions 120 (P→S), 123(T→N) and 161(M→T) were found to affect reactivity of these mAbs.CONCLUSION: Our findings could have important implications for biophysical studies, vaccination strategies and immunotherapy of hepatitis B virus (HBV) mutants.

Production and Characterization of Monoclonal Antibodies to Bluetongue Virus

In the present study,a total of 24 MAbs were produced against bluetongue virus (BTV) by polyethyleneglycol (PEG) mediated fusion method using sensitized lymphocytes and myeloma cells. All these clones were characterized for their reactivity to whole virus and recombinant BTV-VP7 protein,titres,isotypes and their reactivity with 24 BTV-serotype specific sera in cELISA. Out of 24 clones,a majority of them (n = 18) belong to various IgG subclasses and the remaining (n = 6) to the IgM class. A panel of eight clones reactive to both whole BTV and purified rVP7 protein were identified based on their reactivity in iELISA. For competitive ELISA,the clone designated as 4A10 showed better inhibition to hyperimmune serum of BTV serotype 23. However,this clone showed a variable percent of inhibition ranging from16.6% with BTV 12 serotype to 78.9% with BTV16 serotype using 24 serotype specific sera of BTV originating from guinea pig at their lowest dilutions. From the available panel of clones,only 4A10 was found to have a possible diagnostic application.

Evaluation of Efficacy of Stabilizers on the Thermostability of Live Attenuated Thermo-adapted Peste des petits ruminants Vaccines

In this study,thermo-adapted (Ta) PPR vaccines were assessed for their stability at 25,37,40,42 and 45℃ in lyophilized form using two extrinsic stabilizers {lactalbumin hydrolysate-sucrose (LS) and stabilizer E} and in reconstituted form with the diluents (1 mol/L MgSO4 or 0.85% NaCl).The lyophilized vaccines showed an expiry period of 24-26 days at 25℃,7-8 days at 37℃ and 3-4 days at 40℃.LS stabilizer was superior at 42℃ with a shelf-life of 44 h,whereas in stabilizer E,a 40 h shelf-life with a comparable half-life was observed.At 45℃,the half-life in stabilizer E was better than LS and lasted for 1 day.Furthermore,the reconstituted vaccine maintained the titre for 48 h both at 4℃ and 25℃ and for 24-30 h at 37℃.As both the stabilizers performed equally well with regard to shelf-life and half-life,the present study suggests LS as stabilizer as a choice for lyophilization with 0.85% NaCl diluent,because it has better performance at higher temperature.These Ta vaccines can be used as alternatives to existing vaccines for the control of the disease in tropical countries as they are effective in avoiding vaccination failure due to the breakdown in cold-chain maintenance,as this vaccine is considerably more stable at ambient temperatures.

Sofosbuvir/Ribavirin therapy for patients experiencing failure of ombitasvir/paritaprevir/ritonavir + ribavirin therapy: Two cases report and review of literature

BACKGROUND The effectiveness of sofosbuvir/ribavirin(SOF/RBV) combination therapy,which is one of the 1 st-choice therapeutic options for patients with hepatitis C virus(HCV) genotype 2(HCV-G2) in Japan according to the most recent version of the Japan Society of Hepatology guideline, for patients who experienced failure of the ombitasvir/paritaprevir/ritonavir plus ribavirin(OBV/PTV/r+RBV) combination therapy, which was another option for patients with HCV-G2, is unknown.CASE SUMMARY We evaluated the effects of SOF/RBV combination therapy in two patients with genotype 2 a who could not achieve a sustained virological response(SVR) by OBV/PTV/r+RBV combination therapy. One patient was complicated with VogtKoyanagi-Harada(VKH) disease. Resistance-associated variations before SOF/RBV combination therapy were not detected in two patients. Both patients had an SVR at 12 wk after the treatment(SVR12). Regarding adverse events(AEs), itching, chill, a dull feeling in the throat and cough as well as increase of alanine transaminase level were shown in one patient, while a headache and deterioration of light aversion probably due to the recurrence of VKH disease were shown in the other patients. In addition, the latter patient developed arthralgia and morning stiffness approximately 7 wk after the therapy and turned out to be diagnosed with rheumatoid arthralgia.CONCLUSION SOF/RBV therapy might be effective for patients experiencing failure of OBV/PTV/r+RBV therapy, but caution should be taken regarding the AEs.

Cytokines Expression Profile and Kinetics of Peste des petits ruminants Virus Antigen and Antibody in Infected and Vaccinated Goats

The present study deals with the co-ordination of cytokine(IL-4 and IFN-γ) expression and kinetics of peste des petits ruminants(PPR) virus antigen and antibody in PPRV infected and vaccinated goats.The infected animals exhibited mixed cytokine(both T_H1 and T_H2) responses in the initial phase of the disease.The infected and dead goats had increased IFN-γ response before their death;while IL-4 remained at the base level.The cytokine expression in recovered animals was almost similar to that of vaccinated ones,where a unique biphasic response of IL-4 expression was observed with an up-regulation of IFN-γ on 7^(th) days post vaccination(dpv).Analysis of PPR virus antigen and antibody kinetics in different components of blood from infected and vaccinated animals revealed that the PPR virus antigen load was highest in plasma followed by serum and blood of the infected animals,whereas vaccinated animals showed only marginal positivity on 9^(th) dpv.The antibody titer was high in serum followed by plasma and blood in both vaccinated and infected animals.Therefore,it is inferred that the presence of antigen and antibody were significant with the expression of cytokine,and that a decreased response of IL-4 was noticed during intermediate phase of the disease i.e.,7 to 12^(th) days post infection(dpi).This indicates the ability to mount a functional T_H2 response after 14^(th)dpi could be a critical determinant in deciding the survival of the PPR infected

Preliminary Study on a Potential Panel for Quality Assurance of ELISPOT

The ELISPOT assay is increasingly used for assessing cellular immune responses in clinical trials of HIV-1 or cancer vaccines. However, to date, data from clinical trials do not consistently show that immune responses are correlated with clinical endpoints. This is due in part to the lack of assay standardization and validation across laboratories and therefore, a quality control panel is required to establish competency and comparability amongst different laboratories. In this study peripheral blood mononuclear cells (PBMCs) from healthy individuals were screened and frozen in liquid nitrogen. The recovery and viability of the PBMCs and the frequencies of interferon (IFN)-γ-secreting cells after CEF peptide pool stimulation were detected after various intervals in seven different laboratories. The recovery and viability did not differ significantly after different intervals. Although the frequencies of IFN (interferon)-γ-secreting cells among thawed PBMCs (peripheral blood mononuclear cells) fluctuated after CEF peptide pool stimulation at different intervals, they were not significantly decreased compared with those among fresh PBMCs. However, the viabilities, recoveries and frequencies of IFN-γ-secreting cells differed significantly among the seven laboratories. Our results indicate that cryopreserved PBMCs could be used as a quality control panel for ELISPOT. However, the procedures for ELISPOT need to be standardized amongst different laboratories.

Genotypic Distribution of Different Variants of Oncogenic Human Papilloma Virus (HPV) among the Sexually Active HIV-1 Positive Female Population from Manipur, India

A total of 531 sexually active female populations from Manipur (north-eastern India), was chosen for this study. Out of 531 females from Manipur, 111 (20.9%) were HIV positive and 420 were HIV negative (79%). PCR amplification of the MY region of the HPV L1 gene using consensus MY09/11 primer showed 3.7% positivity in Manipur. Interestingly HPV infection among the HIV infected population from Manipur was found to be higher (9%) compared to the non HIV infected population from Manipur (3.7%). Further, single PCR for detecting the 16/18 genotypes and multiplex PCR for the other high risk groups showed equal prevalence of 16 and 18 and other high risk groups (1.3% each) in Manipur. This result was further confirmed by the sequencing and phylogenetic analysis. Thus, our study showed fairly high HPV prevalence rate among the HIV infected population compared to the non HIV cases in Manipur and also equal prevalence of 16/18 genotypes with other high risk groups. According to our knowledge this is the first kind of a genotypic study among the HPV-HIV co-infected population from northeastern states of India.

Microarray analysis of gene expression in the liver of transgenic mouse model of HCV infection

Background: The molecular interactions of hepatitis C virus (HCV) with hepatic tissue have yet to be completely elucidated and understood. The purpose of this study was to compare differential gene expression patterns in the livers of non-transgenic and transgenic mouse model expressing HCV structural proteins Core, Envelope 1 (E1) and Envelope 2 (E2) using complementary DNA (cDNA) microarrays. Results: Total RNA extracted from the livers of HCV transgenic and non-transgenic mice was analyzed with cDNA microarray and differentially expressed genes confirmed by real-time RT-PCR. Relative expression ratios of individual genes were determined by comparing hybridization of Cy5-labelled cDNA from transgenic mouse livers and Cy3-labelled cDNA from non-transgenic mouse livers. The spot array images were quantified using QuantArray software and the outlier spots was normalized and filtered using five different criteria. 15,297 genes were analyzed using three different analytical methods. Depending on these methods, twenty-one genes were found to be differentially expressed at a statistically significant level. From these, 6 genes had a consistent differential expression. Several genes were directly involved in lipid metabolism and lipid β-oxidation. 5-azacytidine induced gene 2 (AZ2), which is involved in the methylation of genes was down regulated in HCV transgenic mice. Altered transcript levels of these 6 genes were confirmed by real-time RT-PCR analysis. Conclusion: Interactions between HCV and hepatocytes not only involve lipid metabolism and redox balance, but this interaction may also influence DNA methylation, indicating a potential association with the development of hepatocellular carcinoma.

Poultry gut health-microbiome functions,environmental impacts,microbiome engineering and advancements in characterization technologies

The gastrointestinal tract(GIT)health impacts animal productivity.The poultry microbiome has functions which range from protection against pathogens and nutrients production,to host immune system maturation.Fluctuations in the microbiome have also been linked to prevailing environmental conditions.Healthy poultry birds possess a natural resistance to infection.However,the exploration of environmental impacts and other relevant factors on poultry growth and health have been underplayed.Since good performance and growth rate are central to animal production,the host-microbiome relationship remains integral.Prior to the emergence of metagenomic techniques,conventional methods for poultry microbiome studies were used and were low-throughput and associated with insufficient genomic data and high cost of sequencing.Fortunately,the advent of high-throughput sequencing platforms have circumvented some of these shortfalls and paved the way for increased studies on the poultry gut microbiome diversity and functions.Here,we give an up-to-date review on the impact of varied environments on microbiome profile,as well as microbiome engineering and microbiome technology advancements.It is hoped that this paper will provide invaluable information that could guide and inspire further studies on the lingering pertinent questions about the poultry microbiome.

Complete Human Immunodeficiency Virus-1 Specific T Lymphocyte Response to Chinese Human Immunodeficiency Virus-1 B/C Chronic Infectors

Objective To characterize the human immunodeficiency virus （HIV） -specific T lymphocyte responses and identify the immunodominant regions in Chinese HIV-1 recombinant subtype B/C chronic infectors at complete genome level. Methods Twenty-five HIV-I B/C recombinant chronic infectors were screened for their specific T lymphocyte responses to a panel of peptides corresponding to the complete HIV-1 subtype B genome by gamma interferon ELISPOT assay. Kruskal-Wallis nonparametric analysis of variance was used to test significant differences across gene regions, and Tukey pairwise analysis was used to identify differences between gene regions. Spearman rank correlation was used to assess the relation between responses. Results The order of recognized frequencies of specific T lymphocyte responses to HIV proteins was Nef〉Vpr〉Gag〉Pol〉Vpu〉Env〉Rev〉Vif〉Tat. When adjusted for protein length, Nef, Vpr, Gag, and Pol were the most intensely targeted proteins and the central region of Nef, Gag p24, Pol RT, and Vpr was most frequently recognized. No significant correlation was observed between the magnitude of IFN-γ production of HIV-l-specific T lymphocyte responses and plasma viremia, breadth of response and CD4 counts. Conclusion The central region of Nef, Gag p24, Pol RT, and Vpr is most frequently targeted in HIV-1 B/C recombinants chronic infectors. HIV-1-specific T lymphocyte responses and plasma viremia or CD4 counts play no protective role at complete genome level in these infectors.

On COVID-19 and Membrane Lipids and Public Health

Coronavirus has a lipid membrane.Whist replication requires hijacking the RNA tools of the host to synthesize virion protein,that then has to be wrapped in a lipid membrane to enable the budding off which extends the infection.Recent studies implicate certain essential fatty acids with replication suppression properties.The lipid membrane is commonly thought of as a fatty barrier to water solubles.It is however highly ordered and compositionally specific to cellular and sub cellular functions.There will likely also be an optimum specificity for the viral coat.Whist DNA,RNA and protein compositions are not affected by diet,the lipid membrane is.Moreover,the greater sensitivity of males over females to inadequacy of these essential fatty acids and membrane integrity has been known since the 1960 s.With evidence that arachidonic and docosahexaenoic acids exhibiting anti-viral,immune,anti-inflammatory,blood pressure control and resolvin activity,their status needs to be urgently examined in relation to the prevention and therapy for Covid-19.It would also be advisable to re-assess food policy.The lipid requirements for the membrane rich systems as in the brain,nervous,vascular and immune systems have not been considered.There is little doubt these were significant in shaping the human genome over several million years.Departure from such conditions would be predicted to put populations at risk to disorder and infection,with males being more at risk than females.

IL-15 increases the frequency of effectormemory CD8^(+) T cells in rhesus monkeys immunized with HIV vaccine

Several studies have suggested that interleukin(IL)-15 is a promising adjuvant that promotes cellular immunity when administered with human immunodeficiency virus(HIV)vaccine.Here we evaluated the effect of IL-15 plasmid on HIV-specific immune responses,especially cellular immunity,in eight rhesus monkeys.These monkeys were immunized three times with HIV DNA vaccine with or without IL-15 plasmid and boosted with recombinant Tiantan strain vaccinia virus-based HIV vaccine(rTV)22 weeks after the first immunization.Although we did not detect any significant differences in the HIV-specific CD81 T-cell response between monkeys with IL-15 coimmunization and monkeys with HIV vaccine alone,our results showed that the frequency of effector CD8^(+) memory T cells in the peripheral blood was significantly higher in monkeys with IL-15 coimmunization than those with HIV vaccine alone at almost all of the time points examined.Furthermore,the titers of anti-HIV antibodies were higher in Group T than those in Group C after rTV boosting.These findings in rhesus monkeys suggest that IL-15 may be useful as a cytokine adjuvant for HIV vaccine.

Glycosylation and an amino acid insertion in the head of hemagglutinin independently affect the antigenic properties of H5N1 avian influenza viruses

Antigenic drift forces us to frequently update influenza vaccines; however, the genetic basis for antigenic variation remains largely unknown. In this study, we used clade 7.2 H5 viruses as models to explore the molecular determinants of influenza virus antigenic variation. We generated eight monoclonal antibodies(MAbs) targeted to the hemagglutinin(HA) protein of the index virus A/chicken/Shanxi/2/2006 and found that two representative antigenically drifted clade 7.2 viruses did not react with six of the eight MAbs. The E131 N mutation and insertion of leucine at position 134 in the HA protein of the antigenically drifted strains eliminated the reactivity of the virus with the MAbs. We also found that the amino acid N131 in the H5 HA protein is glycosylated. Our results provide experimental evidence that glycosylation and an amino acid insertion or deletion in HA influence antigenic variation.

Identification of HIV-1 specific T lymphocyte responses in highly exposed persistently seronegative Chinese

Background Studies of highly exposed persistently seronegative （HEPS） individuals may provide valuable information on mechanisms of protection and on vaccine design. Cellular immune responses play a critical role in containing human immunodeficiency virus. However, the cellular immune responses in HEPS individuals have not been thoroughly assessed at the entire viral genome level. Methods Ten HEPS Chinese with a history of frequent penetrative vaginal intercourse （mean frequency, at least once a week）, with some unprotected sexual contact occurring in the weeks or days immediately before enrollment, 25 HIV-1 seropositive individuals, 10 HIV-1-seronegative healthy individuals with low-risk sexual behavior and no history suggestive of exposure to HIV-1 infection were enrolled. HIV-1-specific T cell responses were comprehensively analyzed by an interferon- 7 Elispot assay against 770 overlapping peptides spanning all HIV-1 proteins. Results HIV-1-specific T-cell responses of interferon- 7 secretion were identified in 3 （30%） out of 10 HEPS individuals; the specific cytotoxic T lymphocytes were targeted at Pol （2/10）, Env （2/10）, and Tat （1/10）. HIV-1-specific T-cell responses of interferon- ~ secretion were identified in 20 （80%） out of 25 seropositive intravenous drug users （IDUs）, revealing that all HIV-1 proteins and protein subunits could serve as targets for HIV-1-specific CD8^＋ T cell responses with 85% recognizing Gag, 80% recognizing Nef, 75% recognizing Pol, 60% recognizing Env, 55% recognizing Vpu, 45% recognizing Vpr, 20% recognizing Vif, 20% recognizing Tat and 15% recognizing Rev in these seropositive individuals. None of the seronegative healthy individuals gave the positive T-cell responses. Conclusions About 30% of HEPS Chinese mounted HIV-1 specific T cell immune responses. Cell-mediated immunity against HIV-1 may be developed through non-productive infections.