BackgroundHepatitis B virus infection is a serious health concern in China. The National Immunization Program has implemented universal hepatitis B vaccination for infants for over two decades. The serological characteristics of hepatitis B virus and the causes of infections among adult blood donors born after 2002 remain undetermined.MethodsQualified blood donors born between 2002 and 2006 from Guangdong were screened for antibodies against hepatitis B surface antigen (anti-HBs) and hepatitis B core antigen (anti-HBc). Donations positive for HBsAg were subjected to various tests, including HBV serological markers, quantitative PCR (qPCR), and nested PCR, to further analyze the molecular characteristics of HBV DNA. Notable mutations were identified through comparison with HBV reference sequences obtained from blood donors.ResultsThis study enrolled 5,487 blood donors with complete results for anti-HBs and anti-HBc testing. Overall, the prevalence of anti-HBs was 47.73% in males and 46.16% in females, while the prevalence of anti-HBc was 10.58% in males and 12.79% in females. Among blood donors born between 2002 and 2006, the prevalence of anti-HBs declined progressively with more recent birth years, decreasing from 54.55% to 40.23%, while the prevalence of anti-HBc remained relatively stable at around 11%. The prevalence (56.54%) and level of anti-HBs in the Pearl River Delta were significantly higher than those in the eastern, western, and northern regions of Guangdong (P<0.001). Quantitative analysis revealed that anti-HBs levels were significantly higher in the anti-HBc+ cohort than in the anti-HBc- group. Furthermore, whole genome sequences genotyping of 72 HBsAg+ samples revealed that 75% (54/72) were genotype B and 25% (18/72) were genotype C. Nine HBV-related point mutations were detected, including four previously reported variants and five novel substitutions. Additionally, six insertions/deletions were observed in genotype B/C. All these mutations were mapped to critical functional regions within the PreS1, Core, X, and S genes.ConclusionOnly 47.13% of young adults vaccinated at birth under the national immunization program retained protective antibodies at the time of testing, indicating this age group vulnerable to HBV infection. This decline has consequently increased the risk of breakthrough infection, necessitating booster vaccination among young adults in Guangdong.

Abstract Background & aims In China, blood donations are routinely screened for both HBsAg and HBV DNA to prevent transfusion-transmitted hepatitis B virus (HBV) infection. However, the benefit of implementing universal anti-HBc screening to intercept occult HBV infection remains debated, primarily due to concerns over donor loss and cost-effectiveness. This study aimed to assess the prevalence of anti-HBc among blood donors in Guangzhou and to evaluate whether the current HBV screening strategy in China requires further optimization. Methods In this study, we enrolled 25,056 voluntary blood donors who tested negative for both HBsAg and HBV DNA. All samples were first screened for anti-HBc using ELISA. A subset of positive samples was then retested with an electrochemiluminescence (ECL) assay. Subsequently, quantitative anti-HBs testing was performed on samples that were positive by both assays, and those that were anti-HBs-negative underwent HBV RNA testing. Results The initial anti-HBc-positive rate was 27.1% (6795/25,056), which exhibited a strong age-dependent increase but showed no association with gender. Of the 1343 samples that underwent retesting, 82.1% (1102/1343) were confirmed positive. Among the double-positive samples, 91.2% (990/1085) were also anti-HBs positive, and 53.5% (580/1085) presented with anti-HBs levels ≥ 200 IU/L. No statistically significant difference in anti-HBs levels was observed between genders or across age groups. Furthermore, HBV RNA was not detected in any of the 95 anti-HBs-negative samples tested. Conclusion The high prevalence of anti-HBc, coupled with a substantial proportion of donors having protective levels of anti-HBs, suggests that the incremental benefit of universal anti-HBc screening in Guangzhou’s current donor population may be limited. While implementing such assays is not considered feasible at present, future declines in prevalence of HBV infection may warrant a re-evaluation and optimization of the screening strategy.

ABSTRACT While occult hepatitis B virus infection is a known risk factor for progressive liver diseases, the mechanistic basis for this association is poorly understood. To investigate this, we conducted a comparative study involving 40 OBI blood donors and 100 healthy blood donors to assess clinical parameters of liver fibrosis, including serum biomarkers, the FIB-4 index, and liver stiffness measurement. Transcriptome sequencing of peripheral blood mononuclear cells from these donors identified IL-17F as significantly upregulated in the OBI group. Mechanistically, we found that IL-17F was chiefly secreted by CD4 + T cells upon HBcAg stimulation and functionally promoted the expression of key fibrosis-related cytokines and markers. Collectively, our study unveils a novel pathway wherein HBcAg induces CD4 + T cells to secrete IL-17F, which in turn accelerates liver fibrogenesis, providing a mechanistic basis for OBI-associated liver disease. IMPORTANCE This study reveals, for the first time, a novel mechanism by which OBI drives liver fibrosis, demonstrating that HBcAg stimulates CD4 + T cells to secrete IL-17F, which in turn activates macrophages and hepatic stellate cells, thereby promoting liver inflammation and fibrosis. This discovery not only elucidates a key molecular pathway underlying OBI-related liver disease progression, but also provides an important theoretical foundation for developing IL-17F-targeted diagnostic approaches and anti-fibrotic therapies.

S gene mutations may lead to undetectable HBsAg, contributing to occult HBV infection (OBI). Nucleic acid testing (NAT) has been widely implemented to reduce transfusion-transmitted OBI. However, distinguishing between false positives and true OBI among NAT+/HBsAg– donors remains challenging, as these individuals frequently exhibit inconsistent or fluctuating test results. This study aims to improve infection status confirmation and donor management by analyzing detection patterns and investigating S gene mutations. Blood donors were detected for HBV using individual-NAT and HBsAg enzyme-linked immunosorbent assays. Routine discriminatory test (dHBV), additional three repeated dHBV (3-repeated dHBV), and serological assays were conducted to confirm the infection status of NAT+/HBsAg– donors. Serial NAT results were analyzed to assess temporal fluctuations. S gene mutations related with OBI were identified through molecular characterization and sequencing analysis. A total of 336,537 blood donors were detected, and 556 (0.16

Introduction:Transfusion-transmitted infections (TTIs) pose significant public health challenges. Screening potential blood donors for hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and Treponema pallidum (TP), along with ongoing monitoring of epidemiological data on TTIs among donors, is essential to ensure blood safety. Methods:This study conducted a retrospective analysis of the seroprevalence of HBsAg, anti-HCV, anti-HIV, and anti-TP among 3,111,265 blood donation events in Guangzhou from 2014 to 2023, investigating their relationships with demographic characteristics such as gender, age, and donation status. Results:The findings indicated that the overall seroprevalence of TTIs among blood donations was 866.21 per 100,000, demonstrating a declining trend over the ten-year period. Seroprevalence rates of transfusion-transmissible infections also exhibited a downward trend across various demographic groups. Notably, male donors, first-time donors, and older donors demonstrated higher seroprevalence rates for TTIs. Conclusions:While the overall seroprevalence rate of transfusion-transmissible infections among blood donors remains relatively low, significant disparities exist among different demographic groups. It is necessary of sustained TTIs monitoring among blood donors to guide public health interventions and donor screening practices.

Hepatitis B virus (HBV), a common blood transmission pathogen worldwide, can lead to viral hepatitis, cirrhosis, liver cancer, and other liver diseases. In particular, occult hepatitis B virus infection (OBI) may be caused by an immune response leading to suppressed virus replication. Gut microbiota can change the immunity status of the human body and, therefore, affect the replication of HBV. Thus, to identify whether there are differences in gut microbiota between HBV carriers and OBI carriers, we collected fecal samples from 18 HBV carriers, 24 OBI blood donors, and also 20 healthy blood donors as negative control. After 16S sequencing, we found that the abundance of Faecalibacterium was significantly reduced in samples from OBI blood donors compared with those from healthy blood donors. Compared with samples from HBV carriers, the samples from OBI blood donors had a significantly increased abundance of Subdoligranulum, which might stimulate immune activation, thus inhibiting HBV replication and contributing to the formation of occult infection. Our findings revealed the potential role of gut microbiota in the formation of OBI and further provided a novel strategy for the treatment of HBV infection.IMPORTANCEOccult hepatitis B virus infection (OBI) is a special form of hepatitis B virus infection with hepatitis B surface antigen (HBsAg) positive and hepatitis B virus (HBV) DNA negative. Gut microbiota may contribute to the immune response leading to suppressed virus replication and, thus, participates in the development of OBI. The study on gut microbiota of OBI blood donors provides novel data considerably advancing our understanding of the immune mechanism for the determination of occult hepatitis B virus infection, which is helpful for improving the strategy of the treatment of HBV infection.

[Objective] To analyze the influence of the cycle length of hepatitis B surface antigen (HBsAg) double reagent positive samples collected from voluntary blood donors in Guangzhou on the detection results. [Methods] A total of 127 044 blood samples from voluntary blood donors at Guangzhou Blood Center from August 10 to December 9, 2023 were selected. Two ELISA reagents were used for HBsAg detection, and samples with HBsAg double reagent positive and S/CO values<10 were tested continuously for 7 days to observe the changes in their S/CO values. [Results] A total of 505 HBsAg double reagent positive samples were detected, of which 52 had S/CO values less than 10. After 7 consecutive days of uninterrupted testing, the S/CO values of Wantai (median 5 decreased to 3) and Xinchuang (median 5 decreased to 3) showed an overall downward trend, and the HBsAg missed detection rate showed an upward trend (from 0 on the first day to 1/10 000 on the seventh day). A total of 13 cases had negative double reagent test results within the 7-day testing cycle. [Conclusion] With the extension of the detection cycle, the S/CO value of HBsAg detection shows a downward trend, and the missed detection rate of HBsAg shows an upward trend. Samples used for HBsAg detection should be tested promptly after sampling to improve the quality of blood testing.

Objective To detect the anti-SARS-CoV-2 antibody levels in blood donors in Guangzhou, so as to provide laboratory data support for the collection and clinical use of convalescent plasma. Methods Anti-SARS-CoV-2 antibodies were measured by ELISA in qualified donors. Among them, 326 donors who gave blood in February 2023 were tested for IgG antibodies, 444 donors were tested for neutralizing antibodies. In July 2023, 398 donors were tested for IgG and IgM. Results 399 of 724 blood samples diluted with normal saline (1∶160) were IgG reactive, with a reactive rate of 55.11%. Chi-square test showed that there was a significant difference in the reactive rate of IgG among samples collected at different times (25.46% in February vs 79.40% in July, χ2=210.74, P<0.01, 95%CI: 7.97, 15.98), but there was no significant difference in the reactive rate between different genders and different age groups. IgM was detected in 5 of 398 blood samples, with a reactive rate of 1.26%. The IgG test results of these five blood donors were all reactive, whereas the nucleic acid test results were negative. Neutralizing antibody was detected in 440 of 444 blood samples, with a reactive rate of 99.10%, and 71.59% of the reactive donors had a neutralizing antibody level of 10 μg/mL or more. Conclusion Blood donors in Guangzhou have a high level of SARS-CoV-2 antibody, which is sufficient to provide convalescent plasma for clinical treatment.

Objective HLA-DRB1 * 11:01, as a class HLA-Ⅱ gene, was reported to be associated with spontaneous clearance of HCV in Han and Li population. Our study was to investigate the effects of viral selection pressure and CD4+T cell epitope on the natural outcome of HCV infection in HLA-DRB1 * 11:01 positive infected patients. Methods The positive selection sites and population growth of E1E2 and NS3 genes of common HCV 6a in HLA-DRB1 * 11:01 positive and negative groups in Guangdong were respectively analyzed. The peptide library covering the conserved regions of common HCV genotypes was used to stimulate HCV spontaneous clearance group and chronic infection group using ELISPOT method. Reactive peptides were obtained according to the number of spot-forming cells per well and the frequency of occurrence in different groups. Results The positive selection sites (PSSs) of E1E2 and NS3 of common HCV 6a in HLA-DRB1 * 11:01 negative group were greater than those in HLA-DRB1 * 11:01 positive group. Furthermore, the number of PPSs in CD4+T cell peptide in HLA-DRB1 * 11:01 negative group were also greater than those in HLA-DRB1 * 11:01 positive group; Both groups of HCV 6a had a population growth in Guangdong, and the expansion trend of HLA-DRB1 * 11:01 negative group was significantly higher than that of HLA-DRB1 * 11 :01 positive group. Compared to HCV chronic infection group, the response rate of HCV spontaneous clearance group to five peptides (C-52 E2691-707, C-119 NS31545-1560, C-134 NS4A1669-1684, C-154 NS4B1912-1927, C-159 NS4B1929-1944) was higher. However, the HCV chronic infection group showed a higher response rate to two of the peptides(C-111 NS31497-1512, C-130 NS31650-1665). When HLA-DRB1 * 11:01 typing was considered, there was no significant difference in HCV-specific immune response generated by PBMCs between HLA-DRB1 * 11:01 positive and HLA-DRB1 * 11:01 negative groups. Conclusion This study revealed the relationship between viral selection pressure of HLA-DRB1 * 11:01 HCV infected persons and CD4+T cell antigen epitopes. At the same time, CD4+ T cell antigen epitopes of HCV pan-genotype were obtained, providing basic data for the development of T cell vaccine suitable for HCV pan-genotype.

Objective To study the application effect of electrochemiluminescence immunoassay (ECLIA) in the screening of human T-lymphotropic virus (HTLV) in blood donors, explore the correlation between COI and confirmed result by western blot (WB), so as to provide scientific evidence for blood banks to select optimal screening strategy and optimize process flows. Methods A total of 1 070 HTLV reactive blood samples by ELISA in preliminary screening in our province from 2016 to 2022 were included in this study. All samples were detected by ECLIA and WB (gold standard), and the receiver operating characteristic curve (ROC) was drawn to find optimal diagnostic critical value of ECLIA. Results Among 1 070 samples, 285 were positive by ECLIA, with a positive rate of 26.64% (285/1 070), with average COI value at 81.92 and the median at 4.98. A total of 70 ECLIA positive samples were confirmed by WB, with a positive rate of 6.54% (70/1 070). According to the results of WB, the median of WB-positive group and WB-negative group was 312.45 vs 2.76, respectively(P<0.01). ROC analysis showed that optimal diagnostic critical value was 72.10 (P<0.01), with concordance rate of the two detection methods at 99.35%. Conclusion ECLIA, with high sensitivity and specificity is suitable for the detection with large sample size. When the diagnostic threshold is properly set, ECLIA results are in good agreement with those of WB, and it has good application prospect in HTLV screening in blood donors.

Abstract Qualified blood from healthy donors may still contain infectious pathogens that could become important threats to human life. The objective of this study was to screen for blood-borne viruses and analyze the virome profiles in plasma via metagenomic sequencing. A total of 1,200 plasma samples were collected and mixed into 12 pools based on sampling time, then DNA and cDNA libraries were constructed for sequencing which was carried out by Illumina NovaSeq 6000 system. After data cleaning and filtering using bioinformatic tools, 2336 viral reads via DNA sequencing and 61985 viral reads via cDNA sequencing were obtained. Seven DNA viruses belonging to three families and one RNA virus were identified from these reads. Most of the viruses found in pooled plasma were considered non- pathogenic, especially anellovirus found in all 12 pools and human pegivirus detected in 7 of the 12 pools. Three pathogenic viruses were found by DNA sequencing, including human herpes virus 6A, human cytomegalovirus and Epstein-Barr virus. All of them belong to the family Herpesviridae and their reads accounted for 7.9% (184/2336). Currently, data on anellovirus and human pegivirus in China are scarce. The herpes virus, which has a high sero-prevalence, is not a mandatory marker for blood screening. This study preliminary elucidated the virome spectra in blood from qualified donors in Guangzhou and contributed to information regarding blood transfusion safety in China.

The residents of Baisha, a county of Hainan Island, mainly composed of Li ethnic population and relatively closed living environment with its unique geographical location. Our previous study showed that Li ethnic population of Baisha is an endemic center for hepatitis C virus, with significantly higher rates than in other parts of China. However, the epidemiology of HBV in this region remains unclear. Therefore, we conducted a comprehensive epidemiological survey of HBV in Baisha County, including 1,682 Li ethnic residents. The total seropositive rate for HBsAg was 10.2% and was higher than other parts of China. HBV-positive status was associated with the 20-40-year-old group (OR = 1.27, 95%CI 1.04-1.39, P < 0.01) and alcohol consumption (OR = 2.17, 95%CI 1.58-2.99, P < 0.01). Phylogenetic analysis showed that HBV subgenotype D3 was predominant in Baisha County which was first discovered in China, followed by C5, C1, B2, and undetermined subgenotypes which were significantly different from other geographical distribution of main genotypes in China. The most recent common ancestor (tMRCA) of the HBV genotype C in the Li ethnic of Baisha County was 1846 (95%CI: 1739-1932), and Baisha-C5 was earlier than Baisha-C1 and Baisha-C2. Most Baisha-D3 sequences were concentrated in one bundle and unrelated to those D3 genome sequences elsewhere in the world. According to the phylogenetic tree, D3 was introduced into Baisha County in 1884 (95%CI: 1816-1993) and became a local endemic virus. In conclusion, HBV infection in the Li ethnic group is characterized by a high prevalence rate in 20-40-year-old individuals and a unique genotype distribution which were significantly different from other geographical distribution of main genotypes in China, and subgenotype D3 was first discovered in China.

Objective Investigate the prevalence of human T-cell lymphotropic virus （HTLV） among blood donors in Guangdong province,so as to improve blood safety and provide support for health authorities to formulate blood screening policies.Method Enzyme linked immunosorbent assay （ELISA） was used to conduct preliminary screening on blood donors in the whole province from January 2018 to October 2021.Those who failed to pass the test were recorded as positive in preliminary screening and further confirmed by Western blotting （WB）.A positive result of confirmation test was considered to be HTLV positive.According to the results of the confirmatory test,the prevalence of HTLV in Guangdong province and each city was figured out,then regional and interannual variations in prevalence rate were analyzed.Result A total of 4 702 539 blood donors were screened in Guangdong province in four years.Therein,1427 blood donors were found to be positive in ELISA but only 74 blood donors were confirmed positive for HTLV,with a prevalence rate of 0.0016% （74/4 702 539）.The prevalence of HTLV in eastern Guangdong is higher than that in northern Guangdong,western Guangdong and the Pearl River Delta （χ~2=5.567,P=0.02;χ~2=13.502,P＜0.001;χ~2=32.612,P＜0.001）.Shanwei in eastern Guangdong had the highest prevalence rate of HTLV in the province （7/56 691,0.0123%）.The prevalence rates of HTLV in the four years were 0.0018% in 2018,0.0012% in 2019,0.0016% in 2020 and 0.0018% in 2021,respectively.The difference was not statistically significant （χ~2=1.965,P=0.58）.Conclusion Guangdong is a low epidemic area of HTLV.In order to improve the quality of blood products,it is suggested to carry out HTLV screening in areas with high prevalence.As for other regions,flexible strategies can be adopted according to the local economic development.

The characteristics of a large sample size of the full-length genome of occult hepatitis B virus (HBV) infection (OBI) have not been extensively explored in China. Voluntary blood donors who were HBsAg-negative/HBV NAT-positive (HBsAg−/HBV NAT+) were identified by blood screening and recruited. Blood samples were tested for HBV serologic markers, viral loads, and PCR to identify OBI. HBV full-length genomes were obtained by amplifying two fragments using nested PCR. The characterization of OBI strains was based on sequence analyses compared with HBsAg+ strains obtained from the same donor population. Of the 50 full-length genomes of 172 identified OBI strains, 33 were classified as genotype B (OBI B ) and 17 strains as genotype C (OBI C ). Significantly higher nucleotide variabilities were observed in the Pre-S2/S promoter region (SP2) and core upstream regulatory sequence (CURS) in OBI B than in their HBsAg+ controls ( P < 0.05). Both OBI B and OBI C showed higher amino acid (aa) variabilities in Pol and Pre-S/S regions than their controls ( P < 0.05). In addition, 19 novel OBI-related mutations were found spanning the four open reading frames (ORFs) of the HBV genome. Four novel deletions and one novel insertion were also found in OBI C strains. Several novel OBI-related mutations spanning the four ORFs of the virus were identified by characterizing a large sample size of the full-length OBI genome, which may affect the production of HBsAg and contribute to the occult infection of HBV.

Hepatitis C virus (HCV) genotype 3 (GT-3) represents 22–30% of all infections and is the second most common genotype among all HCV genotypes. It has two main subtypes, GT-3a and GT-3b, that present epidemiological differences in transmission groups. This report generated 56 GT-3a and 64 GT-3b whole-genome sequences to conduct an evolutionary kinetics and selective force analysis with reference sequences from various countries. Evolutionary analysis showed that HCV GT-3a worldwide might have been transmitted from the Indian subcontinent to South Asia, Europe, North America and then become endemic in China. In China, GT-3a may have been transmitted by intravenous drug users (IDUs) and become endemic in the general population, while GT-3b may have originated from IDUs and then underwent mutual transmission between blood donors (BDs) and IDUs, ultimately becoming independently endemic in IDUs. Furthermore, the spread of GT-3a and GT-3b sequences from BD and IDU populations exhibit different selective pressures: the proportion of positively selected sites (PPSs) in E1 and E2 from IDUs was higher than in BDs. The number of positive selection sites was higher in GT-3b and IDUs. These results indicate that different selective constraints act along with the GT-3a and GT-3b genomes from IDUs and BDs. In addition, GT-3a and GT-3b have different transmission routes in China, which allows us to formulate specific HCV prevention and control strategies in China.

Sexually transmitted infections such as Chlamydia trachomatis can enhance HIV-1 infection. However, the molecular mechanisms modulating the enhancement of HIV-1 infectivity and replication during HIV-1/sexually transmitted infections coinfection remain elusive. In this study, we performed an ex vivo infection of HIV-1 in PBMCs of C. trachomatis-infected patients and observed a significant increase in HIV-1 p24 levels compared with those in cells from healthy donors. Similarly, C. trachomatis-stimulated PBMCs from healthy donors showed enhanced susceptibility to HIV-1. C. trachomatis-stimulated CD4 T cells also harbored more HIV-1 copy numbers. RNA sequencing data revealed the upregulation of CCL3L1/CCL3L3, a paralog of CCL3 in C. trachomatis-stimulated CD4 T cells infected with HIV-1. Furthermore, an increase in CCL3L1/CCL3L3 expression levels correlated with HIV-1 replication in C. trachomatis-stimulated cells. However, the addition of exogenous CCL3L1 reduces HIV-1 infection of healthy cells, indicating a dual role of CCL3L1 in HIV-1 infection. Further investigation revealed that a knockout of CCL3L1/CCL3L3 in Jurkat T cells rescued the increased susceptibility of C. trachomatis-stimulated cells to HIV-1 infection. These results reveal a role for CCL3L1/CCL3L3 in enhancing HIV-1 replication and production and highlight a mechanism for the enhanced susceptibility to HIV-1 among C. trachomatis-infected patients.

Hepatitis C virus (HCV) is a highly diverse pathogen that frequently establishes a chronic long-term infection, but the origins and drivers of HCV diversity in the human population remain unclear. Previously unidentified strains of HCV genotype 6 (gt6) were recently discovered in chronically infected individuals of the Li ethnic group living in Baisha County, Hainan Island, China. The Li community, who were early settlers on Hainan Island, has a distinct host genetic background and cultural identity compared to other ethnic groups on the island and mainland China. In this report, we generated 33 whole virus genome sequences to conduct a comprehensive molecular epidemiological analysis of these novel gt6 strains in the context of gt6 isolates present in Southeast Asia. With the exception of one gt6a isolate, the Li gt6 sequences formed three novel clades from two lineages which constituted 3 newly assigned gt6 subtypes and 30 unassigned strains. Using Bayesian inference methods, we dated the most recent common ancestor for all available gt6 whole virus genome sequences to approximately 2767 bce (95 per cent highest posterior density (HPD) intervals, 3670-1397 bce), which is far earlier than previous estimates. The substitution rate was 1.20 x 10(-4) substitutions/site/year (s/s/y), and this rate varied across the genome regions, from 1.02 x 10(-5) s/s/y in the 5'untranslated region (UTR) region to 3.07 x 10(-4) s/s/y in E2. Thus, our study on an isolated ethnic minority group within a small geographical area of Hainan Island has substantially increased the known diversity of HCV gt6, already acknowledged as the most diverse HCV genotype. The extant HCV gt6 sequences from this study were probably transmitted to the Li through at least three independent events dating perhaps from around 4,000 years ago. This analysis describes deeper insight into basic aspects of HCV gt6 molecular evolution including the extensive diversity of gt6 sequences in the isolated Li ethnic group.

Objective To study the CD4 T cell epitopes in Core and NS3 protein of genotype 1(GT1) and 6(GT6) of hepatitis C virus(HCV). Methods A total of 298 overlapping peptides(16-mer) spanning Core and NS3 protein of GT1 and GT6 HCV were synthesized. Peripheral blood mononuclear cells(PBMCs) from 17 HCV+ and 7 healthy blood donors were stimulated by peptide pools, followed by evaluating T cell response by IFN-γ ELISPOT, by which 21 peptides with positive results were found. These peptides were further applied to individually stimulate 20 HCV+ and 18 healthy PBMCs. The differences of responsive frequencies to the 21 positive peptides between the two study groups were compared. Results Pooled and individual peptide stimulation tests showed that HCV+ PBMCs were responsive to the stimulation of 5 peptides(GT1 NS31273-1288 and NS31315-1330; GT6 NS31033-1048, NS31087-1102 and NS31351-1366), with a responsive frequency ranging 18.9%-27.0%. In contrast, healthy PBMCs were not or low responsive(0%-4.0%) to these five peptides. The responsive frequencies were statistically different between the two groups(P<0.05). No reported epitopes in IEDB were found identical with these 5 peptides via sequence alignment. Conclusion Our study identified novel CD4 T cell epitopes in NS3 protein of GT1 and GT6 HCV, which has potential application value for the research and development of HCV vaccine.

Objective To learn the situation of the evolution process of HCV virus population and the selection pressure of HCV NS5B in intravenous drug users (IDUs) in Guangdong. Methods 141 blood samples from hepatitis C virus (HCV) RNA-positive blood donors and 58 from HCV patients in Guangdong were randomly collected for HCV NS5B sequence amplification, combined with HCV NS5B sequences from blood donors and IDUs obtained by sequencing previously(between 2009 and 2011). Homology analysis was performed by Molecular Evolutionary Genetics Analysis (MEGA) software, evolutionary analysis were performed by Bayesian Evolutionary Analysis Sampling Trees (BEAST) software package. Selection pressure analysis was performed on sequences isolated from IDUs by Datamonkey online software package with Mixed Effects Model Evolution (MEME) method, and the population expansion of species were analyzed using Tajima and Fu neutrality test by Arlequin software. Results The comparison results of internal homology among different subtypes of IDUs in this group were as follows : HCV-3b had the highest homology (97%), followed by HCV-3a (96%), HCV-6a (95%) and HCV-1b (94%); HCV evolution rate analysis showed that HCV-1b had the fastest evolution rate [2.17E-03 substitutions/site/year (y/y/y)], followed by HCV-3b (2.12E-0 y/y/y), HCV-3a (1.58E-03 y/y/y) and HCV-6a (1.28E-03 y/y/y). The analysis on effective population of HCV: 1980~1990 was rapid growth period for HCV-6a, 1990~1995 period for HCV-1b, and 2000~2007 period for HCV-3a. HCV population genetic characteristics was as follows: HCV-1b, 3a, 3b and 6a experienced population expansion, among which 3a and 3b were the most obvious. As to the analysis of HCV selection pressure, two positive selection sites (235 and 243)were found in the 339 nucleotide fragment of the NS5B sequence in injecting drug users, but mutation only occurred at position 316 [mutation rate 1.24% (14/1 130)] among 5 direct antiviral drug (DAA) sites in this gene. Conclusion The evolution of HCV-3b in Guangdong has showed an obvious trend of population expansion, with a high proportion and homology especially in the local IDUs. HCV-3b should be the focus of HCV prevention and control in this region. Given that the positively selected sites of the HCV NS5B gene region of IDUs in Guangdong are non-DAA binding sites, DAA is expected to demonstrate a good effect on these patients.