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.

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 explore the effectiveness of using donor plasma reinfusion to flush the leukoreduction system (LRS) chamber during the final reinfusion phase with the Trima Accel automated blood collection system in preventing the reduction of CD4+ and CD8+ T lymphocytes. Methods A longitudinal and cross-sectional study was designed. CD4+ count<200 cells/μL and CD8+ count<125 cells/μL were considered as the criteria for deficiency. Eighteen first-time platelet donors were followed up. The lymphocyte count was measured at 0, 3-6 and 7-14 times of blood donation in the last 300 days. 170 healthy blood donors who have not donated blood were selected as the control group. According to the cut-off point(October 2021), 88 blood donors who mainly used automatic blood collection system to donate platelet apheresis in the last 365 days(median blood donation times ≥17.5)were divided into three groups(A, B and C)and blood samples were obtained. The time for Groups A, B and C started donating platelet apheresis were as follows: Group A: before October 2019, Group B: from October 2019 to September 2021, Group C: after October 2021. Blood samples were analyzed to obtain blood counts including CD4 + and CD8 + T lymphocytes. Blood samples were analyzed to obtain blood cell counts including CD4+ and CD8+ T lymphocytes. Through a comparative analysis, this study aimed to determine if there are any statistical differences in the detection indices between the follow-up groups with varying frequencies of blood donation, the control group, and groups A, B, and C. This approach was employed to infer the efficacy of donor plasma reinfusion in flushing the leukoreduction system (LRS) chamber for preventing the decline of CD4+ and CD8+ T lymphocytes. Results Eighteen first-time blood donors who were converted to regular platelet apheresis donors did not show a decrease of CD4 + and CD8 + T lymphocytes in the 5 th and 11 th blood donation (median number of blood donation), and there was no significant difference between the above indexes and those in the 0 th blood donation. Among the previous frequent blood donors, the CD4+ and CD8+ T lymphocyte counts in Group B and Group C are both higher than the standard value, showing no statistical difference from the control group. Among regular blood donors, the CD4+ and CD8+ T lymphocyte counts in groups B and C were higher than the criteria values, and had no statistical difference compared to the control group.The CD4+ T lymphocyte count in Group A was normal, with only one donor in Group A having a CD8+ T lymphocyte count below 125 cells/μL. This donor has donated 281 times of platelet apheresis, and the group he belongs to has started blood donation 2-21 years(median of 5 years) before the adjustment of reinfusion mode. The CD4+ and CD8+ T lymphocyte counts in Group A showed significant differences compared to the control group, with median counts (Group A/Control Group) of 359/521 and 257/372, respectively, P<0.001. In Group A, 0%(0/35) had a CD4+ count below 200 cells/μL, and 2.85%(1/35) of donors had a CD8+ count below 125 cells/μL, which was far lower than the proportion of CD4+ and CD8+ T cell deficiency found in regular apheresis donors by John M. Gansner and Mahboubeh Rahmani. The study showed that the adjustment of the plasma reinfusion mode did not further reduce the T lymphocyte counts in blood donors, but instead further restored the T lymphocyte counts in regular blood donors. This indicated that after the adjustment of plasma reinfusion mode, blood donors might not have lost CD4+ and CD8+ T lymphocytes during blood donation, or only lost a small amount, and can recover even if they donate platelet apheresis frequently. Conclusion Trima Accel automated blood collection system has a good effect on preventing CD4 + and CD8 + T lymphocytes from being reduced by flushing the LRS chamber with donor plasma.

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.

Blood donors not only save the lives of patients but also play an important role in the development of medical and health services. Therefore, it is particularly important to pay attention to the blood health of blood donors who are at a high risk of iron deficiency. Detection of serum ferritin and transferrin is an important basis for the diagnosis of iron deficiency anemia. However, to the best of our knowledge, the levels of serum ferritin and transferrin, and the influencing factors, such as age and type of donation, in blood donors have not been clarified. In the present study, the serum ferritin and transferrin levels of donors from two blood centers were investigated. Demographic data were collected from the donors, and their serum ferritin and transferrin levels were tested. A total of 1,817 donors were enrolled and were eligible for evaluation. Reference intervals (RIs) for ferritin and transferrin were obtained from blood donors, and it was revealed that the ferritin and transferrin levels of blood donors were associated with age. Furthermore, serum transferrin levels were associated with the type of donation; the serum transferrin RI level was significantly higher in platelet-only donors compared with in whole blood donors. It was also demonstrated that ferritin levels were negatively associated with transferrin levels. The present study identified RIs for ferritin and transferrin levels in blood donors, and indicated that age and type of donation were important factors affecting ferritin and transferrin levels in blood donors. These findings may prove useful for blood donation recruitment and screening strategies in China, and could promote the health of blood donors.

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) 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.

• Currently, HCV 6a has replaced 1b as the most prevalent subtype in blood donors in Guangdong. • HCV 6a was the predominant subtype in males and older donors, while 1b predominated in females and younger donors. • HCV 6a may expand from Guangdong to other districts of China, and is worthy of attention.

Objective To investigate the CMV-IgG positive yeild among blood donors in Guangzhou and explore the differences in the efficacy of three test reagents, aimed at improving blood safety and service capacity of blood centers. Methods A total of 630 blood samples from eligible blood donors from July to October 2020 in our center were randomly selected and screened for CMV-IgG by one ELISA reagent.Among them, 180 samples were tested in parallel using three reagents (two ELISA reagents and one ECLIA reagent), and those tested negative were conducted quantitative CMV-DNA detection.The test results of different reagents were compared and analyzed. Results Out of the 630 samples, a total of 598 positive samples were screened out, including 180 samples yielded by three reagents, 171 and 175 by the two ELISA reagents, respectively, and 175 by ECLIA.The results given by three reagents were consistent (Kappa>0.4), and no significant difference in the positive yeild by three reagents was found.In the 180 samples, 11 were negative, among which 3, 2 and 6 samples were negative by all three reagents, two reagents and one reagent (ELISA), respectively.All the 11 samples were tested negative for CMV-DNA. Conclusion The yeild of positive CMV-IgG in blood donors was 94.9% (598/630), suggesting a high prevalence of CMV in Guangzhou. CMV serologically negative blood should be considered when providing blood products to immunocompromised patients to improve the safety of recipients.The detection results of ELISA reagents and ECLIA reagent for CMV- IgG are consistent, but ECLIA reagent has better detection efficacy.

There is little known of immunologic factors leading to the occurrence of occult HBV infection (OBI). Specific cellular immune response to hepatitis B virus (HBV) core/pol peptides was compared between blood donor populations, including 37 OBIs, 53 chronic HBV infections (CHB), 47 resolved infections, and 56 non-infected controls, respectively. The rate of CD4+/CD8+ T cell proliferation in OBI or CHB carriers was higher than in HBV resolved and non-infected individuals (P < 0.05). The intensity of IFN-γ-secretion T-cell response of OBI carriers was highest, followed by CHB and resolved infections, and non-infected individuals (P < 0.05). The frequency of intracellular IFN-γ and IL-17A CD4+/CD8+ and IL-21 CD4+ T-cell responses was significantly higher in resolved infections than in OBI or CHB carriers (P < 0.05), while the level of extracellular IL-17A of peripheral blood mononuclear cells (PBMCs) was higher in OBI and CHB carriers than in resolved infections (P < 0.01). The frequency of intracellular IL-10 CD4+ T-cell response in CHB, OBI, and resolved infections was higher than in HBV non-infected individuals (P < 0.01). Intracellular IL-10 CD8+ T cell and extracellular IL-10 T-cell responses were higher in CHB than in OBI (P = 0.012) or HBV resolved infections (P < 0.01). In conclusion, the higher level of effective T-cell response with IFN-γ, IL-17A, and IL-21 contributes to resolved infection outcome, while higher levels of suppressive T-cell response with IL-10 result in HBV chronicity. OBI is an intermediary status between HBV resolved and chronic infections, in which IL-21 effector and IL-10 suppressor T-cell responses play an important role in directing the outcome of HBV infection.

The mechanism of occult hepatitis B infection (OBI) has not yet been fully clarified. Our previous research found that novel OBI-related mutation within S protein, E2G, could cause the hepatitis B surface antigen (HBsAg) secretion impairment, which resulted in intracellular accumulation in OBI of genotype B. Here, to further explore the role of E2 site mutations in the occurrence of OBI, we analyzed these site mutations among 119 OBI strains identified from blood donors. Meanwhile, 109 wild-type HBV strains (HBsAg positive/HBV DNA positive) were used as control group. Furthermore, to verify the E2 site mutations, two conservative 1.3-fold full-gene expression vectors of HBV genotype B and C (pHBV1.3B and pHBV1.3C) were constructed. Then, the E2 mutant plasmids on the basis of pHBV1.3B or pHBV1.3C were constructed and transfected into HepG2 cells, respectively. The extracellular and intracellular HBsAg were analyzed by electrochemical luminescence and cellular immunohistochemistry. The structural characteristics of S proteins with or without E2 mutations were analyzed using relevant bioinformatics software. E2 mutations (E2G/A/V/D) existed in 21.8% (26/119) of OBIs, while no E2 mutations were found in the control group. E2G/A/V/D mutations could strongly affect extracellular and intracellular level of HBsAg (p < 0.05). Notably, unlike E2G in genotype B that could cause HBsAg intracellular accumulation and secretion decrease (p < 0.05), E2G in genotype C could lead to a very significant HBsAg decrease both extracellularly (0.46% vs. pHBV1.3C) and intracellularly (11.2% vs. pHBV1.3C) (p < 0.05). Meanwhile, for E2G/A mutations, the relative intracellular HBsAg (110.7-338.3% vs. extracellular) and its fluorescence intensity (1.5-2.4-fold vs. with genotype-matched pHBV1.3B/C) were significantly higher (p < 0.05). Furthermore, N-terminal signal peptides, with a typical cleavage site for peptidase at positions 27 and 28, were exclusively detected in S proteins with secretion-defective mutants (E2G/A). Our findings suggest that: (1) E2G/A/V/D mutations were confirmed to significantly influence the detection of HBsAg, (2) the underlying mechanism of OBI caused by E2G mutation is quite different between genotype B and genotype C, and (3) E2G/A could produce a N-terminal truncated S protein, which might attribute to the HBsAg secretion impairment in the OBIs.