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Klebsiella pneumoniae is an important opportunistic pathogen causing community-acquired and hospital-acquired infections. This aim of this study was to analysis the antibiotic-resistance phenotypes, carbapenemase genes, virulence genes, and genotypes the 62 K. pneumoniae clinical isolates, and to explore the correlations between these isolates. The antimicrobial susceptibility profiles were determined using the BD Phoenix-100 system. Carbapenemase and virulence genes were detected using multiplex PCR. Out of the 62 K. pneumoniae clinical isolates, 79.0% were exhibited resistance to antibiotics, with 69.4% displaying multi-drug resistance. The rate of antibiotic-resistance was highest for penicillin (71.0%), followed by cephalosporins (66.1%), and lowest for carbapenems (29.0%). The detection rates of carbapenemase genes were as follows: KPC (56.5%), VIM (35.5%), and NDM (1.61%). Additionally, seven virulence genes were detected with the highest prevalence rates, of which entB and mrkD were at the top of the carrier rates with 95.2% each. The study classified 62 isolates into 13 clusters and 46 genotypes using ERIC-PCR. Cluster A6 exhibited the highest genetic diversity, comprising 20 strains and 13 genotypes. The statistical analysis revealed a strong correlation between MDR and resistance to penicillin and cephalosporin. Furthermore, genes related to siderophores were closely associated with mrkD. Genotypes identified by ERIC-PCR showed a negative correlation with allS. The study revealed a negative correlation between antibiotic resistance and genes kfu, ybtS, iutA, rmpA, and allS. Conversely, a positive correlation was observed between antibiotic resistance and genes entB and mrkD. The correlations identified in this study provide insights into the occurrence of hospital-acquired infections. The findings of this study may guide the prevention and control of K. pneumoniae outbreaks by utilizing appropriate medication., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: Combination immunotherapy strategies targeting OX40, a co-stimulatory molecule that can enhance antitumor immunity by modulating the proliferation, differentiation, and effector function of tumor-infiltrating T cells, have attracted much attention for their excellent therapeutic effects. In this study, we aimed to evaluate the antitumor efficacy of combined anti-OX40 and hepatitis B core virus-like particles (HBc VLPs) therapy using a mouse colon cancer model., Methods: Humanized B-hOX40 mice were injected subcutaneously with MC38 colon tumor cells and treated with HBc VLPs+anti-hOX40 antibody. Tumor growth was monitored. Flow cytometric analysis was performed to evaluate the populations of T cell subsets in the tumors., Results: The combination of anti-OX40 with HBc VLPs resulted in a significant delay in tumor growth, suggesting that a potent antitumor immunity was induced by the combination therapy. Further studies revealed that HBc VLPs+anti-OX40 treatment induced a significant increase in effector T cells (Teffs) and a significant decrease in regulatory T cells (Tregs) in the tumor microenvironment (TME), which accounted for the synergistic antitumor effect of anti-OX40 in combination with HBc VLPs., Conclusion: Combination therapy of anti-hOX40 and HBc VLPs provides synergistic antitumor activity in colon cancer-bearing mice, which may represent a potential design strategy for cancer immunotherapy., (Copyright © 2024 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

Objective: The objective of this study was to investigate the global research trends in herbal medicine for the treatment of cardiovascular disease (CVD) from 2000 to 2023. A bibliometric approach was employed to analyze international collaborations, knowledge structures, emerging trends, and research frontiers., Method: The Web of Science (WOS) core collection was utilized as the database, employing the search formula (((TS = (traditional Chinese medicine)) OR TS = (Chinese herbal medicinal ingredient)) OR TS = (Chinese herbal medicinal constituent)) AND TS = (cardiovascular disease) to conduct the search. The search period spanned from January 1, 2000, to February 14, 2023, and the literature type included articles and reviews., Results: A total of 1478 papers were included in the analysis after searching the WOS database and excluding conference proceedings, news articles, retractions, editorials, and letters. China demonstrated the highest number of publications, followed by the United States and Taiwan (China). The institution with the highest publications was the Chinese Academy of Medical Sciences. China, the United States, and India were the main countries involved in research in this field, and there was significant collaboration among them. The hotspots related to herbal components for treating cardiovascular diseases from 2000 to 2023 included systematic reviews, ischemic reperfusion injury, global burden, type 2 diabetes, and protection., Conclusion: This paper provides a reference for the future development of herbal research in cardiovascular aspects by revealing the current status, hotspots, and trends of global herbal research in cardiovascular factors over more than 20 years. Identification of potential collaborators and institutions can assist researchers in exploring new directions for future research and discovering new perspectives for potential collaborations in this field., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

Rabies, caused by the rabies virus (RABV), is the most fatal zoonotic disease. It is a neglected tropical disease which remains a major public health problem, causing approximately 59,000 deaths worldwide annually. Despite the existence of effective vaccines, the high incidence of human rabies is mainly linked to tedious vaccine immunisation procedures and the overall high cost of post-exposure prophylaxis. Therefore, it is necessary to develop an effective vaccine that has a simple procedure and is affordable to prevent rabies infection in humans. RABV belongs to the genus Lyssavirus and family Rhabdoviridae. Previous phylogenetic analyses have identified seven major clades of RABV in China (China I-VII), confirmed by analysing nucleotide sequences from both the G and N proteins. This study evaluated the immunogenicity and protective capacity of SYS6008, an mRNA rabies vaccine expressing rabies virus glycoprotein, in mice and cynomolgus macaques. We demonstrated that SYS6008 induced sufficient levels of rabies neutralising antibody (RVNA) in mice. In addition, SYS6008 elicited strong and durable RVNA responses in vaccinated cynomolgus macaques. In the pre-exposure prophylaxis murine model, one or two injections of SYS6008 at 1/10 or 1/30 of dosage provided protection against a challenge with a 30-fold LD 50 of rabies virus (China I and II clades). We also demonstrated that in the post-exposure prophylaxis murine model, which was exposed to lethal rabies virus (China I-VII clades) before vaccination, one or two injections of SYS6008 at both 1/10 and 1/30 dosages provided better protection against rabies virus challenge than the immunization by five injections of commercial vaccines at the same dosage. In addition, we proved that SYS6008-induced RVNAs could neutralise RABV from the China I-VII clades. Finally, 1/10 of the dosage of SYS6008 was able to stimulate significant RABV-G specificity in the T cell response. Furthermore, we found that SYS6008 induced high cellular immunity, including RABV-G-specific T cell responses and memory B cells. Our results imply that the SYS6008 rabies vaccine, with a much simpler vaccination procedure, better immunogenicity, and enhanced protective capacity, could be a candidate vaccine for post-exposure prophylaxis of rabies infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The genotyping of Campylobacter coli was done using three methods, pulsed-field gel electrophoresis (PFGE), Sau-polymerase chain reaction (Sau-PCR), and denaturing gradient gel electrophoresis assay of flagellin gene (fla-DGGE) and the characteristics of these assays were compared. The results showed that a total of 53 strains of C. coli were isolated from chicken and duck samples in three markets. All isolates were clustered into 31, 33, and 15 different patterns with Simpson's index of diversity (SID) values of 0.972, 0.974, and 0.919, respectively. Sau-PCR assay was simpler, more rapid, and had higher discriminatory power than PFGE assay. Fla-DGGE assay could detect and illustrate the number of contamination types of C. jejuni and C. coli without cultivation, which saved more time and cost than Sau-PCR and PFGE assays. Therefore, Sau-PCR and fla-DGGE assays are both rapid, economical, and easy to perform, which have the potential to be promising and accessible for primary laboratories in genotyping C. coli strains., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: To understand the distribution of genotypes and sub-genotypes of HBV in different ethnic groups in China. Methods: The HBsAg positive samples were selected by stratified multi-stage cluster sampling from the sample base of national HBV sero-epidemiological survey in 2020 for the amplification of S gene of HBV by nested PCR. A phylogeny tree was constructed to determine the genotypes and sub-genotypes of HBV. The distribution of genotypes and sub-genotypes of HBV were analyzed comprehensively by using laboratory data and demographic data. Results: A total of 1 539 positive samples from 15 ethnic groups were successfully amplified and analyzed, and 5 genotypes (B, C, D, I and C/D) were detected. The proportion of genotype B was higher in ethnic group of Han (74.52%, 623/836), Zhuang (49.28%, 34/69), Yi (53.19%, 25/47), Miao (94.12%, 32/34), Buyi (81.48%, 22/27). The proportions of genotype C were higher in ethnic groups of Yao (70.91%, 39/55). Genotype D was the predominant genotype in Uygur (83.78%, 31/37). Genotype C/D were detected in Tibetan (92.35%,326/353). In this study, 11 cases of genotype I were detected, 8 of which were distributed in Zhuang nationality. Except for Tibetan, sub-genotype B2 accounted for more than 80.00% in genotype B in all ethnic groups. The proportions of sub-genotype C2 were higher in 8 ethnic groups, i.e. Han, Tibetan, Yi, Uygur, Mongolian, Manchu, Hui and Miao. The proportions of sub-genotype C5 were higher in ethnic groups of Zhuang (55.56%, 15/27) and Yao (84.62%, 33/39). For genotype D, sub-genotype D3 was detected in Yi ethnic group and sub-genotype D1 was detected in both Uygur and Kazak. The proportions of sub-genotype C/D1 and C/D2 in Tibetan were 43.06% (152/353) and 49.29% (174/353). For all the 11 cases of genotype I infection, only sub-genotype I1 was detected. Conclusions: Five genotypes and 15 sub-genotypes of HBV were found in 15 ethnic groups. There were significant differences in the distribution of genotypes and sub-genotypes of HBV among different ethnic groups.

Campylobacter is regarded as the leading cause of zoonotic diseases and Campylobacter jejuni (C. jejuni) is one of the predominant pathogenic species. To track C. jejuni infections, various genotyping methods have been used. In this study, amplified intergenic locus polymorphism (AILP) was used to type C. jejuni for the first time. To confirm its feasibility, pulsed-field gel electrophoresis (PFGE) was performed as a control, and the results obtained by the AILP and PFGE methods were compared. Fifty-one isolates were resolved into 34 and 29 different genotypes with Simpson's indices of 0.976 and 0.967 using the AILP and PFGE methods, respectively. The adjusted Rand coefficient of the two approaches was as high as 0.845. In summary, the data showed that the two genotyping methods were similar for discriminating isolates and were both appropriate methods to distinguish whether two isolates were indistinguishable, but the AILP was faster and less costly than PFGE. Therefore, the AILP is a reliable, rapid, and highly discriminative method to genotype C. jejuni collected from poultry meat, which is helpful to effectively monitor C. jejuni., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objective: To study the effects of estradiol (E2) on alleviating myocardial ischemia/reperfusion(I/R) injury through estrogen receptorβ(ERβ) mediated extracellular regulated protein kinases(ERK) pathway activation. Methods: Eighty-four adult female SD rats were ovariectomized and randomly divided into control group, NC siRNA adeno-associated virus (AAV) group received sham operation, the myocardial I/R injury model was prepared by ligation of the left anterior descending coronary artery in I/R group, E2+I/R group, NC siRNA AAV+I/R group, NC siRNA AAV+E2+I/R group and ERβ-siRNA AAV+E2+I/R group. E2+I/R group, NC siRNA AAV+E2+I/R group and ERβ-siRNA AAV+E2+I/R group were treated with E2 0.8 mg/kg by gavage for 60 days before modeling. NC siRNA AAV+I/R group, NC siRNA AAV+E2+I/R group, and ERβ-siRNA AAV+E2+I/R group were treated with AAV by caudal vein injection 24 h before modeling. After 120 min of reperfusion, the contents of serum lactate dehydrogenase (LDH), phosphocreatine kinase (CK), phosphocreatine kinase isoenzyme (CK-MB), myocardial infarction area and the expressions of ERβ, p-ERK, the contents of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1 β), malondialdehyde (MDA) and total antioxidant capacity (T-AOC) in myocardium were measured. Results: The contents of serum LDH, CK, CK-MB, myocardial infarction area and the contents of TNF-α, IL-1 β, MDA in myocardium of I/R group were higher than those of the control group, the expression levels of ERβ and p-ERK and the content of T-AOC were lower than those in the control group ( P ＜0.05). The contents of serum LDH, CK and CK-MB, myocardial infarction area and the contents of TNF-α, IL-1 β and MDA in myocardium of E2+I/R group were lower than those of the I/R group, the expression levels of ERβ and p-ERK and the content of T-AOC were higher than those of the I/R group( P ＜0.05). After knockdown ERβ by caudal vein injection of ERβ-siRNA AAV, the contents of serum LDH, CK and CK-MB, myocardial infarction area and the contents of TNF-α, IL-1 β and MDA in myocardium of ERβ-siRNA AAV+E2+I/R group were higher than those of NC-siRNA AAV+E2+I/R, the expression levels of ERβ and p-ERK and the content of T-AOC were lower than those of NC-siRNA AAV+E2+I/R( P ＜0.05). Conclusion: E2 has protective effects on myocardial I / R injury in ovariectomized rats, which are related to the promotion of ERβ mediating the activation of ERK pathway, reducing inflammatory and oxidative stress responses.

Objective: To analyze the genotype distribution of acute hepatitis B virus in China. Methods: A total of six hundred and twenty acute Hepatitis B cases reported to China Information System for Diseases Control and Prevention from 2015 to 2017 were selected. First, the full-length HBV genome was obtained by nested PCR amplification. In addition, the HBV genotype was determined by constructing a phylogeny tree. Finally, using primarydata, HBV genotype distribution was analyzed. Results: A total of 519 (83.71%, 519/620) sequences were obtained genotype of 620 acute hepatitis B cases, including A (0.19%, 1/519), B (27.17%, 141/519), C (62.04%, 322/519), D (9.06%, 47/519), I (0.77%, 4/519) and C/D (0.77%, 4/519); B2(95.03%, 134/141) and C2 (72.67%, 234/322) were the two major subgenotypes. Genotypes were distributed differently in seven regions of China. The proportion of genotype C appeared higher in Northeast China (94.55%, 52/55), North China (93.85%, 61/65), East China (78.87%, 56/71), and South China (58.14%, 50/86). The proportion of genotype B was higher in Central China (58.07%, 36/62) and Southwest China (52.94%, 45/85), the proportion of genotype D was the highest in Northwest China (48.42%, 46/95). A total of 515 cases were classified as serotypes, including 'adr' (57.48%, 296/515), 'adw' (30.87%, 159/515), 'ayr' (0.19%, 1/515), and 'ayw' (11.46%, 59/515). Genotype B was dominated by 'adw' serotype (92.14%, 129/140), genotype C was dominated by 'adr' serotype (91.88%, 294/320),all genotype D were 'ayw' serotype. The genotype of acute hepatitis B was correlated with serotype, 'adw' was dominant in genotype B, 'adr' was dominant in genotype C and 'ayw' was dominant in genotype D.In different gender and age group, there was no statistical significance ingenotype distribution ( P >0.05). Conclusions: The genotype of acute hepatitis B in China from 2015 to 2017 was mainly B, C, and D; genotype C was dominant in the Northeast China,North China, East China and South China; B and C were common in Central and Southwest China, and genotype B was dominant. Genotype D was primarily distributed in Northwest China. The genotype of acute hepatitis B was correlated with serotype, 'adw' was dominant in genotype B, 'adr' was dominant in genotype C and 'ayw' was dominant in genotype D. There was no difference in the distribution of acute hepatitis B genotypes among different genders and age groups.

Without approved vaccines and specific treatments, COVID-19 is spreading around the world with above 26 million cases and approximately 864 thousand deaths until now. An efficacious and affordable vaccine is urgently needed. The Val308 - Gly548 of spike protein of SARS-CoV-2 linked with Gln830 - Glu843 of Tetanus toxoid (TT peptide) (designated as S1-4) and without TT peptide (designated as S1-5) were expressed and renatured. The antigenicity and immunogenicity of S1-4 were evaluated by Western Blotting (WB) in vitro and immune responses in mice, respectively. The protective efficiency was measured preliminarily by microneutralization assay (MN50). The soluble S1-4 and S1-5 protein was prepared to high homogeneity and purity. Adjuvanted with Alum, S1-4 protein stimulated a strong antibody response in immunized mice and caused a major Th2-type cellular immunity supplemented with Th1-type immunity. Furthermore, the immunized sera could protect the Vero E6 cells from SARS-CoV-2 infection with neutralizing antibody titer 256. Recombinant SARS-CoV-2 RBD with a built in T helper epitope could stimulate both strong humoral immunity supplemented with cellular immunity in mice, demonstrating that it could be a promising subunit vaccine candidate., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: To analyze the epidemiological distribution of Hepatitis B virus (HBV) genotype in the mainland of China following the implementation of effective preventive measures., Methods: Five hundred and seventeen HBsAg-positive subjects aged 1-29 years surveyed in the 2014 national HBV sero-survey in the mainland of China were enrolled in the study. The full-length HBV genome was obtained by PCR amplification and sequencing. The HBV genotype was determined by phylogenetic analysis. Combined with questionnaire information, HBV genotype distribution was analyzed., Results: Of the 517 HBsAg-positive subjects, 369 (71.4%) were included in the analysis. HBV genotypes found were B (45.0%), C (36.6%), D (6.0%), C/D (9.8%), B/C (2.2%), and I (0.5%). Geographic differences in HBV genotype were significant for seven regions. Three serotypes were found: adw (47.2%), adr (35.5%), and ayw (17.3%). B2 (43.9%) and C2 (25.2%) were the two major subgenotypes. The predominant genotypes differed between the Han group and the other ethnic groups. No statistical differences in genotype distribution were found by gender, age group, or hepatitis B (HepB) vaccination history., Conclusion: The prevalence of HBV genotype B was higher than that of genotype C with subgenotypes B2 and C2 endemic in 1-29-year-olds in the mainland of China, after HBV prevalence has reduced significantly due to the implementation of preventive measures. HepB vaccination or other factors did not interfere with HBV genotype distribution. The surveillance of HBV genotype was essential for responding to the potential changes and impact on the preventive policies in the future., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

SARS-CoV-2 is the cause of the worldwide outbreak of COVID-19 that has been characterized as a pandemic by the WHO. Since the first report of COVID-19 on December 31, 2019, 179,111 cases were confirmed in 160 countries/regions with 7426 deaths as of March 17, 2020. However, there have been no vaccines approved in the world to date. In this study, we analyzed the biological characteristics of the SARS-CoV-2 Spike protein, Pro330-Leu650 (SARS-CoV-2-SPL), using biostatistical methods. SARS-CoV-2-SPL possesses a receptor-binding region (RBD) and important B (Ser438-Gln506, Thr553-Glu583, Gly404-Aps427, Thr345-Ala352, and Lys529-Lys535) and T (9 CD4 and 11 CD8 T cell antigenic determinants) cell epitopes. High homology in this region between SARS-CoV-2 and SARS-CoV amounted to 87.7%, after taking the biological similarity of the amino acids into account and eliminating the receptor-binding motif (RBM). The overall topology indicated that the complete structure of SARS-CoV-2-SPL was with RBM as the head, and RBD as the trunk and the tail region. SARS-CoV-2-SPL was found to have the potential to elicit effective B and T cell responses. Our findings may provide meaningful guidance for SARS-CoV-2 vaccine design., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background : To assess the long-term protection conferred by plasma-derived hepatitis B vaccine at 20-31y after primary immunization during infancy in Chinese rural community. Method : Participants born between 1986 and 1996, who received a full course of primary vaccination with plasma-derived hepatitis B vaccine and had no experience with booster vaccination were enrolled. An epidemiological investigation was performed, and blood samples were collected to detect hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (anti-HBs), and hepatitis B core antibody (anti-HBc). The positive rate of HBsAg, anti-HBs, and anti-HBc were calculated to evaluate the long-term protection of the plasma-derived hepatitis B vaccine. Results : A total of 949 participants were enrolled in the final analysis. Six subjects were detected to be HBsAg-positive, resulting in a HBsAg carrier rate of 0.63% (6/949). A total of 468 (52.41%) participants maintained a level of anti-HBs antibody ≥10 mIU/mL, with a GMC of 112.20 mIU/mL (95%CI: 97.72 ~ 128.82 mIU/mL). A significant downtrend was observed in the anti-HBs positive rate ( P < .001). The average anti-HBc positive rate was 5.90% (56/949), increased with prolongation of immunization ( P < .001). Conclusions : The plasma-derived hepatitis B vaccine maintained satisfactory protection at 20-31 y after primary immunization. These results indicate that a booster dose is not necessary. Further studies on the immune memory induced by the plasma-derived hepatitis B vaccine are needed.

Objective: To analyze the epidemiological characteristics of hepatitis E Virus antibody (anti-HEV) in people aged 1-29 years in China in 2014. Methods: Based on database of the national serologic survey of hepatitis B in people aged 1-29 years in China, in 2014, the sample size was estimated. The serum samples of the people surveyed were randomly selected to detect anti-HEV IgG by using enzyme- linked immunosorbent assay (ELISA). Statistical software SAS 9.1.3 was used to calculate the positive rate of anti-HEV and 95 % confidence interval ( CI ) in different age, gender groups, urban and rural areas and geographic areas by using the Taylor series linear method with sampling weight. The difference was determined by comparing 95 %CI . Results: A total of 14 106 serum samples were detected from people aged 1-29 years, including 6 996 males (49.60 % ), 7 013 urban residents (49.72 % ). The positive rate of anti-HEV was 8.12 % (95 %CI : 7.19-9.15) in people aged 1-29 years. There was no statistical difference between the men and women, between urban area and rural area. The positive rates of anti-HEV in western area(11.36 % , 95 %CI : 9.45-13.62) was higher than those in eastern and central areas. The positive rates of anti-HEV were 2.46 % , 2.24 % , 4.50 % , 7.58 % , 11.89 % and 17.27 % in people aged 1-, 5-, 10-, 15-, 20- and 25-29 years, respectively. As the age increased, the positive rate of anti-HEV gradually increased. In different ethnic groups, the positive rate of anti-HEV was higher in Tibetan (18.32 % , 95 %CI : 12.02-26.90), Zhuang (9.54 % , 95 %CI : 4.33-19.73) ethnic groups. Conclusion: The positive rate of anti-HEV declined slightly in China in 2014. It is still necessary to pay close attention to the HEV infection, morbidity of hepatitis E and risk factors in people aged 1-29 years.

Objective: To evaluate the effect of intranasal immunization with CTA1-DD as mucosal adjuvant combined with H3N2 split vaccine., Methods: Mice were immunized intranasally with PBS (negative control), or H3N2 split vaccine (3 μg/mouse) alone, or CTA1-DD (5 μg/mouse) alone, or H3N2 split vaccine (3 μg/mouse) plus CTA1-DD (5 μg/mouse). Positive control mice were immunized intramuscularly with H3N2 split vaccine (3 μg/mouse) and alum adjuvant. All the mice were immunized twice, two weeks apart. Then sera and mucosal lavages were collected. The specific HI titers, IgM, IgG, IgA, and IgG subtypes were examined by ELISA. IFN-γ and IL-4 were test by ELISpot. In addition, two weeks after the last immunization, surivival after H3N2 virus lethal challenge was measured., Results: H3N2 split vaccine formulated with CTA1-DD could elicit higher IgM, IgG and hemagglutination inhibition titers in sera. Furthermore, using CTA1-DD as adjuvant significantly improved mucosal secretory IgA titers in bronchoalveolar lavages and vaginal lavages. Meanwhile this mucosal adjuvant could enhance Th-1-type responses and induce protective hemagglutination inhibition titers. Notably, the addition of CTA1-DD to split vaccine provided 100% protection against lethal infection by the H3N2 virus., Conclusion: CTA1-DD could promote mucosal, humoral and cell-mediated immune responses, which supports the further development of CTA1-DD as a mucosal adjuvant for mucosal vaccines., (Copyright © 2019 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

Background: To assess the immune persistence conferred by a Chinese hamster ovary (CHO)-derived hepatitis B vaccine (HepB) 17 to 20 years after primary immunization during early life., Methods: Participants born between 1997 and 1999 who received a full course of primary vaccination with HepB (CHO) and who had no experience with booster vaccination were enrolled. Blood samples were required from each participant for measurement of hepatitis B surface antibody (anti-HBs), surface antigen and core antibody levels. For those who possessed an anti-HBs antibody < 10 mIU/mL, a single dose of HepB was administered, and 30 days later, serum specimens were collected to assess the booster effects., Results: A total of 1352 participants were included in this study. Of these, 1007 (74.5%) participants could retain an anti-HBs antibody ≥10 mIU/mL, with a geometric mean concentration (GMC) of 57.4 mIU/mL. HBsAg was detected in six participants, resulting in a HBsAg carrier rate of 0.4% (6/1352). Of those participants with anti-HBs antibodies < 10 mIU/mL, after a challenge dose, 231 (93.1%) presented an anti-HBs antibody ≥10 mIU/mL, with a GMC of 368.7 mIU/mL. A significant increase in the anti-HBs positive rate (≥ 10 mIU/mL) after challenge was observed in participants with anti-HBs antibodies between 2.5 and 10 mIU/mL and participants boosted with HepB (CHO), rather than those with anti-HBs antibodies < 2.5 mIU/mL and those boosted with HepB (SC)., Conclusion: Since satisfactory immune protection against HBV infection conferred by primary vaccination administered 17-20 years ago was demonstrated, there is currently no urgent need for booster immunization.

In this study, a real-time reverse transcription-polymerase chain reaction (real time RT-PCR) assay targeting 2 genetic segments was established to detect HDV RNA. Utilizing the World Health Organization International Standard for Hepatitis D Virus RNA, the lower limit of detection was 575 IU/mL, and the linearity of quantification ranged from 575,000 IU/mL to 575 IU/mL. 384 HBsAg-positive samples collected from China were tested by this method and HDV antibody detection. Eleven samples were positive for anti-HDV IgG which may persist after HDV resolution, 6 samples were HDV RNA positive, and 5 samples were positive for anti-HDV IgM. This assay showed more sensitivity than the detection of anti-HDV IgM. These data demonstrate that the real-time RT-PCR assay for HDV RNA could be implemented in the clinical detection of HDV infection in chronic HBV-infected patients in China., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Ebola virus (EBOV) disease (EVD) leads to lethal hemorrhagic fever with a case fatality rate as high as 90%, thus posing a serious global public health concern. However, while several vaccines based on the EBOV glycoprotein have been confirmed to be effective in animal experiments, no licensed vaccines or effective treatments have been approved since the first outbreak was reported in 1976. In this study, we prepared the extracellular domain of the EBOV GP protein (designated as N20) by prokaryotic expression and purification via chromatography. Using CTA1-DD (designated as H45) as a mucosal adjuvant, we evaluated the immunogenicity of N20 by intranasal administration and the associated protective efficacy against mouse-adapted EBOV challenge in mice. We found that intranasal vaccination with H45-adjuvanted N20 could stimulate humoral immunity, as supported by GP-specific IgG titers; Th1 cellular immunity, based on IgG subclasses and IFN-γ/IL-4 secreting cells; and mucosal immunity, based on the presence of anti-EBOV IgA in vaginal lavages. We also confirmed that the vaccine could completely protect mice against a lethal mouse-adapted EBOV (MA-EBOV) challenge with few side effects (based on weight loss). In comparison, mice that received N20 or H45 alone succumbed to lethal MA-EBOV challenge. Therefore, mucosal vaccination with H45-adjuvanted N20 represents a potential vaccine candidate for the prevention of EBOV in an effective, safe, and convenient manner., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: To eliminate the side effects of aluminum adjuvant and His-tag, we constructed chimeric VLPs displaying the epitope of EV71 (SP70) without His-tagged. Then evaluating whether the VLPs could efficiently evoke not only humoral but also cellular immune responses against EV71 without adjuvant., Methods: The fusion protein was constructed by inserting SP70 into the MIR of truncated HBcAg sequence, expressed in E. Coli, and purified through ion exchange chromatography and density gradient centrifugation. Mice were immunized with the VLPs and sera were collected afterwards. The specific antibody titers, IgG subtypes and neutralizing efficacy were detected by ELISA, neutralization assay, and EV71 lethal challenge. IFN-γ and IL-4 secreted by splenocytes were tested by ELISPOT assay., Results: HBc-SP70 proteins can self-assemble into empty VLPs. After immunization with HBc-SP70 VLPs, the detectable anti-EV71 antibodies were effective in neutralizing EV71 and protected newborn mice from EV71 lethal challenge. There was no significant difference for the immune efficacy whether the aluminum adjuvant was added or not. The specific IgG subtypes were mainly IgG1 and IgG2b and splenocytes from the mice immunized produced high levels of IFN-γ and IL-4., Conclusion: The fusion proteins without His-tagged was expressed and purified as soluble chimeric HBc-SP70 VLPs without renaturation. In the absence of adjuvant, they were efficient to elicit high levels of Th1/Th2 mixed immune response as well as assisted by aluminum adjuvant. Furthermore, the chimeric VLPs have potential to prevent HBV and EV71 infection simultaneously., (Copyright © 2018 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

Objective: To evaluate the effect of hepatitis B prevention and control by comparative analysis on the results of HBsAg, anti-HBs and anti-HBc prevalence from national hepatitis B seroepidemiological surveys in 1992 and 2014 in different epidemic regions of China. Methods: Data was from the national seroepidemiological surveys of hepatitis B conducted in 1992 and 2014. The survey in 1992 was conducted in 145 disease surveillance points of 30 provinces (excluding Hong Kong, Macao Special Administrative Region and Taiwan province) in China. The survey in 2016 was conducted in 160 disease surveillance points of 31 provinces (excluding Hong Kong, Macao Special Administrative Region and Taiwan province) in China. In the two surveys, face-to-face interviews with the subject by door to door or on the investigation site were conducted by trained staff using standard questionnaires to obtain basic information including birth date, gender, ethnicity, resident place and so on. And then 5 ml venous blood was collected to test the sero-markers of HBsAg, anti-HBs and anti-HBc. We analyzed unweighted point prevalence and 95 % CI of HBsAg, anti-HBs and anti-HBc in 1992 which had no design weighting, and analyzed weighted point prevalence and 95 %CI of HBsAg, anti-HBs and anti-HBc in 2014 which had design weighting. Results: 34 291 and 31 713 people aged 1-29 years were involved in 1992 and 2014 national serosurveys of China, respectively. For the people aged 1-29 years, HBsAg prevalence was 2.64% (95 %CI: 2.28%-3.06%) in 2014 and decreased by 73.92% as compared with the rate 10.13% (95 % CI: 9.81%-10.45%) in 1992. Anti-HBc prevalence was 13.01% (95 %CI: 12.09%-14.00%) in 2014 and decreased by 71.61% as compared with the rate 45.84% (95 % CI: 45.31%-46.37%) in 1992. Anti-HBs prevalence was 57.79% (95 %CI: 56.33%-59.25%) in 2014 and ascended by 127.41% as compared with the rate 25.41% (95 % CI: 24.95%-25.87%) in 1992. In high, medium and low epidemic region, for the people who born during 1992-2001 when hepatitis B vaccine was introduced in routine immunization management, HBsAg prevalence was 4.74% (95 %CI: 3.79%-5.69%), 1.59% (95 %CI: 1.09%-2.10%) and 2.53% (95 %CI: 1.66%-3.39%), respectively, and anti-HBs prevalence was 64.25% (95 % CI: 62.11%-66.39%), 56.34% (95 % CI: 54.50%-58.57%), 54.49% (95 %CI: 51.75%-57.23%), respectively, and anti-HBc prevalence was 15.16% (95 %CI: 13.56%-16.76%), 11.07% (95 %CI: 9.80%-12.33%), 7.61% (95 %CI: 6.15%-9.07%), respectively. In high, medium and low epidemic region, for the people who born during 2002-2013 the duration which hepatitis B vaccine was integrated in expanded immunization program born during when HBsAg prevalence was 0.88% (95 %CI: 0.66%-1.11%), 0.37% (95 %CI: 0.24%-0.49%)and 0.71% (95 %CI: 0.48%-0.94%), respectively, and anti-HBs prevalence was 60.74% (95 %CI: 59.57%-61.90%), 59.46% (95 %CI: 58.44%-60.49%), 52.56% (95 % CI: 51.20%-53.92%), respectively, and anti-HBc prevalence was 3.30% (95 % CI: 2.87%-3.72%), 1.91% (95 %CI: 1.63%-2.20%), 2.25% (95 %CI : 1.85%-2.66%), respectively. Conclusion: China had made great achievement in hepatitis B prevention and control. HBsAg prevalence among people aged 1-29 years old in 2014 decreased dramatically as compared with that in 1992. Since hepatitis B vaccine was integrated into expanded immunization program, China reduced HBsAg prevalence to less than 1% among people aged 1-12 years in 2014 in different epidemic region.

Longan County is considered a highly endemic area for hepatitis B virus (HBV). The plasma-derived vaccine has been used in newborns in this area since 1987. A cross-sectional survey was conducted to evaluate the long-term effectiveness of this vaccine. In total, 1634 participants born during 1987-1993 and who had received a series of plasma-derived HB vaccinations at ages 0, 1, and 6 months were enrolled. Serological HBV markers were detected and compared with previous survey data. Overall the prevalence of hepatitis B surface antigen (HBsAg) in all participants was 3·79%; 3·47% of subjects who had received the first dose within 24 h were HBsAg positive, and 8·41% of subjects who had received a delayed first dose were also HBsAg positive. There were 1527 subjects identified who had received the first dose within 24 h and whose HBsAg and anti-HBc prevalence increased yearly after immunization, while the anti-HBs-positive rate and vaccine effectiveness declined. The geometric mean concentration of antibody in the anti-HB-positive participants was 55·13 mIU/ml and this declined after immunization. Fewer than 2·0% of participants had anti-HB levels ⩾1000 mIU/ml. The data show that the protective efficacy of the plasma-derived vaccinations declined and administration of HB vaccine within 24 h of birth was very important. To reduce the risk of HBV infection in this highly endemic area, a booster dose might be necessary if anti-HBs levels fall below 10 mIU/ml after age 18 years. Furthermore, studies on the immune memory induced by plasma-derived HB vaccine are needed.

Objective: To determine the hepatitis B immunoprophylactic failure rate in infants born to hepatitis B virus (HBV) infected mothers and to characterize HBV genes., Methods: HBV-serological testing was conducted for pregnant women and infants. The complete genomes of 30 HBV isolates were sequenced, and genetic characteristics were analyzed using MEGA 5 software., Results: The immunoprophylactic failure rate for infants who had completed the scheduled hepatitis B vaccination program was 5.76% (32/556). High sequence homology (99.8%-100%) was observed in 8 of the 10 mother-infant pairs. We identified 19 subgenotype C2 strains, 9 subgenotype B2 strains, and 2 subgenotype C1 strains. Three serotypes were detected: adr (19/30), adw (9/30), and ayw (2/30). The frequency of amino acid mutation of the 'a' determinant region was 16.67% (5/30), including that of Q129H, F134Y, S136Y, and G145E. We detected 67 amino acid mutations in the basal core promoter, precore, and core regions of the genome., Conclusion: The immunoprophylactic failure rate in infants born to HBV-infected mothers is low in the regions of China examined during this study. Moreover, HBV mutation in the 'a' determinant region could not account for immunoprophylactic failure for all infants., (Copyright © 2016 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

Objective: Hepatitis Delt a Virus (HDV) antigen is widely used as a capture antigen in ELISAs for the identification of HDV infection; large amounts of recombinant HDV antigen with active antigenicity are required for this purpose., Methods: Reconstruct the gene of HDV antigen based on the bias code of Escherichia coli, the recombinant protein expresses by high-density fermentation with fed-batch feeding strategy, and purify by immobilized metal chromatography. The sensitivity and specificity of this antigen detect by ELISA method., Results: The expression of HDV antigen can reach 20% of the total cell mass in the soluble form. The recombinant HDV antigen can be conveniently purified (98%) by immobilized metal ion affinity chromatography (IMAC) using the interaction between a His-tag and nickel ions. Production of recombinant HDV antigen can reach 0.5 g/L under conditions of high-density cell fermentation. Applied to the diagnostic ELISA method, the recombinant HDV antigen shows excellent sensitivity (97% for IgM and 100% for IgG) and specificity (100% for IgG and IgM) for the detection of anti-HDV antibodies., Conclusion: Expression and purification the recombinant HDV antigen as a candidate protein for application in a diagnostic ELISA for HDV infection. Large-scale production of the protein can be achieved using the high-density fermentation strategy., (Copyright © 2016 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

Objectives: Hepatitis A virus (HAV), transmitted mainly through the fecal-oral route, is one of the major causes of acute viral hepatitis worldwide. HAV is endemic in China. This study performed genetic and evolutionary analysis of HAV isolates circulated in the country., Methods: Clinical samples were collected and HAV nucleotide and deduced amino acid sequences were analyzed. 70 representative sequences of HAV VP3-VP1-2A regions sampled from 1988 to 2014 were compared and characterized using the Bayesian Markov Chain Monte Carlo approach (BEAST software, Version1.7.5)., Results: All isolates from China in this study belonged to genotype I, with most of the samples clustering in subgenotype IA, while several unique amino acid variants were observed. The estimated mean substitution rate was 5.56×10(-4) substitutions / site / year, the time to the most recent common ancestor of genotype I isolates in China was calculated to be around 180 years ago. Skyline plots showed the incidence of HAV went down gradually from the mid-1990s., Conclusions: The evolution estimations were consistent with the laboratory and epidemiological results. Several isolates from China showed amino acid changes close to the immunodominant sites, which needs to be further analyzed. The study results have indicated the effectiveness of improving economic and sanitation levels together with HAV vaccination to control HAV-related infections in China., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The highly-pathogenic EV71 strain is the primary cause of mortality in hand-foot-and-mouth disease (HFMD) associated with neurological symptoms, for which no clinically effective drugs or vaccines exist. This study aimed to construct infectious cDNA clones of the EV71 highly-pathogenic strain and the cell-culture adapted strain using a reverse genetics approach. The genomic RNAs of EV71 parent strains were used as the templates for RT-PCR amplification, and then the PCR products that overlapped the full-length genome were connected into the pBR322 vector to produce infectious clones of pEV71 (HP) and pEV71 (CCA), respectively. The results showed that the HP strain propagated much more quickly than the CCA strain. The rescued viruses derived from the infectious clones not only maintained their consistency with their parent strains in terms of genomic sequences, but also retained their respective biological phenotypes. This research will contribute to our understanding of EV71 pathogenesis and the development of novel vaccines against HFMD.

Objective: To construct full-length hepatitis B core particles presenting preS1 aa 21-47 epitope and truncated core particles presenting preS1 aa 37-45 epitope on their surface and compare their antigenicity., Methods: PreS1 aa21-47 epitope and aa 37-45 epitope were inserted respectively into full-length hepatitis B core (aa 1-183) and truncated HBcAg (aa 1-144), between the 78th (Asp) and 79th (Pro). The genes synthesized after the codon optimization were ligated to the pET43. 1a vector with the same cohesive terminal (NdeI and XhoI) and expressed in the E. coli expression system. The morphology of the proteins of interest were observed by electron microscope and characterized by ELISA and Western Blotting., Results: The morphology of the virus-like particles were confirmed by electron microscope. H2 were solid particles with a diameter of (31.61 +/- 1.27) nm, while H3 were hollow particles with a diameter of (28.46 +/- 1.16) nm. Statistical analysis showed that H2 is larger than H3 in the diameter (P < 0.01). The antigenicity of the inserted epitopes and carrier protein were identified by ELISA and Western Blotting., Conclusion: Chimeric hepatitis B core particles presenting the preS1 neutralizing epitopes on their surface have been expressed, purified and identified, which lays the foundation for its application in vaccine research.

Objective: To overexpress hepatitis B virus S gene in CHO cells cultured in serum-free media., Method: Plasmid was constructed by cloning of HBV S gene and then it was transfected into CHO cells. After cell screen, the positive clones were identified and isolated into a serum-free media followed by the serological and morphological characterization of the expression product., Result: CHO cell strains which can express HBsAg efficiently and stably were obtained. Spherical and filamentous HBsAg could be detected under electronic microscope. The titer of the expression product was up to 1:5000., Conclusion: Serum-free media cultured CHO cell strain for overexpression of HBsAg was successfully constructed and the expression product was high antigenic.

Objective: To express the recombinant D protein in prokaryotic expression system solubly and make preparation for producing D-carrier conjugate vaccine next step., Methods: The hpd gene fragment removed of signal peptide from genomic DNA of Hib CMCC was inserted into pET43. 1a. The recombinant plasmid was transformed to competent E. coli BL21 (DE3) for expression under induction of IPTG. The expressed recombination protein was precipitated with ammonium sulfate, purified by DEAE anion exchange column chromatography and identified for reactogenicity by Western Blot., Results: The expressed recombination protein, in a soluble form, constained about 50% of total somatic protein and showed specific reaction with the HIB antisera after preliminary purification., Conclusion: The D protein recombined expression plasmid was constructed successfully and expressed D protein in prokaryotic cells in a solube form.

Objective: To investigate the risk of HBV infection among the spouses of hepatitis B virus surface antigen (HBsAg) carriers and to provide a reference for developing strategies on hepatitis B control and prevention., Methods: A case-control study including HBsAg carriers aged 20 - 45 years-old from the nationwide sero-epidemiological survey for Hepatitis B in both Guangdong and Jiangxi provinces in 2006, together with their spouses were selected as case group, while. HBsAg negative persons and their spouses were among the control groups, under the same residential areas, gender, age and age of marriage to the HBsAg carriers. Questionnaire survey and hepatitis B serological markers detection were carried out, together with the HBV genotype detection among the HBsAg positive couples between husband and wife by PCR., Results: Among the spouses of HBsAg carriers, the positive rate of HBsAg was 13.21%, while the rate was 6.29% for the spouse of HBsAg negative population, with difference statistically significant (χ² = 4.23, P < 0.05). HBsAg positive rate among spouses of the case group was higher than that in the control group. Among the spouses of HBsAg carriers, the HBsAg rate was positively correlated with the age of marriage, frequency of sexual intercourse and condom use. There were 21 pairs of HBsAg carriers between husband and wife, and HBV were isolated among 13 pairs, and there were 11 pairs carrying the same HBV genotype, accounting for 84.62%. HBV genotypes would include 8 pairs of type B and 3 pairs of type C. However, only 2 pairs were infected with different HBV genotype., Conclusion: High risks of HBV infection existed in the spouses of HBsAg carriers. It was important to ask the HBsAg carriers to take the initiative in informing their spouses, and carrying out the appropriate measures, such as safe sex or timely hepatitis B vaccination for the spouse of HBsAg carriers etc., so as to reduce the HBV transmission between husband and wife.

The receptor-binding domain(RBD) protein of HCoV-NL63 is a major target in the development of diagnostic assay and vaccine, it has a pivotal role in receptor attachment, viral entry and membrane fusion. In this study, we prepared 2 purified recombinant HCoV-NL63 RBD proteins using in E. coli system and identified the proteins by Western blotting. We first optimized codon and synthesized the RL (232-684aa)coding gene, then amplified the RL or RS(476-616aa) coding gene via PCR using different primers . The RL or RS coding gene was cloned into the pM48 expression vector fused with TrxA tag. The RBD (RL and RS) of HCoV-NL63 were expressed majorly as inclusion body when expressed in E. coli BL21pLys S under different conditions. The expressed products were purified by affinity chromatography then analyzed by SDS-PAGE and Western blotting. Our results showed that the recombinant RBD proteins were maximally expressed at 37 degrees C with 0. 8mM IPTG induction for 4h. RL or RS protein with 95 % purity was obtained and reacted positively with anti-sera from mice immunized with the recombinant vaccinia virus (Tiantan strain) in which HCoV-NL63 RL or RS protein was expressed. In conclusion, the purified recombinant RBD proteins(RL and RS)derived from E. coli were first prepared in China and they might provide a basis for further exploring biological role and vaccine development of HCoV-NL63.

Objective: To establish a novel improved loop-mediated isothermal amplification (LAMP) technique to detect hepatitis A virus (HAV)., Methods: A novel improved LAMP assay based on the addition of an acceleration primer was developed for hepatitis A virus nucleotide acid detection., Results: Precision and reproducibility analysis proved its high stability and reliability. Comparison between the improved and conventional LAMP assays revealed that the former was more sensitive with a detection limit of 5 TCID50/ml. The novel detection method displayed 100% consistency with the TaqMan real-time PCR assay when applied to clinical specimens collected from patients with confirmed acute HAV infection., Conclusion: This novel technique is widely applicable as a simple diagnostic tool in the clinical field as well as for the surveillance and investigation of the infectious disease in developing countries where HAV is endemic.

Objective: To establish a specific TaqMan-based Real-time PCR assay for the detection of hepatitis E virus (HEV)., Methods: According to the references, primers-probe sets which were located in ORF2, the conservative part of HEV genome were designed and therefore we established a HEV TaqMan real-time RT-PCR assay with great performance of specificity, sensitivity and reproducibility. And then it was used in the detection of HEV RNA in clinical samples., Results: The HEV Real-time RT-PCR assay established in this study were able to detect HEV RNA with a detection limit of 10 copies/reaction. When the detection of a same sample was repeated for several times, coefficients of variation (CV) was all less than 1.53%. Our data also suggested that there were 1.87 x 10(6)-8.12 x 10(9) RNA copies in 1 ml of the clinical samples., Conclusion: The TaqMan-based Real-time PCR assay established in this study was specific and precise for the rapid detection of HEV RNA. It was applied successfully in the pathogen detection of clinical samples.

Objective: A 12 mer phage display peptide library was used to identify hepatitis A virus mimotopes of antigenic determinants, to provide the feasibility of virus epitope mapping by using this approach., Methods: Using purified anti-hepatitis A virus monoclonal antibody as affinity selective molecule, phage display peptide library was biopanned and positive clones were selected by ELISA, competition assay and DNA sequencing., Results: 10 ELISA positive clones were chosen for DNA sequencing, and the displayed peptide sequences were deduced. 9 of them showed identical nucleotide sequence, and similarity in their amino acid sequence with VP1 of HAV HM175 was found, but no sequence homology was found between the other phage clone and the capsid proteins of HAV. Those peptides may behave as mimotopes of HAV., Conclusion: The mimotope of HAV was selected by using phage display peptide library screening. The results provide the potential of this method to search for the mimotopes of the virus.

Objective: To investigate the seroprevalence of hepatitis D virus in Foshan of Guangdong province, to provide the data for the study about it in China., Methods: ELISA kits from two different companies were used for detecting anti-HDV IgG of all the serum samples, and then RT-PCR was carried out about the selected serum to ensure the results. All the serum samples were collected in 2011 in The First People's Hospital of Foshan., Results: The results from two ELISA kits and RT-PCR were identical. Eight samples were positive., Conclusions: The seroprevalence rate of HDV in Foshan is higher than that in China. It has no statistically significant difference between female and male. Morever, the older with HBsAg are susceptible to HDV.

Objective: To study the effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering., Methods: Based on the colon preference of E. coli, the HDV small antigen original gene from GenBank was optimized. Both the original gene and the optimized gene expressed in prokaryotic cells, SDS-PAGE was made to analyze the protein expression yield and to decide which protein expression style was more proportion than the other. Furthermore, two antigens were purified by chromatography in order to compare the purity by SDS-PAGE and Image Lab software., Results: SDS-PAGE indicated that the molecular weight of target proteins from two groups were the same as we expected. Gene optimization resulted in the higher yield and it could make the product more soluble. After chromatography, the purity of target protein from optimized gene was up to 96.3%, obviously purer than that from original gene., Conclusion: Gene optimization could increase the protein expression yield and solubility of genetic engineering HDV small antigen. In addition, the product from the optimized gene group was easier to be purified for diagnosis usage.

A transient four-plasmid cotransfection system was used to construct avian influenza A (H5N1) pseudotyped viral particle (H5N1Pp) by incorporating hemagglutinin (HA) protein and neuraminidase (NA) protein from H5N1 avian influenza virus onto Murine leukemia virus pseudotyped viral particles, the transmission electron microscopy, infectivity titer assay, hemagglutination assay, neutralization assay of H5N1Pp were studied. We established a pseudotyped H5N1 viral particle at a high titer of 10(8) Pp/mL, the morphology,the hemagglutination activity and neutralization specificity of H5N1Pp is simililar to wild H5N1 virus. The research result sets a platform for studying this virus, including its receptors, the functional analysis of HA and NA, neutralizing antibodies and anti-H5N1 drug development.

To construct a recombinant expression plasmid Bacmid-P1-3CD containing the P1 and 3CD genes of enterovirus 71(EV71), the P1 and 3CD genes were cloned into the same baculovirus shuttle vector (Bacmid). Recombinant AcMNPV-P1-3CD was obtained by transfecting the Bacmid-P1-3CD into the insect cell line of S f9. With the IFA and Western-blot methods for identification of expression products confirmed that the target protein was expressed in interior of infected S f9 cells. Electron microscopy showed that the structural protein capsid P1 was cut by virus-encoded protease 3CD and assembled into EV71 virus like particles (VLPs) about 27nm diameter. Different values of MOI and time points of expression were compared to explore the optimal expression condition, and the results showed that the time point could be a more important factor. Then we used S f9 cells with serum-free medium in CellSTACK-10 Culture Chambers to produce EV71 VLPs in the confirmed condition. After purification of VLPs by density gradient centrifugation, we observed on SDS-PAGE profile the purified sample contained three major proteins whose molecular masses corresponded to those of VP1 (39kD), VP0 (34kD) and VP3 (26kD) as well as the intact structure, which can be greatly used for further study in protein structure and genetic engineering vaccine research.

Objective: To establish a specific TaqMan-based Real-time PCR assay for the detection of hepatitis A virus in serum samples., Methods: According to the references, primers-probe sets which were located in 5'-NCR, the most conservative part of HAV genome were designed and therefore we established a TaqMan real-time RT-PCR assay with great performance of specificity, sensitivity and reproducibility. And then it was used in the detection of HAV RNA in serum from HAV patients., Results: The HAV Real-time RT-PCR assay established in this study were able to detect HAV RNA and its detection limit ranged from 0.1CCID50/reaction to 0.01CCID50/reaction. When the detection of a same sample was repeated for three times, coefficients of varistion (CV) of intra- and inter-assay were calculated and they were all less than 2.0% and 2.6% respectively. Our data suggested that there were 5.18 x 10(2) - 4.93 x 10(7) RNA copies in 1 ml of the serum from acute HAV patients., Conclusion: The TaqMan-based Real-time PCR assay established in this study was specific and precise for the rapid detection of HAV RNA. It was applied successfully in the pathogen detection of clinical samples.

Objective: To prepare HDAg with biological activities as a candidate of diagnostic reagent., Methods: To synthesize HDV gene fragment after codon optimization. To construct a thio-fused recombinant plasmid based on M48 expression vector. To express in E. coli induced by IPTG. To purify the protein by affinity chromatography followed by characterization in ELISA:, Results: Plasmid construction was verified by enzyme digestion. SDS-PAGE indicated the molecular weight of the protein was the same as we expectation. ELISA proved its affinity with HDV antibodies., Conclusion: HDAg was obtained successfully and it will pave the road to the research of HDV diagnostic reagent.

Objective: To evaluate the long-term efficacy of Chinese hamster ovary (CHO) cell derived hepatitis B vaccine in country community in China., Methods: A cross-sectional investigation was carried out. Children who were born between 1997 and 1999 and vaccinated with the three doses of CHO-derived hepatitis B vaccine were selected as study objects. Their serum samples were taken to test for hepatitis B virus (HBV) markers, and the results were compared to that before vaccination. In addition, for HBsAg positive children, their mothers were visited., Results: 1254 Children were enrolled in the study. The prevalence of HBsAg was 0.24% and the vaccine efficacy was 97.0%, similar to that of yeast derived hepatitis vaccines. Among 3 mothers of HBsAg positive children, 2 were HBsAg positive, indicating maternal HBV transmissions., Conclusion: The long-term efficacy of the CHO-derived hepatitis B vaccine is good and after vaccination maternal transmission is the most important route of spreading HBV., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objective: To investigate the seroprevalence of HEV infection in different national human population in Han, Hui and Zang in China., Methods: EIA was used for detecting anti-HEV IgG of the serum samples. All samples were collected in 2006-2008 in Sichuan, Beijing, Heilongjianin, Sandong, Gansuo, Ningxia and Qinghai areas., Results: The total positive rate of anti-HEV IgG was 17.97% (1878/ 10448), 24.32% (1794/7376) in Han national, 3.59% (81/2258) of Hui national and 0.37% (3/814) of Zang national, respectively. The positive rate of Han human at different age group, was 5.19% of < or = 10 year, 11.64% of 11-20 year, 20.08% of 21-30 year, 34.17% of 31-40 year, 41.75% of 41-50 year, 48.58% of 51-60 year, 57.43% of > or = 61 year. The positive rate of Hui human at different age group, was 3.11%, 3.96%, 2.11%, 3.98, 2.52%, 4.57% and 6.67%, respectively. Three positive of Zang human was between 21-60 year., Conclusions: The HEV infection in Han national population was higher than the Hui and Zang national, significantly. The HEV infection was correlation with age significantly, the infection rate was increased with age.

Objective: To investigate the seroprevalence of hepatitis C viruse infection in the human population in six regions of Beijing, Heilongjiang, Shandong, Ningxia, Gansu and Sichuan in China., Methods: ELISA was used for detecting anti-HCV IgG of the serum samples. All sample were collected in 2006-2008 in six areas., Results: 9538 samples were detected. The total positive rate of anti-HCV was 0.39% (37), 0.23% (3/1328) in Beijing, 0.74% (12/1629) in Heilongjiang, 0.26% (5/1962) in Shandong, 0.1% (1/1000) in Ningxia, 0.44% in Gansu (9/2 037) and Sichuan (7/1582), respectively. The 37 positive samples at the sex were 19 (51.35%) of man and 18 (48.65%) of female. At the age group were 1 (2.70%) of < 10 years old, 5 (13.51%) of 10-19 years old, 4 (10.81%) of 20-29 years old, 6 (16.22% ) of 30-39 years old, 9 (24.32%) of 4049 years old, 12 (32.43%) of > or = 50 years old. The positive samples were detected anti-HAV-IgG, anti-HEV-IgG and HBsAg/ HBcAb /HBeAb. The positive Number was 35 (94.59%), 10 (27.03%) and 2 (5.41%) respectively., Conclusions: HCV infection rate in the population was 0.1% -0.74%, 1/3 of > or = 50 years old in HCV positive. Hepatitis C virus co-infection with HAV, HEV and HBV.

Objective: To find a suitable cell line for hepatitis A antigen expressed by vaccinia virus vector and to find a way of inactivation and preservation of the HAV recombinant antigen. Methods Series of cell lines such as K4,143, HEL, Hep-2 and Vero were inoculated with vaccinia virus that can express HAV recombinant antigen. ELISA was used to determine the contents of expression antigen. The characterization of the HAV antigen expressed by vaccinia virus was then analyzed after it was treated with different methods., Results: The expression of HAV recombinant antigen in K4,143 and HEL cell lines was a little more than expression in Hep-2 and Vero cell lines. The antigenicity is obviously higher when HAV recombinant antigen was inactivated by beta-propiolactone other than it was inactivated by formalin. It was best to preserve the prepared HAV recombinant antigen under -40 degrees C condition., Conclusions: The application of vaccinia virus vector in hepatitis A antigen preparation was very useful and promising.

Objective: To better understand and measure the status of hepatitis C virus (HCV) infection, we conducted a sero-epidemiological study using the remaining blood samples and data of the nationwide survey of hepatitis B in Chinese residents which was carried out in 2006., Methods: The anti-HCV reagent was screened out from the reagents by the HCV infection blood serum plate with anti-HCV positives or negatives. This plate recognized the Murex 3.0 and Ortho 3.0 reagents as gold standards. Anti-HCV in the blood samples were tested using this reagent and confirmed by Chiron HCV RIBA 3.0 reagents., Results: Among the population aged 1 year to 59 year-olds, the overall prevalence rate of anti-HCV was 0.43% (95%CI: 0.33% - 0.53%), with the rates of anti-HCV among males and females as 0.46% and 0.40%, respectively. The prevalence rate of anti-HCV in urban area was 0.43%, and in rural area it was 0.43%. The prevalence rate of anti-HCV in the Eastern, Middle and Western areas were 0.37% (95%CI: 0.21% - 0.53%), 0.67% (95%CI: 0.40% - 0.94%) and 0.31% (95%CI: 0.20% - 0.42%) respectively. The prevalence rates of anti-HCV for the three areas did not show significant differences, statistically. The prevalence rate of anti-HCV in the South and North areas were 0.29% (95%CI: 0.21% - 0.52%) and 0.53% (95%CI: 0.38% - 0.64%) respectively., Conclusion: Our data revealed that China was in the low prevalence area for hepatitis C infection and the results also suggested that the comprehensive measures for HCV control and prevention had been successfully achieved in the country.

A universal hepatitis B vaccination programme has been conducted in Long An county since 1986. To investigate the epidemiological changes in hepatitis B virus (HBV) infection we conducted a serosurvey there in 2005. A total of 4686 subjects were enrolled and vaccination history and blood samples collected. HBV infective markers were determined by radioimmunoassay. The results were compared with the data of 1985. Our results show that the overall HBsAg prevalence was 7·5%, less than half of the prevalence reported in 1985. HBsAg and anti-HBc antibody prevalence in people born after 1985 decreased markedly. The gender difference in HBsAg prevalence was abolished in subjects aged <20 years. The administration of a first dose of vaccine within 24 h could reduce the HBsAg prevalence by half. In conclusion, the marked epidemiological changes in HBV prevalence found in this serosurvey indicate that the implementation of HBV vaccination was highly successful.

Objective: To prepare streptavidin-tagged hepatitis C virus (HCV) fusion protein and explore its application for the detection of antibody against HCV infection., Methods: A recombinant plasmid pET-11d-C44P-SA was constructed, which coding a novel HCV diagnostic antigens (C44P) and streptavidin (SA) fusion protein, and the fusion protein was generated with BL21 (DE3) E Coli and identified by Western Blot analysis. Then the fusion protein was purified through the Ni-NTA affinity chromatography and over 90% purity has been achieved. Anti-HCV ELISAs were developed when the fusion protein was used in the biotin-pre-coated microplate or ordinary microplate, and then the sensitivity and specificity of the ELISA were evaluated with confirmed human sera panels., Results: The fusion protein was expressed in high yields and purified successfully, the ELISA detection of anti-HCV with human sera panel indicated that its sensitivity and specificity is higher when SA-tagged HCV antigen (C44P-SA) coated in biotin-pre-coated microplate, compared to C44P or C44P-SA coated in ordinary microplate., Conclusion: The sensitivity and specificity of anti-HCV ELISA can be improved when a novel HCV diagnostic antigen fused to SA combined with the biotin- pre-coated microplate. This study laid a foundation for improving the performance of HCV diagnostics.

Objective: To evaluate the effectiveness of a Chinese hamster ovary cell derived hepatitis B vaccine in a country community in Hebei Province, PR China., Method: A cross-sectional investigation was carried out in 4 of 7 randomly selected country communities in Zhengding County in 2005. All of the children who were born between 1997 and 1999 were selected as study objects. Their serum samples were taken to test for HBV markers, and HBsAg prevalence was compared to that of the same age group before hepatitis B vaccination in 1983. In addition, for HBsAg positive children, their mothers were visited and tested for serum HBV markers, in order to distinguish maternal HBV transmissions., Results: Among the 2205 children of the selected birth cohort, 1696 (76.9%) were visited. The prevalence of HBsAg was 0.53%, and by comparing to that of before hepatitis B vaccination, the effectiveness was 95.3%, similar to that of yeast derived hepatitis B vaccines. Among 7 mothers of HBsAg positive children, 5 were HBsAg positive, indicating maternal HBV transmissions; and although one mother was HBsAg negative, her kid was not vaccinated, which indicates a horizontal transmission. As for the other kid, he was adopted and the HBV infection status of his birth mother was unknown., Conclusion: The effectiveness of the CHO derived hepatitis B vaccine is comparable to yeast derived ones, and after the hepatitis B vaccination maternal transmission is the most important route of spreading HBV., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Molecular typing was conducted according to the reported method for one HBsAg positive carrier who had a physical examination in Yunnan Province. The S gene of this HBV sample was amplified by nested PCR and the PCR products were directly sequenced. Blast searching was done on the Genbank database and the sequence were compared with the HBV reference sequences in database. The phylogenetic tree was constructed. Homology analysis of nucleotide and smino acid were performed between the sequences from the sample and the reference sequences corresponding to HBV genotype A to I. Analysis of nucleotide and amino acid identities suggested that the sample belonged to HBV genotype I. The HBV genotype I is the first reported in China.