Objective To evaluate the adaptability of the “Sen Zhang” master seed strain of tick-borne encephalitis virus(TBEV) during serial passaging in primary hamster kidney cells(PHKCs), screen tick-borne encephalitis(TBE) vaccine candidate strains, and perform serological identification and morphological observation of the virus.Methods The mouse brain-adapted “Sen Zhang” master TBEV strain was serially passaged 10 times in PHKCs. Plaque purification was conducted based on viral titration, and clones with E protein amino acid sequences identical to the parental strain were selected to undergo an additional 10 passages in PHKCs. The virus titers were monitored across generations, and the E protein amino acid sequences from the 1 st and 10 th passages were compared. Experimental vaccines were prepared by 2% formaldehyde inactivation. After 35 female Kunming mice were immunized three times, the mouse brain-derived virus was injected intraperitoneally, and the immunoprotective index was calculated based on the number of mouse deaths within 21 days.Serological identification was performed on the vaccine candidate strains, and the viral morphology was analyzed via transmission electron microscopy(TEM).Results The PHKC-adapted TBEV achieved a maximum titer of 8. 90 lgLD50/mL,stabilizing at(8. 85 ± 0. 059 6) lgLD_(50)/mL. Plaque assays demonstrated uniform visible circular plaques with sharp boundaries. Among 27 purified clones, #7, #14, and #17 retained E protein sequences identical to the parental strain. The virus titers remained 8. 60-8. 90 lgLD_(50)/mL stably during passaging. Clone #7 exhibited minimal E protein mutations and the highest immunoprotective index 2. 3 × 10~6. The serological neutralization index reached 3 162, and TEM revealed spherical enveloped particles with a diameter of about 50 nm, consistent with the vaccine strain.Conclusion A PHKCadapted TBEV strain with high genetic stability was successfully isolated, which meets the critical criteria of vaccine candidate strains and can elicit robust immune responses.

Objective To construct a 293T-CymR cell strain to express cysteine metabolism regulator(CymR) component by gene editing technology, and to regulate the target gene expression with CuO regulatory sequence promoter.Methods Based on clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9 technology, the intracellular β factor receptor, C-C chemokine receptor(CCR5) gene, one of the safe harbors in HEK293T cell genome, was chosen as the target site for inserting the desired gene. The selection gene Pac(denoted as PuroR) encoding puromycin N-acetyltransferase was linked to the 3'end of the CymR gene via an internal ribosome entry site(IRES), sharing a cytomegalovirus(CMV) promoter. The 293 TCymR monoclonal cell strain capable of expressing CymR was screened by Puro resistance and limiting dilution method. The CymR gene position and protein function in the 293T-CymR monoclonal cell strain were verified by PCR, Western blot and fluorescence analysis.Results The pY93-CymR plasmid expressing both the target and resistance genes, was successfully constructed. Following co-transfection with the pX330-CCR5 plasmid into HEK293T cells, four 293T-CymR monoclonal cell strains were obtained via Puro resistance selection and the limiting dilution method. The PCR and Western blot analysis indicated that CymR gene was successfully inserted into the intended CCR5 site and CymR protein was efficiently produced in three of these clones. Subsequent transfection tests demonstrated that the produced CymR could successfully suppress the gene expression under the CMV promoter containing CuO element.Conclusion CymRgene was successfully inserted into the CCR5 locus of HEK293T cell genome, and 293T-CymR monoclonal cell strains capable of expressing CymR and regulating gene expression were obtained.

Objective To participate in the collaborative calibration study of the 2 nd international standard(IS) candidate(code: 23/148) for serum amyloid A(SAA).Methods According to the research plan of the Medicines and Healthcare products Regulatory Agency(MHRA), National Institutes for Food and Drug Control of China, on behalf of Chinese laboratories,organized 12 laboratories(including kit enterprises or testing institutions) for calibration using six chemilumine-scence immu noassay and six latex immunoturbidimetric assay detection kits.Results The SAA geometric mean of the immune potency submitted by Chinese laboratory was 56. 3 μg/ampoule[95% confidence interval(CI): 52. 2-60. 6 μg/ampoule, n = 12,geometric coefficient of variation(GCV): 12. 6%], with a median value of 53. 8 μg/ampoule(95% CI: 51. 9-61. 1 μg/ampoule).A total of 17 laboratories from six countries around the world participated in this study. After analysis, the geometric mean of the immune potency of SAA was 60. 9 μg/ampoule(95% CI: 54. 6-67. 9 μg/ampoule, n = 17, GCV: 23. 5%), with a median value of 55. 8 μg/ampoule(95% CI: 52. 0-60. 0 μg/ampoule).Conclusion After reviewed and approved by the World Health Organization(WHO) Expert Committee on Biological Standards, it is proposed that the candidate preparation coded23/148 is established as the 2 nd IS for SAA, with the median(56 μg/ampoule) as the final value. However, the above study data collectively demonstrates that commercial SAA immunoassays are poorly harmonized at the current time. Manufacturers may be adversely impacted as they transition to use 23/148. Recalibration needs to be performed when necessary, to improve the consistency of test results.

Objective To screen for potential targets of the anti-tuberculosis lead compound M6 using antibody microarray,express Mycobacterium smegmatis(Ms) target protein 2-C-methy1-D-erythriol 4-phosphate cytidylyl-transferase(IspD) in E.coli, and prepare its polyclonal antibodies, in order to provide experimental basis for in-depth study of the protein function and the development of new anti-tuberculosis drugs.Methods The minimum inhibitory concentration(MIC) of M6 against Ms and Corynebacterium glutamicum(Cg) was determined. The protein samples of Cg treated with M6 were captured and analyzed by antibody microarray, and the differential targets were identified and screened by mass spectrometry. The ispD gene of Ms was amplified by PCR and cloned into vector pET28a(+) to construct recombinant plasmid pET28a-ispD. The recombinant plasmid was transformed into E.coli BL21(DE3), and the optimal conditions for protein expression were determined by optimizing the induction temperature(16, 25, 33, 37 ℃) and IPTG concentration(0-1. 5 mmol/L). The recombinant IspD protein was purified by nickel column affinity chromatography. The purified recombinant IspD protein was used as immunogen combined with Freund's incomplete adjuvant to immunize 11 female BALB/c mice to prepare polyclonal antibodies. The titer of antiserum was determined by indirect ELISA, and the antibody specificity was detected by Western blot.Using the prepared polyclonal antibodies, the effect of M6 treatment on the expression level of Ms IspD protein was analyzed by Western blot.Results The MIC of M6 against Cg and Ms was 0. 5 and 32 μg/mL, respectively. IspD protein was one of the primary target proteins of M6. The recombinant expression plasmid pET28a-ispD was constructed correctly as identified by double enzyme digestion, and the IspD protein was expressed in E.coli. The optimal induction conditions were determined to be 1. 0 mmol/L IPTG, 16 ℃ for 14 h. After purification, the purity of recombinant IspD protein reached, and it could specifically bind to mouse anti-His tag monoclonal antibodies. The polyclonal antibodies against IspD protein achieved a high titer of 1∶102 400, which could specifically recognize IspD protein in recombinant bacterial lysate. The expression level of Ms IspD protein did not change significantly after M6 treatment.Conclusion In this study, the potential target IspD protein of M6 was successfully screened by antibody microarray screening, and Ms IspD protein was expressed in E.coli. The polyclonal antibodies against IspD were prepared by immunizing mice, providing essential experimental basic for further functional studies of this protein and the development of novel anti-tuberculosis drugs.

Objective To evaluate the safety and immunogenicity of a booster dose of bivalent types Ⅰ and Ⅲ oral poliovirus vaccine(bOPV) of sugar pill and liquid dosage forms in children who were sequentially vaccinated with oral poliovirus vaccine(OPV) and Sabin strain-based inactivated poliovirus vaccine(sIPV) at the age of 48 months.Methods From January2018 to May 2020, a total of 485 healthy children aged 48 months who completed basic immunization and had paired serum antibody detection results before and after immunization were selected in the phase 3 clinical study of sequential immunization combined with OPV and sIPV in Liujiang District, Liucheng County and Rongan County of Liuzhou City to conduct enhanced immunization study. All the subjects were inoculated with 1 dose of bOPV for enhanced immunization, the occurrence of adverse events within 28 days after enhanced immunization was observed, and serum samples were collected before and 28 days after enhanced immunization to detect neutralizing antibodies against poliovirus typesⅠ,Ⅱ and Ⅲ. The incidence of adverse events, poliovirus neutralizing antibody positive rate, seroconversion rate, and geometric mean titer(GMT) were analyzed.Results A total of 485 subjects were inoculated with bOPV for enhanced immunization, among which, 485 cases were included in safety evaluation, and 342 cases in immunogenicity evaluation. The overall incidence of adverse events at 28 days after enhanced immunization was 31. 75%(154/485), and the incidence of vaccination-related adverse events was 24. 74%(120/485). Among 120 cases of vaccination-related adverse reactions, the severity was mainly grade 1 and grade 2, and no adverse reactions of grade 4 occurred. Before booster immunization, the positive rate of poliovirus type Ⅰ neutralizing antibody was 97. 56% in 2 conventional inactivated poliovirus vaccine(cIPV) + trivalent oral poliovirus vaccine(tOPV)(liquid dosage) group, and 100. 00% in other groups. The positive rate of type Ⅱ poliovirus neutralizing antibody was 75. 00% in sIPV + 2 bOPV(liquid dosage) group, and was more than 90. 00% in other groups. The positive rate of type Ⅲ poliovirus neutralizing antibody was 100% in all groups. At 28 days after booster immunization, the positive rate of typesⅠ, Ⅱ and Ⅲ poliovirus neutralizing antibody was 100. 00% in all groups. The seroconversion rate of typeⅠpoliovirus neutralizing antibody was more than 50. 00% and the seroconversion rate of type Ⅲ poliovirus neutralizing antibody was more than 40. 00%, while the seroconversion rate of type Ⅱ poliovirus neutralizing antibody was relatively low.The GMT of typeⅠpoliovirus neutralizing antibody increased by about 5-20 times, the GMT of typeⅡpoliovirus neutralizing antibody increased by about 1. 5-5 times, and the GMT of type Ⅲ poliovirus neutralizing antibody increased by about5-15 times, compared with that before immunization. The difference of GMT of neutralizing antibodies against poliovirus typesⅠ, Ⅱ and Ⅲ before and after booster immunization was statistically significant(t =-20. 769,-6. 128 and-19. 609,respectively, each P < 0. 01).Conclusion The infants who completed the sequential immunization of OPV and IPV at 2 months of age showed good safety and immunogenicity in enhanced immunization with bOPV at 48 months.

Objective To analyze the rotavirus(RV) vaccine coverage among children in the birth cohorts in Shijingshan District of Beijing from 2015 to 2023, so as to provide a scientific support for RV vaccination.Methods The data were collected through the Beijing Immunization Planning Information Management System, and descriptive epidemiological methods were used for statistical analysis.Results A total of 36 248 doses of RV vaccines were inoculated among children born in Shijingshan District of Beijing from 2015 to 2023, and the overall coverage levels for the first, second, and third doses were 43. 03%, 28. 55% and 18. 72%, respectively. The vaccination rate had shown an overall upward trend over the years(χ~2_(trend)= 4 554. 383, 8 016. 090 and 9 143. 411, respectively, each P < 0. 001). The coverage levels for the first, second, and third doses of Lanzhou lamprotavirus vaccine(LLR) were 22. 91%, 9. 04% and 1. 28% among the 2015-2023 birth cohorts respectively, and the vaccination rate had shown an upward trend followed by a downward trend with the years. The coverage levels for the first, second, and third doses of pentavalent human-bovine reassortant rotavirus vaccine(RV5) among the 2018-2023 birth cohorts were 33. 30%, 32. 30% and 28. 87%, respectively, and the vaccination rate had shown an overall upward trend over the years(χ~2_(trend)= 3 745. 642, 3 402. 195 and 3 237. 052, respectively, each P < 0. 001). The vaccination rates of the first, second, and third doses of LLR and RV5 in different regions showed statistically significant differences(LLR: χ~2=1 244. 175, 1 100. 036 and 44. 453, respectively, each P < 0. 001; RV5: χ~2= 649. 139, 567. 390 and 464. 887, respectively,each P < 0. 001), with a gradual increasing trend from west to east area.Conclusion The vaccination rate of RV vaccine among children born in Shijingshan District of Beijing from 2015 to 2023 is relatively low, and it is necessary to further strengthen publicity and enhance parental awareness, so as to increase the RV vaccination rate for the effective prevention of childhood RV diarrhea.

Objective To understand the current risk factors of hepatitis B virus(HBV) transmission in Jilin City, in order to provide a scientific basis for formulating targeted prevention and control strategies.Methods A total of 111 patients with hepatitis B were randomly selected as the case group from the China Information System for Disease Control and Prevention during the period from January 1, 2021 to December 31, 2022 in Jilin City, at the same time, one family member of every patient who lived in the same household and was not infected with HBV was selected as the control group. A retrospective questionnaire survey was conducted to collect data on demographic characteristics, clinical information, hepatitis B vaccination history, knowledge of HBV transmission, and potential infection risk behaviors inside and outside the family. The related factors of HBV infection were analyzed by using χ2 test, univariate and multivariate Logistic regression models.Results The case and control groups were comparable in demographic characteristics. In the case group, 55. 86% were HBsAg positive,21. 88% had abnormal liver function, and 14. 41% had clinical symptoms. The definite vaccination rate of hepatitis B vaccine in the case group(5. 41%) was lower than that in the control group(11. 71%). The overall accurate awareness rate of the two groups for HBV transmission routes was only 45. 50%, and 82. 89% of the patients did not know the source of their own infection or had misconceptions. Univariate analysis showed that the risk factors for transmission within the family included sharing toothbrushes(OR = 6. 363, P = 0. 011) and tooth cups(OR = 4. 253, P = 0. 004), and the protective factor was complete vaccination(OR = 0. 028, P = 0. 001). Risk factors for out-of-family transmission included a history of oral cavity(OR = 6. 075, P = 0. 014), a history of other diseases(OR = 3. 178, P = 0. 008), and a history of acupuncture(OR =1. 693, P = 0. 016) and beauty treatment(OR = 1. 657, P = 0. 033). Multivariate Logistic regression analysis determined that the dental history(OR = 5. 075, 95% CI: 1. 665-10. 587) and other medical history(OR = 2. 178, 95% CI: 2. 050-5. 633)were risk factors for HBV infection, while having a history of vaccination was a protective factor(OR = 0. 609, 95% CI: 0. 388-1. 081).Conclusion The vaccination rate of hepatitis B vaccine and the awareness rate of prevention and control knowledge of adult hepatitis B patients and their families in Jilin City had been both low. Sharing personal hygiene products(toothbrushes and tooth cups) in the family and invasive medical services and beauty behaviors outside the family(such as dental treatment and acupuncture) are important risk factors for local HBV transmission. Hepatitis B vaccination is an effective protective measure. According to these risk factors, health education and behavioral intervention for key populations should be strengthened, and the vaccination coverage rate of hepatitis B vaccine should be improved.

Objective To analyze the current status of human papillomavirus(HPV) vaccination among women aged 9-45 years in Chaoyang District of Beijing, and to provide a basis for further promoting population vaccination.Methods The HPV vaccination(including HPV2, HPV4 and HPV9) data among women aged 9-45 in Chaoyang District of Beijing as of December 31, 2024 in Beijing's immunization planning information system were derived, and the estimated HPV vaccination rate in Beijing's female population was obtained by data analysis.Results By December 31, 2024, the estimated first dose vaccination rate of HPV vaccine among women aged 9-45 years in Chaoyang District of Beijing had been 50. 88%, and the estimated full dose vaccination rate was 48. 66%. The estimated coverage of the first dose and the full dose among girls aged9-15 years was 4. 62% and 2. 87%, respectively. The estimated full dose coverage rate was 62. 73% in subdistrict and 37. 33%in district.Conclusion The estimated HPV vaccination rate of 9-45-year-old women in Chaoyang District of Beijing is higher than that in other regions of China, but the estimated vaccination rate of adolescent women aged 9-15 is significantly lower than that of other age groups. It is recommended to improve the HPV vaccination rate through multiple joint efforts to achieve the action goal of accelerating the elimination of cervical cancer in China and the world as soon as possible.

Objective To establish a rapid method for extracting genomic DNA(gDNA) from various samples by using the adsorbent material NZVI-GO prepared by combining graphene oxide(GO) with epoxy functional groups and nanoscale zerovalent iron(NZVI), so as to meet the requirements of subsequent molecular experiments.Methods Magetic nanomaterial NZVI-GO was prepared via hydrothermal method and characterized. NZVI-GO was then used to extract gDNA from various biological samples(blood, bacteria, animal and plant tissues). During the gDNA extraction process, the quantities of magnetic nanomaterials(human whole blood and Yersinia pseudotuberculosis: 10, 15, 20 and 25 mg; animal and plant: 20, 35, 50, 65 and 80 mg), SDS buffer concentration(2%, 4%, 6%, 8% and 10%), TC buffer pH(2, 4, 6, 8 and 10) and polyethylene glycol6000 concentration(5%, 10%, 20%, 30% and 40%) were optimized. The gDNA of four biological samples was extracted by the established method, and compared with the nucleic avid extraction kits produced by two manufacturers.Results Fourier transform infrared(FTIR) spectroscopy analysis was performed on GO particles, NZVI particles, and NZVI-GO, revealing the presence of interactions between NZVI and GO. Subsequently, scanning electron microscopy(SEM) observations showed that GO acted as a dispersant, embedding the nano-iron particles uniformly. The spherical structure formed by GO and NZVI protected the hydroxyl and carboxyl functional groups on the NZVI surface from oxidation. The optimal extraction conditions were as follows: 20 mg of magnetic beads, 6% of SDS, pH 10 and 20% of polyethylene glycol 6000. Compared with the commercially available nucleic acid extraction kit 1, the gDNA concentration of human whole blood, bacteria, animal and plant samples extracted by NZVI-GO method increased, but the difference was not statistically significant(t = 1. 236-1. 935,each P > 0. 05). Compared with the commercial nucleic acid extraction kit 2, the gDNA concentration extracted by NZVI-GO was all significantly higher, with statistically significant difference(t = 4. 568-15. 200, each P < 0. 01).Conclusion The established gDNA extraction method based on NZVI-GO has the advantages of simple and rapid operation, high efficiency,and broad-spectrum applicability, which can meet the requirements of subsequent molecular experiments.

Objective To prepare specific antibodies against VP8 proteins of rotavirus(RV) genotypes P[8], P[6], and P[4], and to initially establish and verify an ELISA method for the detection of recombinant RV VP8 protein antigens.Methods Female BALB/c mice were immunized with recombinant P[8], P[6], and P[4]type VP8 proteins at a dose of60 μg per mouse with three mice for each type, and booster immunizations were administered via intramuscular injection in the leg at the same dose for 4 times at intervals of 2 to 4 days. Splenocytes from the immunized mice were fused with SP2/0 cells to screen for monoclonal antibodies(McAbs) against P[8], P[6], and P[4]type VP8 proteins. Polyclonal antibodies were obtained by immunizing rabbits. A double-antibody sandwich ELISA method for detecting recombinant RV VP8 protein antigens was initially established by optimizing the pairing combinations of capture and detection antibodies as well as their working concentration. The linearity and range of the standard curve were determined, and the precision, accuracy, and specificity of the method were verified. The established method was applied to detect intermediate samples(bacterial cell lysate, crude extract, purified solutionⅠ, and purified solutionⅡ) from the preparation and purification processes of three batches of P[8], P[6], and P[4]type VP8 proteins.Results One hybridoma cell line secreting anti-P[8]McAb was screened and named P[8]-19E7; two anti-P[6]McAb-secreting cell lines were obtained, designated as P[6]-4B8 and P[6]-8F11; and one anti-P[4]McAb-secreting cell line was identified, named P[4]-11E3. Additionally, rabbit polyclonal antibodies against P[8], P[6]and P[4]were prepared. For the P[6]antigen detection, both the capture antibody(30 μg/mL)and the detection antibody(1. 0 μg/mL) were mouse McAbs, with a standard curve range of 10. 00 to 640. 00 ng/mL. For the P[8]and P[4]antigen detection, the capture antibodies were McAbs, with the concentration of 5 μg/mL and 2. 5 μg/mL respectively, the detection antibodies were rabbit polyclonal antibodies, both at the concentration of 1. 5 μg/mL, and their standard curve ranges were 0. 63-20. 00 ng/mL and 0. 31-10. 00 ng/mL respectively. The coefficients of variation(CVs) of the six test results for the antigen content of P[8], P[6]and P[4]proteins were all less than 10%, the recovery rates ranged from 80% to 120%, and the specificity was good. The CVs of antigen recovery yield for the in-process products from three batches of purification processes were all less than 30%.Conclusion The screened monoclonal cell lines secrete antibodies that specifically target the P[8], P[6]and P[4]subtypes of VP8 protein without cross-reactivity, and exhibit high antibody titers. The recombinant RV VP8 protein antigen detection method established based on these antibodies demonstrates strong specificity, high sensitivity, and excellent precision.