Objective:To establish a Plaque-reduction Neutralization Test (PRNT) for the detection of neutralizing antibody titers of Human Respiratory Syncytial Virus (HRSV) and optimize the conditions for preliminary application.Methods:The CHO expression system was used to produce palivizumab monoclonal antibody (palivizumab) and the influencing factors such as cell type, cell culture duration, fixation and permeabilization protocols, and blocking agents. The reproducibility of the method was verified and its correlation was verified with conventional PRNT. Finally, the optimized PRNT assay was further used to determine neutralizing antibody titers against HRSV subtypes A and B in BALB/c mouse serum (immunized by intramuscular injection of HRSV fusion proteins).Results:Palivizumab was expressed at approximately 50 mg/L. The optimal working conditions for PRNT were as follows: culturing HEp-2 cells for 2 days, fixing with 4% (V/V) paraformaldehyde at room temperature for 15 min followed by 0.2% (V/V) Triton X-100 permeabilization for 15 minutes as the optimal fixation-permeabilization and removing the blocking step. The overall coefficient of variation (CV) for the reproducibility validation of this method was <15%, showing a good linear relationship with the conventional PRNT. The Spearman correlation coefficient r s was 0.983. This method was used to detect neutralizing antibody titers in mouse sera against HRSV subtype A strain long and subtype B strain 9320, and the fusion proteins combined with AlOH and CpG adjuvant induced the highest neutralizing antibody titers in mice. Conclusion:The HRSV neutralizing antibody assay established in this study is rapid, reproducible, high-throughput, and can be used to detect neutralizing antibodies to HRSV subtypes A and B.

Objective:To establish a Plaque-reduction Neutralization Test (PRNT) for the detection of neutralizing antibody titers of Human Respiratory Syncytial Virus (HRSV) and optimize the conditions for preliminary application.Methods:The CHO expression system was used to produce palivizumab monoclonal antibody (palivizumab) and the influencing factors such as cell type, cell culture duration, fixation and permeabilization protocols, and blocking agents. The reproducibility of the method was verified and its correlation was verified with conventional PRNT. Finally, the optimized PRNT assay was further used to determine neutralizing antibody titers against HRSV subtypes A and B in BALB/c mouse serum (immunized by intramuscular injection of HRSV fusion proteins).Results:Palivizumab was expressed at approximately 50 mg/L. The optimal working conditions for PRNT were as follows: culturing HEp-2 cells for 2 days, fixing with 4% (V/V) paraformaldehyde at room temperature for 15 min followed by 0.2% (V/V) Triton X-100 permeabilization for 15 minutes as the optimal fixation-permeabilization and removing the blocking step. The overall coefficient of variation (CV) for the reproducibility validation of this method was <15%, showing a good linear relationship with the conventional PRNT. The Spearman correlation coefficient r s was 0.983. This method was used to detect neutralizing antibody titers in mouse sera against HRSV subtype A strain long and subtype B strain 9320, and the fusion proteins combined with AlOH and CpG adjuvant induced the highest neutralizing antibody titers in mice. Conclusion:The HRSV neutralizing antibody assay established in this study is rapid, reproducible, high-throughput, and can be used to detect neutralizing antibodies to HRSV subtypes A and B.

Objective:This study comprehensively analyzed the genomic characterizations of human parainfluenza virus type 3 (HPIV3) strains circulating in six provinces and cities of China (Beijing, Henan, Jilin, Anhui, Gansu, and Shandong) during the period of 2019-2020. The aim was to elucidate the intricate genetic variations and molecular evolutionary trends within the HPIV3 genome.Methods:Based on genotypic differentiation, genetic divergence, and spatial and temporal distribution, 12 representative HPIV3 strains (including 7 of C3a subtype, 2 of C3b subtype and 3 of C3f subtype) were selected from the aforementioned provinces, and the complete genome sequence was successfully obtained by overlapping amplification of fragments using nested RT-PCR. Subsequently, a complete genome database of global representative HPIV3 strains was constructed and analyzed using bioinformatics tools.Results:The length of complete genome of the 12 HPIV3 strains in the present study varied between 15 227 bp and 15 370 bp, the G+ C content ranged from 35.1% to 35.3% and the nucleotide identity intermediated from 97.6% to 99.6%. Compared with the prototype strain (GenBank accession number: NC_001796.2), the nucleotide identity of 12 HPIV3 strains ranged from 94.2% to 94.5%. Analysis of the complete genome of HPIV3 available in China and globally showed that the genomic variation of HPIV3 was mainly shaped by substitution mutations, and no base deletions or gene recombination were observed.Only a six-base insertion (ATTAAA) was found in the F gene’s 3′UTR region of a representative strain originating from Jilin province (CHN/Jilin036/2019/C3b) in this study, and its potential pathogenic significance needs to be further investigated. Amino acid analysis of the encoded proteins revealed that the C3a lineage of HPIV3, widely prevalent both in China and worldwide, exhibits lineage-specific mutation sites in the N, P and L proteins. Furthermore, within the Chinese prevalent C3a strains, a distinctive mutation site (N216S) in L protein was also identified. Notably, specific variant sites have not been found in Chinese C3b and C3f branch strains. Based on the complete genome, the comprehensive evolutionary analysis showed that the time to the most recent common ancestor (tMRCA) of global HPIV3 strains was estimated to 1927 (95% HPD: 1901-1945), with an average molecular evolutionary rate of 5.29 × 10 -4 substitutions/site/year, while the average molecular evolutionary rate of HPIV3 strains in China is 5.24 × 10 -4 substitutions/site/year. In addition, each gene of HPIV3 was subjected to negative selection pressure, with the P, HN and F genes showing the most significant nucleotide variation and higher rates of molecular evolution than the other genes. Conclusions:This study reveals that the complete genome of HPIV3 strains circulating in six provinces and cities of China tend to evolve conservatively. Moreover, substitution emerge as the main driving force for molecular evolution of HPIV3.

Objective:To analyze the effects of microvascular invasion (MVI) and anatomical hepatectomy on early recurrence and survival of patients with hepatocellular carcinoma (HCC).Methods:The data of 246 patients with HCC admitted to 215 Hospital of Shaanxi Nuclear Industry, Chinese PLA General Hospital and Beijing Tsinghua Chang Gung Hospital from July 2008 to June 2019 were retrospectively analyzed, including 208 males and 38 females, aged (53.8±9.6) years. According to the occurrence of MVI, 246 patients were divided into the MVI group ( n=83) and control group ( n=163, without MVI). Hepatitis B virus (HBV) infection, preoperative alpha-fetoprotein (AFP), maximum tumor diameter, intraoperative blood loss were compared between the two groups. The recurrence-free survival and cumulative survival were compared between the two groups before and after the inverse probability weighted correction for propensity score. Results:The propensity score was calculated by logistic regression model. After inverse probability weighted correction, the virtual sample size was 247 cases (82 cases in MVI group and 165 cases in control group). The proportion of HBV infection, with a serum level of AFP > 200 μg/L, the maximum diameter of tumor and the intraoperative blood loss were higher in MVI group (all P<0.05). The risk of early recurrence in patients undergoing anatomical hepatectomy ( n=107) was lower than that in patients undergoing non-anatomical hepatectomy ( n=139) (univariate Cox regression analysis of HR=1.60, 95% CI: 1.06 to 2.42, P=0.020), but the overall survival was comparable (univariate Cox regression analysis of HR=1.66, 95% CI: 0.80 to 3.42, P=0.200). The recurrence-free survival (RFS) of MVI group was lower than that of the control group, and the postoperative cumulative survival rate was also lower before the inverse probability weighted correction of the tendency score. The RFS in MVI group was lower than that in control group after the tendency score was adjusted by inverse probability weighting ( HR=2.62, 95% CI: 1.61 to 4.27, P<0.001). There was no significant difference in the cumulative survival between the MVI and control group ( HR=2.09, 95% CI: 0.89 to 4.93, P=0.050). Conclusion:MVI is associated with early postoperative recurrence in patients with HCC, and the early recurrence rate after anatomical hepatectomy is lower than that after non-anatomical hepatectomy.

Objective:To analyze the gene variation and evolutionary characteristics of human parainfluenza virus 3 (HPIV3) circulating in China based on hemagglutinin-neuraminidase (HN) gene.Methods:Multiple qPCR was used to screen nucleic acid of common pathogens in throat swabs from acute respiratory tract infection cases in five provinces (Beijing, Zhejiang, Anhui, Henan, Hunan) from 2007 to 2020. Subsequently, the HN gene sequence of HPIV3 positive samples was determined and compared with the HN gene sequence of HPIV3 strains from 10 provinces in China and abroad in the GenBank database. The molecular evolution analysis was carried out using a variety of bioinformatics method.Results:A total of 49 strains of HPIV3 HN gene sequences were obtained from 5 provinces, with nucleotide homology ranging from 96.6% to 100%. Among them, 48 strains were subtype C3 and 1 strain was subtype C5. Phylogenetic analysis showed that there were co-epidemics of C1, C3 and C5 strains in 12 provinces of China during 2003—2020, and the nucleotide and amino acid homology among the strains were 95.4%-99.8% and 97.9%-100%, respectively. Among them, C3 is the dominant subtype in China, which is divided into five evolutionary branches of C3a, C3b, C3c, C3e and C3f, and the C3f has the widest range and time of spread. The evolutionary analysis of the C3 subtype showed that the estimated time to the most recent common ancestor (tMRCA) of it dated back to 1990, and its effective population size tended to be stable after expansion from 2002 to 2010. The HN gene evolution rate of the evolutionary branches of C3 HPIV3 varied from 3.69×10 -4 to 5.82×10 -4 substitutions/sites/year; the HN protein of C3 subtype strains shared four potential N-glycosylation sites N308, N351, N485 and N523, and the selection pressure was mainly negative. Conclusions:The C3 is an endemic dominant genotype, which has been widely spread and continuously circulating in China, and has formed different evolutionary clades during epidemic.

Objective:To understand the common viral infection among the surveillance cases of fever respiratory syndrome (FRS) in nine provinces in China.Methods:The research data were obtained from nine provinces (Anhui, Beijing, Guangdong, Hebei, Hunan, Jilin, Shandong, Shaanxi and Xinjiang) in the "Infectious Disease Surveillance Technology Platform Information Management System" of the Chinese Center for Disease Control and Prevention from January 2009 to June 2021. Finally, 8 243 FRS cases with nucleic acid detection results of eight viruses [human influenza virus (HIFV), human respiratory syncytial virus (HRSV), human adenovirus (HAdV), human parainfluenza virus (HPIV), human rhinovirus (HRV), human metapneumovirus (HMPV), human coronavirus (HCoV) and human Boca virus (HBoV)] were included in the study. The χ 2 test/Fisher exact probability method was used to analyze the difference of virus detection rate in different age groups, regions and seasons. Results The M ( Q1, Q3) age of 8 243 FRS cases was 4 (1, 18) years old, and 56.56% (4 662 cases) were children under 5 years old. Males accounted for 58.1% (4 792 cases) of all cases. All cases were from outpatient/emergency department (2 043 cases) and inpatient department (6 200 cases). The virus detection rates of FRS cases from high to low were HRSV, HIFV, HPIV, HRV, HAdV, HMPV, HCoV and HBoV. Two or more viruses were detected simultaneously in 524 cases, accounting for 15.66% of virus-positive cases. The difference of the virus detection rate in different age groups was statistically significant (all P values<0.05), and the virus detection rate in children<5 years old was higher (49.96%). The positive rate of any virus in south China was higher than that in north China ( P<0.001). The virus-positive FRS cases were detected throughout the year. The detection rate of HRSV was higher in autumn and winter. The detection rate of HIFV was higher in winter. The detection rate of HMPV was higher in winter and spring. The detection rates of HPIV, HRV, HCoV and HBoV were higher in summer and autumn, while there was no significant difference in the detection rate of HAdV in different seasons. Compared with 2009-2019, the detection rate of any virus in 2020-2021 decreased from 41.37% to 37.86%. The detection rate of HIFV decreased sharply from 10.62% to 1.37%. The detection rate of HPIV decreased from 8.24% to 5.88%. The detection rate of HRV and HBoV increased from 5.43% and 1.79% to 9.67% and 3.19%, respectively. Conclusion:HRSV and HIFV infections are more common among FRS cases in nine provinces in China from 2009 to 2021, and the epidemiological characteristics of eight common respiratory viruses vary in different age groups, regions and seasons.

Objective:To understand the common viral infection among the surveillance cases of fever respiratory syndrome (FRS) in nine provinces in China.Methods:The research data were obtained from nine provinces (Anhui, Beijing, Guangdong, Hebei, Hunan, Jilin, Shandong, Shaanxi and Xinjiang) in the "Infectious Disease Surveillance Technology Platform Information Management System" of the Chinese Center for Disease Control and Prevention from January 2009 to June 2021. Finally, 8 243 FRS cases with nucleic acid detection results of eight viruses [human influenza virus (HIFV), human respiratory syncytial virus (HRSV), human adenovirus (HAdV), human parainfluenza virus (HPIV), human rhinovirus (HRV), human metapneumovirus (HMPV), human coronavirus (HCoV) and human Boca virus (HBoV)] were included in the study. The χ 2 test/Fisher exact probability method was used to analyze the difference of virus detection rate in different age groups, regions and seasons. Results The M ( Q1, Q3) age of 8 243 FRS cases was 4 (1, 18) years old, and 56.56% (4 662 cases) were children under 5 years old. Males accounted for 58.1% (4 792 cases) of all cases. All cases were from outpatient/emergency department (2 043 cases) and inpatient department (6 200 cases). The virus detection rates of FRS cases from high to low were HRSV, HIFV, HPIV, HRV, HAdV, HMPV, HCoV and HBoV. Two or more viruses were detected simultaneously in 524 cases, accounting for 15.66% of virus-positive cases. The difference of the virus detection rate in different age groups was statistically significant (all P values<0.05), and the virus detection rate in children<5 years old was higher (49.96%). The positive rate of any virus in south China was higher than that in north China ( P<0.001). The virus-positive FRS cases were detected throughout the year. The detection rate of HRSV was higher in autumn and winter. The detection rate of HIFV was higher in winter. The detection rate of HMPV was higher in winter and spring. The detection rates of HPIV, HRV, HCoV and HBoV were higher in summer and autumn, while there was no significant difference in the detection rate of HAdV in different seasons. Compared with 2009-2019, the detection rate of any virus in 2020-2021 decreased from 41.37% to 37.86%. The detection rate of HIFV decreased sharply from 10.62% to 1.37%. The detection rate of HPIV decreased from 8.24% to 5.88%. The detection rate of HRV and HBoV increased from 5.43% and 1.79% to 9.67% and 3.19%, respectively. Conclusion:HRSV and HIFV infections are more common among FRS cases in nine provinces in China from 2009 to 2021, and the epidemiological characteristics of eight common respiratory viruses vary in different age groups, regions and seasons.

Objective:To explore the related factors influencing the length of hospital stay(LOS) of pregnant women with heart disease (PWHD) after cesarean section.Methods:A total of 306 patients with PWHD who underwent cesarean section from January 2012 to March 2019 were collected. Among them, 203 patients had not undergone heart surgery (uncorrected group) and 103 patients who had undergone heart surgery (corrected group) during the same period. Demographic, perioperative and postoperative data were recorded. Predictors associated with postoperative LOS were determined using univariate and multivariate linear regression analysis models.Results:(1) The median LOS after cesarean section in the uncorrected group was 6 days (5-8 days). The results of univariate linear regression analysis showed that 38 parameters had significant impact on LOS ( P<0.05). The results of multivariate linear regression analysis showed that 5 parameters were independent risk factors for prolonged LOS in the uncorrected group; among them, the median LOS in uncorrected group with hypertensive disorders of pregnancy was 3 days longer than that in patients with PWHD alone [7 days (5-8 days) vs 4 days (4-5 days), β=0.195, P=0.001]; the median LOS in uncorrected group with high serum creatinine was 3 days longer than normal patients [7 days (5-13 days) vs 4 days (4-5 days), β=0.145, P=0.015]; the LOS of patients who chose general anesthesia was 2 days longer than that of patients who chose spinal anesthesia [6 days (4-8 days) vs 4 days (4-5 days), β=0.154, P=0.007]; the LOS of patients with postoperative pulmonary infection was 4 days longer than that of patients without pulmonary infection [8 days (5-15 days) vs 4 days (4-5 days), β=0.269, P<0.01]; the LOS of patients who admitted to ICU after surgery was 2 days longer than that not admitted patients [6 days (5-8 days) vs 4 days (4-5 days), β=0.268, P<0.01]. (2) The median LOS after cesarean section in corrected group was 4 days (4-5 days). The results of univariate linear regression analysis showed that 8 parameters had significant impact on the LOS (all P<0.05). The results of multivariate linear regression analysis showed that 2 parameters, which were American Society of Anesthesiologists (ASA) grade ( β=0.198, P=0.028) and intraoperative blood loss ( β=0.285, P=0.003), were the independent risk factors for prolonged LOS in corrected group. Conclusion:Preoperative with hypertensive disorders of pregnancy, preoperative creatinine increase, intraoperative general anesthesia, postoperative pulmonary infection, and postoperative admission to ICU are independent predictors of prolonged LOS in uncorrected patients with PWHD; ASA classification and intraoperative bleeding are independent predictor of prolonged postoperative LOS in patients with corrected PWHD.

Objective To establish an indirect ELISA method for detection of measles virus antibody based on the nucleoprotein of measles virus expressed in prokaryotic system selected as the coating antigen.Methods The working conditions were screened and optimized to determine the operation process of indirect ELISA.The anti-measles virus IgG antibody in 157 sera of healthy children and mothers of newborn infants was detected and compared with the commercial measles virus ELISA IgG antibody detection kit.Results The result showed that the coefficient of variation of intra and inter assay repeatability of the indirect ELISA was less than 10％.Compared with the result of the commercial kit,the total concordance rate of serum samples was 95.5％,and the sensitivity and specificity were 94.8％ and 98.3％,respectively.There was no significant difference between the two methods (x2 =0.313,P＞0.05;x2 =0.000,P＞ 0.05) in the positive rate of measles virus IgG antibody in 85 healthy children at the age of 0～ 15 years.The level of measles virus antibody detected by ELISA established in this study showed the same increasing and decreasing trend as that detected by the commercial kit in different age groups.The correlation coefficient r of the quantitative result was 0.893 (P＜ 0.001),which showed the quantitative potential of the method.Conclusions The ELISA method established in this study has good stability,high sensitivity and specificity,and there was no significant difference between the detection results of commercial kit and the ELISA method.It can be used for the seroepidemiological investigation of measles virus and the evaluation of antibody level of the population after vaccination.

Objective: The genetic characteristics of the human adenovirus type 53 (HAdV-53) strains isolated from Taiyuan city of Shanxi Province were studied to obtain the baseline data of their molecular characteristics. Methods: Conjunctival swabs (n=79) were collected from epidemic keratoconjunctivitis (EKC) patients in Shanxi eye Hospital in 2016, and five HAdV-53 strains were obtained after virus isolation and identification based on the three major capsid genes sequences including Penton base, Hexon and Fiber gene. And the corresponding sequences of global epidemic HAdV-53 strains and the strains with the same genetic origin as HAdV-53 were also downloaded from GenBank database, and then the three gene database were established, respectively. With the database, phylogenetic tree was constructed, and the genetic and molecular evolutionary characteristics were analyzed with bioinformatics software. Results: Five HAdV-53 strains in Shanxi Province in 2016 showed high consistency with the HAdV-53 strains prevalent in other countries in 1996-2014 (>99.8%). All HAdV-53 strains were in the same evolutionary branch with their recombinant source genotypes (HAdV-37 and HAdV-8) in Penton base and Fiber gene, respectively, and maintained a high degree of consistency in gene sequences. In Hexon gene, HAdV-53 strains were more closed to its recombinant source genotype HAdV-22, the nucleotide and amino acid sequences between two types were highly homologous, while HAdV-53 and HAdV-22 belonged to different evolutionary branches, and the evolution rate of HAdV-53 based on Hexon gene was 3.51×10^-5 substitution/site/year. Conclusion HAdV-53 has become an important new ocular infectious pathogen of Taiyuan. HAdV-53 strain are relatively conservative and stable based on Penton base, Hexon, and Fiber gene.