Objective:To understand the epidemiology of rubella and the genetic characteristics of the virus circulating during the period 2021-2022, providing basic scientific data for rubella prevention and control in China.Methods:National rubella incidence data for the period 2021-2022 were obtained from the Infectious Disease Surveillance System module and the Surveillance Report Management module of the China′s Disease Prevention and Control Information System. Positive rubella virus(RuV)isolates were obtained from the National Measles/Rubella Laboratory Network. Two nucleotide (nt) fragments [F1-480 (8 633-9 112 nt) and F2-633 (8 945-9 577 nt)] located in the E1 gene were amplified and determined by reverse transcription polymerase chain reaction (RT-PCR), and the target gene (E1-739) was obtained after collating and splicing. The sequences obtained in this study were used to construct a phylogenetic tree with the reported reference strains for genotype and lineage identification. Additionally, the phylogenetic analysis was performed to assess their genetic relatedness of RuV strains prevalent in China during 2018-2020 from GenBank database.Results:In 2021-2022, the rubella incidence in China was 0.06/100, 000 (2021: 840 cases; 2022: 784 cases), with cases primarily concentrated in the western and southern provinces. Age distribution analysis showed that rubella cases in 2021-2022 was mainly in children under 5 years of age (2021: 34.17%, 287/840; 2022: 42.09%, 330/784), with the highest proportion in children aged 0-2 years. Further analysis of the immunization history of cases revealed that in the 8-23 months age group, a significant proportion of cases had received only one dose of rubella containing vaccine (RCV); cases in the 2-14 years age group were mainly among children who had received two or more doses of RCV; however, cases over 15 years of age were primarily found in individuals who had not received RCV or had unknown immunization history. National virological surveillance data showed that totally 22 RuV virus isolates were obtained, from 6 provinces in China during 2021-2022, which belonged to lineage 1E-L2 (11 strains) and 2B-L2c (11 strains). And these viruses displayed high genetic homology with RuV prevalent from 2018 to 2020.Conclusions:The incidence of rubella in China was maintained at a low level during 2021-2022, and the prevalent RuV strains were lineage 1E-L2 and 2B-L2c.

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.

Human parainfluenza viruses (HPIVs) comprise four serotypes (HPIV1-4) and are the second most common pathogens responsible for severe acute respiratory tract infections in infants worldwide. Additionally, HPIVs play a significant role as etiological agents of lower respiratory tract infections in organ transplant recipients, immunocompromised individuals, patients with chronic diseases, and the elderly. HPIVs belong to the Paramyxoviridae family. Among them, HPIV1 and HPIV3 belong to the genus Respirovirus, while HPIV2 and HPIV4 belong to the genus Rubulavirus. The viral genome of HPIV1-4 is approximately 15-17 kb in length and encodes six major structural proteins, including the nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, hemagglutinin-neuraminidase protein, and large RNA-dependent RNA polymerase. In addition, different serotypes of HPIVs also encode several small proteins involved in viral replication. This review summarizes current research advances in the structure and function of genome-encoded proteins, regulatory mechanisms of viral genome transcription and replication, and interactions with host cells to deepen the understanding of the function of HPIVs genome-encoded proteins.

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 genetic characteristics of human adenovirus (HAdV) in severe acute respiratory infection (SARI) cases in Luohe city, Henan province, China.Methods:After viral isolation of HAdV-positive specimens identified in SARI cases from October 2017 to February 2019, the Loop2 region of the Hexon gene was amplified and determined to initially identify the virus type. Then, based on the preliminary screening result, the full length of the sequences of the three target genes (Penton base, Hexon and Fiber) of the viral strains were amplified using specific primers for each HAdV type, and phylogenetic and sequence homology analyses were performed with the prototype strains and the representative strains of the corresponding types at home and abroad to identify the types of viral strains and understand their genetic characteristics.Results:A total of 18 viral isolates were obtained from 27 HAdV-positive throat swab specimens from 783 SARI cases in Luohe city, Henan province, and the molecular typing result showed that these strains belonged to species B (HAdV-3, HAdV-7 and HAdV-55), species C (HAdV-1, P1H2F2, Px1/Ps3H5F5, P89H5F5 and HAdV-6) and species E (HAdV-4). Among them, the highest positive detection rate was found for species C HAdV-1 isolates (33.3%), followed by species B HAdV-3 (22.2%) and species C P1H2F2 (11.1%). The four HAdV strains in this study (HAdV-3, HAdV-4, HAdV-7 and HAdV-55) were characterized by significantly conserved and stable in time and space; while three patterns of genetic recombination (P1H2F2, Px1/Ps3H5F5 and P89H5F5) were identified for HAdV-C strains in this study, and their potential public health significance needed to be confirmed by further studies.Conclusions:The HAdV infection of SARI cases in Luohe city, Henan province during 2017-2019 was dominated by species C, followed by species B and species E. These data provided a scientific basis for the prevention and control of local adenovirus-associated infectious diseases.

Objective:To evaluate the effect of synthetic CpG oligodeoxynucleotide (CpG-ODN) as adjuvant on immune response induced by inactivated human adenovirus (HAdV)-55 antigen in BALB/c mice.Methods:HAdV-55 virus QS prototype strain was purified by plaque to construct a seed bank of vaccine candidate strain. The amplified product of vaccine candidate strain was inactivated by 0.05%β-propiolactone, and purified to prepare perfect virus particle antigen. The purified HAdV-55 antigen was mixed with the same volume CPG-ODN and aluminum hydroxide adjuvant in low-dose group (0.2 mg/ml) and high-dose group (1 mg/ml), respectively, and inoculated BALB/c mice after emulsification. Meanwhile, the control group was set with PBS, and the immunization was enhanced once every 21 days. Respectively on primary immune 21 and 35 days after collecting venous blood in mice and separation of serum, serum was collected at the end of the time of separating spleen lymphocytes in mice. The levels of HAdV-55 specific IgG antibody and neutralization antibody in serum of immunized mice were observed by ELISA and micro-neutralization test, and the levels of lymphocytes secreting IL-4 and IFN-γ cytokines were detected by ELISpot.Results:No matter with or without adjuvant, along with the increase of the number of immunization and vaccination dose of inactivated HAdV-55 antigen induced BALB/c mice virus specific IgG antibody was also significantly increased. However, neutralizing antibody can reach detectable level only after enhanced immunity, and the geometric mean titer (GMT) of neutralizing antibody is between 1: 11 and 1: 23. Different adjuvants have significant effects on the immune response of mice. Low dose antigen combined with CPG-ODN and aluminum hydroxide mixed adjuvant can induce higher humoral and cellular immune responses in mice, and the levels of specific IgG antibody and neutralizing antibody are 2.2 and 1.8 times higher than those in the aluminum hydroxide adjuvant group, respectively. The number of lymphocytes secreting IFN-γ was 2.3 times that of the group immunized with aluminum hydroxide adjuvant.Conclusions:The novel CPG-ODN adjuvant significantly increased the immunogenicity of the inactivated HAdV-55 whole virus antigen in BALB/c mice and directed the cellular immune response toward Th1 type.

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.