Objective:To analyze the epidemiologic characteristics of avian influenza virus and the molecular characteristics and genetic evolution of H9N2 subtype avian influenza viruses in the live poultry markets in Fujian province in 2021-2023.Methods:Six types of specimens were collected from five cities in Fujian province. The specimens were subjected to nucleic acid detection for influenza A viruses, subtypes H5, H7, and H9 by fluorescence quantitative PCR, and the results were analyzed statistically with χ2 test. Specimens with Ct value less than 31 were screened for targeted amplification and next-generation sequencing of the hemagglutinin (HA) and neuraminidase (NA) genes. Reference sequences were downloaded from the databases, and the characteristics of molecular variation and genetic evolution were analyzed by using bioinformatics softwares. Results:From 2021 to 2023, a total of 1 853 specimens were collected from five cities, with a positive rate of 50.94% for influenza A viruses, including 684 specimens of H9 subtype, 23 specimens of H5 subtype, 1 specimen of H7 subtype, 28 specimens of H5 and H9 subtype, 1 specimen of H7 and H9 subtype, and 207 specimens of unclassified A-type. There were significant differences in the positive detection rates of influenza A viruses in different cities( χ2=461.82, P<0.001). Statistically significant differences in influenza A virus positivity rates across years and quarters( χ2=12.26, P=0.002; χ2=30.12, P<0.001), with higher rates of 56.39% and 55.34% in the first and third quarters, respectively. And the differences in the positive rates of influenza A viruses in different types of specimens were statistically significant( χ2=23.05, P<0.001), with specimens on the cage surface having a positivity rate of 56.09%, which was the highest of all types of specimens. A total of 24 strains of HA and NA genes of H9N2 subtype were fully sequenced. Compared with the sequence of NCBI database, the highest identity of HA gene nucleotide sequences ranged from 97.03% to 99.87%, and the highest identity of NA gene nucleotide sequences ranged from 97.50% to 99.78%.Twenty-three strains with both HA and NA genes belonged to the G57 genotype in the Y280-like evolutionary branch of the Eurasian lineage, and one strain belonged to the Y439-like evolutionary branch of the Eurasian lineage. The cleavage sites of all the strains showed the characteristic of low pathogenicity, and most strains had receptor binding sites characterized by avian-derived and human receptors. Conclusions:The overall positive rate of avian influenza viruses in live poultry markets in Fujian province was relatively high, especially in the first and third quarters, with H9 subtype accounting for the main proportion. Most of H9N2 subtype belonged to the Y280-like G57 genotype with a small number of Y439-like evolutionary branches, suggesting the possibility of genetic recombination and the risk of human infection. Thus, surveillance of avian influenza viruses in the live poultry markets as well as mutation analysis should continue to be strengthened.

This study was aimed at comprehensively understanding the epidemic status of acute hemorrhagic conjunctivitis(AHC)and influencing factors related outbreak in Fujian Province,to provide valuable insights for AHC prevention and control in this region.We conducted a descriptive statistical analysis of epidemiological data for AHC cases.Additionally,clinical samples collected from a 2023 outbreak were subjected to pathogen detection,determination of full-length nucleotide sequences within the VP1 region,genotype identification,and comprehensive analysis.The peak incidence of AHC in 2023 was observed in September and accounted for a substantial proportion(86.39%,5 205/6 025)of the total cases throughout the year.The three areas with the highest incidence rates were Sanming(118.57/100 000),Longyan(53.83/100 000)and Zhangzhou(16.02/100 000).From 2011 to 2022,the inci-dence of AHC in Longyan City was the highest for consecutive 12 years.During the peak AHC outbreak period in Fujian Province in 2023,all 62 conjunctival swabs collected were identified as CVA24v,and 60 complete VP1 sequences were obtained through se-quencing.The nucleotide sequence identity among these 60 sequences ranged from 98.7%to 100%,whereas the amino acid identity ranged from 98.4%to 100%.The highest nucleotide sequence identity was observed for the 2023 strain isolated from Zhongshan,Guangdong Province(rang:99.0%～100%).The 2023 strains in Fujian Province belonged to the GIV genotype,and L16I represented a potential unique amino acid variation within the sequence.CVA24v was the causative agent responsible for this AHC epidemic,and its GIV genotype is currently the dominant type in China.Future efforts should prioritize AHC prevention and control measures cen-tered in key cities.Monitoring and early warning of AHC in the whole province should be strengthened before September each year,and the variation of pathogen nucleic should also be tracked in time.

To analyze the epidemiological characteristics of COVID-19 in Fujian Province from the 49th week of 2022 to the 5th week of 2023,after further optimization of China's COVID-19 prevention and control measures on December 7,2022(the 49th week of 2022),this study used multi-dimensional surveillance data to dynamically assess population infection levels and their changing trends.The aim of the study was to provide a scientific basis for early warning of epidemic risk,medical resource allocation,and evalu-ation of socio-economic impact.A multi-source data surveillance system was constructed,encompassing surveillance of fever clinics at medical institutions(weekly collection of visits,positive nucleic acid and antigen test results,inpatients,and severe cases in sec-ondary or above hospitals),population nucleic acid test monitoring(weekly person-times and positivity rates of single-tube tests from the provincial system),sentinel hospital monitoring(weekly proportion of influenza-like illness visits at 18 sentinel hospitals and re-lated viral testing data),and monitoring of novel coronavirus variants(weekly systematic collection of genomic sequences of local and imported cases).Line charts were plotted weekly,and time series analysis,molecular epidemiological methods,and an improved SEIAR model were used to simulate epidemic spread.During the study period,the COVID-19 epidemic in Fujian Province exhibited three distinct stages.In the infection peak stage(52nd week of 2022),the provincial fever clinic visits reached 606 893 person-times,and a 49.2%positivity rate in population single-tube nucleic acid tests and 63.8%positivity rate in sentinel hospital monitoring were observed.In the medical load peak stage(2nd week of 2023),274 460 inpatients and 28 487 severe cases were recorded.In the epidemic decline stage(4th to 5th weeks of 2023),fever clinic visits decreased by 96.3%with respect to the peak,the single-tube nucleic acid test positivity rate decreased to 6.3%,and the sentinel hospital COVID-19 nucleic acid test positivity rate was 6.4%.All 508 sequenced local cases were Omicron variants,predominantly BA.5.2 and its sub-lineages(67.4%).Among 56 imported se-quenced cases,BA.5.2 and its sub-lineages accounted for 50.0%,and 16.1%comprised nine variants of interest including XBB and BQ.The model predicted the infection peak in the 52nd week of 2022,whereas the hospitalization peak lagged by approximately 10.6 days.Multi-source data monitoring revealed a three-stage development of the COVID-19 epidemic in Fujian.The BA.5.2 strain was dominant during the epidemic.The combination of multi-source monitoring data and modeling provides important references for epi-demic prevention and control,and highlights the need to improve the monitoring system in follow-up.

To analyze the epidemiological characteristics of COVID-19 in Fujian Province from the 49th week of 2022 to the 5th week of 2023,after further optimization of China's COVID-19 prevention and control measures on December 7,2022(the 49th week of 2022),this study used multi-dimensional surveillance data to dynamically assess population infection levels and their changing trends.The aim of the study was to provide a scientific basis for early warning of epidemic risk,medical resource allocation,and evalu-ation of socio-economic impact.A multi-source data surveillance system was constructed,encompassing surveillance of fever clinics at medical institutions(weekly collection of visits,positive nucleic acid and antigen test results,inpatients,and severe cases in sec-ondary or above hospitals),population nucleic acid test monitoring(weekly person-times and positivity rates of single-tube tests from the provincial system),sentinel hospital monitoring(weekly proportion of influenza-like illness visits at 18 sentinel hospitals and re-lated viral testing data),and monitoring of novel coronavirus variants(weekly systematic collection of genomic sequences of local and imported cases).Line charts were plotted weekly,and time series analysis,molecular epidemiological methods,and an improved SEIAR model were used to simulate epidemic spread.During the study period,the COVID-19 epidemic in Fujian Province exhibited three distinct stages.In the infection peak stage(52nd week of 2022),the provincial fever clinic visits reached 606 893 person-times,and a 49.2%positivity rate in population single-tube nucleic acid tests and 63.8%positivity rate in sentinel hospital monitoring were observed.In the medical load peak stage(2nd week of 2023),274 460 inpatients and 28 487 severe cases were recorded.In the epidemic decline stage(4th to 5th weeks of 2023),fever clinic visits decreased by 96.3%with respect to the peak,the single-tube nucleic acid test positivity rate decreased to 6.3%,and the sentinel hospital COVID-19 nucleic acid test positivity rate was 6.4%.All 508 sequenced local cases were Omicron variants,predominantly BA.5.2 and its sub-lineages(67.4%).Among 56 imported se-quenced cases,BA.5.2 and its sub-lineages accounted for 50.0%,and 16.1%comprised nine variants of interest including XBB and BQ.The model predicted the infection peak in the 52nd week of 2022,whereas the hospitalization peak lagged by approximately 10.6 days.Multi-source data monitoring revealed a three-stage development of the COVID-19 epidemic in Fujian.The BA.5.2 strain was dominant during the epidemic.The combination of multi-source monitoring data and modeling provides important references for epi-demic prevention and control,and highlights the need to improve the monitoring system in follow-up.

This study was aimed at comprehensively understanding the epidemic status of acute hemorrhagic conjunctivitis(AHC)and influencing factors related outbreak in Fujian Province,to provide valuable insights for AHC prevention and control in this region.We conducted a descriptive statistical analysis of epidemiological data for AHC cases.Additionally,clinical samples collected from a 2023 outbreak were subjected to pathogen detection,determination of full-length nucleotide sequences within the VP1 region,genotype identification,and comprehensive analysis.The peak incidence of AHC in 2023 was observed in September and accounted for a substantial proportion(86.39%,5 205/6 025)of the total cases throughout the year.The three areas with the highest incidence rates were Sanming(118.57/100 000),Longyan(53.83/100 000)and Zhangzhou(16.02/100 000).From 2011 to 2022,the inci-dence of AHC in Longyan City was the highest for consecutive 12 years.During the peak AHC outbreak period in Fujian Province in 2023,all 62 conjunctival swabs collected were identified as CVA24v,and 60 complete VP1 sequences were obtained through se-quencing.The nucleotide sequence identity among these 60 sequences ranged from 98.7%to 100%,whereas the amino acid identity ranged from 98.4%to 100%.The highest nucleotide sequence identity was observed for the 2023 strain isolated from Zhongshan,Guangdong Province(rang:99.0%～100%).The 2023 strains in Fujian Province belonged to the GIV genotype,and L16I represented a potential unique amino acid variation within the sequence.CVA24v was the causative agent responsible for this AHC epidemic,and its GIV genotype is currently the dominant type in China.Future efforts should prioritize AHC prevention and control measures cen-tered in key cities.Monitoring and early warning of AHC in the whole province should be strengthened before September each year,and the variation of pathogen nucleic should also be tracked in time.

Objective:To analyze the epidemiologic characteristics of avian influenza virus and the molecular characteristics and genetic evolution of H9N2 subtype avian influenza viruses in the live poultry markets in Fujian province in 2021-2023.Methods:Six types of specimens were collected from five cities in Fujian province. The specimens were subjected to nucleic acid detection for influenza A viruses, subtypes H5, H7, and H9 by fluorescence quantitative PCR, and the results were analyzed statistically with χ2 test. Specimens with Ct value less than 31 were screened for targeted amplification and next-generation sequencing of the hemagglutinin (HA) and neuraminidase (NA) genes. Reference sequences were downloaded from the databases, and the characteristics of molecular variation and genetic evolution were analyzed by using bioinformatics softwares. Results:From 2021 to 2023, a total of 1 853 specimens were collected from five cities, with a positive rate of 50.94% for influenza A viruses, including 684 specimens of H9 subtype, 23 specimens of H5 subtype, 1 specimen of H7 subtype, 28 specimens of H5 and H9 subtype, 1 specimen of H7 and H9 subtype, and 207 specimens of unclassified A-type. There were significant differences in the positive detection rates of influenza A viruses in different cities( χ2=461.82, P<0.001). Statistically significant differences in influenza A virus positivity rates across years and quarters( χ2=12.26, P=0.002; χ2=30.12, P<0.001), with higher rates of 56.39% and 55.34% in the first and third quarters, respectively. And the differences in the positive rates of influenza A viruses in different types of specimens were statistically significant( χ2=23.05, P<0.001), with specimens on the cage surface having a positivity rate of 56.09%, which was the highest of all types of specimens. A total of 24 strains of HA and NA genes of H9N2 subtype were fully sequenced. Compared with the sequence of NCBI database, the highest identity of HA gene nucleotide sequences ranged from 97.03% to 99.87%, and the highest identity of NA gene nucleotide sequences ranged from 97.50% to 99.78%.Twenty-three strains with both HA and NA genes belonged to the G57 genotype in the Y280-like evolutionary branch of the Eurasian lineage, and one strain belonged to the Y439-like evolutionary branch of the Eurasian lineage. The cleavage sites of all the strains showed the characteristic of low pathogenicity, and most strains had receptor binding sites characterized by avian-derived and human receptors. Conclusions:The overall positive rate of avian influenza viruses in live poultry markets in Fujian province was relatively high, especially in the first and third quarters, with H9 subtype accounting for the main proportion. Most of H9N2 subtype belonged to the Y280-like G57 genotype with a small number of Y439-like evolutionary branches, suggesting the possibility of genetic recombination and the risk of human infection. Thus, surveillance of avian influenza viruses in the live poultry markets as well as mutation analysis should continue to be strengthened.

Objective:To profile the distribution of avian influenza virus in poultry-related environment in poultry industry developed area in Fujian province, an investigation was conducted in Nanping city from Dec.2021 to Dec.2023.Methods:The samples from multiple types of external environment related to poultry in Nanping city were collected from Dec. 2021 to Dec. 2023, and the real-time fluorescence quantitative RT-PCR used to detect and subtype the influenza A virus (FluA). SPSS 26.0 software was used to analyze the distribution characteristics of FluA in poultry-related environment and the differences in time, places and sample types.Results:The overall positive rate of FluA in samples from poultry-related environment was 49.16% (1 435/2 919). The positive rates of H3, H5, H9 and H10 subtypes were 0.72% (21/2 919), 9.42% (275/2 919), 33.20% (969/2 919), 0.89% (26/2 919) respectively, and no H7 subtype was detected. The positive rate of mixed type (more than one subtype of FluA detected in a same sample) was 6.51% (190/2 919), and the positive rate of unknown subtype (positive for FluA but negative for H3/5/7/9/10) was 11.58% (338/2 919). The higher positive rate of FluA mainly occurred in autumn-winter season (September to February of the following year). In live poultry markets and slaughterhouses, the positive rates of FluA, H9 subtype, mixed type and unknown subtype were significantly higher than that in poultry farms. The positive rate of FluA in poultry drinking water and feces was higher than samples of other types, most of the positive samples were H9 subtype.Conclusions:The positive rate of FluA in poultry-related environment in Nanping city was higher in autumn-winter season. The investigation also showed that higher FluA positive rate in drinking water and feces sample and diversity of the virus existed in the place of multiple types of poultry clustered, such as live poultry markets and slaughterhouses.

Objective:To investigate the molecular epidemiological characteristics of coxsackievirus A16 (CVA16) in Fujian province from 2020 to 2023.Methods:The epidemiological characteristics of CVA16 associated hand, food and mouth disease (HFMD) in Fujian province from 2020 to 2023 was analyzed. The complete VP1 gene of CVA16 was amplified by RT-PCR and then sequenced, and genetic evolution was analyzed by MEGA X and other softwares.Results:From 2020 to 2023, there were 13 120 cases of HFMD in Fujian province, and the proportion of HFMD which caused by CVA16 was 16.5% (2 160/13 120). From 2020 to 2023, the proportion of accounted cases was 4.7% (94/2 019), 14.1% (457/3 243), 47.6% (1 521/3 199) and 1.9% (88/4 659) respectively. HFMD caused by CVA16 was mainly concentrated in children aged 1 to 5 years, and most of them were 3 years old. The genetic evolution and genotype analysis of 92 complete VP1 gene sequences obtained from 2020 to 2023 showed that the genetic distance between CVA16 strains in Fujian province and the prototype strain was far away. The CVA16 genotype in Fujian province from 2020 to 2023 has three clusters of B1a, B1b and B1c, among which the composition ratio of B1a and B1b in Fujian province in 2020 was 40% and 60% respectively. In 2021, B1a and B1b accounted for 81.8% and 18.2% respectively. Only B1a in 2022; in 2023, there were B1a, B1b and B1c, which respectively accounted for 44.4%, 7.4% and 48.2%. During the period from January to September, B1a was the main cluster. After October we observed an emergence of B1c cluster, which had never been found in Fujian province and was rare in China, was detected and became the dominant cluster.Conclusions:The evolutionary cluster of CVA16 dominant changed from B1b in 2020 to B1a in 2021-2023. After October 2023, the newly discovered B1c became the dominant cluster in Fujian province.

Objective:To establish a nucleic acid detection method for human parainfluenza virus (HPIV) based on reverse transcription recombinase-aid amplification (RT-RAA) combined with CRISPR/Cas12a.Methods:The type-specific primer pairs of RT-RAA and CRISPR RNA (crRNA) targeted on conserved sequence of nucleocapsid protein (N) gene of HPIV were designed. Fluorescence intensities from cleavage of fluorophore labeled probes mediated by Cas12a were measured for screening of crRNA and concentration optimization of crRNA, Cas12a as well as the probe. With the RNA transcribed in vitro and clinical specimen, the lower limit of detection and specificity of RT-RAA combined CRISPR/Cas12a detection were evaluated. Results:The crRNA specific to each type of HPIV 1-4, with strongest cleavage activity, were screened out. With the optimal concentration of crRNA, Cas12a as well as the probe, the lower limit of detection could reach 10 copies of target gene per reaction on fluorescence intensity measurement. No cross-reaction was found in clinical samples of eight other respiratory viruses detected by this method .Conclusions:The established HPIV1-4 fluorescence CRISPR nucleic acid detection method is rapid, specific, and does not require professional nucleic acid detection equipment.

Objective:To improve the efficiency and success rate of human adenovirus 3 (HAdV-3) whole genome sequencing, a high-throughput sequencing method for the whole genome of HAdV-3 based on multiplex PCR was established.Methods:Multiplex PCR primers suitable for the whole genome amplification of HAdV-3 were designed. The whole genome sequence of HAdV-3 was amplified by multiplex PCR, and the specificity of the amplification product was preliminarily verified by agarose gel electrophoresis. High-throughput sequencing of the multiplex PCR products was performed using Illumina second-generation sequencing. After obtaining the sequence, software such as CLC and IGV were used to analyze the effective data amount, average sequencing depth, and whole genome coverage obtained by high-throughput sequencing, then the sequencing quality was evaluated. Based on the whole genome sequencing result, a phylogenetic tree was constructed to confirm the virus type and analyze homology of the sequences, and then the accuracy of this method was evaluated.Results:A total of 70 (35 pairs) multiplex PCR amplification primers for the whole genome of HAdV-3 were designed, with amplicon size of approximately 1 200 bp. And the expected whole genome coverage could reach 99.8% (with a total genome length of approximately 35 240 bp). Agarose gel electrophoresis analysis showed that the size of the multiplex PCR products was consistent with expectations, and the amplification specificity was high. The high-throughput sequencing result showed that the whole genome sequences obtained by this method were complete and intact, and the genome coverage was consistent with expectations. Sequence quality analysis showed that the high-throughput sequencing method based on multiplex PCR could obtain more effective data and greater sequencing depth, resulting in more uniform whole genome coverage. Phylogenetic analysis showed that the evolutionary typing result of viral DNA sequenced after multiplex PCR amplification were consistent with those of viral DNA sequenced directly and had high homology, indicating that the multiplex PCR method had high amplification fidelity and the results obtained in combination with high-throughput sequencing were accurate.Conclusions:A high-throughput sequencing method for the whole genome of HAdV-3 based on multiplex PCR was established in this study successfully. This method could improve the efficiency and success rate of HAdV-3 whole genome sequencing, aiming to provide better technical support and reference for HAdV-3 pathogen surveillance and epidemic source-tracing based on whole genome sequencing.