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 confirm the cross-provincial long-distance transmission events of Coronavirus Disease 2019 (COVID-19) by lorry drivers, the origin of infections of the cases and the transmission routes were tracked.Methods:Nasopharyngeal swab specimens from five lorry driver cases of COVID-19, found in Zhangzhou city in March, 2022 when the local outbreaks occurred in adjacent Quanzhou city, Fujian province, were collected to perform 2019 novel coronavirus (2019-nCoV) targeted genome amplification and followed by next-generation sequencing. The sequences were submitted to online 2019-nCoV analysis platforms to classify the type of variant and mutation sites. Phylogenetic tree for the viruses were constructed by phylogenetic analysis software. Combined with the epidemiological investigation, the origin of infections of the cases and the transmission routes were deduced.Results:Five complete genome sequences, with 29 770-29 839 bp in length and 99.53% average genomic coverage, of 2019-nCoV were successfully obtained. The viruses were all Omicron variants and further divided into three different subclades of BA.2. Of the five strains of 2019-nCoV, three were highly similar to the viruses of two distinct lineages co-circulated in Quanzhou city during the period of local outbreak of COVID-19, respectively. Phylogenetic analysis also revealed that the viruses from three infected lorry drivers were highly homologous to that from local outbreaks in Quanzhou city. The viruses from the rest two cases had seven to fourteen nucleotide mutations (corresponding to 5-7 amino acid substitutions) when compared with the viruses in local outbreaks in Quanzhou city, which excluded the involvement of the two cases into the transmission chains of local outbreaks. Combined with the field epidemiological investigations, the result revealed that the origin of infection of 2019-nCoV of the two sporadic lorry driver cases was outside of Fujian province.Conclusions:With the aid of high-throughput sequencing and bioinformatics technology combined with field epidemiological investigations, we speculated in this study that at least three origins of infection of 2019-nCoV in five lorry driver cases and cross-provincial long-distance transmission via two sporadic cases infected outside Fujian province when they returned.

Objective:To analyze the epidemiologic characteristics of environmental samples of avian influenza virus in Fujian province from 2017 to 2021, and provide a reference for the prevention and control of avian influenza.Methods:Six types of specimens were collected from four types of environments in six cities in Fujian province. And the specimens were subjected to nucleic acid detection for influenza A, subtypes H5, H7 and H9 by fluorescence quantitative PCR, and the results were analyzed statistically with descriptive epidemiological methods.Results:From 2017 to 2021, a total of 4 214 samples were collected from 6 cities, of which the positive rate of avian influenza virus was 41.53%, and the positive rates of H5, H7 and H9 subtypes were 2.33%, 1.16% and 23.16%, respectively. The positive rate for mixed subtypes of H5 and H7 was 0.05%, the positive rate for mixed subtypes of H5 and H9 was 1.83%, the positive rate for mixed subtypes of H7 and H9 was 0.83%, the positive rate for mixed subtypes of H5, H7 and H9 was 0.09%, and the positive rate of A-type unclassified was 12.08%. The difference in avian influenza virus detection among different monitoring places ( χ2=517.57, P<0.001), different types of specimens( χ2= 51.58, P<0.001), and different cities ( χ2=458.34, P<0.001) was statistically significant. Among different monitoring places, the positive rate of avian influenza virus in urban and rural live poultry markets was the highest. The highest rate of positive detection was found in specimens from the cage surface, cleaning poultry sewage and poultry chopping board surface, with 48.09%, 47.34% and 45.66%, respectively. In terms of different cities, Sanming city had the highest positivity rate (56.00%), while Zhangzhou city had the lowest positivity rate(3.34%). And the positive rate was higher from November to February of the next year and June to August each year. Conclusions:The overall positive rate of avian influenza viruses in Fujian province was relatively high, with H9 subtype accounting for the main proportion. The monitoring of avian influenza viruses in winter, spring and summer should be strengthened. And effective measures should be taken to deal with avian influenza especially in urban and rural live poultry markets.

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.

Objective:To understand the genome characteristics of group A rotavirus (RVA) strains among hospitalized children under 5 years of age with acute diarrhea in Fuzhou sentinel hospital in 2020.Methods:The ELISA method was used for screening RVA-positive stool samples of hospitalized children under 5 years of age, then 11 gene segments of RVA-positive samples were sequenced and typed after amplification by RT-PCR, and their homology and phylogenetic analysis were performed by Molecular Evolutionary Genetics Analysis (MEGA).Results:Twenty RVA whole genome sequences were successfully obtained, including 4 kinds of G/P gene combinations-G9P[8] (55%), G3P[8] (25%), G8P[8] (15%) and G2P[4] (5%). DS-1-like reassortant strains accounted for 40% of the whole genomes. Strains of the same type have high sequence homology, are closely related to the virus strains that currently circulating in the world. There are mutations at multiple important antigenic sites on VP7, VP4 and NSP4 fragments. The variation of amino acid substitutions of VP7, VP4 and NSP4 fragments is complicated, and there are many amino acid substitutions in the antigenic regions.Conclusions:G3P[8] and G8P[8] DS-1-like reassortants were detected for the first time in Fuzhou, amino acid substitutions were observed in the antigenic regions of the VP7, VP4 and NSP4 gene. Due to the emergence of uncommon DS-1-like reassortant strains and multiple important antigenic regions substitutions, it is necessary to continuously monitoring genome characteristics of RVA strains to provide scientific evidence for the pandemic prevention and vaccine immunization strategies.

Objective:To investigate the whole genome characteristics and mutation of imported 2019-nCoV and trace potential source at the genomics level, the viral genomes from the specimens of a cluster of COVID-19 cases which were imported from a freighter were directly sequenced and determined by high-throughput sequencing technology and bioinformatics analysis.Methods:Throat swab specimens from the 8 confirmed cases of COVID-19 from the same freighter were collected to perform the whole genome sequencing for 2019-nCoV using the targeted genome amplification, combined with Ion S5 next-generation sequencing (NGS) technology. Varieties of online virus analysis platforms was used to classify the viruses and analyze mutation sites in the whole genome. Phylogenetic analysis software was used to construct a phylogenetic tree for the viruses, combined with the epidemiological data of the case, to speculate about the source of the viruses.Results:Eight complete genome sequences of 2019-nCoV was successfully obtained. The complete genomes of the viruses were 29, 822-29, 865 bp in length, with the average sequencing depth of 11 928×-33 588× and the coverage of 99.73%-99.87%; the result of Pangolin classification showed that all the eight 2019-nCoV genomes belong to VOC/Delta (B.1.617.2) lineage. The result of whole genome mutation analysis showed that compared with the Wuhan reference strain, the median number of nucleotide mutations in the eight 2019-nCoV genome sequences was 35 (31 to 38), and the median number of amino acid mutations was 26 (24-28); the mutation sites were distributed in 8 gene coding regions (ORF1a, ORF1b, S, ORF3a, M, ORF7a, ORF8, N). Further analysis revealed that eight 2019-nCoV genomes contained 23 characteristic mutation sites belonging to the Delta (B.1.617.2·AY.2) variants of 2019-nCoV. Since the mutation sites among the eight 2019-nCoV genomes were not completely overlapped, and the epidemiological survey report showed that the freighter stopped at multiple ports and had personnel alternation, it was speculated that the clustered COVID-19 cases might have different origins. Phylogenetic analysis showed that all the eight 2019-nCoV genomes were on the AY.2 sub-lineage of the B. 1.617.2 lineage. This result was consistent with the result of Pangolin classification and mutation analysis.Conclusions:In this study, 8 whole genome sequences of Delta variants were obtained through NGS technology from the clustered COVID-19 cases which were imported from a freighter. The sequencing method and analysis result in this study could provide reference for the 2019-nCoV mutation analysis and tracing the source of the COVID-19 cases for the prevention and control of the COVID-19 epidemic.

Objective:To study the epidemiological and molecular characteristics of coxackievirus A2 (CV-A2) associated with hand, foot and mouth disease (HFMD) in Fujian province, 2011-2020.Methods:Descriptive epidemiological method was used to analyze the epidemiological characteristics of CV-A2. Case information was collected in the laboratory of Fujian Provincial Center for Disease Control and Prevention from 2011 to 2020. The VP1 gene obtained by first-generation sequencing and the complete genome obtained by second-generation sequencing were made phylogenetic analysis and recombination analysis by Mega X and Simplot3.5.1.Results:From 2011 to 2020, there were 35 HFMD-associated CV-A2 cases in Fujian province, most of whom aged 1 to 2 years (28 / 35), and the gender ratio of male to female was 2.5∶1 (25 / 10). Phylogenetic tree of VP1 region showed that CV-A2 in Fujian province was mainly distributed in the evolutionary branches of D1 and C1 subgenotype, only one strain (2019FJPT064) belonged to C1 subgenotype and the other 27 strains were grouped under D1 subgenotype. Among them, 4 isolates from 2011 to 2012 belonged to cluster 1 of D1 subgenotype, and the other two isolates from 2011 to 2012 and those after 2012 belonged to cluster 2 of D1 subgenotype. The complete genome length of 6 CV-A2 strains were about 7 393 to 7 402 bp. The homology of the nucleic acid sequences in the VP1 region of 6 CV-A2 strains and the CV-A2 prototype strain were 80.3%-81.7%, while in the other regions were 72.7%-86.2%. The phylogenetic tree of P2 region showed that 6 CV-A2 strains had a close evolutionary distance with the coxackievirus A4 (CV-A4) and the phylogenetic tree of P3 region showed that 2011FJFZ027, 2012FJQZ311 and 2020FJPTN040 had close evolutionary distance with coxackievirus A6 (CV-A6), coxackieviru A14 (CV-A14), coxackievirus A8 (CV-A8), respectively. Recombination analysis showed that 2011FJFZ027, 2012FJQZ311 and 2020FJPTN040 maybe evolved from recombinant CV-A6 strain (GenBank accession number: KR706309), CV-A14 strain (GenBank accession number: KP036482) and CV-A8 strains (GenBank accession number: KM609475 and MT648786) in non-structural protein region.Conclusions:HFMD-associated CV-A2 was sporadic in Fujian province from 2011 to 2020. The D1 subgenotype of CV-A2 was persistently circulating in Fujian province. From 2011 to 2012, the cluster 1 of D1 subgenotype was the dominant type, and then it shifted to the cluster 2 to take its place after 2012. CV-A2 strains in Fujian were frequently recombined in diversity patterns.

Objective:To investigate the gene characteristics and recombination of coxsackievirus A5(CV-A5)in Fujian province.Methods:The VP1 regions of CV-A5 strains from positive samples of hand, foot and mouth disease (HFMD) collected from all cities in Fujian province from 2011 to 2020 were amplified by reverse transcription nested polymerase chain reaction (RT-nPCR), then sequenced and identified the subgenotypes. CV-A5 strains were isolated in rhabdomyosarcoma (RD) cells. The isolated virus strains were sequenced by next generation sequencing to obtain the whole genome sequences. MEGA7.0 was used for phylogenetic analysis. RDP4 and SimPlot 3.5.1 softwares were used to analyze viral gene recombination.Results:Total thirty-one HFMD related CV-A5 cases were detected in Fujian province from 2011 to 2020, mainly in children aged 1 to 2 years (16/31), with a male/female ratio of 2.1∶1 (21/10), including 3 severe cases and 1 deceased case. CV-A5 subgenotyping was successful in 27 cases, all of them belonged to E1 subgenotype. The whole genome sequences of six CV-A5 strains were obtained, and the homology with the prototype strain Swartz was 81.1%-81.6%. Phylogenetic analysis results based on the sequences of whole genome, P1, P2 and P3 regions showed that the virus strain 2017FJFZ239 is a suspected recombinant strain, which was distributed in the same branch as CV-A5 reference strains (GenBank accession number: MW079817 and MN663160) in P2 and P3 regions. Analyzing with RDP4 showed that the virus strain 2017FJFZ239 had the same recombination event with CV-A5 reference strains (GenBank accession number: MW079817 and MN663160) and the potential parental sequences were CV-A2 (GenBank accession number: KX595284) and CV-A5 (GenBank accession number: KP289362). Further analyzing with SimPlot showed that the virus strain 2017FJFZ239 may recombine with CV-A2 strain (GenBank accession number: KX595284) in non-structural protein 2B, 2C and P3 regions.Conclusions:From 2011 to 2020, HFMD related CV-A5 in Fujian province belongs to E1 subgenotype, and there is a recombinant strain.