Objective:To identify a novel reassortant H3N2 avian influenza virus using nanopore sequencing technology and analyze its genetic characteristics.Methods:The positive samples of the H3N2 avian influenza virus, collected from the external environment in the farmers' market of Guangzhou, were cultured in chicken embryos. The whole genome was sequenced by targeted amplification and nanopore sequencing technology. The genetic characteristics were analyzed using bioinformatics software.Results:The phylogenetic trees showed that each gene fragment of the strain belonged to the Eurasian evolutionary branch, and the host source was of avian origin. The HA gene was closely related to the origin of the H3N6 virus. The NA gene was closely related to the H3N2 avian influenza virus from 2017 to 2020. The PB1 gene was closely related to the H5N6 avian influenza virus in Guangxi Zhuang Autonomous Region and Fujian Province from 2016 to 2022 and was not related to the PB1 gene of the H5N6 avian influenza epidemic strain in Guangzhou. The other internal gene fragments had complex sources with significant genetic diversity. Molecular characteristics indicated that the strain exhibited the molecular characteristics of a typical low pathogenic avian influenza virus and tended to bind to the receptors of avian origin. On important protein sites related to biological characteristics, this strain had mutations of PB2-L89V, PB1-L473V, NP-A184K, M1-N30D/T215A, and NS1-P42S/N205S.Conclusions:This study identified a novel reassortant H3N2 avian influenza virus by nanopore sequencing, with the PB1 gene derived from the H5N6 avian influenza virus. The virus had a low ability to spread across species, but further exploration was needed to determine whether its pathogenicity to the host was affected.

To monitor and analyze the molecular variation of the H3N2 influenza virus in Guangzhou during the COVID-19 pandemic, respiratory samples of influenza-like cases from influenza monitoring sentinel hospitals were collected from influenza monitoring sentinel hospitals for virus isolation and whole genome sequencing. The results showed that during COVID-19, there was only one peak of H3N2 influenza in the second quarter of 2022 in Guangzhou (the positive rate was 52.23%), and the epidemic intensity and duration were both higher than those in 2019. The HA gene and NA gene of the epidemic strain in Guangzhou in 2022 belonged to the 3C.2a1b. 2a. 1a. 1 branch, which had a good antigenic site matching with the vaccine strain (A/Cambodia/e0826360/2020) from 2021 to 2022 and had no antigen drift. In 2022 strains, the variation of antigen determinant mainly occurred in the I48T of C region, while no variation occurred in the A, B, D, and E regions. The binding site of the HA protein receptor was consistent with the vaccine strain (A/Cambodia/e0826360/2020). Most of the strains in 2022 carried 13 glycosylation sites on the HA protein, but an outbreak of strains caused a loss of glycosylation sites at 24-NST. In conclusion, the strains that caused the epidemic of H3N2 influenza in Guangzhou in 2022 were not evolved or transmitted from the local strains in 2019 during the COVID-19 pandemic.

Objective:To establish a rapid pipeline for whole genome sequencing of Chikungunya virus (CHIKV) by combining imbricated multiplex-PCR amplification and Illumina high-throughput sequencing platform.Methods:The primary reference sequences of CHIKV were downloaded from the National Center for Biotechnology Information (NCBI) database, covering all genotypes of CHIKV. After multiple alignments using the Mafft software and phylogenetic analysis, the 20 CHIKV references were selected for primer design. The Primal Scheme tool and Geneious Prime software were used to design, evaluate and optimize the primer panel. Finally, seven CHIKV-positive samples were involved in the validation of the primer panel.Results:All the amplicons of the designed panel were generated successfully. The consensuses generated from the mapping results could cover 100.00% of the coding region of the CHIKV genome when the Ct-value of the sample was less than 33, as the percentage would decrease to 99.38% when the Ct-value reached 35. The mapping percentage could be increased by 5.70%-25.43% when using the stepwise correction mapping strategy.Conclusion:The multiplex-PCR amplification method for CHIKV whole genome sequencing is relatively simple and convenient, which only requires two tubes of PCR amplification and performs well on CHIKV-positive clinical samples with different concentration levels of virus.

To monitor and analyze the molecular variation of the H3N2 influenza virus in Guangzhou during the COVID-19 pandemic, respiratory samples of influenza-like cases from influenza monitoring sentinel hospitals were collected from influenza monitoring sentinel hospitals for virus isolation and whole genome sequencing. The results showed that during COVID-19, there was only one peak of H3N2 influenza in the second quarter of 2022 in Guangzhou (the positive rate was 52.23%), and the epidemic intensity and duration were both higher than those in 2019. The HA gene and NA gene of the epidemic strain in Guangzhou in 2022 belonged to the 3C.2a1b. 2a. 1a. 1 branch, which had a good antigenic site matching with the vaccine strain (A/Cambodia/e0826360/2020) from 2021 to 2022 and had no antigen drift. In 2022 strains, the variation of antigen determinant mainly occurred in the I48T of C region, while no variation occurred in the A, B, D, and E regions. The binding site of the HA protein receptor was consistent with the vaccine strain (A/Cambodia/e0826360/2020). Most of the strains in 2022 carried 13 glycosylation sites on the HA protein, but an outbreak of strains caused a loss of glycosylation sites at 24-NST. In conclusion, the strains that caused the epidemic of H3N2 influenza in Guangzhou in 2022 were not evolved or transmitted from the local strains in 2019 during the COVID-19 pandemic.

Objective:To establish a rapid pipeline for whole genome sequencing of Chikungunya virus (CHIKV) by combining imbricated multiplex-PCR amplification and Illumina high-throughput sequencing platform.Methods:The primary reference sequences of CHIKV were downloaded from the National Center for Biotechnology Information (NCBI) database, covering all genotypes of CHIKV. After multiple alignments using the Mafft software and phylogenetic analysis, the 20 CHIKV references were selected for primer design. The Primal Scheme tool and Geneious Prime software were used to design, evaluate and optimize the primer panel. Finally, seven CHIKV-positive samples were involved in the validation of the primer panel.Results:All the amplicons of the designed panel were generated successfully. The consensuses generated from the mapping results could cover 100.00% of the coding region of the CHIKV genome when the Ct-value of the sample was less than 33, as the percentage would decrease to 99.38% when the Ct-value reached 35. The mapping percentage could be increased by 5.70%-25.43% when using the stepwise correction mapping strategy.Conclusion:The multiplex-PCR amplification method for CHIKV whole genome sequencing is relatively simple and convenient, which only requires two tubes of PCR amplification and performs well on CHIKV-positive clinical samples with different concentration levels of virus.

Objective:To isolate the influenza A (H3N2) viruses from different sources in Guangzhou in 2019 and analyze these viruses' evolution and variation characteristics.Methods:The hemagglutinin (HA) and neuraminidase (NA) genes of H3N2 isolates from outpatient monitoring, influenza outbreaks, and inpatient severe cases in Guangzhou in 2019 were sequenced. Bioinformatics software analyzed the variations and evolution characteristics of HA and NA genes.Results:The epidemic peaks of influenza A (H3N2) viruses were made up of period Ⅰ (from January to August) and period Ⅱ (from November to December). The positive rate of influenza A (H3N2) in males was 13.46% (703/5 221), which was higher than that in females (11.50%, 510/4 435) ( χ 2=8.43, P=0.00). The group's positive rate of 10-20 years old was the highest (25.18%,665/2 641). The isolates from different sources were highly homologous and closely related to 3C.2a.1 branches, which could be further divided into three small groups of Group 1-3. Gene recombination was observed between different branches. The mutations of HA antigen sites gradually appeared from Group 1 to Group 3, leading to new antigen drift. Variations of HA antigenic sites mainly occurred in the region of A and B. The mutations of receptor binding sites of Group 1 and Group 3 viruses occurred in the anterior and posterior walls. There were two glycosylation sites lacked on region A of HA antigen observed in the isolates of Group 2-3. Conclusions:Genetic variations of H3N2 influenza viruses in Guangzhou included gene mutations and gene recombination. Under the pressure of the vaccine, the evolution of viruses was rapid. Therefore, the monitoring of molecular-related epidemic characteristics of the H3N2 influenza virus was necessary.

Objective By analyzing the epidemic characteristics and related indicators of SARS (2003) and COVID-19(2020), to explore the reasons for the similarities and differences of the two epidemics, so as to provide reference for epidemic prevention and control. Methods The general situation, clinical classification, activity history, contact history, family members’ contact and incidence of the two infectious diseases in Guangzhou were collected and used to analyze the time characteristics, occupational characteristics, age characteristics and other key indicators of the two diseases, including the number of cases, composition ratio (%), mean, median, crude mortality, etc. Results A total of 1 072 cases of SARS (2003) were included in the study. 353 of which were severe cases with the incidence of 30.13%. 43 cases of death were reported with a mortality rate of 4.01%. The average age was 46 years old, and 26.31% of the cases were medical staff. The interval time between first report to continuous zero reports was 129 days. As to COVID-19 (2020), a total of 346 cases were included. 58 of which were severe cases with the incidence of 16.67%. One case of death was reported with a mortality rate of 0.29%. The average age was 38 years old, and no hospital infection among medical staff was reported. The interval time between first report to continuous zero reports was 35 days. Conclusions The prevention and control strategies for COVID-19 (2010) are more effective compared to that of SARS (2003), and the emergency response procedures are worth to be evaluated and summarized.

Objective:To investigate the epidemiological and clinical characteristics of a case infected with avian influenza A (H5N6) virus associated with exposure to aerosol and provide evidence for the prevention and control of human infection with avian influenza virus.Methods:Epidemiological investigation was conducted to identify the history of exposure, infection route, and disease progression. Real-time fluorescent quantitative RT-PCR was used to test the samples collected from the case, close contacts, environment and poultry market.Results:The case had no history of exposure to live poultry and poultry market. But before the onset the case had a history of exposure to the live poultry placed in a car with doors and windows closed. The samples collected from the case’s lower respiratory tract and the remaining frozen chicken meat were all influenza A (H5N6) virus positive.Conclusions:The source of infection was the live poultry, and the infection route might be the exposure to aerosol in a car with doors and windows closed, where the poultry were temporarily stored. It is necessary to promote centralized poultry slaughtering, cold chain distribution and fresh poultry sale, as well as strengthen health education and establish the concept of consuming fresh poultry.

Objective:To analyze the characteristics of spread and genetic evolution of H5 subtype avian influenza virus in Guangzhou from 2014 to 2019.Methods:H5 subtype virus was detected by fluorescence quantitative RT-PCR from the environmental samples in Guangzhou poultry markets. The genes of HA and NA of 48 isolates randomly selected were sequenced, including 46 isolates from environmental samples and 2 isolates from cases. The characteristics of molecular variation and genetic evolution were analyzed by using bioinformatics software.Results:A total of 1 094 strains of H5 subtype avian influenza virus were isolated from 52 284 samples (2.09%). All the strains belonged to Clade 2.3.4.4.C. NA gene belonged to H6N6 of Eurasian lineage. The cleavage sites of all the strains showed the characteristics of highly pathogenicity. Receptor binding sites were avian-derived receptors. However, mutations of S123P, S133A and T156A occurred, which implied that these strains could tend to bind to human receptors. There was an additional glycosylation site at 140 in strains isolated after 2017. The variation of antigen loci mainly occurred in B and E regions.Conclusions:H5 subtype avian influenza virus spread in Guangzhou from 2014 to 2019 with annual increased proportion of positive rate, and the sequencing results indicated that it belonged to Clade 2.3.4.4.C of H5N6 highly pathogenic virus, and genetic evolution and mutation continued, especially the common mutations which could enhance the binding capacity to human receptors. It is necessary to strengthen the surveillance.

Objective:To analyze the contagiousness and secondary attack rate of 2019 novel coronavirus in cluster epidemics in Guangzhou and provide evidence for the prevention and control of COVID-19.Methods:All the individuals identified to be infected with 2019-nCoV in Guangzhou, including confirmed cases and asymptomatic cases, were included and classified as imported cases and local cases. The first case of each cluster epidemic was defined as index case, and the number of subsequent infections was calculated to evaluate the contagiousness and secondary attack rate of 2019 novel coronavirus in the shortest incubation period of 1-3 days.Results:As of 18 February, 2020, a total of 349 cases of 2019-nCoV infection, including 339 confirmed cases (97.13 %) and 10 asymptomatic cases (2.87 %) were reported in Guangzhou. There were 68 clusters involving 217 2019-nCoV infection cases (210 confirmed cases and 7 asymptomatic cases). The median number of subsequent infections caused by an index case in a cluster epidemic was 3, among which 2 were confirmed cases and 1 was asymptomatic cases, respectively. The average number of contagiousness was 2.18 in shorted incubation period of 1-3 days (The average number of infected cases were 2.18 cases by the index case in a cluster epidemic), the average infection number in family members was 1.86, and the infection ratio of family member transmission was 85.32 % (1.86/2.18). The secondary attack rate in close contacts with shortest incubation period of 1-3 days was 17.12 %-18.99 %, the secondary attack rate in family members was 46.11 %-49.56 %. Conclusions:The cluster epidemic of COVID-19 in Guangzhou mainly occurred in families, the contagiousness was high. It is necessary to strengthen the prevention and control to reduce the community transmission of COVID-19.