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BACKGROUND: We investigated the presence of Chlamydia psittaci in poultry and the environment in live poultry wholesale markets in Changsha during 2021-2022 and conducted a phylogenetic analysis to understand its distribution in this market. METHODS: In total, 483 samples were analyzed using real-time polymerase chain reaction and 17 C. psittaci-positive samples using high-throughput sequencing, BLAST similarity, and phylogenetic analysis. RESULTS: Twenty-two out of 483 poultry and environmental samples were positive for C. psittaci (overall positivity rate: 4.55%) with no difference in positivity rates over 12 months. Chlamydia psittaci was detected at 11 sampling points (overall positivity rate: 27.5%), including chicken, duck, and pigeon/chicken/duck/goose shops, with pigeon shops having the highest positivity rate (46.67%). The highest positivity rates were found in sewage (12.5%), poultry fecal (7.43%), cage swab (6.59%), avian pharyngeal/cloacal swab (3.33%), and air (2.29%) samples. The ompA sequences were identified in two strains of C. psittaci, which were determined to bear genotype B using phylogenetic analysis. Thus, during monitoring, C. psittaci genotype B was detected in the poultry and environmental samples from the poultry wholesale market in Changsha. CONCLUSIONS: To address the potential zoonotic threat, C. psittaci monitoring programs in live poultry markets should be enhanced.

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@#Abstract: Objective To analysis the genetic evolution characteristics of hemagglutinin (HA) gene of influenza A(H1N1)pdm virus in Changsha City from 2016-2023, to understand the trend of the HA genetic evolution and the mutations of the amino acid. It provides a scientific basis for the prevention and control of influenza epidemics, as well as the screening of vaccines under the new situation. Methods The A(H1N1)pdm09 virus strains from Changsha City from 2016 to 2023 were isolated using SPF chicken embryos, and then the HA genes were sequenced by MiSeq of Illumina Inc. The homology of HA gene was analyzed by MegAlign of the DNASTAR, and the phylogenetic tree was constructed using the Neighbor Joining (NJ) method in the Molecular Evolutionary Genetics Analysis version 11 (MEGA11). Results The homology of the HA gene of A(H1N1)pdm virus in Changsha from 2016 to 2023 was between 94.8%-99.9%, with the HA gene homology decreasing annually. The homology between the isolated strains of A(H1N1)pdm09 in Changsha City from 2016 to 2023 and the WHO recommended vaccine strain ranged from 96.8% to 99.0%, indicating a relatively good match between the flu isolates and the recommended vaccine strain. The phylogenetic tree of the HA gene of the A(H1N1)pdm09 influenza virus in Changsha City showed that the HA gene evolved into several different branches within the 6B branch, and it had currently evolved to 6B.1A.5a.2a branch. Constant mutations had occurred at the amino acid sites of the four antigenic determinant clusters of HA protein. Currently, amino acid mutations had occurred at 15 antigenic sites within the four antigenic determinant clusters, and the newly emerged A186T antigen mutant site in the isolates from 2023 was worth recent notice. The receptor-binding sites are relatively conserved in loop 130, minor amino acid mutations occurred in loop 220, whether the amino acid mutation site in loop 190 is becoming more stable needs to be further monitored. Taking A/California/07/2009 (CY121680) as the reference strain, most of the A(H1N1) pdm09 isolates in Changsha was increased 162 NQTY glycosylation site and was decreased 276 NTTC glycosylation site, and the glycosylation mutations at these two sites have become more stable recently. Conclusions The HA genes of influenza A(H1N1)pdm virus in Changsha are constantly evolving and mutating, suggesting influenza surveillance should be strengthened continuously.

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BACKGROUND: The H3N8 avian influenza virus (AIV) has been circulating in wild birds, with occasional interspecies transmission to mammals. The first human infection of H3N8 subtype occurred in Henan Province, China, in April, 2022. We aimed to investigate clinical, epidemiological, and virological data related to a second case identified soon afterwards in Hunan Province, China. METHODS: We analysed clinical, epidemiological, and virological data for a 5-year-old boy diagnosed with H3N8 AIV infection in May, 2022, during influenza-like illness surveillance in Changsha City, Hunan Province, China. H3N8 virus strains from chicken flocks from January, 2021, to April, 2022, were retrospectively investigated in China. The genomes of the viruses were sequenced for phylogenetic analysis of all the eight gene segments. We evaluated the receptor-binding properties of the H3N8 viruses by using a solid-phase binding assay. We used sequence alignment and homology-modelling methods to study the effect of specific mutations on the human receptor-binding properties. We also conducted serological surveillance to detect the H3N8 infections among poultry workers in the two provinces with H3N8 cases. FINDINGS: The clinical symptoms of the patient were mild, including fever, sore throat, chills, and a runny nose. The patient's fever subsided on the same day of hospitalisation, and these symptoms disappeared 7 days later, presenting mild influenza symptoms, with no pneumonia. An H3N8 virus was isolated from the patient's throat swab specimen. The novel H3N8 virus causing human infection was first detected in a chicken farm in Guangdong Province in December, 2021, and subsequently emerged in several provinces. Sequence analyses revealed the novel H3N8 AIVs originated from multiple reassortment events. The haemagglutinin gene could have originated from H3Ny AIVs of duck origin. The neuraminidase gene belongs to North American lineage, and might have originated in Alaska (USA) and been transferred by migratory birds along the east Asian flyway. The six internal genes had originated from G57 genotype H9N2 AIVs that were endemic in chicken flocks. Reassortment events might have occurred in domestic ducks or chickens in the Pearl River Delta area in southern China. The novel H3N8 viruses possess the ability to bind to both avian-type and human-type sialic acid receptors, which pose a threat to human health. No poultry worker in our study was positive for antibodies against the H3N8 virus. INTERPRETATION: The novel H3N8 virus that caused human infection had originated from chickens, a typical spillover. The virus is a triple reassortment strain with the Eurasian avian H3 gene, North American avian N8 gene, and dynamic internal genes of the H9N2 viruses. The virus already possesses binding ability to human-type receptors, though the risk of the H3N8 virus infection in humans was low, and the cases are rare and sporadic at present. Considering the pandemic potential, comprehensive surveillance of the H3N8 virus in poultry flocks and the environment is imperative, and poultry-to-human transmission should be closely monitored. FUNDING: National Natural Science Foundation of China, National Key Research and Development Program of China, Strategic Priority Research Program of the Chinese Academy of Sciences, Hunan Provincial Innovative Construction Special Fund: Emergency response to COVID-19 outbreak, Scientific Research Fund of Hunan Provincial Health Department, and the Hunan Provincial Health Commission Foundation.

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