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Zunyi (遵义; Zūnyì) is a city in northern Guizhou province, China. around the Jiangxi Soviet, the CCP and Red Army fled west across Hunan and into Guizhou. Xiangshan Temple: This temple dating from the s is the largest in Zunyi.
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Due to LPMs as a transmission source and even potential incubator for human infections with AIV 28 , 29 , more and more provinces have started to close LPMs 30 , 31 , 32 or take special measures at the human-animal interface to lower the risks of human infection. Similar strategies have since been implemented in other Chinese provinces. Since , the dominant AIV subtypes have substantially changed. Remarkably, despite the widespread circulation of H7N9 AIVs during —17 23 , it almost disappeared in However, these results highlight the distinct change of the dominant AIV subtypes in China and will have a profound influence on prevention strategies against AIVs, including vaccine development and usage.

Including the present study, the existence of impure isolates with different subtypes has also been reported in many studies 36 , In fact, most of the other rare subtypes with diverse genetic constellations were also found in ducks.


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This may be partly due to the more contacts between domestic duck and wild waterfowl, which was considered as the natural reservoir of AIVs. Therefore, the probability of emergence of novel AIVs by reassortment among the diverse genetic constellations may likely be higher in ducks, not only because of the high diversity of AIV genetic constellation in ducks but also the excellent genetic compatibilities among H9N2 and other influenza subtypes including H7N9, pandemic H1N1, H5N1, H5N6, and so on 13 , 16 , 41 , 42 , Therefore, several AIV subtypes potentially infecting humans were circulating in LPMs and intensive surveillance of AIVs particularly among ducks should be performed continuously.

Receptor binding was considered as the first step of influenza infection to host cells 44 , 45 , 46 , All the sequenced H5N6 strains had Q, while some tested strains presented slight preference for human-type receptor, which could be partly explained by the loss of a glycosylation site at the positions — 52 , 53 , However, H6N6 strains with E and G were also found to bind to both receptors.

Taken together, our receptor-binding tests highlight that only few AIV strains showed pure binding abilities to avian-type receptors, whereas the majority presented human-type receptor-binding capacity, particularly the dominant H9 AIVs. Therefore, despite lower positive rate in LPMs, AIVs showed increased abilities and risk to infect humans, which deserves closer attention. Therefore, constant monitoring on AIVs should be more closely conducted for agricultural and public health.

Oropharyngeal and cloacal swabs from apparently healthy poultry, as well as environmental samples, were collected in LPMs in 37 cities and counties across 23 provinces or municipalities or minority municipalities in China. Poultry included chickens, ducks, geese, and pigeons. Environmental samples included swabs from cages, poultry drinking water, defeathering machines, chopping boards, and feces in the LPMs.

Sampling was collected from May to February samples collected once a month, unless the LPM was closed, and there were no samples collected in the corresponding month , a period of 26 months spanning three flu seasons.


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Furthermore, sampling was also performed in several additional provincial level administrative regions, including Xinjiang, Xizang, Qinghai, Guizhou, Hainan, Shaanxi, Shanxi, Ningxia, and Chongqing. Avian influenza viruses were isolated in day-old specific pathogen-free SPF chicken embryos according to the WHO manual Next, the sequencing libraries were prepared.

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The AIV positive samples are the cultured samples including both pure and impure isolates. Repetitive sequences in the two databases were removed by matching strain names using Bioedit version 7. Only full-length genomes were kept and sequence with obvious errors e. The remaining sequences were combined with those generated in the present study, and the sequences of H9Ny, H5Ny, H7Ny, and H6Ny isolates were phylogenetically analyzed.

Multiple sequence alignment was performed using Muscle version 3. Trees were visualized using FigTree version 1. Coveralls, gloves, and N95 masks were used during the working in BSL-2 labs, and all wastes were autoclaved. First, during the sample collection process, all the tubes with samples were placed into different cells in sample box.

In fact, samples from the same site were detected as soon as possible after collection by month, and usually no more than samples were identified in each experiment. Fifth, all the experiments associated with original and cultured samples, as well as RNAs sample handling, virus isolation, PCR system preparation, and NGS library preparation , were performed in biosafety cabinets with tweezers and tips with filters.

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