H9N2 avian influenza viruses are widely prevalent in birds and pose an increasing threat to humans because of their enhanced virulence and transmissibility in mammals. Active surveillance on the prevalence and evolution of H9N2 viruses in different avian hosts will help develop eradication measures. We isolated 16 H9N2 viruses from chickens, green peafowls, and wild birds in eastern China from 2017 to 2019 and characterized their comparative genetic evolution, receptor-binding specificity, antigenic diversity, replication, and transmission in chickens and mice. The phylogenetic analysis indicated that the green peafowl viruses and swan reassortant shared the same ancestor with the poultry H9N2 viruses prevalent in eastern China, while the seven wild bird viruses belonged to wild bird lineage. The chicken, peafowl, and swan H9N2 viruses that belonged to the poultry lineage preferentially recognized α-2, 6-linked sialic acids (human-like receptor), but the wild bird lineage viruses can bind both α-2, 3 (avian-like receptor) and human-like receptor similarly. Interestingly, the H9N2 viruses of poultry lineage replicated well and transmitted efficiently, but the viruses of wild bird lineage replicated and transmitted with low efficiency. Importantly, the H9N2 viruses of poultry lineage replicated in higher titer in mammal cells and mice than the viruses of wild birds lineage. Altogether, our study indicates that co-circulation of the H9N2 viruses in poultry, wild birds, and ornamental birds increased their cross-transmission risk in different birds because of their widespread dissemination.

Chicken products and chickens with colibacillosis are often reported to be a suspected source of extraintestinal pathogenic Escherichia coli (ExPEC) causing several diseases in humans. Such pathogens in healthy chickens can also contaminate chicken carcasses at the slaughter and then are transmitted to humans via food supply; however, reports about the ExPEC in healthy chickens are still rare. In this study, we determined the prevalence and characteristics of ExPEC isolates in healthy chickens in China. A total of 926 E. coli isolates from seven layer farms (371 isolates), one white-feather broiler farm (78 isolates) and 17 live poultry markets (477 isolates from yellow-feather broilers) in 10 cities in China, were isolated and analyzed for antibiotic resistance phenotypes and genotypes. The molecular detection of ExPEC among these healthy chicken E. coli isolates was performed by PCRs, and the serogroups and antibiotic resistance characteristics of ExPEC were also analyzed. Pulsed-field gel electrophoresis (PFGE) and Multilocus sequence typing (MLST) were used to analyze the genetic relatedness of these ExPEC isolates. We found that the resistance rate for each of the 15 antimicrobials tested among E. coli from white-feather broilers was significantly higher than that from brown-egg layers and that from yellow-feather broilers in live poultry markets (p < 0.05). A total of 22 of the 926 E. coli isolates (2.4%) from healthy chickens were qualified as ExPEC, and the detection rate (7.7%, 6/78) of ExPEC among white-feather broilers was significantly higher than that (1.6%, 6/371) from brown-egg layers and that (2.1%, 10/477) from yellow-feather broilers (p < 0.05). PFGE and MLST analysis indicated that clonal dissemination of these ExPEC isolates was unlikely. Serogroup O78 was the most predominant type among the six serogroups identified in this study, and all the six serogroups had been frequently reported in human ExPEC isolates in many countries. All the 22 ExPEC isolates were multidrug-resistant (MDR) and the resistance rates to ampicillin (100%) and sulfamethoxazole-trimethoprim (100%) were the highest, followed by tetracycline (95.5%) and doxycycline (90.9%). bla_CTX-M was found in 15 of the 22 ExPEC isolates including 10 harboring additional fosfomycin resistance gene fosA3. Notably, plasmid-borne colistin resistance gene mcr-1 was identified in six ExPEC isolates in this study. Worryingly, two ExPEC isolates were found to carry both mcr-1 and bla_NDM, compromising both the efficacies of carbapenems and colistin. The presence of ExPEC isolates in healthy chickens, especially those carrying mcr-1 and/or bla_NDM, is alarming and will pose a threat to the health of consumers. To our knowledge, this is the first report of mcr-1-positive ExPEC isolates harboring bla_NDM from healthy chickens.

Identification of the pathogenic mechanism of highly pathogenic avian influenza viruses in mammals is helpful to develop novel anti-influenza virus strategies. Two amino acid mutations (D30N and A215T) in M1 were found to collectively attenuate H5N1 influenza viruses in mice, but the underlying mechanism remained unknown.

The emerging avian influenza reassortants and mutants in birds pose an increasing threat to poultry and public health. H10 avian influenza viruses are widely prevalent in wild birds, poultry, seals, and minks and pose an increasing threat to human health.