Epidemiological survey and genetic evolution of H3N2 subtype influenza viruses from stray dogs in Shanghai, China.

An avian-origin canine influenza virus (CIV) has recently emerged in dogs and is spreading in China. Given that humans have frequent contact with dogs, this has prompted an increased emphasis on biosafety. In this study, we collected 693 nasal swab samples and 800 blood samples from stray dogs in animal shelters to survey canine influenza epidemiology and characterize the evolution of CIV H3N2 in Shanghai. We tested samples for canine influenza antibodies and canine influenza RNA in January-May, 2019, and the results showed that the positive rate was 17.62% by ELISA, 15.75% by microneutralization (MN) assay, and 18.51% by real time RT-PCR, respectively. We also performed phylogenetic and genomic analysis on six H3N2 CIV isolates. The H3N2 viruses which prevailed in Shanghai originated from Beijing and Jiangsu isolates. Phylogenetic analysis showed that the sequences of CIV isolates have multiple amino acid antigenic drifts, deletions, and substitutions. The time of the most recent common ancestor (TMRCA) of HA and NA was 2004 and 2005, respectively. Notably, the substitution, 146S, in hemagglutinin and the deletion in the neuraminidase (NA) stalk region we found in this study warrant attention because they have frequently been identified in human influenza viruses. The potential adaptation of this CIV H3N2 clade to mammals and its public health threat should be further evaluated.

Epidemiological survey and genetic evolution of H9 subtype influenza viruses in Shanghai, China, from 2006 to 2010.

The H9N2 influenza virus is endemic in poultry. We report its occurrence in live-poultry markets, fair-trade markets and poultry farms in the Shanghai region between September 2006 and December 2010. An analysis of partial sequences of the HA, NA, PB1, PB2 and NP genes of eleven distinct H9N2 isolates revealed that all carried an RSSR motif at the cleavage site of HA, diagnostic of low pathogenicity in chickens. A phylogenetic analysis indicated that these isolates are derived from the lineage represented by Duck/HK/Y280/97, but they have evolved a range of reassortments. Their PB1 and NP sequences resembled those of H5N1 strains, indicating a hybrid origin involving both H9 and H5 strains. The HA and NA sequences present in all eleven isolates resembled those of the Duck/HK/Y280/97-like lineage. Infection by H9N2 is commonplace in Shanghai live-poultry markets, allowing the viruses to have evolved rapidly.

Genetic evolution of H9 subtype influenza viruses from live poultry markets in Shanghai, China.

H9N2 influenza viruses have become established and maintain long-term endemicity in poultry. The complete genomes of seven avian H9N2 influenza viruses were characterized. These seven influenza virus isolates were obtained from live poultry markets in Shanghai, China, in 2002 and from 2006 to 2008. Genetic analysis revealed that all seven isolates had an RSSR motif at the cleavage site of hemagglutinin (HA), indicating low pathogenicity in chickens. Phylogenetic analyses indicated that the seven avian H9N2 viruses belonged to the lineage represented by Duck/Hong Kong/Y280/97 (H9N2), a virus belonging to the Chicken/Beijing/1/94-like (H9N2) lineage, and that they are all quadruple reassortants consisting of genes from different lineages. The six internal genes of the isolates possessed H5N1-like sequences, indicating that they were reassortants of H9 and H5 viruses. All of the viruses had nonstructural (as well as HA and neuraminidase) genes derived from the Duck/Hong Kong/Y280/97-like virus lineage but also had other genes of mixed avian virus origin, including genes similar to those of H5N1 viruses (Gs/GD-like). The infected chickens showed no signs of disease. These results show the genetic and biological diversity of H9N2 viruses in Shanghai and support their potential role as pandemic influenza agents.