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Waterfowl, such as ducks, are natural hosts for avian influenza viruses (AIVs) and act as a bridge for transmitting the virus to humans or susceptible chickens. Since 2013, chickens and ducks have been threatened by waterfowl-origin H5N6 subtype AIVs in China. Therefore, it is necessary to investigate the genetic evolution, transmission, and pathogenicity of these viruses. In this study, we determined the genetic characteristics, transmission, and pathogenicity of waterfowl-origin H5N6 viruses in southern China. The hemagglutinin (HA) genes of H5N6 viruses were classified into the MIX-like branch of clade 2.3.4.4h. The neuraminidase (NA) genes belonged to the Eurasian lineage. The PB1 genes were classified into MIX-like and VN 2014-like branches. The remaining five genes were clustered into the MIX-like branch. Therefore, these viruses belonged to different genotypes. The cleavage site of the HA proteins of these viruses was RERRRKR/G, a molecular characteristic of the H5 highly pathogenic AIV. The NA stalk of all H5N6 viruses contained 11 amino acid deletions at residues 58–68. All viruses contained 627E and 701D in the PB2 proteins, which were molecular characteristics of typical bird AIVs. Furthermore, this study showed that Q135 and S23 viruses could replicate systematically in chickens and ducks. They did not cause death in ducks but induced mild clinical signs in them. All the infected chickens showed severe clinical signs and died. These viruses were shed from the digestive and respiratory tracts and transmitted horizontally in chickens and ducks. Our results provide valuable information for preventing H5N6 avian influenza outbreaks.

Since 2017, the new H7N9 highly pathogenic avian influenza viruses (HPAIVs) have been responsible for more than 200,000 cases of chicken infection and more than 120,000 chicken deaths in China. Our previous study found that the Q26 was chicken-origin H7N9 HPAIV. In this study, we analyzed the genetic characterization of Q24, Q65, Q66, Q85, and Q102 H7N9 avian influenza viruses isolated from Guangdong, China in 2017. Our results showed that these viruses were highly pathogenic and belonged to two different genotypes, which suggested they occurred genetic reassortant. To investigate the pathogenicity, transmission, and host immune responses of H7N9 virus in chickens, we selected Q24 and Q26 viruses to inoculate chickens. The Q24 and Q26 viruses killed all inoculated chickens within 3 days and replicated effectively in all tested tissues. They were efficiently transmitted to contact chickens and killed them within 4 days through direct contact. Furthermore, we found that the expressions of several immune-related genes (e.g., TLR3, TLR7, MDA5, MAVS, IFN-β, IL-6, IL-8, OAS, Mx1, MHC I, and MHC II) were upregulated obviously in the lungs and spleen of chickens inoculated with the two H7N9 viruses at 24 h post-inoculation (HPI). Among these, IL-6 and IFN-β in lungs were the most upregulated (by 341.02–381.48-fold and 472.50–500.56-fold, respectively). These results suggest that the new H7N9 viruses isolated in 2017, can replicate and transmit effectively and trigger strong immune responses in chickens, which helps us understand the genetic and pathogenic variations of H7N9 HPAIVs in China.

The protein inhibitor of the activated STAT2 (PIAS2) has been implicated in many cellular processes and can also regulate viral replication in mammals. However, the role of PIAS2 in the highly pathogenic avian influenza virus (HPAIV) H5N1 replication in ducks is still unclear. Through liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay, we identified that duck PIAS2 (duPIAS2) was one protein that interacted with the nucleoprotein (NP) from the H5N1 HPAIV strain of DK212. Through confocal microscopy images and Co-IP assay, we confirmed NP could interact with duPIAS2. Overexpression of duPIAS2 in primary duck embryo fibroblast (DEF) cells was shown to promote DK212 replication, and knockdown of duPIAS2 could repress DK212 replication. We further found duPIAS2 could promote NP SUMOylation through duck SUMO1 (duSUMO1), and the potential SUMOylation sites of NP were at lysines 7, 48, and 87. Furthermore, duPIAS2 promoted the replication of DK212, here relying on the activity of its SUMO E3 ligase. Duck SENP1 (duSENP1), a deSUMOylation enzyme, could repress NP SUMOylation and also inhibit DK212 replication. Together, we identified duPIAS2 could interact with NP and that duPIAS2 promoted H5N1 HPAIV replication, which might be related to NP SUMOylation.

Clade 2.3.4.4 H5 avian influenza viruses (AIVs) are widely prevalent and of significant concern to the poultry industry and public health in China. Nowadays, the clade 2.3.4.4 H5N6 virus has become a dominant AIV subtype among domestic ducks in southern China. We found that waterfowl-origin clade 2.3.4.4 H5N6 viruses (A/goose/Guangdong/16568/2016, GS16568 and A/duck/Guangdong/16873/2016, DK16873) isolated from southern China in 2016 could replicate in multiple organs of inoculated ducks. DK16873 virus caused mild infections and killed 2/5 of inoculated ducks, and GS16568 virus did not kill inoculated ducks. In addition, the two viruses could be transmitted via direct contact between ducks. DK16873 and GS16568 viruses killed 2/5 and 1/5 of contact ducks, respectively. Furthermore, ducks inoculated with the two H5N6 viruses exhibited different expressions of immune-related genes in their lungs. The expression of RIG-I, TLR3 and IL6 was significantly upregulated at 12 h post-inoculation (HPI) and most of the tested immune-related genes were significantly upregulated at 3 days post-inoculation (DPI). Notably, the expression of RIG-I and IL-6 in response to DK16873 virus was significantly higher than for GS16568 virus at 12 HPI and 3 DPI. Our research have provided helpful information about the pathogenicity, transmission and immune-related genes expression in ducks infected with new H5N6 AIVs.

H5Nx viruses have continuously emerged in the world, causing poultry industry losses and posing a potential public health risk. Here, we studied the phylogeny, pathogenicity, transmission, and immune response of four H5N6 avian influenza viruses in chickens and mice, which were isolated from waterfowl between 2013 and 2014. Their HA genes belong to Clade 2.3.4.4, circulated in China since 2008. Their NA genes fall into N6-like/Eurasian sublineage. Their internal genes originated from different H5N1 viruses. The results suggested that the four H5N6 viruses were reassortants of the H5N1 and H6N6 viruses. They cause lethal infection with high transmission capability in chickens. They also cause mild to severe pathogenicity in mice and can spread to the brain through the blood−brain barrier. During the infection, the viruses result in the up-regulation of PRRs and cytokine in brains and lungs of chickens and mice. Our results suggested that the high viral loads of several organs may result in disease severity in chickens and mice; there were varying levels of cytokines induced by the H5N6 viruses with different pathogenicity in chickens and mice.

There were five outbreaks of H7N9 influenza virus in humans in China since it emerged in 2013, infecting >1000 people. The H7N9 low pathogenic influenza virus was inserted into four amino acids in the HA protein cleavage site to mutate into the H7N9 highly pathogenic virus. This emerging virus caused 15 outbreaks in chickens from the end of 2016 to date. Two H7N9 avian influenza virus (AIV) strains, A/chicken/Guangdong/A46/2013 (LPAIV) and A/chicken/Guangdong/Q29/2017 (HPAIV), were selected to compare the pathogenicity and transmissibility between H7N9 LPAIVs and HPAIVs in chickens. We inoculated 3- to 4-week-old specific-pathogen-free (SPF) chickens with 6 log10EID50/0.1 mL viruses via the ocular-nasal route and co-housed four chickens in each group. The inoculated chicken mortality rate in the A46 and Q29 groups was 1/5 and 5/5, respectively. Q29 virus replication was more efficient compared to the A46 virus in inoculated chickens. Infected chickens initiated viral shedding to naïve contact chickens through respiratory and digestive routes. Both viruses transmitted between chickens by naïve contact, but the Q29 virus had a higher pathogenicity in contact chickens than the A46 virus. Compared with early H7N9 LPAIVs, the pathogenicity and transmissibility of the emerging H7N9 HPAIV was stronger in chickens, indicating that H7N9 influenza virus may continue to threaten human and poultry health.

Highly pathogenic avian influenza H5N6 viruses have been circulating in poultry in Asia since 2013 and producing serious diseases in chickens. Here, we analyzed the genetic properties of 10 H5N6 subtypes AIVs from geese in 2015−2016 in Guangdong province. Phylogenic analysis showed that all HA genes of the 10 viruses belonged to clade 2.3.4.4, and their genes including HA, PA, PB1, M, NP, and NS all derived from Mix-like 1 (CH, VN, LS). Their PB2 genes come from Mix-like 2 (CH, VN, JP). The NA genes were classified into a Eurasian lineage. Therefore, the 10 viruses likely originate from the same ancestor and were all recombinant viruses between different genotypes. We selected A/Goose/Guangdong/GS144/2015(H5N6) (GS144) and A/Goose/Guangdong/GS148/2016(H5N6) (GS148) viruses to inoculate 5-week-old chickens intranasally with 104 EID50/0.1 mL dose intranasally to assess their pathogenicity and transmissibility. Inoculated chickens showed that the GS144 virus caused systematic infection with a lethality of 100%, but the lethality of GS148 virus was 0%. The two viruses were efficiently transmitted to contact chickens. The lethality of GS144 and GS148 virus in contact with chickens was 87.5% and 0%, respectively, which suggests that the transmissibility of GS144 virus was stronger than GS148 virus in chickens. Thus, different H5N6 viruses from the same waterfowl can show different pathogenicity and transmissibility in chickens. Continued surveillance and characteristic analysis of the H5N6 viruses will help us to keep abreast of evolution and variation in avian influenza viruses in the future.