Isolation And Characterization And Pathogenicity Analysis Of An H6N6 Subtype Avian Influenza Virus Isolated From Ducks

Avian influenza virus(AIV) is a member of the orthomyxovirus family that can cause the infection of avian species and most of the mammals.. According to the antigenic differences between HA and NA, AIV can be divided into 18 HA subtype(H1-H18) and 11 NA subtype(N1-N11). Waterfowl is considered to be a huge natural reservoir for AIV, and almost all subtypes of AIV can be isolated from the waterfowls. Studies showed that the H6 subtype AIV has the highest isolation rate among the low pathogenic AIV in recent years.However, recent studies have found that human H5N6 infection is mainly caused by the genetic rearrangements between H5N1 and H6N6 viruses. Therefore, It is important to strengthen the monitor and analysis of H6 subtype AIV. In this study, an H6N6 subtype AIV isolated from apparently healthy Sansui Sheldrake ducks in November 2014 was used to study its genetic variation and pathogenicity. We aimed at providing reliable data for the epidemiology and genetic evolution of H6 subtype AIV in ducks.1. Isolation and characterization and genetic evolution analysis of H6N6 subtype AIVTo investigate the epidemiology and genetic evolution of H6 subtype AIV in Guizhou province, an H6N6 subtype AIV named A/duck/Guizhou/013/2014(H6N6) was isolated from apparently healthy Sansui Sheldrake ducks through chicken embryo inoculation, serology and RT-PCR method. The biological characteristics, full-length genome and the genetic evolution of this isolate was studied. The results showed that the MDT and ICPI of the isolate were 98.4hrs and 0.415, respectively, and the cleavage site of HA was 339PQIETRG345, which was the typical characteristic of low pathogenic AIV. Sequence alignment showed that the HA gene had the nucleotide identity of 97.1%with A/chicken/Hunan/S4191/2009(H6N2), and the NA gene showed 97% nucleotide similarity with A/duck/Eastern China/48/2010(H6N6). However,the PB2 gene displayed high similarity with A/duck/Yamagata/061004/2014(H6N6), the Mand NP gene showed close relationship with A/duck/Jiangsu/4/2010(H3N6) and A/duck/Hokkaido/Vac-1/04(H5N1), respectively. In conclusion, these results demonstrated that the H6N6 AIV isolate was a low pathogenic AIV and was possibly a reassortant virus derived from H6N2, H3N6 and H5N1 subtype AIV.2. Pathogenicity analysis of H6N6 subtype AIV in ducksIn order to understand the pathogenicity of duck origin H6N6 subtype AIV to ducks, 363-week-old non-immunized ducks were randomly divided into two groups: the experimental group was infected with the H6N6 virus at a dose of 107.0 EID50/0.1 mL, and the control group received 0.1 mL PBS. The laryngeal and cloacal swabs and ducks organs including heart, liver,spleen, lung, kidney, brain, bursa of Fabricius were collected at 1, 3, 5, 7, 9 and 11 days post-infection. In addition, quantitative Real-time RT-PCR and pathological test were used to detect the virus load in the tissue organs and observe the histopathological lesions. The results showed that the infected ducks had no obvious clinical symptoms. Viruses were not detected in the laryngeal and cloacal swabs, but could be detected with low virus load in some parts of the tissues on 3, 5 and 7 days with the exception of high virus load in lungs on the 3 day.Histopathological experiments found that the tissues had no obvious histopathological changes except the lungs with small amount of inflammatory cells infiltration. Together, these results demonstrated that the pathogenicity and replication ability of H6N6 subtype AIV in ducks were low.3. The effect of H6N6 infection on the transcriptional level of cytokines in duck immune organsTo understand the effect of H6N6 AIV infection on the transcriptional level of IL-2,IL-10 and IFN-? in duck immune organs, real-time RT-PCR assay was used to evaluate the dynamic change of mRNA expression of IL-2, IL-10 and IFN-? in virus-infected duck immune organs. The results showed that the established real-time RT-PCR method could be successfully used to detect the mRNA expression level of duck of IL-2, IL-10 and IFN-?. IL-2and IL-10 in duck spleen, thymus and bursa and IFN-? in duck spleen and bursa all increased firstly and then decreased at 1, 3 and 5 days post-infection. However, the mRNA expression of IFN-a in duck thymus showed downward trend. Moreover, the cytokines in different immune organs had different expression levels at 3 days post-infection, showing that thehighest expression level in duck spleen, thymus and bursa was IFN-?, IL-10 and IL-2,respectively. These results demonstrated that H6N6 AIV infection had some certain effects on the transcriptional level of IL-2, IL-10 and IFN-? in duck immune organs.In summary:The isolated H6N6 subtype AIV was a low pathogenic virus and might be a gene rearrangement virus derived from H6N2, H3N6 and H5N1. Ducks infected with the isolate had no obvious clinical symptoms and histopathological changes. Virus could not be detected in the throat and cloaca of infected ducks and could cause slight damage to immune organs accompanied by some certain effects on the transcriptional levels of duck IL-2, IL-10 and IFN-?.