Abstract
Clade 2.3.4.4 H5Nx highly pathogenic avian influenza viruses (HPAIVs) have caused outbreaks in poultry in the world. Some of these viruses acquired internal genes from other subtype avian influenza viruses (AIVs) such as H9 and H6 for the generation of novel reassortant viruses and continually circulated in poultry. Here, we applied a duck-origin virus DK87 and a chicken-origin virus CK66 to assess the biological characteristics of novel reassortant H5N6 HPAIVs and its pathogenesis in ducks. A genetic analysis indicated that the HA genes of the two H5N6 HPAIVs were closely related to the H5 viruses of clade 2.3.4.4 circulating in Eastern Asia and classified into H5 AIV/Eastern Asia (EA)-like lineage. Their NA genes fell into Eurasian lineage had close relationship with those of H5N6 viruses circulating in China, Laos, Vietnam, Japan, and Korea. All internal genes of DK87 were aggregated closely with H5 AIV/EA-like viruses. The internal genes (PB1, PA, NP, M, and NS) of CK66 were derived from H9N2 AIV/SH98-like viruses and the PB2 were derived from H5 AIV/EA-like viruses. These results indicate that clade 2.3.4.4 H5N6 AIVs have continually evolved and recombined with the H9N2 viruses circulating in Southern China. Pathogenicity test showed that the two viruses displayed a broader tissue distribution in ducks and caused no clinical signs. These results indicated that ducks were permissive for the replication of the chicken-origin reassortant virus CK66 without prior adaptation, but the duck-origin virus DK87-inoculated ducks showed significantly higher viral titers in some organs than the CK66-inoculated ducks at 5 day post-inoculated (DPI). The recovery of viruses from oropharyngea and cloacal swabs of contacted ducks indicated that they transmitted in native ducks by direct contact. Quantitative reverse transcription PCR (qRT-PCR) results revealed that the immune-relative genes (PRRs, IFNs, Mx-1, IL-6, and IL-8) in the lungs of inoculated ducks were expressed regardless of virus origin, but the expression of these genes was significantly higher in response to infection with the DK87 virus compared to the CK66 virus at 3 DPI. Overall, we should provide further insights into how clade 2.3.4.4 H5N6 AIVs undergo genetic and pathogenic variations to prevent outbreaks of this disease.
Highlights
Avian influenza viruses (AIVs) are enveloped, segmented, singlestranded negative sense RNA viruses belonging to the family Orthomyxoviridae (Swayne and Suarez, 2000)
The HA genes of our viruses were closely related to the H5 viruses of clade 2.3.4.4 circulating in Eastern Asia and classified into H5 AIV/Eastern Asia (EA)like lineage (Figure 1)
The amino acid sequence of the cleavage site in the HA protein of the DK87 was PLKERRRKR/GLF, and the CK66 virus was PLRERRRKR/GLF, which is characteristic of highly pathogenic viruses (HPAIVs) (Supplementary Table S1)
Summary
Avian influenza viruses (AIVs) are enveloped, segmented, singlestranded negative sense RNA viruses belonging to the family Orthomyxoviridae (Swayne and Suarez, 2000). With growing number of isolates, clade 2 H5 subtype AIVs have expanded into second- (e.g., clade 2.1) and third-order (e.g., clade 2.3.4) clades during 2006–2008. Since 2008, H5 HPAIVs bearing the genetic backbone of clade 2.3.4.4 H5N1 have been identified in China and evolved into different subtypes including H5N1, H5N2, H5N5, H5N6, and H5N8 by acquiring NA gene from other AIVs (Yamaji et al, 2020). Among these reassortments, H5N6 AIVs have become dominantly prevalent subtypes in Southern China especially in waterfowl since 2014 (Bi et al, 2016). Rapid evolution and circulation of the H5 subtype AIVs of clade 2.3.4.4 pose an increased threat to poultry
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