Background
There were large outbreaks of high pathogenicity avian influenza (HPAI) caused by clade 2.3.4.4e H5N6 viruses in the winter of 2016–2017 in Japan, which caused large numbers of deaths among several endangered bird species including cranes, raptors, and birds in Family Anatidae. In this study, susceptibility of common Anatidae to a clade 2.3.4.4e H5N6 HPAI virus was assessed to evaluate their potential to be a source of infection for other birds. Eurasian wigeons (Mareca penelope), mallards (Anas platyrhynchos), and Northern pintails (Anas acuta) were intranasally inoculated with 106, 104, or 102 50% egg infectious dose (EID50) of clade 2.3.4.4e A/teal/Tottori/1/2016 (H5N6).
Results
All birds survived for 10 days without showing any clinical signs of infection. Most ducks inoculated with ≥ 104 EID50 of virus seroconverted within 10 days post-inoculation (dpi). Virus was mainly shed via the oral route for a maximum of 10 days, followed by cloacal route in late phase of infection. Virus remained in the pancreas of some ducks at 10 dpi. Viremia was observed in some ducks euthanized at 3 dpi, and ≤ 106.3 EID50 of virus was recovered from systemic tissues and swab samples including eyeballs and conjunctival swabs.
Conclusions
These results indicate that the subject duck species have a potential to be a source of infection of clade 2.3.4.4e HPAI virus to the environment and other birds sharing their habitats. Captive ducks should be reared under isolated or separated circumstances during the HPAI epidemic season to prevent infection and further viral dissemination.
Although verogenic Newcastle disease viruses (NDVs) generally cause subclinical infection in waterfowls such as ducks, NDVs with high virulence in waterfowl have been sporadically reported.We previously reported that the NDV d5a20b strain, which is obtained by serial passaging of the velogenic 9a5b strain in domestic ducks, showed increased virulence in ducks (Hidaka et al., 2021).The d5a20b strain had 11 amino acid substitutions in its P/V, M, F, HN, and L proteins as compared to 9a5b. In the present study, we generated a series of recombinant (r) NDVs with these amino acid substitutions to identify the molecular basis of virulence of NDV in ducks, and evaluated their influences on virulence and in vitro viral properties. Each of the single amino acid substitutions in either the F protein I142M or the M protein Q44R contributed to the enhancement of intracerebral and intranasal pathogenicity in domestic ducks. The cell-cell fusion activity of the virus with F I142M was five times higher than that of the parental r9a5b. The virus with M Q44R rapidly replicated in duck embryo fibroblasts. Additionally, the rM+F+HN strain, which has the same amino acid sequences as d5a20b in M, F, and HN proteins, showed the highest level of virulence and replication efficiency among the generated recombinant viruses, nearly comparable to rd5a20b. These results suggest that multiple factors are involved in the high growth ability of NDV in duck cells, leading to increased virulence in vivo.
KEYWORDduck, Newcastle disease virus (NDV), virulence, waterfowl
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