A Turkey-origin H9N2 Avian Influenza Virus Shows Low Pathogenicity but Different Within-host Diversity in Experimentally Infected Turkeys, Quail and Ducks

Abstract

Avian influenza virus (AIV) is a highly diverse and widespread poultry pathogen. Its evolution and adaptation may be affected by multiple host and ecological factors, which are still poorly understood. In the present study, a turkey-origin H9N2 AIV was used as a model to investigate the within-host diversity of the virus in turkeys, quail and ducks in conjunction with the clinical course, shedding and seroconversion. Ten birds were inoculated oculonasally with a dose of 106 EID50 of the virus and monitored for 14 days. Virus shedding, transmission and seroconversion were evaluated, and swabs collected at selected time-points were characterized in deep sequencing to assess virus diversity. In general, the virus showed low pathogenicity for the examined bird species, but differences in shedding patterns, seroconversion and clinical outcome were noted. The highest heterogeneity of the virus population as measured by the number of single nucleotide polymorphisms and Shannon entropy was found in oropharyngeal swabs from quail, followed by turkeys and ducks. This suggests a strong bottleneck was imposed on the virus during replication in ducks, which can be explained by its poor adaptation and stronger selection pressure in waterfowl. The high within-host virus diversity in quail with high level of respiratory shedding and asymptomatic course of infection may contribute to our understanding of the role of quail as an intermediate host for adaptation of AIV to other species of poultry. In contrast, low virus complexity was observed in cloacal swabs, mainly from turkeys, showing that the within-host diversity may vary between different replication sites. Consequences of these observations on the virus evolution and adaptation require further investigation.

Low Pathogenicity H7N3 Avian Influenza Viruses Have Higher Within-Host Genetic Diversity Than a Closely Related High Pathogenicity H7N3 Virus in Infected Turkeys and Chickens

“…Nonetheless, it has been shown that the overall substitution rate and dN/dS ratio of AIVs is lower in wild birds than in domestic poultry species and is likely a reflection of its AIV reservoir status [ 72 , 73 ]. Furthermore, experimental infections of a turkey-derived H9N2 virus has also shown that the highest number of nonsynonymous changes occurred in samples from infected ducks, most likely reflecting shifts in host species [ 11 ]. Similarly, experimental passage of avian, canine, or seasonal human influenza A viruses in embryonating eggs or immortalized cell lines have shown the preponderance of nonsynonymous changes that has been attributed to shifts in viral replication from its natural host to an experimental culture system [ 7 , 74 , 75 ].…”

“…Viral diversity can be described through the number of iSNVs in each sample, or a metric such as Shannon entropy and nucleotide diversity [ 4 , 7 , 11 , 13 , 44 , 81 , 82 ]. In this study, we observed that LPAIV-1 and LPAIV-2 had higher iSNV diversity than the HPAIV ( Figure 4 ).…”

“…Further studies will be needed to determine whether these changes are potentially related to virus adaptation to gallinaceous species. It has been previously shown that within-host diversity of AIVs can vary depending on the host, species, route of exposure, and virus replication site [10,11]. Within-host diversities were thus compared for dose (2, 3, 4, 5, or 6 log 10 EID 50 ), route of exposure (inoculated or contact-exposed), and time after exposure for each virus (LPAIV-1, LPAIV-2, or HPAIV), but no significant differences were found.…”

“…Moreover, deep sequencing has allowed investigators to identify small low-frequency variations across a substantial number of samples, particularly single nucleotide variations (SNVs). Within-host SNVs, also known as intra-host SNVs (iSNVs), have been examined for a number of virus species, including influenza viruses [ 6 , 7 , 8 , 9 , 10 , 11 , 12 ], Newcastle disease virus [ 13 ], human immunodeficiency virus (HIV) [ 14 , 15 , 16 ], hepatitis C virus (HCV) [ 17 , 18 ], foot-and-mouth disease virus (FMDV) [ 19 ], severe acute respiratory disease coronavirus 2 (SARS-CoV-2) [ 20 ], and herpes simplex virus (HSV-1) [ 21 ]. Examination of within-host diversity has provided insights on the emergence of phenotypic traits such as antiviral drug resistance [ 14 , 17 , 18 , 22 ], the transmission of viruses between hosts [ 8 , 9 , 19 , 20 ], and viral spread to multiple tissues in a single host [ 23 ].…”

Airborne Transmission of Avian Origin H9N2 Influenza A Viruses in Mammals

“…IAVs of the H9N2 subtype are widespread in several species of wild waterfowl, shorebirds, and poultry, such as chickens, turkeys, and quails, among others [17][18][19][20][21][22]. H9N2 IAVs are the most prevalent LPAIVs, and are enzootic in poultry in parts of Asia, the Middle East, and Africa (reviewed in [3]).…”