Previous observations indicated that quails are a potential intermediate host for generating novel reassortant IAVs with pandemic potential [30
]. With the emergence of the pH1N1 virus, concerns about novel highly pathogenic reassortant pH1N1 generated in quail necessitated detailed studies of pH1N1 infection in this species. A few reports are available on susceptibility of quails to pH1N1 infection [5
]. However, those investigations were restricted to reporting only virus shedding and did not offer direct comparison with IAVs endemically circulating in other species. In the present study, the susceptibility and pathogenicity of pH1N1 in quails were determined and directly compared to that associated with swine (swH1N1) and avian (dkH3N2) influenza viruses. We confirmed that the quails were susceptible to pH1N1 infection that led to a prolonged virus-shedding period without the development of any clinical signs. Viral pathogenesis was restricted to the respiratory and intestinal tracts but the main mode of virus transmission to contact birds was through an OP route. Importantly, our study revealed that the quails were more susceptible to infection with pH1N1 and dkH3N2 compared to the endemic swH1N1 strain.
As observed in previous studies [5
], the pH1N1 swine influenza virus and LPAIV did not produce clinical signs in either the infected and contact quails. Furthermore, our results were in agreement with ones from previous studies demonstrating that quails inoculated with pH1N1 are asymptomatic but shed virus mainly from the respiratory tract [5
]. This finding corresponded to data from another study showing high expression of α-2,6-linked receptors in quail trachea [30
]. In addition, our data agreed with those of previous investigations showing that LPAIV- and swine influenza virus-infected quails do not develop clinical signs, and shed virus predominantly from the respiratory tract that is poorly transmitted to contact quails [10
]. In the present study, low or undetectable levels of virus shedding from the cloaca were observed in quails infected with all three viruses, indicating that these viruses replicated more efficiently in the respiratory tract than in the intestinal tract.
Our results showed that quails inoculated with pH1N1 could shed virus from the oropharynx for up to 7 DPI. This finding was different from those of previous studies indicating that quails can shed pH1N1 for only 4 or 5 days [5
]. However, the levels of virus shedding were slightly higher compared to those observed in our study. This difference might be related to the strain of pH1N1, route of inoculation, and viral doses. The swH1N1 used in our study was classified as an avian-like virus since most of its genes belonged to an avian-like swine lineage. However, lower levels of OP shedding were detected in quails inoculated with swH1N1 compared to birds exposed to classical swH1N1 virus as previously reported [11
]. Moreover, our study demonstrated that lower levels of dkH3N2 from a Eurasian lineage were shed from the oropharynx of infected quails compared to Eurasian H3 virus previously described in the literature [10
]. Another study showed that mammalian H1 viruses, including pH1N1 and swH1N1 viruses, have a typical human-like preference for the α-2,6-linked receptor [5
]. However, swH1N1 replicates poorly in quail respiratory tract whereas pH1N1 could replicate more efficiently despite the presence of α-2,6-linked receptors in quail trachea. Therefore, the limited replication of swH1N1 compared to pH1N1 could not be explained based only on receptor distribution. This might be due instead to a combination of internal pH1N1 genes not shared with swH1N1 that may have enabled pH1N1 to replicate in the quail respiratory tract. Overall, our study showed that quails inoculated with pH1N1 and dkH3N2 shed higher virus titers in OP swabs compared to birds inoculated with swH1N1. However, levels of virus were relatively low compared to ones reported in previous studies [5
]. These data confirmed that all three viruses examined in our study replicated at low levels in quails.
pH1N1, swH1N1, and dkH3N2 caused pathological changes mainly in the respiratory organs and intestines of infected quails. Both sites are known to be the primary site for LPAIV replication in avian species [13
]. However, minimal IAV antigen staining was only observed in the intestinal tract of a quail inoculated with dkH3N2. This finding is in agreement with observations in many avian species in which a minimal or lack of IAV antigen staining in tissues from LPAIV-infected birds was observed [7
]. In addition, histopathological lesions observed in this study were similar to those associated with LPAIV infection in many avian species [13
]. Therefore, it can be assumed that these lesions may be related to IAV infection. Pathological changes in pH1N1-infected bird observed in our investigation were different from a previous study in which only heterophillic-to-lymphocytic rhinitis and positive IAV antigen staining within the mucosa of the nasal cavity were noted [27
]. This difference might be attributed to the route of inoculation and the swine-adapted strain of pH1N1 used in this study. Overall, the results from our study showed that pH1N1 and dkH3N2 were more pathogenic in quails than swH1N1. However, our findings also indicated that all three viruses are of low pathogenicity in quails.
Serological results confirmed the seroconversion of quails infected with pH1N1 and swH1N1 viruses and the corresponding contact quails. Our study detected HI antibodies against pH1N1 in pH1N1-inoculated quails a week earlier than a previous study [27
]. On the other hand, none of the dkH3N2-inoculated or contact quails seroconverted 7 DPI, although virus shedding and pathological changes were observed in dkH3N2-inoculated quails. A previous study [4
] determined that HI assays using CRBCs fail to detect serum HI antibodies in ring-necked pheasants and chukar partridges infected with avian influenza A viruses (AIVs) unlike those using horse red blood cells (HRBCs). This is because AIVs prefer to bind to α-2,3-linked receptors, which are predominantly found on HRBCs [26
]. Thus, the absence of HI titers in the dkH3N2-infected quails may have been due to the use of CRBCs in the HI test. In addition, quails may need more time to develop HI antibodies against dkH3N2.
In conclusion, the present study demonstrated that quails were susceptible to infection with pH1N1, swH1N1, and dkH3N2 viruses. The replication and pathogenicity levels of pH1N1 and dkH3N2 in quails were much higher than that of swH1N1. However, none of the viruses replicated efficiently in quails and caused only mild lesions, indicating that these viruses act as LPAIV in quails. Overall, our findings revealed that the quails were susceptible to infection with all three different virus lineages yet appeared tolerant to the disease caused by these viruses. These data indicate important roles of quails as a susceptible host for mammalian and AIVs, and an intermediate host of IAVs. Importantly, the possibility of quails transmitting IAVs to other hosts as well as quail producers (in the case of farm-raised birds) can easily be overlooked since these birds are essentially asymptomatic while shedding IAVs. Awareness of IAV interspecies transmission and continued monitoring of IAVs in quails is thus crucial for IAV control and pandemic preparedness.