Abstract
Equine influenza virus (EIV) causes a highly contagious disease in horses and other equids. Recently, we isolated an H3N8 EIV (A/equine/Kyonggi/SA1/2011) from a domestic horse in South Korea that exhibited symptoms of respiratory disease, and found that the EIV strain contained a naturally mutated NS gene segment encoding a truncated NS1 protein. In order to determine whether there was an association between the NS gene truncation and viral virulence, a reverse genetics system was applied to generate various NS gene recombinant viruses using the backbone of the H1N1 A/Puerto Rico/8/1934 (PR/8) virus. In a mouse model, the recombinant PR/8 virus containing the mutated NS gene of the Korean H3N8 EIV strain showed a dramatically reduced virulence: it induced no weight loss, no clinical signs and no histopathological lesions. However, the mice infected with the recombinant viruses with NS genes of PR/8 and H3N8 A/equine/2/Miami/1963 showed severe clinical signs including significant weight loss and 100% mortality. In addition, the levels of the pro-inflammatory cytokines; IL-6, CCL5, and IFN-γ, in the lungs of mice infected with the recombinant viruses expressing a full-length NS1 were significantly higher than those of mice infected with the virus with the NS gene from the Korean H3N8 EIV strain. In this study, our results suggest that the C-terminal moiety of NS1 contains a number of virulence determinants and might be a suitable target for the development of a vaccine candidate against equine influenza.
Highlights
Equine influenza virus (EIV), which is a member of the genus Orthomyxovirus, family Orthomyxoviridae, contains a single-stranded, negative-sense RNA in the following eight gene segments: PB2, PB1, PA, HA, NP, NA, M, and NS
As a first attempt to characterize the three recombinant viruses, their sequences were checked, and we confirmed that cells infected with the recombinant virus rPR/8 × KYGNS virus expressed a ~15 kDa NS1; full-length NS1protein of approximately 26 kDa was revealed for the rPR/8 and rPR/8 × MINS viruses by western blotting (Figure 1A)
We found that all recombinant viruses grow to a similar titer in both cell lines at each time point, indicating that the truncated NS gene did not substantially affect the replicative capacity of these viruses in cell culture (Figures 1B and C)
Summary
Equine influenza virus (EIV), which is a member of the genus Orthomyxovirus, family Orthomyxoviridae, contains a single-stranded, negative-sense RNA in the following eight gene segments: PB2, PB1, PA, HA, NP, NA, M, and NS. This virus is the causative agent of serious respiratory diseases in horses [1]. It was demonstrated that H3N8 EIV was responsible for the emergence of H3N8 canine influenza A virus (CIV) in North America in 2004 [3]. We isolated from a domestic horse in South Korea an H3N8 EIV strain (A/equine/Kyonggi/ SA1/2011; KYG11) belonging to the Florida sublineage clade 1 [2]. The KYG11 strain possessed an NS gene segment that harbored a deletion of nucleotides
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