Attenuation of the virulence of a recombinant influenza virus expressing the naturally truncated NS gene from an H3N8 equine influenza virus in mice

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.

Development of Live Vaccine Candidates for Canine Influenza H3N2 Using Naturally Truncated NS1 Gene

“…IFNs are a group of signaling proteins that are produced by cells in response to viral infections, and they play a key role in activating the host immune system to fight off the virus [28]. Truncation of the effector domain of the NS gene can affect viral replication by altering the ability of the virus to evade the host immune response [15,16,29]. The effector domain of NS1 includes the PKR binding site, p85β-binding site, CPSF30-binding site, and PDZ domain, which act to evade the host antiviral In the 2nd week after the 2nd vaccination, mice were euthanized to obtain the spleen (n = 5) and lungs (n = 5).…”

“…In the case of the SA1 strain, which has a naturally truncated NS gene, it was revealed that the virus showed significantly increased expression of antiviral and pro-inflammatory genes in vitro [16]. Furthermore, in a mouse model, the recombinant PR/8 virus containing the concerned truncated NS gene showed dramatically reduced virulence, and the level of IFN-g cytokines was statistically elevated [15].…”

“…In a previous study, we isolated A/canine/Korea/01/2007 (CIV) and A/equine/Kyonggi/SA1/ 2011 (SA1) and also found that when the NS gene of SA1 is inserted into human H1N1 influenza A virus (A/Puerto Rico/ 8/1934), viral virulence was attenuated in mice [3,15]. To develop CIV LAIV, reverse genetics technology was used to generate CIV SA1 strain.…”

“…Among 11 proteins, nonstructural protein1 (NS1 protein) plays a key role in infection through a multitude of molecular interaction partners, host cell activities, and structural plasticity or polymorphism [14]. According to previous studies that analyzed roles of NS gene using recombinant or truncated NS genes [15][16][17], partially truncated NS1 protein has reduced activation of Akt kinase, whereas it increased phosphorylation of interferon regulatory factor 3 (IRF3) in cells and it showed dramatically attenuation of virulence in mice. The partially truncated NS1 protein expresses an impaired function of inhibiting host antiviral responses, resulting in the attenuation of viral replication.…”

Acetylation at K108 of the NS1 protein is important for the replication and virulence of influenza virus

“…The mRNA levels of IFN-β, IL-1β, and TNF-α in mouse lungs of the WSN-NS1-K108R-infected group were significantly higher than those in the other two groups at 3 dpi, which indicated that NS1-108R was less efficient at inhibiting the production of innate antiviral cytokines at 3 dpi in mice. The NS1 protein of influenza virus is a virulence factor that inhibits the antiviral immunity of the infected host, and C-terminal truncation has been widely used as a strategy to generate attenuated virus vaccine candidates [39]. One mechanism used by NS1 to inhibit the IFN response is through direct binding and sequestration of RNA as well as direct interaction with TRIM25 and complex formation with the RNA sensor RIG-I, resulting in inhibition of the activation of the RIG-I CARD and hence inhibition of IRF3 activation [12].…”