Mutations and phylogenetic analysis of the equine influenza virus (H3N8) nucleoprotein isolated in Morocco

The equine influenza (EI) is a highly contagious respiratory viral disease of equines. The aim of the present study was to determine the amino acid mutation sequences of the partial nucleoprotein (NP), which includes four epitopes of the equine leucocyte antigen (ELA) of A/equine/Nador/1/1997 (H3N8). These epitopes are critical for their binding to major histocompatibility molecule complex (MHC) class I and recognition by specific Cytotoxic T Lymphocytes (CTLs). The isolate was subjected to RT-PCR amplifications followed by sequencing analysis. Phylogenetic analysis showed that Moroccan isolate belongs to equine host-specific lineage and more closely related to Italian strains A/equine/Rome/5/1991 and A/equine/Italy/1062/1991. Amino acid sequence comparison of the NP showed that the strain A/equine/Nador/1/1997 has twelve substitutions at the residues T/284/A, A/286/T, R/293/K, I/299/V, V/312/I, N/319/K, S/344/L, V/353/I, M/374/I, C/377/N, N/397/S and R/452/K. All substitutions concerned both the interaction domains NP–NP and NP–PB2. However, the mutation N319 K enhances the NP–PB2 interaction and polymerase activity in mammalian infected cells. S/344/L mutation was located on the FEDLRVSNFI epitope (aa 338–347), this substitution is likely to help the virus to overcome the barrier of cell-mediated immunity of the host. The identified mutations were grouped into two groups, one included residues that facilitate the adaptation and evolution of influenza viruses within the equine lineage such as A/286/T, R/293/K, S/344/L, V/353/I and R/452/K, while the second contained the substitutions which enhance the virulence as polymerase activity (N319K) and mutations that affect CTL epitopes, resulting in an escape from immune surveillance by specific CTLs (S/344/L)