Biological Significance Of The Five Amino Acid Deletion From 80-84 And 92E And/or 97E In NS1 Protein Of H5N1 Avian Influenza Viruses

H5N1-Highly Pathogenic Avian influenza(H5N1-HPAI)is a serious infectious disease caused by influenza A virus within the family Orthomyxoviridae. Office International Des Epizooties (OIE) once classified this disease as list A disease and now notifiable disease. The genome of Avian Influenza Virus (AIV) consists of eight single-stranded, negative sense, segmented RNAs in which the NS gene is smallest. In 2000, NS gene of H5N1 subtype AIVs showed up a 15nt deletion of nucleotide sequence from 263-277 (5 aa deletion at residues 80-84). Since then, the viruses with the 15nt deletion in NS gene have been the predominant strains circulating in poultry based on the analysis of NS gene sequences available on line. However, the biological significance of this phenomenon remains unknown until now.There are many studies on the virulent sites of NS1 protein in H5N1 virus. It has been reported that glutamic acid (Glu) at position 92 or 97 in NS1 play a key role in the high pathogenicity of AIV. In order to elucidate the effect of amino acids deletion from 80-84, 92E and/or 97E in NS1 on viral replication and virulence, a series of mutant viruses were generated via reverse genetics technique, and their biological characteristics and virulence were measured.1. The effect of the deletion from 263-277 in NS gene on the competiting-replication ability of H5N1 virusesTo verify the postulation that the HPAIV with 15nt deletion in NS gene has obtained stronger propagation ability in competing with the non-deletion virus, two recombinant H5N1 subtype AIVs, m248 with 15nt deletion and 248 without deletion, were used in this study, they shared seven other genes. The two viruses, with 103 PFU each, were mixed and successively passaged for ten times in MDCK, COS-1, Vero and CEF, respectively. The RNAs of the 0, 1st, 5th and 10th generations from different cells were prepared, and NS genes were amplified by RT-PCR, and cloned into T-vector. The proportions of m248 and 248 in these generations were measured by colony multiplex PCR. In the 1st generation, no significant difference of the propagation between m248 and 248 was observed in the four cell types. However, for the 5th and 10th generations, compared with the similar growth ability of these two viruses in IFN-α/β–incompetent cell line (Vero), m248 exhibited higher propagation ability than 248 in IFN-α/β–competent cell lines (MDCK and CEF), indicating that the HPAIVs with the 15nt deletion, circulating in poultry in recent years, obtained a stronger propagation ability in the infected cells when compared with the non-deletion viruses. It was further demonstrated that this deletion can improve anti-IFN ability of AIV. In the 10th generation, the difference of proportions in COS-1(IFN-α/β–competent cell) and Vero (IFN-α/β–incompetent cell) was not detected, suggesting the less IFN secretion in COS-1 cells.2. The effect of amino acids deletion from 80-84, 92 E and/or 97E on viral biological characeristicsMany mutations with deletion from 80-84, 92E and/or 97E in NS1 protein were made in this study, and a series of mutant viruses were generated by reverse genetics technique. In vitro growth experiment, each mutation, such as the deletion of residues 80-84, D92E or E97D, can increase the viral replication ability in cells. And synergistic effect in this ability could be detected when any two kinds of these mutations coexisted. Based on analysis of biological properties of these viruses, the substitution of Asp for Glu at positions of 92 or 97 resulted in increased viral virulence in chickens, mice and fertilized eggs. Although the deletion of residues 80-84 increased virulence in mice and fertilized eggs, it had no effect on the virulence in non-immunized chikens. It was supposed that the physical ability of non-immunized chikens may be better than SPF chickens,thus the significant difference in the virulence of these reassortant viruses could not be observed. It was also found that they induced mice to produce high-level antibodies that can protect mice against fatal-infection. The three-dimensional structures of NS1 proteins from these reassortants were also predicted and analyzed in this paper. The substitution of Asp for Glu at residue 92 probably decreased the tortuosity on N-teriminal domains, however, this change did not alter the hydrophilicity, antigenic index and surface probability of NS1 protein.In conclusion:(1) The virus with a deletion of nucleotide sequence from 263 to 277 in NS gene obtained stronger anti-IFN ability compared with non-deletion virus.(2) The deletion of residues 80-84, and mutation of D92E or E97D all increased the viral replication ability in cells, and cooperating effect on this ability could be detected when any two factors co-existed.(3) The deletion at residues 80-84 and glutamic acid (Glu) at residues 92 or 97 in NS1 protein were associated with the AIV virulence.(4) The reassortant viruses with inner genes of human influenza viruses can induce mice to produce high-level antibodies.