GENETICS AND PATHOGENICITY OF THREE H5N1 AVIAN INFLUENZA A VIRUS ISOLATES OF CHICKEN IN SOUTHWESTERN NIGERIA

Abstract

Highly Pathogenic Avian influenza A (HPAI) H₅NI outbreak has been reported in several African countries including Nigeria. The outbreak continues to pose a risk to human health and livelihood. The genetics and pathogenicity of three Nigerian H₅NI avian influenza virus isolates were therefore investigated. Carcasses of chicken were obtained from Ogun State for post mortem examination. Nasopharyngeal and cloacal swabs, lung, brain, intestine, heart and tracheal tissues were collected for virus isolation in embryonated chicken eggs. Three virus isolates were obtained and identified serologically with haemagglutination-inhibition assay. Viral RNA was extracted with phenol and guanidinium isothiocyanate and the genes were amplified using polymerase chain reaction, cloned into pGEM-T vector for mini preps, sequenced and analysed phylogenetically. The non-structural genes were transfected into 293T cells to assess their effects on interferon production in mammalian cells. Intravenous and intranasal pathogenicity teas were carried out in eight 4-week old specific pathogen free White Leghorn chickens. Tissue samples were collected for virus isolation and titration. Gross and histopathological examinations and immunohistochemical staining were also carried out. The isolates were designated influenza chicken/Nigeria/228-5/2006, A/chicken/Nigeria/228-6/2006 and A/chicken/Nigeria/228-10/2006. Sequence analyses confirmed the presence of multibasic amino acids in the haemagglutinin cleavage site as PQGERRKK which has been associated with high virulence. The neuraminidase did not have histidine substitution for tyrosine at position 274 (H274Y) which is associated with antiviral drug (oseltamivir) resistance. The non-structural protein 1 (NSI) open reading frame encoded a five-amino acid deletion at positions 80 to 84 in the three isolates while A/chicken/Nigeria/228-10/2006 had a 7 amino acid C-terminal extension. Intravenous and intranasal pathogenicity tests revealed virulence and high pathogenicity with mean death time of 1.4 and 2.2 days respectively. Gross lesions included congestion of the lungs, petechiae in epicardial fat, in the gizzard and swollen kidneys. Histologically, the most severe lesions were severe interstitial pneumonia with edema, myocyte degeneration and necrosis in the heart and moderate nonsuppurative encephalitis. Tissues with lesions coincided with sites of virus replication and indicated a severe systemic infection. This was confirmed by immunohistochemical staining of the viral antigen. The sequence changes did not affect the ability of NSI to block interferon induction when expressed transiently in 293T cells. The PB2 protein also had a lysine residue at position 627, which has been implicated in mammalian adaptation of H₅NI avian viruses. The sequence changes observed in NSI did not influence the ability of the virus to establish in mammalian cells. Based on the H₅NI Evolution Working Group classification, the three Nigerian H₅NI isolates are highly pathogenic and belong to clade 2.2.2 as other viruses from Africa, Asia, Middle East and Europe.