Abstract
BackgroundSince the end of 2009, H9N2 has emerged in Tunisia causing several epidemics in poultry industry resulting in major economic losses. To monitor variations of Influenza viruses during the outbreaks, Tunisian H9N2 virus isolates were identified and genetically characterized.MethodsThe genomic RNA segments of Tunisian H9N2 strains were subjected to RT-PCR amplifications followed by sequencing analysis.ResultsPhylogenetic analysis demonstrated that A/Ck/TUN/12/10 and A/Migratory Bird/TUN/51/10 viruses represent multiple reassortant lineages, with genes coming from Middle East strains, and share the common ancestor Qa/HK/G1/97 isolate which has contributed internal genes of H5N1 virus circulating in Asia. Some of the internal genes seemed to have undergone broad reassortments with other influenza subtypes. Deduced amino acid sequences of the hemagglutinin (HA) gene showed the presence of additional glycosylation site and Leu at position 234 indicating to binding preference to α (2, 6) sialic acid receptors, indicating their potential to directly infect humans. The Hemagglutinin cleavage site motif sequence is 333 PARSSR*GLF341 which indicates the low pathogenicity nature of the Tunisian H9N2 strains and the potential to acquire the basic amino acids required for the highly pathogenic strains. Their neuraminidase protein (NA) carried substitutions in the hemadsorption (HB) site, similar to those of other avian H9N2 viruses from Asia, Middle Eastern and human pandemic H2N2 and H3N2 that bind to α -2, 6 -linked receptors. Two avian virus-like aa at positions 661 (A) and 702 (K), similar to H5N1 strains, were identified in the polymerase (PB2) protein. Likewise, matrix (M) protein carried some substitutions which are linked with increasing replication in mammals. In addition, H9N2 strain recently circulating carried new polymorphism, "GSEV" PDZ ligand (PL) C-terminal motif in its non structural (NS) protein.Two new aa substitutions (I) and (V), that haven't been previously reported, were identified in the polymerase and matrix proteins, respectively. Nucleoprotein and non-structural protein carried some substitutions similar to H5N1 strains.ConclusionConsidering these new mutations, the molecular basis of tropism, host responses and enhanced virulence will be defined and studied. Otherwise, Continuous monitoring of viral genetic changes throughout the year is warranted to monitor variations of Influenza viruses in the field.
Highlights
Since the end of 2009, H9N2 has emerged in Tunisia causing several epidemics in poultry industry resulting in major economic losses
Phylogenetic analyses of surface genes of H9N2 viruses To determine evolutionary relationships between Tunisian H9N2 isolates and those selected from the Genbank (Table 2), phylogenetic analyses were carried out for 6 viral gene segments (i.e., the genes for hemagglutinin (HA), neuraminidase (NA), polymerase (PB2), nucleoprotein (NP), matrix (M) and non structural (NS)) (Figure 1)
Phylogenetic analyses of internal genes Four internal genes (PB2, NP, M and NS) of Tunisian H9N2 strains showed more than 96% nucleotide identity with those of the Middle Eastern strains isolated from 2001 and 2008 (Table 3)
Summary
Since the end of 2009, H9N2 has emerged in Tunisia causing several epidemics in poultry industry resulting in major economic losses. By 1997, H9N2 viruses have been isolated in multiple avian species including chickens, ducks, turkeys, quail, geese and pigeons, throughout Asia, the Middle East, Europe and Africa and for the first time from humans in Hong Kong and China, in 1999 [5,6,7]. They could emerge as human pathogens through reassortment in intermediate hosts, such as pigs [6] and in avian species, or through direct adaptation in human host [8]. The A/quail/Hong Kong/G1/97 is thought to be the donor of 6 internal genes to the poultry and the human H5N1 viruses isolated in 1997 [8]
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