| Background and Objective: The Hong Kong incidents that avian influenza viruses H5N1 and H9N2 can infect humans without any intermediate mammal host in 1997 and 1999 respectively remind us that the next influenza pandemic is imminent. There were three lineages of H9N2 influenza viruses circulating in avian species in southern China, which were represented by Qa/HK/Gl/97, Dk/HK/Y280/97, Dk/HK/Y439/97 respectively. Gl-like viruses were also isolated from two children with influenza-like illness in 1999. Cocirculation of avian Y280-like H9N2 viruses and human Sydney/97-like H3N2 virus was also documented in 1998. Matrosovich et al reported that avian H9N2 virus circulating in southern China contained human-like receptor specificity recently. Thus, H9 influenza viruses are considered as a most possible candidate for the next human pandemic. However, previous studies mainly focused on H9 subtype viruses in terrestrial poultry in this region. There are no reports related to H9 subtype viruses in aquatic birds, influenza natural gene pool. Present studies are designed to understand the interrelationship between the H9 aquatic origins and those circulating in terrestrial poultry through systemic surveillance. The data generated will help to reveal whether those H9 subtype viruses from aquatic birds are the precursors of Gl-like and Y280-like lineages prevalent in terrestrial poultry.Materials and Methods: Fecal, cloacal and tracheal swabs from different types of poultry were collected in 6 live-bird retail markets once a week. They were inoculated into 9-11 days embryonated chicken eggs and incubated in 35"C for 72 hours. HI and Nl assays were performed to detect the subtype of viruses if HA test were positive(AD^ 16) and contamination test were negative. Twelve H9N2 and two H9N1 influenza viruses were selected for further antigenic and genetic analysis. Viral RNAs were extracted from virus-infected allantoic fluids using QIAamp Mini-extraction Kits. After reverse transcription, cDNA was amplified using specific primers for each gene segment. PCR products were purified with Qiaquick PCR Purification Kit, followed by sequencing using the Big Dye Terminator Cycle Sequencing Ready Reaction Kits used withAmpliTaq DNA polymerase FS. Samples were electrophoresised and analyzed on a Perkin-Elmer model 377XL DNA sequencer. All sequence data were assembled and edited using Wisconsin Package, version 10.2. Phylogenetic analyses were carried out using PA UP, version 4.1. Alignment and homology analyses were performed with GeneDoc, version 2.3, to quantitate the nucleotide and amino acid substitutions. Results:1. Epidemiology: Influenza viruses circulated in different types of poultry year round. Almost all subtype viruses were isolated from ducks, while the main body of the isolates were H9, H6 subtypes in terrestrial poultry i.e. chickens and quails. It was noteworthy that an H5N1 subtype virus was isolated from quails for the first time in southern China.2. Analysis of H9 subtype:(1) Seven out of fourteen H9 strains tested shared similar reaction patterns with Dk/HK/Y280/97 or Ck/HK/G9/97 virus, while the rest showed almost no inhibition and low reactivity to all reference antisera. Thus at least two distinct groups of H9 subtype viruses were circulating in domestic duck in southern China during 2000-2001.(2) Although phylogenetic analysis of H9 HA1 section shows that all of the duck H9N2 viruses cluster within the lineage represented by Ck/HK/G9/97 or Dk/HK/Y280/97, they appear closely related to H9N2 viruses isolated from chickens at different points of time during their evolutionary process. Four viruses are more closely related to the earlier chicken virus Ck/Bei/1/94, while five and three viruses are more closely related to Dk/HK/Y280/97 and recent H9N2 viruses isolated in 1999 respectively. Similar relationships are also observed in N2 gene. These suggest that the H9N2 viruses circulating in chickens in southern China may have, at intervals, transmitted back into domestic ducks. After trans...
|
PDF Full Text Request