| Background and Objective:Influenza is a zoonotic disease caused by a constantly varying RNA virusresulting in a need for continuous surveillance to update human vaccine. It has been confirmed that outbreaks of influenza in mammals, including human being, originate from influenza viruses of avian directly or indirectly. At present, H9N2 subtype influenza viruses are prevalent in terrestrial poultry worldwide. What is more, the introduction of these new types of terrestrial poultry was in southern China, a hypothetical epicenter for the emergence of pandemic influenza viruses. It is, therefore, more important to carry out influenza surveillance, especially in those newly forming poultry in this region. Here we report that the genetic identification of H9N2 influenza viruses from those newly forming poultry in this region, partridge and chukka. Our results suggest that those H9N2 influenza viruses isolated from these types of terrestrial poultry are not sorted into the following two genotypes, i.e., Qa/HK/Gl/97-like and Dk/HK/Y280/97-like virus lineages. Genetic analysis revealedthat some of them are reassortment viruses between above two lineages. The information generated in current study suggests that the developing influenza ecosystem in southern China region may play an important role in the process of emerging novel influenza viruses, even directly impact the genesis of pandemic influenza strains.Materials and Methods:Fecal, cloacal and tracheal swabs from different types of poultry were collected inlive-bird retail markets once a week. They were inoculated into 9-11 days embryonated chicken eggs and incubated in 35 for 72 hours. HI and NI assays were performed to detect the subtype of viruses if HA test was positive(AD 16) and contamination test was negative. Eighteen H9N2 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 with AmpliTaq 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 PAUP, version 4.1. Alignment and homology analyses were performed with GeneDoc, version 2.3, to quantitate the nucleotide and amino acid substitutions.Results:1. H9N2 subtype of Influenza A viruses is the dominant subtype in newly-formed terrestrial poultry year round.2. Antigenic characterization showed that all viruses isolated from the Partridge/Chukka were closely related to the Ck/HK/G9/97 and Sw/HK/9/98, and were distinguishable from Qa/HK/Gl/97 and HK/1073/99. However, there was antigenic diversity among these H9N2 viruses.3. Multiple genotypes existed in Partridge/Chukka, and at least one genotype has been established in these species.4. Several strains from contemporary aquatic birds were genetically homologous to some genotypes from Partridge / Chukka, namely, they have the same gene source, may be produced through crossing species barrier.Discussion:1. Stable lineage: Its origin?2. Several genotypes occurred in Pa/Cu. Where the genetic reassortments took place? In Partridge, Chukka, Duck, Chicken, Quail, or others?3. Several strains from contemporary Duck /Wild Duck are genetically homologous to some viruses from Partridge / Chukka, namely, they have the same gene source, may be produced through crossing species barrier. How this happened? From Duck to Partridge / Chukka, or indirectly through Chicken? Whether the reverse transmission happened, too?4. Multiple genotypes emerged and waned, which means reassortments still occurred frequently and these reassortments competed t... |
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