| Background and Objective :Southern China - was considered to be a hypothetical - "epicenter " as most of human pandemic strains of last century was first emerged from this region. Aquatic birds have been recognized to be the " natural reservoirs" of influenza viruses since all known influenza virus subtypes could be isolated from these hosts and genetic analysis sugguested that all influenza viruses residential in other hosts were originally derived from those viruses from birds. Thus, it's essential to understand the epidemiological informantion of influenza virus in aquatic birds from southern China. In 1970's , influenza surveillance studied by K.F.Shortridge showed the major subtypes isolated from aquatic birds, i.e ,domestic ducks, in southern China were H3 , H4, and H6 , while H5N3 subtype of viruses provide the main body of H5 subtype.Italy, located in southern European, continent , has some similarity in influenza ecosystem with southern China. Since 1997 onwards, several highly pathogenic avian influenza viruses caused outbreaks in poultry of Italy . Genetic and antigenic studies revealed that - those outbreaks were associated with inter-species transmissions of influenza virus from wild birds to domestic poultry.Since 2002 , H5N3 subtype of influenza viruses has been isolated from domestic ducks in several provinces of southern China,while two H5N3 viruses were also isolated from fecal material of migratory birds in Hong Kong during late 2004. Currently , highly pathogenic H5N1 influenza virus became endemic in aquatic birds in southern China , Thus, our project would focus on to understandthe origin and evolution of those newly emerged H5N3 subtype viruses - in attempt to answer those questions,including : CD Whether the newly H5N3 virus strains were derived directly - from those 1970's H5 viruses ? ?Whether migratory birds have been involved in the emergency of those novel H5N3 influenza virus from southern China ? (3) Are there any "gene flow " exchanges between recently H5N3 subtype viruses and H5N1 subtype viruses or others subtype influenza viruses in aquatic birds ? (4) Are there any clues or indirect evidence to explain the genesis of the GS/GD/l/96-like virus in this region ?Materials and Methods :Firstly, routinely treated the samples(including recently collected samples from several provinces in southern China, 1970's Hong Kong samples and samples kindly provided by Italian colleagues ) need to be inoculated. All the samples were inoculated into 9-11 days embryonated chicken eggs and incubated at 35°C for 48 hours. Hemagglutinin inhibition test (HI test) and Neuraminidase inhibition test (NI test) were performed to detect the subtype of viruses if HA test was positive(AD>16) and contamination test was negative. Twenty six of H5 influenza viruses strains were selected as representative for further molecular epidemiological analysis. Viral RNA 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 3700 DNA sequencer. All sequence data were assembled and edited using Lasergene , version 6.0 software package . Phylogenetic analyses were carried out using Mega version 2.0. Alignment and homology analyses were performed with GeneDoc, version 2.3, to quantify the nucleotide and amino acid substitutions.Results :1. During 70's , H5N3 was the major H5 subtype in southern China's aquatic birds and no H5N1 subtype of influenza virus was isolated from aquatic birds . Currently, H5N1 subtype became endemic in domestic ducks of southern China, while the isolation rate of H5N3 subtype is still relatively low .2. Antigenic analysis indicated recently H5N3 influenza viruses have the similar reaction patterns to anti-H5 monoclonal antibodies with those 70's viruses and Italian viruses . While , there was dramatically difference between genotype Z H5N1 influenza viruses and recently H5N3 influenza viruses.3. Genetic analysis reveals that the HA and NA genes of the novel H5N3 viruses from southern China are closely related to the Italian H5 viruses both from migratory birds and domestic poultry .4. Deduced amino-acid sequence analysis of HA proteins showed that all those recently H5N3 influenza viruses were not highly pathogenic for chickens since they are lack of the basic motif at their connecting peptide required for highly pathogenic , i.e., R-X-R/K-R. Animal experimental test confirmed that they are low or non-pathogenic for chicken.5. Phylogenetic analysis showed HA and NA gene of these H5N3 influenza viruses were derived from migratory birds , while the internal genes donors including viruses from migratory birds , ducks and H5N1 viruses .Conclusions :1. H5N1 subtype viruses and H5N3 subtype viruses co-circulated in southern China' s aquatic poultry .2. Recently isolated H5N3 subtype influenza viruses were reassortants and generated by recently interspecies transmission from migratory birds to ducks and others subtype influenza viruses circulated in southern China .3. Multiple genotypes of H5N3 influenza viruses circulated in southern Chinasuggesting that the viruses and their hosts are in the process of host adaptation or virus fitness selection. Thus, biological properties of those novel viruses are still unstable . They may continue evolving toward adaptation or link to new interspecies transmission event and even pathogenic variation .4. The findings of our study suggested that migratory birds may play an important role in the evolution and establishment of influenza virus in Eurasian continent.5. The influenza ecosystem in southern China is multiplex and complex and the "gene flows" are multiple directions . However, understanding the role and position of aquatic birds from southern China are still essential.
|
PDF Full Text Request