| H5N1 avian influenza virus can infect not only birds but also human and other mammalian animals, crossing the species barrier, posing severe threat to human health. But up to now, we have known a lithe about the mechanism of H5N1 virus infection and pathogenesis to human.The plaque forming characteristic is one of the important phenotypes for avian influenza virus. And it's different for dfferent H5N1 strains, and plaques with different size and morgh can be formed for same human H5N1 strain. If there were difference in pathogenesis for variants with different plaques, it should be easier to identify the molecular determinants of pathogenesis difference of them, because there are few differences in the genome sequences of these viruses. Large-plaque virus and small-plaque virus with invariant plaque forming characteristic can be isolated from same human H5N1 strain with heterogeneity in plaque forming by plaque forming assay. Then the pathogenesis of large-plaque virus and small-plaque virus to mice and the determinants of the pathogenesis in virus sequences can be proved by contrasting the differences in genome sequences, pathogenicity and replication efficiency of the two viruses.I. Difference in plaque forming characteristics of H5N1 avian influenza viruses from different hostsPlaque forming assay was built firstly, and significant difference was proved in plaque forming for H5N1 strains isolated from birds and human by the plaque assay in MDCK cells of H5N1 strains isolated from human and birds: small and punctiform plaques in MDCK cells were formed by A/GbhGull/QH/02/06 and A/Bhgoose/QH/ 01/06 isolated from wild waterfowls post a longer period inoculation; while mainly large and round plaques, mixed with small ones which were more than two times smaller than the large plaques in MDCK cells were formed by A/Vietnam/1194/2004 and A/Beijing/01/03 isolated from human. And it indicates that there are a correlation between the difference in plaque morgh and hosts of H5N1 avian influenza viruses and maybe also a a correlation between the difference in plaque morgh and the transmission crossing host barriers and the evolution of the virus.To further research into whether there are differences in pathogenicity, replication efficiency and so on between large-plaque virus and small-plaque virus isolated from human H5N1 strain A/Vietnam/1194/2004, large-plaque virus and small-plaque virus were isolated successfully from human H5N1 strain A/Vietnam/1194/2004 forming mixed plaques by plaque isolation assay. And the plaque forming characteristics of the purified large-plaque virus and small-plaque virus were invariant post passages. And this was the prerequisite for this research to further determining the difference in pathogenesis to animals of large-plaque virus and small-plaque virus.II. The pathogenesis of large-plaque virus and small-plaque virus isolated from same human H5N1 avian influenza virusTo determine whether there was difference in pathogenesis of the large-plaque virus and the small-plaque virus isolated from human H5N1 avian influenza virus, the pathogenicity to chickens and mice of the two viruses was determined. And it was proved that the IVPI to chickens of the large-plaque virus and the small-plaque virus be 2.98 and 2.99, indicating both viruses were highly pathogenic to chickens and there was no significant difference between each other. The small-plaque virus was higher pathogenic than large-plaque virus to mice according to the change in mice body weight, average life time, mortality rate, and LD50 of the two viruses in a mice infection model.The large-plaque virus could replicate more efficiently than the small-plaque virus according to the results of replication efficiency assay of the large-plaque virus and small-plaque virus in mammalian cells. It is indicated that there be a correlation between plaque size and virus replication efficiency, while no correlation between virus pathogenicity and virus replication efficiency according to above results, and the large-plaque virus passably be a mutatant of small-plaque virus or evolved from the small-plaque virus post the small-plaque virus transmission to human crossing the species barrier. All above results help to illuminate the pathogenesis to human and evolution of H5N1 avian influenza virus post transmission to human.It was proved that the small-plaque virus had higher tropism to mice central nerve system than the large-plaque virus: (1) virus could be detected in mice infected with the small-plaque virus, while not in mice infected with the large-plaque virus; (2) only small-plaque virus could be detected in mice infected with the original A/Vietnam/1194 /2004. (3) Variant isolated from mice infected with original A/Vietnam/1194/2004 and the plaque-purified small plaque virus isolated in MDCK cells were equally higher pathogenic to mice than the large-plaque virus. We can conclude that the small-plaque virus should have a high troposm to mice central nerve system, while the large-plaque virus couldn't invade the mice central nerve system, there should be a correlation between the high pathogenicity to mice of the small-plaque virus and the invasiveness of the virus.In further research, virus could be detected in blood and brain of mice infected intranasally or intravenously with wild A/Vietnam/1194/2004 strain and the small-plaque virus, and there was a parallelism in tropism to mice blood and mice brain; while virus could be only detected in the blood, not in the brain of mice post inoculation intranasally or intravenously with the large-plaque virus; mice inoculated intravenously with the small-plaque virus or wild virus had higher mortality rate, shorter average life time than mice inoculated intranasally with those virus separately; no death happened to mice inoculated intranasally or intravenously with the large-plaque virus (virus could be detected in infected mice lung). It is indicated according to above results that there be a significant correlation between the inoculation path and the pathogenicity of the small-plaque virus, and mice inoculated intravenously with the small-plaque virus had a more severe clinical process than mice inoculated intranasally with the virus. It seemed that the small-plaque virus can invade mice central nerve system crossing the blood-brain barrier.III. Molecular determinants in differences in plaque forming characteristic, pathogenicity and so on of the large-plaque virus and small-plaque virus isolated from human H5N1 avian influenza virus.To determine the difference in genome sequences of the large-plaque virus and the small-plaque virus, genome sequencing of the two viruses were performed, post determining the significant differences in plaque forming, replication efficiency, pathogenicity and so on of the two viruses. The results showed that there were 21 different amino acid sites in polymerase complex (PB2, PB1 and PA), NP protein, membrane protein (HA and NA). And according to the function of these proteins, the higher replication efficiency maybe relate to the mutated sites in the polymerase complex, and its less pathogenicity maybe relate to the decrease in tropism to mice central nerve system due to the mutation of 2 (N to D) and 315 (C to R) .To further determine the correlation between the virus pathogenicity, replication efficiency and so on and different amino acid sites of the two viruses. We constructed the recombinant plasmids of eight genome fragments of A/Vietnam/1194/2004 and pHW2000, then the recombinant plasmids of PB2, PB1, PA, HA, NA, NP and pHW2000 were constructed by site-specific recombination. And all these work provide important prerequisites to further virus rescue, plaque forming assay and determination of the molecular of pathogenicity of recombinant viruses.In a summary, all above results throw lights on the mechanism of avian influenza virus transmission accrossing species barrier and virus evolution. It's a novel and effect method to research into the pathogenicity and evolution of avian influenza virus by determine the differences genome sequences, pathogenicity and replication efficiency of viruses forming different plaques isolated from same H5N1 avian influenza strain. |
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