| Tibet, located in the southwest of China, borders on many countries and areas with its long borderline. On the migration route of birds, it's one of the important gathering places of wild birds in China. Up to now, avian influenza viruses have been detected in wild birds in many countries and areas. As the species with the highest altitude geographical distribution, Tibetan chickens have great adaptability to high altitude, low oxygen and stocking. Tibetan chickens have some small local original breeds and were listed into the National Variety Resources Protection List in 2000. With the adjustment of agricultural and husbandry industrial structure in Tibet, the level of intensive farming of Tibetan chickens has risen a lot over the recent years. The number of large-scaled chicken farms is continually growing and chicken farming has become one of the significant ways for Tibetan farmers and herdsmen to get rich. Due to the lack of a comprehensive and systemic prevention and treatment technology, the incidence of various infectious poultry disease remains rather high. What's worse, the global influenza outbreak and pandemic have brought substantial economic loss to the farming of Tibetan chickens, which severely dampen the enthusiasm of farmers and herdsmen. This situation poses a serious threat to Tibetan poultry industry.In this study, avian influenza virus and Newcastle virus were isolated and identified for the first time. Then the biological characteristics of both viruses were studied in order to understand the genetic evolution pattern. The comparative proteomics analysis was used to screen the proteins differentially expressed by the representative NDV strains after infecting chicken embryo fibroblasts. The results revealed the interaction between viruses and host cells, as well as the pathogenic molecular mechanism. Our study provides basis for the formulation of prevention and control strategy of avian influenza in Tibet, and lays the foundation for the prevention and control of NDV outbreak and prevalence.1. Whole genome sequencing and analysis of avian influenza virus subtype H9N2 isolated in Tibet provinceThe main purpose of our study is to sequence and analyze the whole genome of the three strains of avian influenza virus (AIV) subtype H9N2 isolated from Tibet. The method of the study is to design eight pairs of primers according to the sequences of AIV subtype H9N2 released in GenBank, then they were used in RT-PCR to get the complete nucleotide sequences of A/chicken/Tibet/S1/2009, A/duck/Tibet/S2/2009 and A/chicken/Tibet/ S4/2009. After cloning and sequencing, obtained gene sequences were analyzed on homology and heredity evolution. The result suggesting that the split site of HA gene is RSSR/G,which is a character of low pathogenic AIV; The nucleotide homology of the three strains islolated from Tibet was from 98%to 99%presumably they might come from the same source; the genetic distance of HA,NS and NA gene in each islolated strains and A/Chiken/Beijing/1/94,a branch belonged to Asian&eEurope branch is the closest,however,the genetic distance of M, NP, PA, PB1,PB2 gene is closer to A/Quail/HongKong/G1/97,another branch of Asian&eEurope branch. It is conclude that the braches to which each gene of the three strains belongs is different, indicating that the three strains of avian influenza virus (AIV) subtype H9N2 isolated from Tibet in our study possibly are the natural gene rearrangment products of the process of different subseries of AIV infecting the same host.2. The biological characterization and phylogenetic analysis of Newcastle disease virues isolated in Tibetstrains of virus with hemagglutination activity were isolated from the dead chickens or ducks in Tibet and were identified Newcastle disease virus (NDV) by the hemagglutination inhibition test, RT-PCR and sequencing, furthermore it proved that the viruses were pathogenic to chickens by pathogenicity test; the classical pathogenicity test, including MDT, EID50, IVPI and ICPI of these isolated strains were 120h,10-8.44/ml,0.5 and 0.6. The hemagglutinating free and hemagglutining thermal stability test showed that: hemagglutinating free time of these strains was short, and the thermal stability of hemagglutinin was low, in line with the characteristics of avirulent NDV. The phylogenetic analysis of F genes showed nucleotides sequence homology between these strains was 99%, the homology with the vaccine strain LaSota and standard virulent strain F48E8 was 90%and 81%respectively. Deduced amino acid sequence analysis showed that the cleavage site amino acids of F protein were 112G-K-Q-G-R-L117, similar to the characteristics of attenuated NDV, which were consistent with the pathogenicity test. This paper first reported the genetic evolution and biological characteristics of NDV strains isolated in Tibet which is contributed to further study the biological characteristic changes of NDVviruses in high altitude, hypoxic environment,.For study the interaction of Tibetan chicken-NDV and host cells, the comparative protomices analysis was used for CEF which was infected by Tibetan chicken-NDV. And differentially abundant cellular proteins were detected by 2-DE and identified by MALDI-TOF MS/MS at the 24,48 and 60 hpi. The analysis of different expression in Two-dimensional gel mapping showed that a total of 50 spots were found to be changed. 38 spots were successfully identified, which were mapped 29 proteins including 17 up-regulated proteins and 12 down-regulated proteins.It was notable that 8 phosphproteins of NDV were identified in Tibetan chicken-NDV infected CEF. In the 8 phosphproteins,7 proteins (CJ02, CJ03, CJ05, CJ07, CJ08, CJ20, CJ23) have same molecular weights but different isoelectric point. However, the other phosphprotein (CJ14) was different from the 7 phosphproteins in molecular weight and isoelectric point. In present study, some of the actin were up-regulation (actin, a cardiac muscle 1 isoform 2 and cytoskeletal?actin), and some other actins were down-regulation. keratin 18 and cytokeratin 8 were both down-regulation cells. Though it was not indentified the differential expression of tubulin and Hsp70 because of the not perfect technology of 2-DE in this experiment, western-blot confirmed the down-regulation of?-tubulin and Hsp70. In the present study, Hsp90 and Hsp27 were great down-regulation. The research found out that the protein of VDAC-2 was significant up-regulation. Moreover, the expression of hnRNPs was found change. hnRNP C was down-regulation, but hnRNP K was up-regulation.Through the signal network model of biological software, it was found that signal network was based by HSPs and transcriptional regulation metabolism of protein. However, some differentially expressed protein interacted with cellular inflammatory and immune regulated factors. It demonstrated the cellular complex biological changes in CEF cells infected by NDV. For further revealing the pathogenesis of NDV, more research should be performed to study the interaction of cellular proteins and NDV. |
PDF Full Text Request