الفهرس يوجد فقط 14 صفحة متاحة للعرض العام
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المستخلص
Through 2-3 years of HPAI H5N1 circulation in Egyptian environment, there have been 23 human deaths out of 54 infections. In addition, more than 1084 outbreaks were reported in poultry. Moreover Egypt is ranked the worst affected country after Indonesia and Vietnam, and the worst affected country in the eastern Mediterranean region and Africa. The results obtained from this study can be summarized in the following points,
1) Cloacal swabs were collected from diseased animals having typical symptoms of avian influenza.
2) Isolated samples were characterized by using rapid antigen strips as a field screening tool for rapid diagnosis of avian influenza viruses and H5 viruses in the field.
3) Characterized isolates by rapid antigen strips were confirmed by RT-PCR amplification of specific fragment amplification of H5 and N1 and further sequencing and phylogenetic analysis of complete HA sequence. This indicated that the isolated strain is HPAI (H5N1).
4) Confirmed positive H5N1 sample was propagated on MDBK cells to increase viral concentration and titrated using plaque infectivity assay.
5) Propagated virus harvest was used for in vitro infection of different cell lines obtained from different hosts.
6) Significant CPE was observed microscopically through the different cell lines used. Viral infection and propagation was confirmed by RT-PCR amplification of conserved gene fragment of H5, SDS-PAGE, and Western blotting of the infected and control cell lines´ harvests. Previously mentioned tests showed clearly that the tested cell lines were susceptible to infection with H5N1, and the virus may be able to infect other hosts of no history to be susceptible to infection with H5N1 virus such as rabbits (RK13), baby hamster (BHK), bovine (MDBK) and monkey (Vero).
7) Quantitative measurement of different cell lines’ susceptibility to infection with Egyptian isolate of H5N1 by Enzyme linked immunosorbent assay (ELISA), Plaque infectivity assay and Real time PCR of different infected cell lines in comparison to uninfected ones showed differences in susceptibility between the infected cell lines. This is due to the difference in viral particle counts and genome copies indicating that not all cells mediate virus replication in the same manner and there are host cell factors that mediate or retard virus replication depending on the characteristics of each cell lines.
8) Propagated stock HPAI-H5N1 virus harvest was used for in vitro inoculation of different types of water to study avian influenza H5N1 isolate persistence in different types of waters. This persistence was detected by microscopical examination of inoculated cell culture sheets and plaque assay of water samples collected at weekly base interval. The results revealed that the persistence of the virus is inversely proportional with salinity and bacterial load because virus persistence was found to be lower at sea water and raw water than that at tap water and Nile surface water. Moreover, the viral isolate persists for longer periods in well water than surface water samples incubated at similar temperature and less persistent in natural waters and other media compared to same media that have been sterilized.
9) Propagated stock HPAI-H5N1 virus harvest was also used for experimental infection of Biomophlaria alexandrina snails in water. After this exposure, the snails were washed and transferred to fresh water. The RT-PCR amplification of the H5 gene of viral RNA extracted from fresh water and the detection of viral antigens in such water as well as homogenates prepared from the snail tissues by enzyme linked immunosorbent assay (ELISA) revealed that Biomophlaria alexandrina might support propagation and shedding of HPAI-H5N1 virus in the snail tissues.
10) To confirm the previous evidences that support the hyposis of the susceptibility of B. alexandrina to infection, zero time snail water samples collected before transfer, immediately after transfer and each 3 days of transfer of snails exposed to infection from virus containing water to fresh water were subjected for real-time RT-PCR.
11) Zero time snail water do not exceed the cycle-threshold, while the output figure showed narrow variation in cycle-threshold (RNA copies) among samples collected after transferring of exposed snails to H5N1 infection into fresh water (3days and 6 days). Such results of real-time RT-PCR ensure the susceptibility of the snails to infection and shedding of virus in water.
12) from previously mentioned, we can conclude that this study can direct us to many approaches.
A) Firstly, Studying the host range using an easy, controlled, economic and rapid in-vitro cell line system instead of highly costive experimental infection of animals. B) Secondly, the importance of elevation of public awareness to deal with infected and dead poultry, and not improperly dispose bird carcasses in the Nile. C) Thirdly, Further studies on the susceptibility of different marine hosts especially the most popular in our environment, especially Biomophlaria alexandrina snails, to infection with H5N1 isolate of Egypt.