Study Of The Aerosol Transmission Of Avian Influenza Virus H9N2Subtype Mediated By Annexin â…¡

Avian influenza virus H9N2subtyp e was first isolated from the chicken poultry in China in1994and then became more and more endemic. The hemagglutinin (HA) is a glycoprotein and the major antigen found on the surface of the avian influenza viruses, also can recognise vertebrate cells, and has to be made cleavage into the HA1and HA2subunits which is essential for the avian influenza viruses to become infectious. The conversion of plasminogen (PLG) into plasmin (PL) which mediated by Annexin II (A2) can make the plasmin possess a trysin-like protease activity, provide an alternative way for the cleavage of the HA and then improve the ability of the replication of the avian influenza. The inhibiting activity of plasminogen would limit the replication of the avian influenza viruses. In this study, A/Chicken/Shanghai/7/2001(H9N2) AIV (62strain), which can spread heavily through aerosol, and A/Chicken/Shanghai/14/2001(H9N2) AIV (70strain), which cannot spread through aerosol, were selected to study the mechanism of the aerosol transmission of avian influenza virus H9N2subtype mediated by Annexin II with and without PLG in vitro (Madin-Darby canine kidney cells, MDCK cells) and with and without6-aminohexanoic acid(6-AHA) in vivo (4-week-old SPF chickens). 1. Study in vitro of the aerosol transmission of avian influenza virus H9N2subtype mediated by Annexin â…¡The62strain and70strain were purified by ultracentrifugation methods and A2could be detected in both purified viruses and the unpurified viruses, and the A2protein expressed in62strain was significantly higher than that of the70strain; With the different concentrations of PLG, the tissue culture infective dose50(TCID50) of the70strain was decreased obviously, but increased for62strain at72h after inoculation MDCK cells, indicating that the ability of the conversion of plasminogen into plasmin mediated by A2in62strain was stronger than that in70strain; The TCID50of60strain and70strain in added PLG group was significantly higher than that in none-added PLG group, suggestting that PLG play a important role in the replication of virus, so the conversion of plasminogen into plasmin mediated by A2can improve the replication and increase the possibility of the aerosol transmission. To further confirm these conclusion, To prove the above views further, the PL protein expressed in MDCK cells infected with62strain and70strain respectively with PLG and PLG plus6-AHA ead detected by western blot. The titer of PL from MDCK cells in adding PLG group was higher than that in PLG plus6-AHA group for both62strain and70strain, and the expression of PL in MDCK cells infected with62strain was visibly higher than that in MDCK cells infected with70strain with PLG only. These results fully proved that the efficiency of the conversion of PLG into PL play a important role in the replication of H9N2subtype of avian influenza virus, and the ability of the conversion of plasminogen into plasmin mediated by A2in62strain was stronger than that in70strainDue to A2was detected in virus, to explore the role of A2in the efficiency of the conversion of PLG into PL, the expression of A2protein in the MDCK cells infected with62and70respectively was analyzed by western blot in two different group, one group was with PLG and the other was with PLG plus6-AHA. The expression of A2in the supernatant of MDCK cells infected with62and70separately with PLG was distinctly lower than that with PLG plus6-AHA, showed that A2in both62strain and70strain could promote the conversion of PLG into PL, 2.Study in vivo of the aerosol transmission of avian influenza virus H9N2subtype mediated by Annexin â…¡The transmission experiments that the4-week-old SPF chichens were infected with avian influenza viruses H9N2subtype62strain and70strain showed that the62strain have the ability of the aerosol transmission, but70strain not. The chicken in infection group and the aerosol transmission group were fed with the6-AHA in water, the ability of replication in trachea and cloaca in70and62strain infection group were monitored more less; especially that of the aerosol transmission group of62decreased significantly, indicating that6-AHA could inhibit the activity of PLG and the aerosol transmission capacity of62strain. The less virus antigen in lung was observed in chickens inoculated with62stran and70stran and fed with6-AHA by immunohistochemistry compared to fed without6-AHA. It suggested that6-AHA could inhibit PLG into PL mediated by A2, and stop the HA cleavage of influenza virus into HA1and HA2in vivo and then reduce the spread influenza virus of by aerosol.