Inhibitory Effect Of Oseltamivir On Various Subtypes Of Influenza Virus And Screening Of Chinese Herbal Monomers Of Drug-Resistant Strains

Objective:1.To establish an avian influenza virus infection model in mice to evaluate the inhibitory effect of oseltamivir on five HA subtypes of influenza viruses.2.To analyze the NA gene resistance sites of seasonal influenza A（H1N1）viruses and evaluate the inhibitory effect of several Chinese herbal monomer compounds on H1N1 subtype influenza viruses at cellular level and to explore new methods of anti-influenza viruses.Methods:1.Culture human seasonal influenza viruses H3N2,H1N1 and avian H6N1,H9N2,H5N6 and H3N2（adaptive strains of avian mice）by chicken embryo reproduction virus technology.The influenza virus infection model of mice was established by intranasal inoculation.The preventive group,treatment group and virus group were treated with oseltamivir by intragastric administration.The control group was treated with saline of equal volume.The clinical manifestations were observed daily,and the weight change and death of mice were recorded.Lung tissues of each group were taken every 3 days after intranasal inoculation of influenza virus.The titers of viral lung tissues were measured and the pathological changes were observed by HE staining.2.H1N1 subtype influenza virus was amplified and cultured by MDCK cell line,and the full sequence of NA gene was obtained for drug resistance site analysis.Neuraminidase inhibitor oseltamivir and monomer compounds of Chinese medicine theaflavin,rutin,hyperoside,hydroxysafflower yellow A,jatrorrhizine hydrochloride and patchouli alcohol were used to evaluate the inhibitory effects of H1N1 subtype influenza virus through direct and indirect action,respectively.Results:1.Five influenza virus infection models were successfully constructed in mice.The clinical manifestations and pathological sections showed that human seasonal influenza virus H3N2 had no obvious clinical manifestations but could replicate in the lungs of mice.Human seasonal influenza virus H1N1 could cause death in mice,and the titer of virus in lung tissue was as high as 6.167 log10EID50/0.1ml.Severe inflammatory changes were observed in the lungs.H5N6 avian influenza virus and H3N2 avian influenza adaptive strain can cause death and high level of pulmonary viral titer in mice;H6N1 influenza virus and H9N2 influenza virus can not be replicated in lungs after intranasal inoculation in mice.2.A human seasonal influenza virus H1N1 cell infection model was successfully constructed.It was determined that 100%of the inoculated holes could be hemagglutinated by inoculating 96-well single-layer MDCK cells with 100 TCID₅₀.3.It was found that human seasonal influenza virus H3N2,H5N6 avian influenza virus and H3N2 avian influenza mouse adapted strains could reduce the viral titer of lung tissue,alleviate pulmonary inflammation,or improve the survival rate of mice after the prevention and treatment of oseltamivir by intragastric administration.The related evaluation indexes of H1N1 influenza virus in the prevention and treatment of oseltamivir did not improve,which was similar to the viral group.No difference.4.No mutation of H1N1 influenza virus strain was found by comparing the whole gene sequence of NA coding region.It was found that theaflavins could inhibit H1N1influenza virus when the concentration of theaflavins was above 0.125 mg/ml,rutin could inhibit the virus when the concentration of rutin was above 2 mg/ml,and jatrorrhizine hydrochloride could inhibit the virus when the concentration of theaflavins was above 2 mg/ml.The viral titer could be reduced when the concentration reached 1mg/ml.Hyperoside,hydroxysafflower yellow A and patchouli alcohol have not yet shown the effect of anti-H1N1 influenza virus.Conclusion:The infection models of human seasonal influenza virus H3N2,H1N1,avian H5N6 and avian H3N2 strains in mice were successfully constructed.It was confirmed that avian H6N1 subtype influenza virus and H9N2 subtype influenza virus could not replicate in lung tissue of mice.The inhibiting effects of oseltamivir on human seasonal influenza virus H3N2,H1N1,avian H5N6 and avian H3N2 strains in mice were evaluated by preventive and therapeutic methods.It was found that oseltamivir could effectively inhibit human seasonal influenza virus H3N2,avian H5N6 and avian H3N2 strains in mice.It was found that oseltamivir could not inhibit H1N1 subtype influenza virus both in vivo and at the cellular level in mice infected with the virus.However,theaflavin,rutin and jatrorrhizine hydrochloride,the monomer compounds of traditional Chinese medicine,had better inhibitory effect on the strain.