| Influenza is a disease caused by influenza virus. Influenza viruses hold generic status in the Orthomyxoviridae family and are classied into types A, B or C based on antigenic differences of their nucleo- and matrix proteins. Avian influenza viruses (AIV) belong to type A. On the basis of the antigenicity of these glycoproteins, influenza A viruses currently cluster into sixteen H (H1—H16) and nine N (N1—N9) subtypes. These clusters are substantiated when phylogenetically analysing the nucleotide and deduced amino acid sequences of the HA and NA genes, respectively . The highly pathogenic form of avian influenza has been caused to date by influenza A viruses of the H5 and H7 subtypes exclusively.Vaccination has been widely used in prophylaxis for many years, it can cut down morbidity and mortality in high risk group, also can relieve symptoms and complications.Influenza mainly occurred during december to the next february, it belongs to "hidden exogenous seasonal disease", "acute seasonal febrile disease" category(ASF). ASF was caused by winter cold,incubating and stagnation internalfor a long time , then transmit into febrile evil.Until spring comes, the insidious febrile emit from Qi fen or Ying fen.During influenza pandemic,sufferers presented symptoms as fever,sore-throat, cough, myosalgia, hypodynamia, headche, chill, sweating, coma and delirium.Theses symptoms onset suddenly,pathogenetic conditions are hazardous,even to die within a few days. A ccording to these records , influenza pandemic maily onset from Qi fen and Yingfen of Traditional Chinese Medicine therory. Ye Tianshi said that febrile evil canbe transferred from Ying back to Qi fen. Therapeutic principle is cooling Ying fen and disperse Qi febrile,the typified formula is Cooling Ying Soup(CYS). Objectives:1, According to the ASFtheory, to establish the CYS therapy of influenza, in vivo and vitro experiments to verificated the antivirus effects.2, To approach the mechanism of CYS in antivirus action.3, To clone the HA and NA genes of A/Fort Monmouth/1/47(H1N1),sequencing and find variation nucleotide sites, compared between the correlated subtypes. Methods: 1, Influenza virus A/F M/1(H1N1) was passaged in embryonated egg allantoic cavity,incubated in 37 "Cfor 48hours then collected allantoic fluid to infected mice , tissuebomogenated lung for infecting mice, repeated three times, then determinatedhemagglutinin titre and medial lethal dose(LD50).2^ NIH mice randomly divied into five groups named A ,B,C,D. A is model groupwhich lavaged distilled water, B is Ribavilin groupwhich was lavaged 70mg/kg/d,C andD are high and low dose of CYS which were lavaged lOg/kg/d and 5g/kg/d. all thegroups were ethylether mild anesthesia, nasal dropped virus 15LD50, 96 hours laterdissedted to take lungs,weighed,calculated lung-body weight index and index inhibitionratio.3^ NIH mice randomly divied into five groups named A ,B>C,D,and E.A is modelgroup which lavaged distilled water,B is Ribavilin groupwhich was lavaged70mg/kg/d,C and D are high and low dose of CYSwhich were lavaged lOg/kg/d and5g/kg/d. all the groups were ethylether mild anesthesia, nasal dropped virus 2LD50,survery and record the dead amount, 15days later calculated the death-protective ratioand life span prolong ratio.4> In vitro, inoculate virus into 96 holes template contain MDCK cell. CYS wasmultipile proportion diluted into 8 concentration, misce bene, survery the inhibitoryaction of CYS on viral multiplication and cytopathogenic.5> NIH mice randomly divied into five groups named A ,B,C,D,and E.A is modelgroup which lavaged distilled water, B is Ribavilin groupwhich was lavaged70mg/kg/d,C and D are high and low dose of CYSwhich were lavaged lOg/kg/d and5g/kg/d. all the groups were ethylether mild anesthesia, nasal dropped virus 15LD50,seven days later picked eyeballs to take blood.centifuging for serum,determine theserum SOD and MDA.6> Virus inoculated into 9-day-old SPF embryonated egg allantoic cavity,incubated in37 °C for 48 hours then collected allantoic fluid,extract RNA,referenced the sequences ingenbankto design primer, RT-PCR amplifyed HANAgene,retrieve the DNA,lingasepMD18-T vector,transfected JM109 competent cell,delivered to Invitrogen technologyCo.sequencing.Analysis the nucleotide sequence by ORF finder and BLAST online.Results:1> Lung index inhibition ratio of Ribavilin group ,high and low dose of CYS groupsare 30.54%,33.46%,36.73%,compared with the model group, there significantdifference (p<0.01).2 n Four days after infected virus,the mice were lofty wool,wriggly, hair-withered.reduce appetite.body weight loss,gradully failure,most of which died afterseven days.the death-protective rate of high and low dose CYS are:40%, 40%, lifespan prolong rate are 69% and 38%.3^ CYS had no inhibitory action on influenza virus in vitro study.4^ High and low dose of CYS influenced the mice serum SOD MDA.which was SOD176.36±35.47U/mL , MDA % 13.64±2.69nmol/mL in model group;SOD 298.98±32.53U/mL , MDA4.98±1.73nmol/mL high dose of CYS;SOD 207.85±37.79U/mL, MDA % 6.80±2.76nmol/mL in low dose of CYSoConclusionK In vivo study indicated that CYS could relieve the mouce pneumonia and prolongthe life span of mice infected influenza virus.2n In vitro showed that it had no inhibitory effect on the replication of influenza virus.3n In vivo study, CYS could increase serum SOD, decrease MDA, which indicated thatCYS could reduce lipid peroxidation damage thus reduce inflammatory reaction to treatinfluenza.
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