The Establishment Of The Animal Model Infected By Avian Influenza Virus In Mice And The Effect Of Interference Avian Influenza Virus Of The Chinese Herbal Compound

Avian influenza(AI) is an infection and/or a diseased syndrome in poultry and wildfowl,which is caused by influenza type A virus.Human avian influenza (HAI) is a zoonosis which manifest respiratory tract infection and mucous hyperemia,it results from contacting with excrements and secretions contaminated by avian influenza virus(AIV).Some patients infected with highly pathogenic AI strains can show Acute Respiratory Distress Syndrome and Multiple Organ Failure,and the mortality is high.AI has been classified as A-type infectious diseases by OIEAI has been considered as the largest human influenza gene pool all along, and is the source of new genes of human influenza viruses mutation.In recent years,AIV has spread in many countries,and is a great threat to human's health.According to the statistics made by the World Health Organization,now 405 people from 15 different countries had been infected with highly pathogenic AIV,and 94 people are dead.In China,38 cases of highly pathogenic AIV infection has been reported yet.AIV has been proved that it can spread from fowl to human,but at present there is no sufficient evidence to confirm that whether AIV can be transmitted among persons or not.It still needs researchers to analyze an question,why AIV can infect human and cause human to death.AIV is able to evade identification of the human immune system,but the mechanism remains unclear.It has been reported that AIV from human source or poultry source could successfully infect the bird,mice,rat,pig,cat,ferret and monkey.Because the mice has the feature of small body,many strain,easily obstained,simply controlled and sensitivity.Mice will become important laboratory animals used for researching the pathogenesis of AIV infection in mammal.Because of continual drift and variation of AIV antigen,threat to poultry and human,we speculate that AIV can transmit from person to person eventually. So we need to establish the laboratory animal model and go on basal and precautionary research on AIV so that we can master the pathogenic characteristics,pathogenesis and the precautionary measures of it.At present,western medicines prevent AI mainly through cutting off viral replication or transmission,but the main shortages of them are low bioavail-ability of the amantadine,drug vice-effect of the Duffy,and teratogenicity from the ribavirin.The vaccines used in controlling AIV mainly make use of eliminating alive virus or of virus so that organism can produce antibodies against the similar virus infection,but they also have shortages.For example,AIV antigen are varied continually,furthermore the vaccines can be affected by the birds species,age,gender,complicated infection,virulence of AIV strains,and other environmental factors.Therefore,regardless of the traditional inactivated vaccine,genetically engineered vaccines,or DNA vaccine,they can not produce cross-protection of AIV.The AIV may break out again in the vaccinated chicken flock.Traditional Chinese medicine(TCM) has emphasized the rules about "the dialectical control" and "the prevention is better than the treatment".The control strategy of TCM is to strengthen body resistance and eliminate pathogenic factors.TCM has many advantages,for example,TCM contain a lot of active components,which can kill virus by different mechanism.TCM also has a role in immune regulation,and can improve and enhance the immune function.TCM can control virus infection significantly.Therefore,we should strengthen studying on TCM about prevention and treatment against AIV,which are to make up for the deficiencies of biological medicine,chemical medicine(western medicine) in the treatment of AIV.According to the features that HAI commonly happens in winter or spring,HAI belongs to the epidemic cold of the plague in TCM,the pre-experiment suggested that the disease quickly spread from external to internal and fever was no obvious after the mice were infected by virus of AIV.So after doing many CHC(Chinese Herbal Compound) pre-experiment,we eventually selected the Guizhi Decoction with the two-way regulation effect that could control the temperature,immune reaction and gestrointestinal function;the Huoxiang-Zhengqi powder that could treat gestrointestinal cold;the Shangju Drink that could treat the initial wind-head cold.These Chinese Herbal Compound would interfere the animal model infected by AIV.[Objectives]In the studies,we will compare the sensitivity of laboratory animals infected with AIV,and develop animal models of AIV by using the most sensitive laboratory animals,we also evaluate the effect of the Chinese Herbal Compound about Anti-AIV.[Methods]There are four experiments in the second part of the paper:1.To determine LD50 of AIV infection in BALB/c mice by Reed and Muench's method.2.After anesthesia,SPF mice:KM,ICR,NIH,BALB/c and Clean rat:F344,SD, Conventional Gerbils,were inoculated intranasally with AIV 100μL per 20g weight.The rabbits were infected with 2mL AIV intratracheally by thyrocricoid puncture.Then we observed the clinical symptoms,changes of body temperature, body weight,mortality and the pathological changes to evaluate the pathogenic sensitivity of lab-animals infected with AIV.3.30 BALB/c mice were inoculated intranasally with 100μL AIV,another 30 BALB/c mice as control group were inoculated intranasally with normal allantoic fluid.Then we observed the clinical symptoms,changes of body temperature,body weight,mortality,and pathological changes.4.110 BALB/c mice were divided into 6 groups randomly:HuoxiangZhengqi powder group(26.4g/kg,20mice);Guizhi Decoction group(15.6 g/kg,20mice);the ShangJu Drink group(34.4g/kg,20mice);positive medicine control group (ribavirin 70mg/kg,20mice);a model control group(0.4mL saline,20mice); allantoic fluid control group(0.4mL saline,10mice).Before infection with AIV, the mice were given different types of medicine 2 days ahead respectively through inoculated into the stomach.On the day infected AIV,the drugs were inoculated into the stomach in the morning,while those group except for the allantoic fluid control group were infected intranasally with 100μL AIV in the afternoon.The drugs were still given for 2 days after infection,we would observed the body temperature,body weight,survival time,mortality and food consumptions of the groups.[Results]1.MLD₅₀ of AIV in BALB/c mice is 10^-0.375/0.1mL.2.The result of the pathogenic experiment about the eight lab-animals infected by AIV revealed that KM,NIH,ICR,BALB/c mice and Gerbils could replicate different degree clinical features of AIV.Firstly there are different latency,the latency of the KM mice is long and the BALB/c mice is short,disease happens quickly.Secondly the clinical symptoms and mortality of the kinds of mice infected by AIV are different.BALB/c,ICR and NIH mice showed a development progression of the disease,More than half in these animals died with serious clinical symptoms,and the remainder recovered and cured from the disease,which is contribute to the evaluation of the model.The KM mice revealed only the slight clinical symptoms,the body temperature and body weight decreased transiently and recovered quickly.SD and F344 rats showed no clinical symptoms and were separated no AIV.The rabbit showed the slight change of the body temperature and lung pathology and were separated no AIV.So the KM mice,rats and rabbit were not fit for making the animal model of AIV.Acording to the statistical analysis of the result,The body temperature changes of KM,NIH,ICR,BALB/c mice and SD,F344 rats,Gerbils decreased apparently than the allentoic fluid Group in the statistic.the temperature of BALB/c mice significantly reduced the most.The body weight changes of KM,NIH,ICR,BALB/c mice and Gerbils decreased apparently than the allentoic fluid Group in the statistic.The mortality of the different strains or species of animals infected with H5N1 AIV were as follows:BALB/c mice is 80%(8/10)＞Gerbil 54%(7/13)＞ICR mice 50%(5/10)＞NIH mice 44%(4/9)＞KM mice 10%(1/10)＞SD rats and F344 rats.The important model index of animals infected by the virus is that we can separate the same virus in the infected animals.In our studing,the results of separated AIV from the lung of the KM,NIH,ICR,BALB/c mice and Gerbils infected by AIV confirmed that those clinical symptoms were produced by AIV infection.This also can be confirmed by the results of separated no AIV from the lung of the rats and rabbits.To sum up,AIV can produce different pathogenicity in different lab-animal's body.The clinical symptoms,changes of body temperature and weight,mortality,pathological changes from the BALB/c mice infected is most obvious in all testing animals.3.The experiment results illustrated that the AIV could lead to the inflammation pathological changes of many organs,tall mortality,the recoverd antibody produced in BALB/c mice.There was a complete development and progression of the disease in infected BALB/c mice,so it could become the better AIV animal model.The six model indexes were as follows:â‘ The clinical features:The progression of the disease in infected BALB/c mice were classified as the latency,onset period and convale--scence.The specific clinical manifestations consist of short latency,acute onset,turning up the respiratory tract symptoms,developing fastly the disease such as pneumonia,multiple organ failure and die.During the onset period,the lung was observed to serious hemorrhage,edema,necrosis,the epithelial degeneration and detachment in lung catheter,with hyperemia and numerous inflammatory cell infiltration that also showed in the lung alveolar and pulmonary interestitial.â‘¡The changes of body temperature:the BALB/c mice infected showed the progression of the body temperature decreased.â‘¢The changes of body weight:the BALB/c mice infected showed the progression of thebody weight reduced.â‘£The viscera index:the lung and liver index increased in BALB/c mice infected.⑤The mortality:the mortality of BALB/c mice infected was high.â‘¥The virus replicate:The virus was separated successfully from the lung,beart,liver and spleen in the BALB/c mice infected.The rate of virus separated from the lung was higher than other.The lung was regarded as the target organ infected by AIV,the macrophages and tracheal epithelial cells in lung were regarded as the target cells infected by AIV.The antibodies could be observed in the bodies of recovered mices.The establishment of the animal model Infected by AIV in mice provide us the small mammalian inbred strain lab-animals that have easily obstaining,stable,clear genetic backg round features to study the pathogenesis of AIV and observe the effects of drug treatment.4.Huoxiangzhengqi powder administrated on mice infected by AIV showed the most obvious protective effect of body temperature,the following was Guizhi Decoction and Shangju Drink,but Ribavirin group showed no protective effect.Body weight lost in the group treatment with western and Chinese medicine was slower than those of model control group.The mices in Huoxiangzhengqi powder group and Guizhi Decoction group resumed taking foods at 5-6 days post-infection,which demonstrated that the two groups can restore the function of stomach,and Guizhi groupwas the best.The spleen and stomach are the fundamental physical organs after birth,the stomach has the functions of digestion,general health and nutrition status,the body's compensatory and immune.The body's organs are intrinsic gas collection in stomach. Therefore,Huoxiangzhengqi powder and Guizhi Decoction can regulate the decreased body temperature of mice infected AIV,protect the stomach,and enhance the mice's ability of anti-virus.Huoxiangzhengqi powder and Guizhi Decoction can help the mices rapfdly restore the health and appetite,which according with the slow weight loss of mice treated with the two Chinese Herbal Compound respectively.By Chi-square test,The death-protection rate among Huoxiangzhengqi powder group,Guizhi Decoction group,Shangju Drink group,and Ribavirin group was significantly different.The death-protection rates sorted from high to low were as follows:Guizhi Decoction group,Huoxiangzhengqi powder group,Shangju Drink group,Ribavirin group.Inhibitory rate of lung index of Huoxiangzhengqi powder group,Guizhi Decoction group,Shangju Drink group and Ribavirin group were positive,which indicated that the degree of pulmonary pathologic change of the treatment group was lighter than that of model control group.Chinese Herbal Compound had better effect to reduce lung lesions than western medicine,which showed that Huoxiangzhengqi powder group and Guizhi Decoction group can resist H5N1 AIV infection and protect animals.[Conclusions]Firstly,the result of the pathogenic experiment about the eight different lab-animal infected by AIV revealed that BALB/c mice was the most sensitive animal to H5N1 AIV infection in this study.The clinical symptoms,body weight changes,mortality,pathological changes in BABL/c mice are the most obvious. Other strains of mice also showed a certain pathogenic.The rats and rabbit showed no clinical symptoms after infected AIV.Secondly,the whole animal model about BALB/c mice infected by AIV has been established successfully.We have mastered the six model indexes including clinical features,body temperature changes,body weight changes,mortality, viscera index and virus replicate.The establishment of the animal model and the model indexes can help us take advantage of the small mammalian inbred strain lab-animals which have easily obstaining,stable,clear genetic back-ground features to study the pathogenesis of AIV and observe the effects of drug treatment.Thirdly,the study results on effect of interference H5N1 AIV in mice by using three Chinese Herbal Compound were as follows:1.Huoxiangzhengqi powder and Guizhi Decoction had a warming effect on lower body temperature of the mice infected.2.Guizhi Decoction and Huoxiangzhengqi powder could increased the body weight of the infected mice.3.Guizhi Decoction and Huoxiangzhengqi powder could increased the death-protection rates of the infected mice.4.Huoxiangzhengqi powder and Guizhi Decoction could inhibit the lung lesion.5.Guizhi Decoction and Huoxiangzhengqi powder could regulate gastro-intestinal function and restore the function of the stomach.Guizhi Decoction and Huo Xiang Zheng Qi San focus on different function in prevention and treatment of H5N1 AIV.Since the clinical features of AIV will be changed,we should change prescription of Chinese Herbal Compound.It will be helpful to improve the effect of prevention and treatment of Chinese Herbal Compound against H5N1 AIV.