禽流感病毒感染小鼠模型及中药复方干预作用的研究
|
摘要
禽流感(Avian Influenza,AI)是由A型流感病毒引起的家禽或野禽的一种感染和/或疾病综合征。人禽流感(human avian influenza,HAI)是人接触被禽流感病毒污染的排泄物、分泌物或气溶胶而感染,并出现以呼吸道感染、黏膜充血等为主要表现的人禽共患病,部分感染高致病性禽流感毒株的患者可出现呼吸衰竭和多器官损害,死亡率高。禽流感已被国际兽医局列为A类烈性传染病。
禽流感历来被认为是人类流感的最大基因库,是人流感病毒发生变异的新基因来源,近年来禽流感病毒正在世界多个国家传播,已对人类健康构成了很大的威胁。据世界卫生组织统计,目前,全球共有15个国家405人感染了高致病性禽流感病毒,其中94人死亡,我国至今发生38例人感染高致病性禽流感。
禽流感病毒(Avian Influenza virus,AIV)从禽类感染人已被证实,但目前还无充分证据证实禽流感病毒从人到人的感染。禽流感病毒为什么能感染人并对人具有致死性仍是研究人员在深入探讨的问题。禽流感病毒能逃避人体免疫系统的识别,但其具体机制仍不清楚。
已有人源或禽源禽流感病毒引起不同鸟类、大小鼠、猪、家猫、雪貂和非人灵长类动物致病性的研究报道,但从实用性考虑,小鼠有体形小、品系多、易获得、操作方便、对禽流感较敏感等突出优点,小鼠将成为研究禽流感病毒感染哺乳动物发病机制的重要实验动物。
面对禽流感病毒抗原的不断漂移与变异,面对禽流感病毒对禽类和人类构成的严重威胁,面对禽流感病毒直接感染人,最终将出现人传人的现象。我们有必要建立实验动物模型并开展禽流感病毒的基础性和预防性研究,掌握禽流感病毒的病原特性、致病机理及防治措施。
目前,西药防治禽流感病毒主要是通过切断病毒复制或传播的途径来进行治疗,其主要不足是金刚烷胺口服时生物利用度太低,“达菲”易产生严重副作用,病毒唑有致畸性。
疫苗防治禽流感病毒主要是利用灭活病毒或病毒的免疫原使机体产生相应抗体,抵抗同类型病毒的感染。主要不足是由于病毒抗原的多变性,加上受禽类的种类、年龄、性别、并发感染、所感染毒株的毒力和其它环境因素变化的影响,无论传统灭活疫苗和弱毒苗,还是基因工程疫苗和核酸疫苗,都不能对所有禽流感病毒实现交叉保护,很多免疫过的鸡群还会爆发禽流感。
中医强调“辩证论治”和“预防胜于治疗”的原则。中药的防治策略是扶正祛邪或祛邪扶正。中药防治的主要优势是中药含有多种活性成分,对病毒的杀灭作用机制多样,同时还具有免疫调节作用,可改善和提高机体的免疫功能,因而中药对多种病毒有效,对病毒的防治作用明显。因此,加强中药预防和治疗禽流感的研究是弥补生物医药、化学药(西药)治疗缺陷的有效方法。
根据人禽流感主要发生在冬春季节和引起急性呼吸道症状等特点,将其归属于中医瘟疫中的时行感冒范畴。预实验提示:禽流感病毒感染小鼠后,病邪由表入里迅速传变,病情急骤,发热过程不明显。因此,我们在经过很多中药复方的预试后,选择了对体温、免疫、胃肠道等功能具有双向调节功能的桂枝汤,治疗胃肠型感冒和暑瘟的藿香正气散,治疗风热感冒初期的桑菊饮三种中药复方对禽流感病毒动物模型进行干预研究。
目的:本论文将比较禽流感病毒对多种实验动物的致病敏感性,利用最敏感的实验动物建立禽流感病毒疾病动物模型,在建立模型指标的基础上,开展中药复方对禽流感病毒的干预研究。
方法:本论文第二部分共四个实验。
实验一、按Reed和Muench氏法测定禽流感病毒对BALB/c小鼠的LD_(50)。
实验二、麻醉后,对SPF级KM小鼠、ICR小鼠、NIH小鼠、BALB/c小鼠和清洁级F344大鼠、SD大鼠、沙鼠接毒,。每20g动物接种100μL禽流感病毒原液,用移液枪滴入接毒组大小鼠、沙鼠鼻腔。通过“环甲膜穿刺术”经气管注射2mL禽流感病毒感染兔。观察临床症状、体温变化、体质量变化、死亡情况、病理改变等指标。比较禽流感病毒对各实验动物的致病敏感性。
实验三、将100μL禽流感病毒原液用移液枪滴入30只BALB/c小鼠鼻腔,另30只对照组接种正常鸡胚尿囊液。观察临床症状、体温变化、体质量变化、死亡情况、病理改变等。
实验四、将110只BALB/c小鼠分为6组:藿香正气散组(灌胃藿香正气散26.4g/kg)、桂枝汤组(灌胃桂枝汤15.6g/kg)、桑菊饮组(灌胃桑菊饮34.4g/kg)、病毒唑组(灌胃病毒唑70mg/kg)、模型对照组(灌胃0.4mL生理盐水),每组小鼠20只,鸡胚尿囊液对照组(灌胃0.4mL生理盐水)10只,各组小鼠均为雌雄各半。接毒前2天,各组小鼠每天灌胃不同类型的治疗方药,接毒当天上午给药,下午对藿香正气散组、桂枝汤组、桑菊饮组、病毒唑组和模型对照组接种100μL禽流感病毒原液。接毒后,再给药2天。尿囊液对照组在接毒当天接种100μL鸡胚尿囊液,再灌胃0.4mL生理盐水2天。以小鼠体温、体质量、存活时间、死亡保护率、食料消耗量为观察指标。
结果:
实验一、禽流感病毒感染BALB/c小鼠的LD_(50)为10~(-0.375)/0.1mL。
实验二、本实验研究了禽流感病毒对8种不同实验动物的致病敏感性,结果显示KM、NIH、ICR、BABL/c等小鼠和沙鼠均不同程度的复制出了禽流感病毒感染后的临床特点。首先,有不同的潜伏期,KM小鼠的潜伏期长,而BABL/c小鼠的潜伏期最短,发病急。第二,不同品系小鼠在禽流感病毒感染后,其临床症状轻重表现不一、死亡率及死亡高峰出现时间也存在差别,禽流感病毒对于不同品系小鼠的致病性是不同的。BABL/c、ICR和NIH小鼠有一个可观察到的疾病发生发展过程,临床症状严重的动物出现接近半数或半数以上的死亡,另一部分好转痊愈,有助于模型评价。KM小鼠感染禽流感病毒后只出现轻微的临床表现,体温、体质量出现一过性的下降后,迅速恢复正常;SD和F344大鼠感染禽流感病毒后没有出现感染表现,未分离到病毒;兔感染禽流感病毒后仅观察到轻度体温变化和肺脏病理变化,未分离到病毒,因此,KM小鼠、大鼠和兔不适合制作禽流感病毒动物模型。
从实验结果的统计分析可以看出,KM、NIH、ICR、BABL/c小鼠和F344、SD大鼠,沙鼠在接毒后都出现体温下降,而且与尿囊液对照组相比,体温下降幅度有统计学差异,BALB/c小鼠体温降低最显著。KM、NIH、ICR、BABL/c小鼠和沙鼠在接毒后都出现体质量减轻,与尿囊液对照组相比,都有统计学意义。
不同品系的小鼠和沙鼠感染禽流感病毒后出现不同的死亡率,禽流感病毒感染BABL/c小鼠的死亡率最高,其次是沙鼠,再次是ICR小鼠,NIH小鼠死亡率较低,KM小鼠有10%的死亡率,大鼠不出现死亡。这说明禽流感病毒对于小鼠和沙鼠有致病性,而且,上述实验动物的死亡率有统计学差异。与禽流感BALB/c小鼠高死亡率对应,病理变化结果也是BALB/c小鼠最严重。
在被感染动物体内分离出同种病毒是病毒感染动物模型成模的重要指标,本实验能在禽流感病毒感染后死亡的KM、NIH、ICR、BABL/c小鼠和沙鼠的肺脏分离到病毒,证实以上临床症状确实是由于禽流感病毒感染机体所致。而在禽流感病毒感染大鼠和兔的脏器没有分离到病毒,这也反证了禽流感病毒感染导致小鼠和沙鼠的发病死亡。
综上所述,禽流感病毒对不同实验动物有不同的致病性,BABL/c小鼠的致病敏感性最强,其临床症状、体温变化、体质量变化、死亡率、病理变化最明显。
实验三、实验结果显示,禽流感病毒感染BABL/c小鼠已具备了禽流感病毒引起小鼠肺等多个脏器炎症病理、发生大量死亡、康复小鼠产生抗体的完整的传染病发生发展和恢复的过程,能成为研究禽流感病毒的较好动物模型。
现将本实验在临床特征、体温变化、体质量变化、死亡率、脏器指数、病毒复制6个方面建立的模型指标综述如下:
①临床特征:禽流感病毒感染BALB/c小鼠后的病程主要表现为潜伏期、发病期和恢复期。具体临床特征表现为潜伏期短、急性起病、出现呼吸道症状,继而病情发展迅速、出现肺炎、多器官功能衰竭、死亡。在发病期肺脏出现严重的出血、水肿、坏死等病变,肺内导管上皮细胞变性、坏死、脱落,并有充血和大量炎性细胞浸润,肺泡中有大量出血和炎性细胞浸润,有的肺泡融合呈气肿状,有的肺泡实变塌陷,肺间质严重充血、水肿伴有大量炎性细胞浸润。
②体温变化:BABL/c小鼠感染禽流感病毒后体温出现下降过程。
③体质量变化:BABL/c小鼠感染禽流感病毒后体质量出现减轻。
④脏器指数:感染禽流感病毒后,BABL/c小鼠的肺和肝指数增高。
⑤死亡率:感染禽流感病毒后,BABL/c小鼠的死亡率较高。
⑥病毒复制:在禽流感病毒感染BABL/c小鼠的肺、心、肝、脾中分离到病毒,而且从肺脏分离病毒的比率较其它器官高,说明禽流感病毒以小鼠肺脏为靶器官,以肺巨噬细胞和气管上皮细胞为其靶细胞。在康复小鼠体内检出抗体。
禽流感病毒感染BABL/c小鼠疾病动物模型的建立,使我们可以利用稳定的、遗传背景清晰、易获得的小型哺乳类近交系实验动物来观察禽流感病毒的发病机制和药物疗效等。
实验四、藿香正气散对禽流感病毒感染小鼠的体温保护效果最明显,其次是桂枝汤组和桑菊饮组,病毒唑组对体温无保护作用。中西药治疗组的小鼠体重下降均比模型对照组慢,藿香正气散组、桂枝汤组在感染禽流感病毒的第5-6天小鼠恢复进食,表明藿香正气散、桂枝汤具有恢复胃气的功能,以桂枝汤恢复胃气的作用最佳。脾胃乃后天之本,胃气包括了消化功能、全身健康营养状况、机体的代偿功能和免疫功能等。人体的五脏六腑皆“禀气于胃”,胃气能养五脏之气;而百病皆由“胃气衰而生”;“胃气不伤,百病皆愈;胃气一败,百药难施”之说。因此,藿香正气散、桂枝汤调节了感染禽流感病毒小鼠下降的体温,同时固护胃气,增强了小鼠的抗病毒的能力。藿香正气散和桂枝汤能使感染禽流感病毒小鼠较快恢复健康和食欲,与藿香正气散组、桂枝汤组小鼠体质量下降缓慢相符合。
经统计学检验,藿香正气散组、桂枝汤组、桑菊饮组、病毒唑组之间死亡保护率有显著性差异,死亡保护率由高到低排序,依次是桂枝汤组＞藿香正气散组＞桑菊饮组＞病毒唑组。藿香正气散组、桂枝汤组、桑菊饮组、病毒唑组肺指数抑制率均为正值,表明给药组动物的肺病变程度比模型组轻,但以中药治疗组减轻肺脏病变疗效较西药佳,表明藿香正气散组、桂枝汤组等有对抗禽流感病毒的作用和保护小鼠机能的作用。
结论:
第一,研究探讨了禽流感病毒对8种不同实验动物的致病敏感性,结果显示,BALB/c小鼠的致病敏感性最强,其临床症状、体温变化、死亡率、病理变化都是最明显的,其它品系小鼠也表现一定致病性,禽流感病毒对大鼠和兔未表现临床症状。
第二,研究探讨了禽流感病毒感染BALB/c小鼠疾病动物模型的建立。研究和掌握了禽流感病毒小鼠模型在临床特征、体温变化、体质量变化、死亡率、脏器指数、病毒复制6个方面稳定的模型指标。禽流感病毒感染BALB/c小鼠疾病动物模型及模型指标的建立,使我们可以利用稳定的、遗传背景清晰、易获得的小型哺乳类近交系实验动物来观察禽流感病毒的发病机制和药物疗效等。
第三,研究探讨了中药复方对禽流感病毒的干预作用,结果表明:
①藿香正气散、桂枝汤对禽流感病毒感染小鼠所致体温降低具有升温作用;
②桂枝汤、藿香正气散能提高禽流感病毒感染小鼠的体质量;
③桂枝汤、藿香正气散能提高禽流感病毒感染小鼠的死亡保护率;
④藿香正气散、桂枝汤对禽流感病毒感染小鼠的肺指数有抑制作用;
⑤桂枝汤、藿香正气散具有调节胃肠和恢复胃气的功能。
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 OIE
AI 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.MLD_(50) 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:
①The 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.
②The changes of body temperature:the BALB/c mice infected showed the progression of the body temperature decreased.
③The changes of body weight:the BALB/c mice infected showed the progression of thebody weight reduced.
④The viscera index:the lung and liver index increased in BALB/c mice infected.
⑤The mortality:the mortality of BALB/c mice infected was high.
⑥The 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.
禽流感历来被认为是人类流感的最大基因库,是人流感病毒发生变异的新基因来源,近年来禽流感病毒正在世界多个国家传播,已对人类健康构成了很大的威胁。据世界卫生组织统计,目前,全球共有15个国家405人感染了高致病性禽流感病毒,其中94人死亡,我国至今发生38例人感染高致病性禽流感。
禽流感病毒(Avian Influenza virus,AIV)从禽类感染人已被证实,但目前还无充分证据证实禽流感病毒从人到人的感染。禽流感病毒为什么能感染人并对人具有致死性仍是研究人员在深入探讨的问题。禽流感病毒能逃避人体免疫系统的识别,但其具体机制仍不清楚。
已有人源或禽源禽流感病毒引起不同鸟类、大小鼠、猪、家猫、雪貂和非人灵长类动物致病性的研究报道,但从实用性考虑,小鼠有体形小、品系多、易获得、操作方便、对禽流感较敏感等突出优点,小鼠将成为研究禽流感病毒感染哺乳动物发病机制的重要实验动物。
面对禽流感病毒抗原的不断漂移与变异,面对禽流感病毒对禽类和人类构成的严重威胁,面对禽流感病毒直接感染人,最终将出现人传人的现象。我们有必要建立实验动物模型并开展禽流感病毒的基础性和预防性研究,掌握禽流感病毒的病原特性、致病机理及防治措施。
目前,西药防治禽流感病毒主要是通过切断病毒复制或传播的途径来进行治疗,其主要不足是金刚烷胺口服时生物利用度太低,“达菲”易产生严重副作用,病毒唑有致畸性。
疫苗防治禽流感病毒主要是利用灭活病毒或病毒的免疫原使机体产生相应抗体,抵抗同类型病毒的感染。主要不足是由于病毒抗原的多变性,加上受禽类的种类、年龄、性别、并发感染、所感染毒株的毒力和其它环境因素变化的影响,无论传统灭活疫苗和弱毒苗,还是基因工程疫苗和核酸疫苗,都不能对所有禽流感病毒实现交叉保护,很多免疫过的鸡群还会爆发禽流感。
中医强调“辩证论治”和“预防胜于治疗”的原则。中药的防治策略是扶正祛邪或祛邪扶正。中药防治的主要优势是中药含有多种活性成分,对病毒的杀灭作用机制多样,同时还具有免疫调节作用,可改善和提高机体的免疫功能,因而中药对多种病毒有效,对病毒的防治作用明显。因此,加强中药预防和治疗禽流感的研究是弥补生物医药、化学药(西药)治疗缺陷的有效方法。
根据人禽流感主要发生在冬春季节和引起急性呼吸道症状等特点,将其归属于中医瘟疫中的时行感冒范畴。预实验提示:禽流感病毒感染小鼠后,病邪由表入里迅速传变,病情急骤,发热过程不明显。因此,我们在经过很多中药复方的预试后,选择了对体温、免疫、胃肠道等功能具有双向调节功能的桂枝汤,治疗胃肠型感冒和暑瘟的藿香正气散,治疗风热感冒初期的桑菊饮三种中药复方对禽流感病毒动物模型进行干预研究。
目的:本论文将比较禽流感病毒对多种实验动物的致病敏感性,利用最敏感的实验动物建立禽流感病毒疾病动物模型,在建立模型指标的基础上,开展中药复方对禽流感病毒的干预研究。
方法:本论文第二部分共四个实验。
实验一、按Reed和Muench氏法测定禽流感病毒对BALB/c小鼠的LD_(50)。
实验二、麻醉后,对SPF级KM小鼠、ICR小鼠、NIH小鼠、BALB/c小鼠和清洁级F344大鼠、SD大鼠、沙鼠接毒,。每20g动物接种100μL禽流感病毒原液,用移液枪滴入接毒组大小鼠、沙鼠鼻腔。通过“环甲膜穿刺术”经气管注射2mL禽流感病毒感染兔。观察临床症状、体温变化、体质量变化、死亡情况、病理改变等指标。比较禽流感病毒对各实验动物的致病敏感性。
实验三、将100μL禽流感病毒原液用移液枪滴入30只BALB/c小鼠鼻腔,另30只对照组接种正常鸡胚尿囊液。观察临床症状、体温变化、体质量变化、死亡情况、病理改变等。
实验四、将110只BALB/c小鼠分为6组:藿香正气散组(灌胃藿香正气散26.4g/kg)、桂枝汤组(灌胃桂枝汤15.6g/kg)、桑菊饮组(灌胃桑菊饮34.4g/kg)、病毒唑组(灌胃病毒唑70mg/kg)、模型对照组(灌胃0.4mL生理盐水),每组小鼠20只,鸡胚尿囊液对照组(灌胃0.4mL生理盐水)10只,各组小鼠均为雌雄各半。接毒前2天,各组小鼠每天灌胃不同类型的治疗方药,接毒当天上午给药,下午对藿香正气散组、桂枝汤组、桑菊饮组、病毒唑组和模型对照组接种100μL禽流感病毒原液。接毒后,再给药2天。尿囊液对照组在接毒当天接种100μL鸡胚尿囊液,再灌胃0.4mL生理盐水2天。以小鼠体温、体质量、存活时间、死亡保护率、食料消耗量为观察指标。
结果:
实验一、禽流感病毒感染BALB/c小鼠的LD_(50)为10~(-0.375)/0.1mL。
实验二、本实验研究了禽流感病毒对8种不同实验动物的致病敏感性,结果显示KM、NIH、ICR、BABL/c等小鼠和沙鼠均不同程度的复制出了禽流感病毒感染后的临床特点。首先,有不同的潜伏期,KM小鼠的潜伏期长,而BABL/c小鼠的潜伏期最短,发病急。第二,不同品系小鼠在禽流感病毒感染后,其临床症状轻重表现不一、死亡率及死亡高峰出现时间也存在差别,禽流感病毒对于不同品系小鼠的致病性是不同的。BABL/c、ICR和NIH小鼠有一个可观察到的疾病发生发展过程,临床症状严重的动物出现接近半数或半数以上的死亡,另一部分好转痊愈,有助于模型评价。KM小鼠感染禽流感病毒后只出现轻微的临床表现,体温、体质量出现一过性的下降后,迅速恢复正常;SD和F344大鼠感染禽流感病毒后没有出现感染表现,未分离到病毒;兔感染禽流感病毒后仅观察到轻度体温变化和肺脏病理变化,未分离到病毒,因此,KM小鼠、大鼠和兔不适合制作禽流感病毒动物模型。
从实验结果的统计分析可以看出,KM、NIH、ICR、BABL/c小鼠和F344、SD大鼠,沙鼠在接毒后都出现体温下降,而且与尿囊液对照组相比,体温下降幅度有统计学差异,BALB/c小鼠体温降低最显著。KM、NIH、ICR、BABL/c小鼠和沙鼠在接毒后都出现体质量减轻,与尿囊液对照组相比,都有统计学意义。
不同品系的小鼠和沙鼠感染禽流感病毒后出现不同的死亡率,禽流感病毒感染BABL/c小鼠的死亡率最高,其次是沙鼠,再次是ICR小鼠,NIH小鼠死亡率较低,KM小鼠有10%的死亡率,大鼠不出现死亡。这说明禽流感病毒对于小鼠和沙鼠有致病性,而且,上述实验动物的死亡率有统计学差异。与禽流感BALB/c小鼠高死亡率对应,病理变化结果也是BALB/c小鼠最严重。
在被感染动物体内分离出同种病毒是病毒感染动物模型成模的重要指标,本实验能在禽流感病毒感染后死亡的KM、NIH、ICR、BABL/c小鼠和沙鼠的肺脏分离到病毒,证实以上临床症状确实是由于禽流感病毒感染机体所致。而在禽流感病毒感染大鼠和兔的脏器没有分离到病毒,这也反证了禽流感病毒感染导致小鼠和沙鼠的发病死亡。
综上所述,禽流感病毒对不同实验动物有不同的致病性,BABL/c小鼠的致病敏感性最强,其临床症状、体温变化、体质量变化、死亡率、病理变化最明显。
实验三、实验结果显示,禽流感病毒感染BABL/c小鼠已具备了禽流感病毒引起小鼠肺等多个脏器炎症病理、发生大量死亡、康复小鼠产生抗体的完整的传染病发生发展和恢复的过程,能成为研究禽流感病毒的较好动物模型。
现将本实验在临床特征、体温变化、体质量变化、死亡率、脏器指数、病毒复制6个方面建立的模型指标综述如下:
①临床特征:禽流感病毒感染BALB/c小鼠后的病程主要表现为潜伏期、发病期和恢复期。具体临床特征表现为潜伏期短、急性起病、出现呼吸道症状,继而病情发展迅速、出现肺炎、多器官功能衰竭、死亡。在发病期肺脏出现严重的出血、水肿、坏死等病变,肺内导管上皮细胞变性、坏死、脱落,并有充血和大量炎性细胞浸润,肺泡中有大量出血和炎性细胞浸润,有的肺泡融合呈气肿状,有的肺泡实变塌陷,肺间质严重充血、水肿伴有大量炎性细胞浸润。
②体温变化:BABL/c小鼠感染禽流感病毒后体温出现下降过程。
③体质量变化:BABL/c小鼠感染禽流感病毒后体质量出现减轻。
④脏器指数:感染禽流感病毒后,BABL/c小鼠的肺和肝指数增高。
⑤死亡率:感染禽流感病毒后,BABL/c小鼠的死亡率较高。
⑥病毒复制:在禽流感病毒感染BABL/c小鼠的肺、心、肝、脾中分离到病毒,而且从肺脏分离病毒的比率较其它器官高,说明禽流感病毒以小鼠肺脏为靶器官,以肺巨噬细胞和气管上皮细胞为其靶细胞。在康复小鼠体内检出抗体。
禽流感病毒感染BABL/c小鼠疾病动物模型的建立,使我们可以利用稳定的、遗传背景清晰、易获得的小型哺乳类近交系实验动物来观察禽流感病毒的发病机制和药物疗效等。
实验四、藿香正气散对禽流感病毒感染小鼠的体温保护效果最明显,其次是桂枝汤组和桑菊饮组,病毒唑组对体温无保护作用。中西药治疗组的小鼠体重下降均比模型对照组慢,藿香正气散组、桂枝汤组在感染禽流感病毒的第5-6天小鼠恢复进食,表明藿香正气散、桂枝汤具有恢复胃气的功能,以桂枝汤恢复胃气的作用最佳。脾胃乃后天之本,胃气包括了消化功能、全身健康营养状况、机体的代偿功能和免疫功能等。人体的五脏六腑皆“禀气于胃”,胃气能养五脏之气;而百病皆由“胃气衰而生”;“胃气不伤,百病皆愈;胃气一败,百药难施”之说。因此,藿香正气散、桂枝汤调节了感染禽流感病毒小鼠下降的体温,同时固护胃气,增强了小鼠的抗病毒的能力。藿香正气散和桂枝汤能使感染禽流感病毒小鼠较快恢复健康和食欲,与藿香正气散组、桂枝汤组小鼠体质量下降缓慢相符合。
经统计学检验,藿香正气散组、桂枝汤组、桑菊饮组、病毒唑组之间死亡保护率有显著性差异,死亡保护率由高到低排序,依次是桂枝汤组＞藿香正气散组＞桑菊饮组＞病毒唑组。藿香正气散组、桂枝汤组、桑菊饮组、病毒唑组肺指数抑制率均为正值,表明给药组动物的肺病变程度比模型组轻,但以中药治疗组减轻肺脏病变疗效较西药佳,表明藿香正气散组、桂枝汤组等有对抗禽流感病毒的作用和保护小鼠机能的作用。
结论:
第一,研究探讨了禽流感病毒对8种不同实验动物的致病敏感性,结果显示,BALB/c小鼠的致病敏感性最强,其临床症状、体温变化、死亡率、病理变化都是最明显的,其它品系小鼠也表现一定致病性,禽流感病毒对大鼠和兔未表现临床症状。
第二,研究探讨了禽流感病毒感染BALB/c小鼠疾病动物模型的建立。研究和掌握了禽流感病毒小鼠模型在临床特征、体温变化、体质量变化、死亡率、脏器指数、病毒复制6个方面稳定的模型指标。禽流感病毒感染BALB/c小鼠疾病动物模型及模型指标的建立,使我们可以利用稳定的、遗传背景清晰、易获得的小型哺乳类近交系实验动物来观察禽流感病毒的发病机制和药物疗效等。
第三,研究探讨了中药复方对禽流感病毒的干预作用,结果表明:
①藿香正气散、桂枝汤对禽流感病毒感染小鼠所致体温降低具有升温作用;
②桂枝汤、藿香正气散能提高禽流感病毒感染小鼠的体质量;
③桂枝汤、藿香正气散能提高禽流感病毒感染小鼠的死亡保护率;
④藿香正气散、桂枝汤对禽流感病毒感染小鼠的肺指数有抑制作用;
⑤桂枝汤、藿香正气散具有调节胃肠和恢复胃气的功能。
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 OIE
AI 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.MLD_(50) 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:
①The 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.
②The changes of body temperature:the BALB/c mice infected showed the progression of the body temperature decreased.
③The changes of body weight:the BALB/c mice infected showed the progression of thebody weight reduced.
④The viscera index:the lung and liver index increased in BALB/c mice infected.
⑤The mortality:the mortality of BALB/c mice infected was high.
⑥The 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.
引文
[1]甘孟侯.禽流感[M].北京:北京农业大学出版社,1995:28.
[2]Subbarao K,Klimov A,Katz J,et al.Characterization of an Avian Influenza A(H5N1) Virus Isolated from a Child with a Fatal Respiratory Illness[J].Science,1998,279(5349):393-396.
[3]Guan Y,Kennedy F.Molecular Characterization of H9N2 Influenza Viruses:Were They the Donors of the "Internal" Genes of H5N1 Viruses in Hong Kong?[J].PNAS,1999,96(16):9363-9367.
[4]Subbarao K,Shaw MW.Molecular Aspects of Avian Influenza(H5N1) Viruses Isolated from Humans[J].Rev Med Virol,2000,10(5):337-348.
[5]殷震,刘景华主编.动物病毒学(第二版)[M].北京:科学出版社,1997:330.
[6]Yuen KY,Chan PKS,Peiris M,et al.Clinical Features and Rapid Virus Diagnosis of Human Disease Associated with Avian Influenza A(H5N1) Virus[J].Lancet,1998,351:467-471.
[7]Murphy BR,Webster RG.Orthomyxoviruses[M].In:Fields BN,Kurpe DM,Housley PM CPI Plan-Virology.3th.Lippincott-Raven Publishers,Philadelphia,1996 p:1397-1445.
[8]Webster RG.Influenza:an Emerging Disease[J].Emerg.Infec.Dis.1998,4(3):436- 441.
[9]廖明,曹伟胜,罗开健,等.H5N1亚型禽流感病毒对鸡的致病性研究进展[J].华南农业大学学报,2006,27(3):1-5.
[10]Saifym.Diseases of Poultry[M].11thed.Ames:Iowa State Press,2003:135-160.
[11]辛朝安.禽病学[M].第2版.北京:中国农业出版社,2003:47-57.
[12]Swayne DE,Pantin JM.Pathogenicity of Avian Influenza Viruses in Poultry[J].DevBiol(Basel),2006,124(282):61-67.
[13]Perkinsle,Swaynede.Pathobiology of A/chicken/HongKong/220/97(H5N1) Avian Influenza Virus in seven gall in aceousspecies[J].Vet Pathol,2001,38(2):149- 164.
[14]罗开健.不同宿主来源H5N1亚型AIV致病机理与遗传变异研究[D].广州:华南农业大学兽医学院,2005.
[15]Keawcharoen J,Oraveerakul K,Kuiken T,et al.Avian influenza H5N1 in tigers and leopards.Emerg Infect Dis[serial on the Internet].2004 Dec[date cited].Available from http://www.cdc.gov/ncidod/EID/vol10no12/04-0759.htm.
[16]Kuiken T,Rimmelzwaan G,Debby VR,et al.Avian H5N1 Influenza in Cats[J].Science 2004,306(5694):241.
[17]Vahlenkamp TW,Harder TC,Giese M.Protection of Cats against Lethal Influenza H5N1Challenge Infection[J].J.Gen.Virol,2008,89:968-974.
[18]Yen HL,Lipatov AS,Ilyushina NA,et al.Inefficient Transmission of H5N1 Influenza Viruses in a Ferret Contact Model[J]../ Virol.2007,81(13):6890-6898.
[19]Choi YK,Nguyen TD,Ozaki H,et al,Studies of H5N1 Influenza Virus Infection of Pigs by Using Viruses Isolated in Vietnam and Thailand in 2004[J].J.Virol,2005,79(16):10821-10825.
[20]Ungchusak K,Auewerakul P,Douell SF.Probable Person-to-Person Transmission of Avian Influenza A(H5Nl)Viruses[J].N.Engl.J.Med,2005,352(4):333-340.
[21]Connor RJ,Kawaoka Y,Webster RG,et al.Receptor Specificity in Human,Avian,and Equine H2 and H3 Influenza Virus Isolates[J].Virology,1994,205(1):17-23.
[22]Hatta M,Kawaoka Y.The Continued Pandemic Threat posed by Avian Influenza Viruses in Hong Kong[J].Trends of Microbiology,2002,10(7):340-344.
[23]Couceiro JN,Paulson JC,Baum LG,et al.Influenza Virus Strains Selectively Recognize Sialyloligosaccharedes on Human Respiratory Epithelium;the Role of Host Cell in Selection of Hemagglutinin Receptor Specificity[J].Virus Res,1993,29(2):155-165.
[24]Ito T,Couceiro JNSS,Kelm S,et al.Molecular Basis for the Generation in Pigs of Influenza A Viruses with Pandemic Potential[J].J.Virol,1998,72(9):7367-7373.
[25]Claas EC,Osterhaus AD,Van BR,et al.Human Influenza A H5N1 Virus Related to a highly Pathogenic Avian Influenza Virus[J].Lancet,1998,351:472-477.
[26]Naffakh N,Massin N,Escriou B,et al.Genetic analysis of the compatibility between Polymerase Proteins from Human and Avian Strains of Influenza A VirusesLJ].J.Gen.Virol,2000,81:1283-1291.
[27]Subbarao EK,London W,Murphy BR.A Single Amino Acid in the PB2 Gene of Influenza A Virus is a Determinant of Host Range[J].J.Virol,1993,67(4):1761-1764.
[28]Zhou NN,Shortridge KF,Claas EC,et al.Rapid Evolution of H5N1 Influenza Viruses in Chickens in Hong Kong[J].Journal of Virology,1999,73(4):3366-3374.
[29]Massin P,Sylvie WS,Naffakh N.Residue 627 of PB2 is a Determinant of Cold Sensitivity in RNA Replication of Avian Influenza Viruses[J].Journal of Virology,2001,75(11):5398-5404.
[30]Yao YX,Mingay LJ,Mccauley JW,et al.Sequences in Influenza A Virus PB2 Protein that Determine Productive Infection for an Avian Influenza Virus in Mouse and Human Cell Lines[J].Journal of Virology,2001,75(11):5410-5415.
[31]Hatta M,Gao P,Halfmann P,et al.Molecular Basis for High Virulence of Hong Kong H5N1 Influenza A Viruses[J].Science,2001,293(7)1840-1842.
[32]Smirnor YA,Lipatov AS,Van Beek R,et al.Characterization of Adaptation of an Avian Influenza A(H5N2) Virus to a Mammalian Host[J].Acta Virologica,2000,44(1):l-8.
[33]Laver G,Garman E.The Origin and Control of Pandemic Influenza[J].Science,2001,293(5536):1776-1777.
[34]Webster RG.Molecular Whodunit[J].Science,2001,293(7):1773-1775.
[35]Gorman OT,Bean WJ,Gormn OT,et al.Evolution and Ecology of Influenza A Viruses[J].Microbiol Mol Biol Rev,1992,56(1):152-179.
[36]Taubenberger JK,Reid AH,Krafft AE,et al.Initial Characterization of the 1918"Spanish" Influenza Virus[J].Science,1997,275(5307):1793-1796.
[37]Capua I,Alexander DJ.Avian Influenza and Human Health[J].Actatropica,2002,83(1):1-6.
[38]刘树国,周秀珍,李钊华,等.A(H5N1)流感疫情.疾病监测[J],1998,13(7):271-273.
[39]垄震宁.香港甲型流感(H5N1)流行状况[J].疾病监测,1998,13(6):234-236.
[40]Suarez DL,Perdue ML,Cox N,et al.Comparisons of Highly Virulent H5N1 Influenza A Viruses Isolated from Humans and Chickens from Hong Kong[J].Journal of Virology,1998,72(8):6678-6688.
[41]Porter AG,Barber C,CNH,et al.Complete Nucleotide Sequence of an Influenza Virus Hemagglutinin Gene from Cloned DNA[J].Nature,1979,282(5738):471-477.
[42]Bosch FX,Garten W,Klenk HD,et al.Proteolytic Cleavage of Influenza Virus Hemagglutinins:Primary Structure of the Connecting Peptide between HA1 and HA2Determines Proteolytic Cleavability and Pathogencity of Avian Influenza Viurses[J],Virology,1981,113(2):725-735.
[43]Perdue ML,Garcia M,Senne D,et al Virulence-associated Sequence Duplication at the Hemagglutinin Cleavage Site of Avian Influenza Viruses[J].Virus Res,1997,49(2):173-186.
[44]Vey M,Orlich M,Adler S,et al.Hemagglutinin Activation of Pathogenic Avian Influenza Viruses of Serotype H7 Requires the Protease Recognition motif R-X-K/R-R[J].Virology,1992,188(1):408-413.
[45]Goto H,Wells K,Takada A,et al.Plasminogen-binding Activity of Neuraminidase Determines the Pathogenicity of Influenza A Virus[J].Journal of Virology,2001,75(19):9297-9301.
[46]王幼明,黄克和.禽流感的病理学进展[J].中国人兽共患病杂志.2001,17(2):83-86.
[47]To KF,Chan PK,Chan KF,et al.Pathology of Fatal Human Infection Associated with Avian Influenza A H5N1 Virus[J].Journal of Medical Virology,2001,63(3):242-6.
[48]Campen HV,Easterday BC,Hinshaw VS.Virulent Avian Influenza A Viruses:their Effect on Avian Lymphocytes and Macrophages in Vivo and Vitro[J].Journal of Genenal Virology,1989,70(11):2887-95.
[49]Tumpey TM,Lu X,Morken T,et al.Depletion of Lymphocytes and Diminished Cytokine Production in Mice Infected with a Highly Virulent Influenza A H5N1 Virus Isolated from Humans[J].J Virol,2000,74(13):6105-6116.
[50]Dybing JK,Schultz CS,Swaynede,et al.Distinct Pathogenesis of HongKong-origin H5N1 Viruses in Mice Compared to that of Other Highly Pathogenic H5 Avian Influenza Viruses[J].J.Virol,2000,74(3):1443-1450.
[51]Peng G,Watanabe SJ,Ito S,et al.Biological Heterogeneity including Systemic Replication in Mice of H5N1 influenza A virus isolates from humans in HongKong[J].J.Virol,73(4):3184-3189.
[52]Lu XH,Tunpey TM,Morken T,et al.A Mouse Model for the Evaluation of Pathogenesis and Immunity to Influenza A (H5N1) Viruses isolated from Humans[J].Journal of virology,1999,73(7):5903-5911.
[53]Chen H,Deng G,Li Z,et al.The Evolution of H5N1 Influenza Viruses in Ducks in Southern China[J].PNAS,2004.101(28):10452-10457.
[54]Perkinsle,Swaynede.Varied Pathogenicity of a Hong Kong-origin H5N1 Avian Influenza Virus in Four Passerine Species and Budgerigars[J].Journal of Virology,1999,73(2):1146-1155.
[55]Perkinsle,Swaynede.Pathogenicity of a Hong Kong-origin H5N1 Highly Pathogenic Avian Influenza Virus for emus,geese,ducks,and pigeons[J].Avian Dis,2002,46 (l):53-63.
[56]Shortridge KF,Zhou NN,Guan Y,et al.Characterization of Avian H5N1 Influenza Viruses from Poultry in Hong Kong[J].Virology,1998,252(2):331-342.
[57]Ward AC.Virulence of Influenza A Virus for Mouse in Lung[J].Virus Genes,1997,14:187-194.
[58]Hoyle L,Gard S,Hallauer C.Adaptation of Virus to Mice[M].The influenza viruses.Springer-Verlag,1968,p:170-171.
[59]Katz JM,Lu X,Tumpey TM,et al.Molecular Correlates of Influenza A H5N1 Virus Pathogenesis in Mice[J].J.Virol,2000,74(22):10807-10810.
[60]Nishimura H,Itamura S,Iwasaki T,et al.Characterization of Human Influenza A (H5Nl)Virus Infection in Mice:neuro-,pneumo-and adipotropic Infection[J].Journal of General Virology,2000,81(10):2503-2510.
[61]Taronna R,Xui HL,Steven M,et al.Avian Influenza(H5N1) Viruses Isolated from Humans in Asia in 2004 Exhibit Increased Virulence in Mammals[J].J Virol,2005,79(18):11788-11800.
[62]Barber WH,Small PA.Local and Systemic Immunity to Influenza Infections in Ferrets[J].Infect.Immun,1978,21(1):221-228.
[63]Fenton RJ,Clark A,Potter CW.Immunity to Influenza in Ferrets.XIV:Comparative Immunity following Infection or Immunization with Live or Inactivated Vaccine[J].Br.J.Exp.Pathol,1981,62(3):297-307.
[64]Yetter RA,Barber WH,Small PA.Heterotypic Immunity to Influenza in Ferrets[J].Infect.Immun,1980,29(2):650-653.
[65]Lois A,Rowe T,Morken T,et al.Pathogenesis of Avian Influenza A (H5N1) Viruses in Ferrets[J].Journal of Virology,2002,76(9):4420-4429.
[66]Kida H,Ito T,Yasuda J,et al.Potential for Transmission of Avian Influenza Viruses to Pigs.J Gen Virol,1994,75(9):2183-2188.
[67]Enserink M,Kaiser J.Avian Flu Finds New Mammal Hosts[J].Science,2004,305 (5689):1385.
[68]Rimmelzwaan GF,Van RD,BaarsM,et al.Influenza A Virus (H5N1) Infection in Cats Causes Systemic Disease with Potential Novel Routes of Virus Spread within and between Hosts[J].Am J Pathol,2006,168:176-183.
[69]Renegar KB.Influenza Virus Infections and Immunity:a Review of Human and Animal Models[J].Lab.Anim.Sci,1992,42(3):222-232.
[70]Murphy BR,Sly DL,Hosier NT,et al.Evaluation of Three Strains of Influenza A Virus in Humans and in OwL Cebus and Squirrel Monkeys[J].Infect.Immun,1980,28(3):688-691.
[71]Murphy BR,Hinshaw VS,Sly DL,et al.Virulence of Avian Influenza A Viruses for Squirrel Monkeys[J].Infect.Immun,1982,37(3):1119-1126.
[72]Rimmelzwaan GF,Kuiken T,Amerongen G,et al.Pathogenesis of Influenza A (H5N1) Virus Infection in a Primate Model[J].J.Virol,2001,75(14):6687-6691.
[73]Rimmelzwaan GF,Kuiken T,Amerongen G,et al.A Primate Model to Study the Pathogenesis of Influenza A (H5N1) Virus InfectionQ].Avian Diseases,2003,47(s3):931-933.
[74]Rimmelzwaan GF,Baars FM,Beek RV,et al.Induction of Protective Immunity against Influenza Virus in a Macaque Model Comparison of Conventional and Iscom vaccines[J].J.Gen.Virol,1997,78(4):757-765.
[75]Kim CU,Lew W.Influenza Neuraminidase Inhibitors Possessing Novel Hydrophobic Interactions in the Enzyme Active Site:Design,Synthesis,and Structural Analysis of Carbocyclic Sialic Acid Analogues with Potent Anti-Influenza Activity[J].J.Am.Chem.Soc,1997,119(4):681-690.
[76]Klenk HD,Rott R.The Molecular Biology of Influenza Virus Pathogenicity[J].Adv.Virus Res.1988,34:247-281.
[77]Gubcrera LV,Penn R.Inhibition of Replication of Avian Influenza Viruses by the Neuraminidase Inhibitor 4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneurami—nic Acid[J].Virology,1995,212(2):323-330.
[78]陈忠武.抗病毒药物的研究进展[J].国外医学药学分册.1998,25(4):71.
[79]Fynan EF,Robinson HL,Webster RG.Use of DNA encoding Influenza Hemagglutinin as an Avian Influenza Vaccine[J].DNA Cell Biol,1993,12(9):785-9.
[80]Kodihalli S,Sivanandan V,Nagaraja KV,et al.A type-Specific Avian Influenza Virus Subunit Vaccine for Turkeys:Induction of Protective Immunity to Challenge Infection[J].Vaccine,1994,12(15):1467-72.
[81]Swayne DE,Beck JR,Mickle TR.Efficacy of Recombinant Fowl Poxvirus Vaccine in Protecting Chickens against a Highly Pathogenic Mexican-origin H5N2 Avian Influenza Virus[J].Avian Dis,1997,41(4):910-22.
[82]Crawford J,Wilkinson B,Vosnesensky A,et al.Baculovirus-derived Hemagglutinin Vaccines Protect against Lethal Influenza Infections by Avian H5 and H7Subtypes[J].Vaccine,1999,17(18):2265-74.
[83]Webster RG,Kodihalli S,Kobasa DL.Strategies for inducing Protection against Avian Influenza A Virus Subtypes with DNA Vaccines[J].Vaccine,2000,18(23):2592-9.
[84]Kodihalli S,Haynes JR.Cross-protection among Lethal H5N2 Influenza Viruses Induced by DNA Vaccine to the Hemagglutinin[J].Virol,1997,71(5):3391-3396.
[85]乔传玲,姜永萍,李呈军,等.禽流感重组禽痘病毒rFPV-HA-NA活载体疫苗的研究[J].中国免疫学杂志,2003,19(1):46.
[86]乔传玲,于康震.禽流感病毒A/Goose/Guangdong/3/96(H5N1)NA基因的克隆及其重组禽痘病毒转移载体的构建[J].中国兽医学报,2003,23(2):111.
[87]陈吉祥,李广林,陈化兰,等.禽流感HA基因疫苗脂质体的制备及理化性质[J].中国兽医学报,1999,19(3):230.
[88]马文军,张建林,陈化兰,等.禽流感血凝素基因重组鸡痘病毒的构建及其免疫保护性研究[J].中国农业科学,2000,33(5):75.
[89]贾立军,刘红旗,黄勇,等.在疫苗免疫选择压力下H9N2亚型禽流行性感冒病毒HA基因的遗传变异[J].病毒学报,2002,18(2):149-154.
[90]陈化兰,于康震,田国斌,等.DNA免疫诱导鸡对禽流感病毒的免疫保护反应[J].中国农业科学,1998,31(5):63.
[91]陈化兰,于康震,田国斌,等.禽流感病毒血凝素基因的克隆及其DNA疫苗的免疫原性[J].中国兽医学报,1997,17(6):555.
[92]李艳华,于康震,佟恒敏,等.中药抗流感病毒研究概况[J].中国兽医杂志,2003,39(6)35-37.
[93]张卫民,杨增歧,卿素珠.抗病毒的中草药研究进展[J].动物医学进展,2001,22(4):16-21.
[94]张晓菊,麻廷峰.浅论中药抗流感病毒研究概况[J].中兽医医药杂志,2005,3:27-28.
[95]李艳华,王秀荣,田国彬,等.复方中药防治禽流感实验[J].中国兽医杂志, 2005,41(6):34-35.
[96]郭世宁,刘天龙,李守军,等.中药复方口服液体外抗禽流感病毒作用的研究[J].中国兽医杂志,2006,42(7):41-43.
[97]路振香,时维静,钱卫东.20份中草药提取液抗禽流感病毒作用的实验研究[J].中国中医药科技,2006,13(2):97-98.
[98]中医药专家研讨防治人禽流感[EB].[2004-2-12],中国新闻网.
[99]卫生部.人禽流感诊疗方案[J].医药导报,2006,25(2):1-3.
[100]胡天佑.具有抗病毒作用的中草药应用研究[J].中国处方药,2004,29(8):69-70.
[101]中华中医药学会组织专家商讨中医药及早参与防治禽流感[J].中医药管理杂志,2004,(1):37.
[102]人禽流感中医药诊治指导方案(天津)[J].中国中西医结合急救杂志,2005,12(6):327-328.
[103]侯钧.禽流感中医预防小探[J].医学动物防治,2004,20(5):312.
[104]陈剑声.人类禽流感的辨证食疗探讨[J].药膳食疗,2004,(4):17-18.
[105]洪炳根.中医防治流感、非典、人禽流感初探[J].福建中医药,2005,36(4):54-55.
[106]宣文.关于应用中医药防治人感染高致病性H5N1禽流感的可行性分析[J].上海中医药杂志,2006,40(1):18-19.
[107]陈声明,刘丽丽编著.微生物学研究法[M].北京:中国农业科技出版社,1996.127-128.
[108]Shinya K,Shimada A,Ito T,et al.Avian Influenza Virus Intranasally Inoculated Infects the Central Nervous System of Mice through the General Visceral Afferent Nerve[J].Arch Virol.2000,145:187-195.
[109]杨松涛,高玉伟,王承宇,等.虎源H5N1亚型禽流感病毒感染小鼠模型的建立[J].中国病毒学.2006,21(4):353-357.
[110]Kurokawa M,Imakita M,Kumeda CA,et al.Cascade of Fever Production in Mice Infected With Influenza Virus[J].Journal of Medical Virology,1996,50(2):152 -158.
[111]杨仕贵.禽流感临床表现[EB/OL].(查阅日期:2007.10.16)http://www.drugunabuse.com.
[112]Kuken T,Rimmelzwaan GF,Amerongen GV,et al.Pathology of Human Influenza A(H5N1)Virus Infection in Cynomolgus Macaques[J].Vet Pathol,2003,40(3):304-310.
[113]李培生.伤寒底论讲义[M].第1版,上海:科学技术出版社,1985:67.
[114]富杭育,周爱香,查显元,等.桂枝汤对体温双向调节作用机理的探讨-对下丘脑前列腺素E 含量的影响[J].中西医结杂志,1991,11(1):34-35.
[115]霍海如,谭余庆,李晓芹,等.桂枝汤对有效部位A(FR.A)对体温双向调节作用及其机理研究-对下丘脑前腺素E含量的影响[J].中国实验方剂学,1998,4(2):25-28.
[116]富杭育,郭淑英,高英杰,等.桂枝汤对体温双向调节作用的探讨-对下丘脑乙酰胆碱Ach和去甲肾上腺素NE含量的影响[J].中国实验方剂学,1996,(2):8-10.
[117]富杭育,周爱香,郭淑英,霍海如,等.桂枝汤对体温双向调节作用的探讨-对下丘脑蛙皮素含 量的影响[J].中西医结合杂志,1994,11(2):99-100.
[118]富杭育,郭淑英,周爱香,等.桂枝汤对体温双向调节作用的探讨-对下丘脑神经降压素和促肾上腺皮质激素含量的影响[J].中西医结合杂志,1995,(1):11-13.
[119]富杭育,周爱香,姚祥珍,等.桂枝汤对体温、肠蠕动双向调节作用的实验研究[J].中国医药学报,1990,5(2):34-36.
[120]梅全喜主编,现代中药药理临床应用手册[M],中国中医药出版社,2008:44.
[121]国家医药管理局草药情报中心.药物有效成分手册[M].人民出版社,1986:49.
[2]Subbarao K,Klimov A,Katz J,et al.Characterization of an Avian Influenza A(H5N1) Virus Isolated from a Child with a Fatal Respiratory Illness[J].Science,1998,279(5349):393-396.
[3]Guan Y,Kennedy F.Molecular Characterization of H9N2 Influenza Viruses:Were They the Donors of the "Internal" Genes of H5N1 Viruses in Hong Kong?[J].PNAS,1999,96(16):9363-9367.
[4]Subbarao K,Shaw MW.Molecular Aspects of Avian Influenza(H5N1) Viruses Isolated from Humans[J].Rev Med Virol,2000,10(5):337-348.
[5]殷震,刘景华主编.动物病毒学(第二版)[M].北京:科学出版社,1997:330.
[6]Yuen KY,Chan PKS,Peiris M,et al.Clinical Features and Rapid Virus Diagnosis of Human Disease Associated with Avian Influenza A(H5N1) Virus[J].Lancet,1998,351:467-471.
[7]Murphy BR,Webster RG.Orthomyxoviruses[M].In:Fields BN,Kurpe DM,Housley PM CPI Plan-Virology.3th.Lippincott-Raven Publishers,Philadelphia,1996 p:1397-1445.
[8]Webster RG.Influenza:an Emerging Disease[J].Emerg.Infec.Dis.1998,4(3):436- 441.
[9]廖明,曹伟胜,罗开健,等.H5N1亚型禽流感病毒对鸡的致病性研究进展[J].华南农业大学学报,2006,27(3):1-5.
[10]Saifym.Diseases of Poultry[M].11thed.Ames:Iowa State Press,2003:135-160.
[11]辛朝安.禽病学[M].第2版.北京:中国农业出版社,2003:47-57.
[12]Swayne DE,Pantin JM.Pathogenicity of Avian Influenza Viruses in Poultry[J].DevBiol(Basel),2006,124(282):61-67.
[13]Perkinsle,Swaynede.Pathobiology of A/chicken/HongKong/220/97(H5N1) Avian Influenza Virus in seven gall in aceousspecies[J].Vet Pathol,2001,38(2):149- 164.
[14]罗开健.不同宿主来源H5N1亚型AIV致病机理与遗传变异研究[D].广州:华南农业大学兽医学院,2005.
[15]Keawcharoen J,Oraveerakul K,Kuiken T,et al.Avian influenza H5N1 in tigers and leopards.Emerg Infect Dis[serial on the Internet].2004 Dec[date cited].Available from http://www.cdc.gov/ncidod/EID/vol10no12/04-0759.htm.
[16]Kuiken T,Rimmelzwaan G,Debby VR,et al.Avian H5N1 Influenza in Cats[J].Science 2004,306(5694):241.
[17]Vahlenkamp TW,Harder TC,Giese M.Protection of Cats against Lethal Influenza H5N1Challenge Infection[J].J.Gen.Virol,2008,89:968-974.
[18]Yen HL,Lipatov AS,Ilyushina NA,et al.Inefficient Transmission of H5N1 Influenza Viruses in a Ferret Contact Model[J]../ Virol.2007,81(13):6890-6898.
[19]Choi YK,Nguyen TD,Ozaki H,et al,Studies of H5N1 Influenza Virus Infection of Pigs by Using Viruses Isolated in Vietnam and Thailand in 2004[J].J.Virol,2005,79(16):10821-10825.
[20]Ungchusak K,Auewerakul P,Douell SF.Probable Person-to-Person Transmission of Avian Influenza A(H5Nl)Viruses[J].N.Engl.J.Med,2005,352(4):333-340.
[21]Connor RJ,Kawaoka Y,Webster RG,et al.Receptor Specificity in Human,Avian,and Equine H2 and H3 Influenza Virus Isolates[J].Virology,1994,205(1):17-23.
[22]Hatta M,Kawaoka Y.The Continued Pandemic Threat posed by Avian Influenza Viruses in Hong Kong[J].Trends of Microbiology,2002,10(7):340-344.
[23]Couceiro JN,Paulson JC,Baum LG,et al.Influenza Virus Strains Selectively Recognize Sialyloligosaccharedes on Human Respiratory Epithelium;the Role of Host Cell in Selection of Hemagglutinin Receptor Specificity[J].Virus Res,1993,29(2):155-165.
[24]Ito T,Couceiro JNSS,Kelm S,et al.Molecular Basis for the Generation in Pigs of Influenza A Viruses with Pandemic Potential[J].J.Virol,1998,72(9):7367-7373.
[25]Claas EC,Osterhaus AD,Van BR,et al.Human Influenza A H5N1 Virus Related to a highly Pathogenic Avian Influenza Virus[J].Lancet,1998,351:472-477.
[26]Naffakh N,Massin N,Escriou B,et al.Genetic analysis of the compatibility between Polymerase Proteins from Human and Avian Strains of Influenza A VirusesLJ].J.Gen.Virol,2000,81:1283-1291.
[27]Subbarao EK,London W,Murphy BR.A Single Amino Acid in the PB2 Gene of Influenza A Virus is a Determinant of Host Range[J].J.Virol,1993,67(4):1761-1764.
[28]Zhou NN,Shortridge KF,Claas EC,et al.Rapid Evolution of H5N1 Influenza Viruses in Chickens in Hong Kong[J].Journal of Virology,1999,73(4):3366-3374.
[29]Massin P,Sylvie WS,Naffakh N.Residue 627 of PB2 is a Determinant of Cold Sensitivity in RNA Replication of Avian Influenza Viruses[J].Journal of Virology,2001,75(11):5398-5404.
[30]Yao YX,Mingay LJ,Mccauley JW,et al.Sequences in Influenza A Virus PB2 Protein that Determine Productive Infection for an Avian Influenza Virus in Mouse and Human Cell Lines[J].Journal of Virology,2001,75(11):5410-5415.
[31]Hatta M,Gao P,Halfmann P,et al.Molecular Basis for High Virulence of Hong Kong H5N1 Influenza A Viruses[J].Science,2001,293(7)1840-1842.
[32]Smirnor YA,Lipatov AS,Van Beek R,et al.Characterization of Adaptation of an Avian Influenza A(H5N2) Virus to a Mammalian Host[J].Acta Virologica,2000,44(1):l-8.
[33]Laver G,Garman E.The Origin and Control of Pandemic Influenza[J].Science,2001,293(5536):1776-1777.
[34]Webster RG.Molecular Whodunit[J].Science,2001,293(7):1773-1775.
[35]Gorman OT,Bean WJ,Gormn OT,et al.Evolution and Ecology of Influenza A Viruses[J].Microbiol Mol Biol Rev,1992,56(1):152-179.
[36]Taubenberger JK,Reid AH,Krafft AE,et al.Initial Characterization of the 1918"Spanish" Influenza Virus[J].Science,1997,275(5307):1793-1796.
[37]Capua I,Alexander DJ.Avian Influenza and Human Health[J].Actatropica,2002,83(1):1-6.
[38]刘树国,周秀珍,李钊华,等.A(H5N1)流感疫情.疾病监测[J],1998,13(7):271-273.
[39]垄震宁.香港甲型流感(H5N1)流行状况[J].疾病监测,1998,13(6):234-236.
[40]Suarez DL,Perdue ML,Cox N,et al.Comparisons of Highly Virulent H5N1 Influenza A Viruses Isolated from Humans and Chickens from Hong Kong[J].Journal of Virology,1998,72(8):6678-6688.
[41]Porter AG,Barber C,CNH,et al.Complete Nucleotide Sequence of an Influenza Virus Hemagglutinin Gene from Cloned DNA[J].Nature,1979,282(5738):471-477.
[42]Bosch FX,Garten W,Klenk HD,et al.Proteolytic Cleavage of Influenza Virus Hemagglutinins:Primary Structure of the Connecting Peptide between HA1 and HA2Determines Proteolytic Cleavability and Pathogencity of Avian Influenza Viurses[J],Virology,1981,113(2):725-735.
[43]Perdue ML,Garcia M,Senne D,et al Virulence-associated Sequence Duplication at the Hemagglutinin Cleavage Site of Avian Influenza Viruses[J].Virus Res,1997,49(2):173-186.
[44]Vey M,Orlich M,Adler S,et al.Hemagglutinin Activation of Pathogenic Avian Influenza Viruses of Serotype H7 Requires the Protease Recognition motif R-X-K/R-R[J].Virology,1992,188(1):408-413.
[45]Goto H,Wells K,Takada A,et al.Plasminogen-binding Activity of Neuraminidase Determines the Pathogenicity of Influenza A Virus[J].Journal of Virology,2001,75(19):9297-9301.
[46]王幼明,黄克和.禽流感的病理学进展[J].中国人兽共患病杂志.2001,17(2):83-86.
[47]To KF,Chan PK,Chan KF,et al.Pathology of Fatal Human Infection Associated with Avian Influenza A H5N1 Virus[J].Journal of Medical Virology,2001,63(3):242-6.
[48]Campen HV,Easterday BC,Hinshaw VS.Virulent Avian Influenza A Viruses:their Effect on Avian Lymphocytes and Macrophages in Vivo and Vitro[J].Journal of Genenal Virology,1989,70(11):2887-95.
[49]Tumpey TM,Lu X,Morken T,et al.Depletion of Lymphocytes and Diminished Cytokine Production in Mice Infected with a Highly Virulent Influenza A H5N1 Virus Isolated from Humans[J].J Virol,2000,74(13):6105-6116.
[50]Dybing JK,Schultz CS,Swaynede,et al.Distinct Pathogenesis of HongKong-origin H5N1 Viruses in Mice Compared to that of Other Highly Pathogenic H5 Avian Influenza Viruses[J].J.Virol,2000,74(3):1443-1450.
[51]Peng G,Watanabe SJ,Ito S,et al.Biological Heterogeneity including Systemic Replication in Mice of H5N1 influenza A virus isolates from humans in HongKong[J].J.Virol,73(4):3184-3189.
[52]Lu XH,Tunpey TM,Morken T,et al.A Mouse Model for the Evaluation of Pathogenesis and Immunity to Influenza A (H5N1) Viruses isolated from Humans[J].Journal of virology,1999,73(7):5903-5911.
[53]Chen H,Deng G,Li Z,et al.The Evolution of H5N1 Influenza Viruses in Ducks in Southern China[J].PNAS,2004.101(28):10452-10457.
[54]Perkinsle,Swaynede.Varied Pathogenicity of a Hong Kong-origin H5N1 Avian Influenza Virus in Four Passerine Species and Budgerigars[J].Journal of Virology,1999,73(2):1146-1155.
[55]Perkinsle,Swaynede.Pathogenicity of a Hong Kong-origin H5N1 Highly Pathogenic Avian Influenza Virus for emus,geese,ducks,and pigeons[J].Avian Dis,2002,46 (l):53-63.
[56]Shortridge KF,Zhou NN,Guan Y,et al.Characterization of Avian H5N1 Influenza Viruses from Poultry in Hong Kong[J].Virology,1998,252(2):331-342.
[57]Ward AC.Virulence of Influenza A Virus for Mouse in Lung[J].Virus Genes,1997,14:187-194.
[58]Hoyle L,Gard S,Hallauer C.Adaptation of Virus to Mice[M].The influenza viruses.Springer-Verlag,1968,p:170-171.
[59]Katz JM,Lu X,Tumpey TM,et al.Molecular Correlates of Influenza A H5N1 Virus Pathogenesis in Mice[J].J.Virol,2000,74(22):10807-10810.
[60]Nishimura H,Itamura S,Iwasaki T,et al.Characterization of Human Influenza A (H5Nl)Virus Infection in Mice:neuro-,pneumo-and adipotropic Infection[J].Journal of General Virology,2000,81(10):2503-2510.
[61]Taronna R,Xui HL,Steven M,et al.Avian Influenza(H5N1) Viruses Isolated from Humans in Asia in 2004 Exhibit Increased Virulence in Mammals[J].J Virol,2005,79(18):11788-11800.
[62]Barber WH,Small PA.Local and Systemic Immunity to Influenza Infections in Ferrets[J].Infect.Immun,1978,21(1):221-228.
[63]Fenton RJ,Clark A,Potter CW.Immunity to Influenza in Ferrets.XIV:Comparative Immunity following Infection or Immunization with Live or Inactivated Vaccine[J].Br.J.Exp.Pathol,1981,62(3):297-307.
[64]Yetter RA,Barber WH,Small PA.Heterotypic Immunity to Influenza in Ferrets[J].Infect.Immun,1980,29(2):650-653.
[65]Lois A,Rowe T,Morken T,et al.Pathogenesis of Avian Influenza A (H5N1) Viruses in Ferrets[J].Journal of Virology,2002,76(9):4420-4429.
[66]Kida H,Ito T,Yasuda J,et al.Potential for Transmission of Avian Influenza Viruses to Pigs.J Gen Virol,1994,75(9):2183-2188.
[67]Enserink M,Kaiser J.Avian Flu Finds New Mammal Hosts[J].Science,2004,305 (5689):1385.
[68]Rimmelzwaan GF,Van RD,BaarsM,et al.Influenza A Virus (H5N1) Infection in Cats Causes Systemic Disease with Potential Novel Routes of Virus Spread within and between Hosts[J].Am J Pathol,2006,168:176-183.
[69]Renegar KB.Influenza Virus Infections and Immunity:a Review of Human and Animal Models[J].Lab.Anim.Sci,1992,42(3):222-232.
[70]Murphy BR,Sly DL,Hosier NT,et al.Evaluation of Three Strains of Influenza A Virus in Humans and in OwL Cebus and Squirrel Monkeys[J].Infect.Immun,1980,28(3):688-691.
[71]Murphy BR,Hinshaw VS,Sly DL,et al.Virulence of Avian Influenza A Viruses for Squirrel Monkeys[J].Infect.Immun,1982,37(3):1119-1126.
[72]Rimmelzwaan GF,Kuiken T,Amerongen G,et al.Pathogenesis of Influenza A (H5N1) Virus Infection in a Primate Model[J].J.Virol,2001,75(14):6687-6691.
[73]Rimmelzwaan GF,Kuiken T,Amerongen G,et al.A Primate Model to Study the Pathogenesis of Influenza A (H5N1) Virus InfectionQ].Avian Diseases,2003,47(s3):931-933.
[74]Rimmelzwaan GF,Baars FM,Beek RV,et al.Induction of Protective Immunity against Influenza Virus in a Macaque Model Comparison of Conventional and Iscom vaccines[J].J.Gen.Virol,1997,78(4):757-765.
[75]Kim CU,Lew W.Influenza Neuraminidase Inhibitors Possessing Novel Hydrophobic Interactions in the Enzyme Active Site:Design,Synthesis,and Structural Analysis of Carbocyclic Sialic Acid Analogues with Potent Anti-Influenza Activity[J].J.Am.Chem.Soc,1997,119(4):681-690.
[76]Klenk HD,Rott R.The Molecular Biology of Influenza Virus Pathogenicity[J].Adv.Virus Res.1988,34:247-281.
[77]Gubcrera LV,Penn R.Inhibition of Replication of Avian Influenza Viruses by the Neuraminidase Inhibitor 4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneurami—nic Acid[J].Virology,1995,212(2):323-330.
[78]陈忠武.抗病毒药物的研究进展[J].国外医学药学分册.1998,25(4):71.
[79]Fynan EF,Robinson HL,Webster RG.Use of DNA encoding Influenza Hemagglutinin as an Avian Influenza Vaccine[J].DNA Cell Biol,1993,12(9):785-9.
[80]Kodihalli S,Sivanandan V,Nagaraja KV,et al.A type-Specific Avian Influenza Virus Subunit Vaccine for Turkeys:Induction of Protective Immunity to Challenge Infection[J].Vaccine,1994,12(15):1467-72.
[81]Swayne DE,Beck JR,Mickle TR.Efficacy of Recombinant Fowl Poxvirus Vaccine in Protecting Chickens against a Highly Pathogenic Mexican-origin H5N2 Avian Influenza Virus[J].Avian Dis,1997,41(4):910-22.
[82]Crawford J,Wilkinson B,Vosnesensky A,et al.Baculovirus-derived Hemagglutinin Vaccines Protect against Lethal Influenza Infections by Avian H5 and H7Subtypes[J].Vaccine,1999,17(18):2265-74.
[83]Webster RG,Kodihalli S,Kobasa DL.Strategies for inducing Protection against Avian Influenza A Virus Subtypes with DNA Vaccines[J].Vaccine,2000,18(23):2592-9.
[84]Kodihalli S,Haynes JR.Cross-protection among Lethal H5N2 Influenza Viruses Induced by DNA Vaccine to the Hemagglutinin[J].Virol,1997,71(5):3391-3396.
[85]乔传玲,姜永萍,李呈军,等.禽流感重组禽痘病毒rFPV-HA-NA活载体疫苗的研究[J].中国免疫学杂志,2003,19(1):46.
[86]乔传玲,于康震.禽流感病毒A/Goose/Guangdong/3/96(H5N1)NA基因的克隆及其重组禽痘病毒转移载体的构建[J].中国兽医学报,2003,23(2):111.
[87]陈吉祥,李广林,陈化兰,等.禽流感HA基因疫苗脂质体的制备及理化性质[J].中国兽医学报,1999,19(3):230.
[88]马文军,张建林,陈化兰,等.禽流感血凝素基因重组鸡痘病毒的构建及其免疫保护性研究[J].中国农业科学,2000,33(5):75.
[89]贾立军,刘红旗,黄勇,等.在疫苗免疫选择压力下H9N2亚型禽流行性感冒病毒HA基因的遗传变异[J].病毒学报,2002,18(2):149-154.
[90]陈化兰,于康震,田国斌,等.DNA免疫诱导鸡对禽流感病毒的免疫保护反应[J].中国农业科学,1998,31(5):63.
[91]陈化兰,于康震,田国斌,等.禽流感病毒血凝素基因的克隆及其DNA疫苗的免疫原性[J].中国兽医学报,1997,17(6):555.
[92]李艳华,于康震,佟恒敏,等.中药抗流感病毒研究概况[J].中国兽医杂志,2003,39(6)35-37.
[93]张卫民,杨增歧,卿素珠.抗病毒的中草药研究进展[J].动物医学进展,2001,22(4):16-21.
[94]张晓菊,麻廷峰.浅论中药抗流感病毒研究概况[J].中兽医医药杂志,2005,3:27-28.
[95]李艳华,王秀荣,田国彬,等.复方中药防治禽流感实验[J].中国兽医杂志, 2005,41(6):34-35.
[96]郭世宁,刘天龙,李守军,等.中药复方口服液体外抗禽流感病毒作用的研究[J].中国兽医杂志,2006,42(7):41-43.
[97]路振香,时维静,钱卫东.20份中草药提取液抗禽流感病毒作用的实验研究[J].中国中医药科技,2006,13(2):97-98.
[98]中医药专家研讨防治人禽流感[EB].[2004-2-12],中国新闻网.
[99]卫生部.人禽流感诊疗方案[J].医药导报,2006,25(2):1-3.
[100]胡天佑.具有抗病毒作用的中草药应用研究[J].中国处方药,2004,29(8):69-70.
[101]中华中医药学会组织专家商讨中医药及早参与防治禽流感[J].中医药管理杂志,2004,(1):37.
[102]人禽流感中医药诊治指导方案(天津)[J].中国中西医结合急救杂志,2005,12(6):327-328.
[103]侯钧.禽流感中医预防小探[J].医学动物防治,2004,20(5):312.
[104]陈剑声.人类禽流感的辨证食疗探讨[J].药膳食疗,2004,(4):17-18.
[105]洪炳根.中医防治流感、非典、人禽流感初探[J].福建中医药,2005,36(4):54-55.
[106]宣文.关于应用中医药防治人感染高致病性H5N1禽流感的可行性分析[J].上海中医药杂志,2006,40(1):18-19.
[107]陈声明,刘丽丽编著.微生物学研究法[M].北京:中国农业科技出版社,1996.127-128.
[108]Shinya K,Shimada A,Ito T,et al.Avian Influenza Virus Intranasally Inoculated Infects the Central Nervous System of Mice through the General Visceral Afferent Nerve[J].Arch Virol.2000,145:187-195.
[109]杨松涛,高玉伟,王承宇,等.虎源H5N1亚型禽流感病毒感染小鼠模型的建立[J].中国病毒学.2006,21(4):353-357.
[110]Kurokawa M,Imakita M,Kumeda CA,et al.Cascade of Fever Production in Mice Infected With Influenza Virus[J].Journal of Medical Virology,1996,50(2):152 -158.
[111]杨仕贵.禽流感临床表现[EB/OL].(查阅日期:2007.10.16)http://www.drugunabuse.com.
[112]Kuken T,Rimmelzwaan GF,Amerongen GV,et al.Pathology of Human Influenza A(H5N1)Virus Infection in Cynomolgus Macaques[J].Vet Pathol,2003,40(3):304-310.
[113]李培生.伤寒底论讲义[M].第1版,上海:科学技术出版社,1985:67.
[114]富杭育,周爱香,查显元,等.桂枝汤对体温双向调节作用机理的探讨-对下丘脑前列腺素E 含量的影响[J].中西医结杂志,1991,11(1):34-35.
[115]霍海如,谭余庆,李晓芹,等.桂枝汤对有效部位A(FR.A)对体温双向调节作用及其机理研究-对下丘脑前腺素E含量的影响[J].中国实验方剂学,1998,4(2):25-28.
[116]富杭育,郭淑英,高英杰,等.桂枝汤对体温双向调节作用的探讨-对下丘脑乙酰胆碱Ach和去甲肾上腺素NE含量的影响[J].中国实验方剂学,1996,(2):8-10.
[117]富杭育,周爱香,郭淑英,霍海如,等.桂枝汤对体温双向调节作用的探讨-对下丘脑蛙皮素含 量的影响[J].中西医结合杂志,1994,11(2):99-100.
[118]富杭育,郭淑英,周爱香,等.桂枝汤对体温双向调节作用的探讨-对下丘脑神经降压素和促肾上腺皮质激素含量的影响[J].中西医结合杂志,1995,(1):11-13.
[119]富杭育,周爱香,姚祥珍,等.桂枝汤对体温、肠蠕动双向调节作用的实验研究[J].中国医药学报,1990,5(2):34-36.
[120]梅全喜主编,现代中药药理临床应用手册[M],中国中医药出版社,2008:44.
[121]国家医药管理局草药情报中心.药物有效成分手册[M].人民出版社,1986:49.