季节性流感病毒H1N1和H3N2在BALB/c小鼠中的传代适应与分子机理研究

目的:建立季节性流感病毒H1N1和H3N2的BALB/c小鼠致死性动物模型,并探索其鼠肺适应的分子机理。方法:将季节性流感病毒A/Guangdong/51/2008(H1N1)(简称为H1N1-GDwt)和A/Anhui/137/2008(H3N2)(简称为H3N2-AHwt)分别滴鼻感染BALB/c小鼠,于病毒增殖高峰期制备肺组织悬液,连续传40代,并对野生型与鼠肺适应株在小鼠中的致死性与全基因组序列进行比较。结果:H3N2-AHwt感染BALB/c小鼠后各代次鼠肺悬液均未检测到病毒;而H1N1-GDwt感染小鼠后第4 d肺部病毒滴度达到高峰,病毒滴度随传代次数的增加而呈现"波浪型"升高,鼠肺适应株对BALB/c小鼠的致死性与致病性明显强于野生型病毒,全基因组序列分析发现鼠肺适应株在血凝素(HA)、酸性聚合酶(PA)、核蛋白(NP)及非结构蛋白(NS)中共发生了10个氨基酸突变。结论:H3N2-AHwt难以在BALB/c小鼠中有效复制与适应;而H1N1-GDwt能够在BALB/c小鼠中有效复制,其毒力可以通过连续鼠肺传代而提高,HA、PA、NP及NS蛋白的突变与其鼠肺适应密切相关。     

艾氏腹水癌克隆细胞株E2G8建立小鼠动物模型生物学特性研究

目的　比较不同品种 (系 )小鼠接种E2G8细胞株的某些生物学特性。方法　将E2G8细胞稀释成不同浓度 ,接种KM、DBA 2、6 15、C57BL 6及BALB c小鼠皮下或腹腔 ,观察小鼠出现可见肿块 腹水时间及小鼠生存时间 ;测量肿块直径 (cm)及瘤重 ;荷瘤小鼠腹腔注射环磷酰胺 (CTX) ,观察E2G8细胞肿瘤模型对CTX治疗的敏感性。结果　E2G8接种BALB c、6 15小鼠皮下毒性最大 ,DBA 2、C57BL 6小鼠次之 ,接种KM小鼠皮下毒性最小 ;接种DBA 2、C57BL 6、6 15小鼠腹腔毒性较大 ,致死性高 ,对BALB c和KM小鼠的致死性弱 ;E2G8瘤细胞在KM和 4种纯系小鼠皮下均能很好生长肿瘤 ,其中在KM小鼠皮下肿瘤生长最大 ,在BALB c小鼠皮下肿瘤最小 ;KM、C57BL 6、6 15、DBA 2、BALB c小鼠皮下接种瘤细胞第 12天瘤重分别为 (2 4 0± 1 30 ) ,(0 90± 0 4 8) ,(1 2 0± 0 38) ,(1 10± 0 2 9) ,(0 80± 0 30 )g ;E2G8细胞接种 5个不同品种 (系 )小鼠制备的肿瘤模型 ,用CTX治疗 ,抑瘤率由高到低依次为6 6 7% (6 15 ) ,5 5 0 % (BALB c) ,5 3 3% (C57BL 6 ) ,5 0 % (KM) ,36 4 (DBA 2 ) ,其中 6 15小鼠建立的模型对CTX的治疗最敏感。结论　E2G8细胞株接种 5个不同品种 (系 )小鼠 ,所测生物学特性不完全相同 ,但是从腹腔 皮下接种、CTX     

A/California/7/2009与A/California/4/2009病毒感染力比较

目的甲型H1N1流感病毒A/California/7/2009与A/California/4/2009病毒序列比较同源性在99%以上,本实验旨在比较两株病毒感染BALB/c小鼠研究感染力强弱。方法分别将A/California/7/2009（CA7）与A/California/4/2009（CA4）两株病毒分别连续10倍稀释后,对4~6周龄雌性BALB/c小鼠经乙醚麻醉后进行滴鼻攻毒,每个稀释度接种10只实验小鼠,测定CA7 MLD50为101.24/0.05 mL,检测小鼠感染、致病的多项指标,观察期为14 d。结果相同TCID50的CA7和CA4病毒感染小鼠,CA4感染小鼠后14 d内死亡率为20%,而CA7感染小鼠后8 d内死亡率为100%。CA7 106TCID50感染的小鼠病理表现为重度弥漫性间质性肺炎,CA4 106TCID50感染的小鼠病理表现为中度-重度间质性肺炎。结论在相同条件下,CA7感染力明显强于CA4。     

虎源H5N1亚型禽流感病毒感染小鼠模型的建立

为研究H5N1亚型禽流感病毒的病原特性、致病机理及对其疫苗与救治药物效果评价提供平台,利用本室分离鉴定的虎源A/Tiger/Harbin/01/2002株(简称HAB/01)H5N1亚型禽流感病毒进行连续10倍稀释后,对4～6周龄雄性BALB/c小鼠经乙醚麻醉后进行滴鼻攻毒,每个稀释度接种10只实验小鼠,测定其MLD50,检测小鼠感染、致病的多项指标,观察期为14d。结果感染小鼠呈现出规律的以肺炎为主的临床症状、病理变化及病死率;测得该病毒对小鼠的MLD50为10-7.1/0.05mL。成功建立了虎源H5N1亚型禽流感病毒感染BALB/c小鼠的实验模型。     

虎源H5N1亚型禽流感病毒感染小鼠模型的建立

为研究H5N1亚型禽流感病毒的病原特性、致病机理及对其疫苗与救治药物效果评价提供平台,利用本室分离鉴定的虎源A/Tiger/Harbin/01/2002株(简称HAB/01)H5N1亚型禽流感病毒进行连续10倍稀释后,对4～6周龄 雄性BALB/c小鼠经乙醚麻醉后进行滴鼻攻毒,每个稀释度接种10只实验小鼠,测定其MLD50,检测小鼠感染、致病的多项指标,观察期为14d.结果感染小鼠呈现出规律的以肺炎为主的临床症状、病理变化及病死率;测得该病毒对小鼠的MLD50为10-7.1/0.05mL.成功建立了虎源H5N1亚型禽流感病毒感染BALB/c小鼠的实验模型.     

博尔纳病病毒对BALB/c小鼠感染性检测

目的分析博尔纳病病毒（Borna disease virus,BDV）H1766株对BALB/c小鼠的感染性。方法选择病毒滴度为2.0×107FFU/ml的BDV病毒液分别对新生和成年BALB/c小鼠进行脑内接种,并用相同病毒液对原代培养的新生BALB/c小鼠脑细胞进行接种。经过一定时间的病毒作用后分别提取总RNA,采用巢式RT-PCR方法检测BDV-p40基因,并通过免疫组化方法检测脑内接种脑组织中BDV-P40蛋白。结果脑内接种病毒的小鼠脑组织中可以检测到BDV-p40基因和BDV-P40蛋白,培养的小鼠脑细胞中可以检测到BDV-p40基因。结论BDVH1766株可以感染新生和成年的BALB/c小鼠。     

EV71不同临床株的新生小鼠毒力研究

目的探索不同EV71毒株在新生鼠的毒力。方法临床轻症、重症和死亡来源的EV71毒株,接种1日龄BALB/c乳鼠,观察14 d小鼠的症状,于感染后1、3、5、7 d和9 d,取小鼠组织RT-QPCR测病毒载量,HE染色观察病变。结果重症株与轻症株症状严重程度差异无显著性(P=0.693),且两组均较死亡株组重(P=0.000,P=0.000);轻症、重症和死亡株组小鼠生存率分别为77.2%、81.7%和97.8%,差异有显著性(P=0.0010,P=0.001,P=0.0004);轻症株组肺出血最重,脑、肌肉、脾和肠病理定性差异无显著性;轻症株组各组织病毒量最高,死亡株组最低。结论临床轻症毒株在乳鼠症状最重,死亡株最轻,与人的临床结局不一致,可能与病毒基因、宿主等因素相关。     

Tim-1分子在约氏疟原虫感染模型中的表达分析

Tim-1分子表达在T细胞和调节性B细胞表面,具有促进Th2应答的作用。为探讨Tim-1分子在抗疟疾免疫应答中的作用,利用致死型约氏疟原虫(Plasmodium yoelii)感染鼠疟模型研究了Tim-1分子表达与小鼠感染结局和免疫应答模式的关系。结果显示,BALB/c小鼠对P.yoelii易感,在感染后6～7 d全部死亡,感染后第3、5天脾内细胞因子IFN-γmRNA水平明显低于对P.yoelii抵抗的DBA2小鼠,而脾IL-10 mRNA水平显著高于DBA2小鼠。BALB/c小鼠脾内Tim-1+T、B细胞百分比在感染后第3、5天显著升高,而DBA2小鼠感染前后脾内Tim-1+T、B细胞百分比没有显著变化。结果提示,Tim-1分子参与抗P.yoelii免疫应答的调节,可能与易感鼠产生高水平Th2型细胞因子有关。     

Listr1遗传位点影响小鼠对约氏疟原虫易感性的研究

探讨Listr1遗传位点影响小鼠对疟原虫易感性的可能性及其初步作用机制。致死型约氏疟原虫(Plasmodiumyoelii17XL,P.y17XL)感染C.B6By-Listr1小鼠(BALB/c小鼠基因背景下引入C57BL/6小鼠Lis-tr1遗传位点的同类系小鼠)和BALB/c小鼠,分析二者生存率和感染率的差异;HE染色分析P.y17XL感染后第5天C.B6By-Listr1小鼠和BALB/c小鼠肝脏组织损伤情况;定量PCR法检测P.y17XL感染后小鼠肝脏组织第1、2、5天IL-1β、IL-6、TNF-α、IFN-γ的表达含量。结果显示,P.y17XL感染后4~8dC.B6By-Listr1小鼠虫血症水平显著高于BALB/c小鼠(P<0.05)。C.B6By-Listr1小鼠于感染后7~9d全部死亡;而半数BALB/c小鼠于感染后8~9d死亡,其余BALB/c小鼠至感染后第13天仍存活,两种小鼠生存率差异具有统计学意义(P<0.01)。C.B6By-Listr1小鼠P.y17XL感染后第5天肝组织HE染色可见到明显的疟色素沉积;而BALB/c小鼠全片基本未见到疟色素的沉积。P.y17XL感染后第2天BALB/c小鼠肝组织IL-6(P<0.05)、TNF-α(P<0.05)mRNA水平显著高于C.B6By-Listr1小鼠。结果表明Listr1遗传位点可以影响小鼠对疟原虫的易感性,与肝脏固有免疫相关。     

CD4+CD25+T细胞在夏氏疟原虫感染鼠中的应答差异

为探讨CD4 CD25 调节性T细胞(Treg细胞)在夏氏疟原虫感染过程中的活化特点及其与疾病进展的相关性,用夏氏疟原虫分别感染BALB/c和DBA/2小鼠,Giemsa染色制备薄血膜,镜检计数红细胞感染率;以流式细胞术检测脾细胞悬液中Treg细胞百分含量;ELISA测定脾细胞培养上清IFN-γ水平。BALB/c小鼠原虫血症于感染后第8d达到峰值34.4%后迅速下降,于感染后第15d左右小鼠自愈;其IFN-γ水平和Treg细胞百分含量均在感染后第5d明显增加后开始下降(P<0.01);DBA/2小鼠原虫血症于感染后第9～11d一直维持在峰值38%左右,并在感染后约第10d小鼠死亡;其IFN-γ水平在感染后第3d明显升高后迅速回落(P<0.01),Treg细胞百分含量在感染后的前8d平稳升高,但于感染后第8～11d出现骤然上升。结果提示,CD4 CD25 调节性T细胞数量异常变化与宿主感染结局呈密切相关性。     

Comparison of lung virus titers in susceptible and resistant inbred mouse strains against Sendai virus infection

Susceptibility of inbred mouse strains to Sendai virus (Mol strain) infection was studied. Although some mouse strains showed age differences in susceptibility between 3-to 4-week-old and 7-to 8-week-old mice, such age differences in susceptibility were not observed in susceptible DBA/2N and resistant BALB/cA mice. In 7-to 8-week-old mice, remarkable strain differences were observed in mortality and intensity of the lung lesions, but not in lung virus titers and serum antibody, between resistant BALB/cA and susceptible DBA/2N mice.     

Inhibition of nitric oxide synthesis increases mortality in Sindbis virus encephalitis.

Sindbis virus (SV) is an alphavirus that causes acute encephalomyelitis in mice. The outcome is determined by the strain of virus and by the age and genetic background of the host. The mortality rates after infection with NSV, a neurovirulent strain of SV, were as follows v: 81% (17 of 21) in BALB/cJ mice; 20% (4 of 20) in BALB/cByJ mice (P < 0.001); 100% in A/J, C57BL/6J, SJL, and DBA mice; and 79% (11 of 14) in immunodeficient scid/CB17 mice. Treatment with Nomega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthetase (NOS) inhibitor, increased mortality to 100% (P < 0.05) in NSV-infected BALB/cJ mice, to 95% (P < 0.001) in BALB/cByJ mice, and to 100% in scid/CB17 mice. BALB/cJ and BALB/cByJ mice had similar levels of inducible NOS mRNA in their brains, which were not affected by L-NAME or NSV infection. Brain NOS activity was similar in BALB/cJ and BALB/cByJ mice before and after infection and was markedly inhibited by L-NAME. NSV replication in the brains of BALB/cJ mice, BALB/cByJ mice, and mice treated with L-NAME was similar. Treatment of N18 neuroblastoma cells with NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside in vitro before infection increased cell viability at 42 to 48 h compared with untreated NSV-infected N18 cells with little effect on virus replication. These data suggest that NO protects mice from fatal encephalitis by a mechanism that does not directly involve the immune response or inhibition of virus growth but rather may enhance survival of the infected neuron until the immune response can control virus replication.     

Differential resistance/susceptibility patterns to pneumovirus infection among inbred mouse strains

Respiratory syncytial virus (RSV) is a prominent cause of airway morbidity in children under 1 yr of age. It is assumed that host factors influence the severity of the disease presentation and thus the need for hospitalization. As a first step toward the identification of the underlying genes involved, this study was undertaken to establish whether inbred mouse strains differ in susceptibility to pneumonia virus of mice (PVM), the murine counterpart of RSV, which has been shown to accurately mimic the RSV disease of children. With this purpose in mind, double-chamber plethysmography and carbon monoxide uptake data were collected daily for 7 days after inoculation of PVM in six inbred strains of mice. In parallel, histological examinations and lung viral titration were carried out from day 5 to day 7 after inoculation. Pulmonary structure/function values reflected the success of viral replication in the lungs and revealed a pattern of continuous variation, with resistant, intermediate, and susceptible strains. The results suggest that SJL (resistant) and 129/Sv (susceptible) strains should be used in crossing experiments aimed at identifying genes controlling pneumovirus replication by the positional cloning approach. Similarly, crossing experiments using BALB/c or C57BL/6 (resistant) and DBA/2 or 129/Sv (susceptible) will allow the identification of the genes involved in the control of pulmonary inflammation during pneumovirus infection.     

DBA/2 mouse as an animal model for anti-influenza drug efficacy evaluation

Influenza viruses are seasonally recurring human pathogens. Vaccines and antiviral drugs are available for influenza. However, the viruses, which often change themselves via antigenic drift and shift, demand constant efforts to update vaccine antigens every year and develop new agents with broad-spectrum antiviral efficacy. An animal model is critical for such efforts. While most human influenza viruses are unable to kill BALB/c mice, some strains have been shown to kill DBA/2 mice without prior adaptation. Therefore, in this study, we explored the feasibility of employing DBA/2 mice as a model in the development of anti-influenza drugs. Unlike the BALB/c strain, DBA/2 mice were highly susceptible and could be killed with a relatively low titer (50% DBA/2 lethal dose = 10^2.83 plaque-forming units) of the A/Korea/01/2009 virus (2009 pandemic H1N1 virus). When treated with a neuraminidase inhibitor, oseltamivir phosphate, infected DBA/2 mice survived until 14 days post-infection. The reduced morbidity of the infected DBA/2 mice was also consistent with the oseltamivir treatment. Taking these data into consideration, we propose that the DBA/2 mouse is an excellent animal model to evaluate antiviral efficacy against influenza infection and can be further utilized for combination therapies or bioactivity models of existing and newly developed anti-influenza drugs.     

Behavioural profiles of inbred mouse strains used as transgenic backgrounds. II: cognitive tests

One of the characteristic manifestations of several neurodegenerative diseases is the progressive decline in cognitive ability. In order to determine the suitability of six mouse strains (129S2/Sv, BALB/c, C3H/He, C57BL/6j, CBA/Ca and DBA/2) as transgenic background strains, we investigated the performance on a variety of tasks designed to identify subtle changes in cognition. In addition, a test of exploratory behaviour was used to probe the level of underlying anxiety in these mouse strains, as anxiety can be a confounding factor on behavioural performance generally. The C3H/He mice exhibited the least anxiogenic behavioural profile spending most time on the open arms of the maze, in contrast to the 129S2/Sv mice which spent the least amount of time in this location and were the quickest to move into a closed arm. The C3H/He mouse strain failed to acquire a visual discrimination task and failed to demonstrate learning on a water maze spatial learning task, in contrast to the CBA/Ca, DBA/2 and C57BL/6j strains which demonstrated a degree of learning in both tasks. No significant strain differences were identified on the object recognition task. These data, taken together, suggest that care must be taken when choosing cognitive tasks to be used with particular mouse strains and that task sensitivity must be considered as a critical element to research protocols with regard to these mouse strains.     

Resistance/susceptibility to lethal Sendai virus infection genetically linked to a mucociliary transport polymorphism.

Linkage was tested between a mucociliary transport polymorphism and resistance/susceptibility to lethal Sendai virus infection in segregant hybrid mice of C57BL/6J and DBA/2J parents. The distribution of paired phenotypes for tracheal mucociliary transport rates and susceptibility to lethal Sendai virus infection in 171 F1 X DBA/2J mice showed strong interaction of the parental phenotypes.     

副粘病毒Tianjin株NP蛋白的表达及分析

1999年,本实验室从群体性暴发的急性呼吸道感染素致死的普通棉耳绒猴肺组织中分离到一株副粘病毒,命名为副粘病毒 Tianjin株.经研究发现该动物中心的工作人员都有此病毒抗体,且正常人群(献血员)抗体阳性率高达46%,急性呼吸道感染的患儿抗体阳性率为19.28%,提示此病原体与人类可能有密切的关系.     

Genetic resistance to lethal Sendai virus pneumonia: virus replication and interferon production in C57BL/6J and DBA/2J mice

Resistant C57BL/6J and susceptible DBA/2J mice were exposed to aerosols of Sendai virus and killed at intervals to 12 days. Lungs were removed and assayed for infectious virus and interferon. Mean virus titers were 6 to 400 times higher in DBA/2J mice than in C57BL/6J mice 3 to 10 days after exposure. Mean interferon titers were 10 to 140 times higher in DBA/2J mice than in C57BL/6J mice 4 to 7 days after exposure. These results suggest that genetic resistance to the lethal effects of Sendai virus is expressed through control of viral replication within the first 72 hours of infection and that early expression of inherited resistance is not regulated by interferon.     

Mouse strain differences in Gurmarin-sensitivity of sweet taste responses are not associated with polymorphisms of the sweet receptor gene, Tas1r3

Gurmarin (Gur) is a peptide that selectively inhibits responses of the chorda tympani (CT) nerve to sweet compounds in rodents. In mice, the sweet-suppressing effect of Gur differs among strains. The inhibitory effect of Gur is clearly observed in C57BL/6 mice, but only slightly, if at all, in BALB/c mice. These two mouse strains possess different alleles of the sweet receptor gene, Sac (Tas1r3) (taster genotype for C57BL/6 and non-taster genotype for BALB/c mice), suggesting that polymorphisms in the gene may account for differential sensitivity to Gur. To investigate this possibility, we examined the effect of Gur in another Tas1r3 non-taster strain, 129 X 1/Sv mice. The results indicated that unlike non-taster BALB/c mice but similar to taster C57BL/6 mice, 129 X 1/Sv mice exhibited significant inhibition of CT responses to various sweet compounds by Gur. This suggests that the mouse strain difference in the Gur inhibition of sweet responses of the CT nerve may not be associated with polymorphisms of Tas1r3.     

Sendai virus infection of mice with protein malnutrition.

Sendai virus pneumonia was produced in BALB/c mice fed protein-deficient diets in an effort to understand the severity of viral pneumonia in infants in developing countries. Animals on the deficient diet became clinically malnourished, and some aspects of cellular immunity were altered. In protein-deprived animals, the 50% lethal dose of intranasally administered Sendai virus was over 1,000-fold lower, pulmonary virus titers were higher, the infection was prolonged, and lung infection was established at a lower inoculum than in normal animals.