从蚤体内分离流行性出血热病毒的研究

本研究从流行性出血热(EHF)病毒抗原阳性的褐家鼠体表收集缓慢细蚤(Leptop-sylla segnis)、开皇客蚤(Xenopsylla cheopis),经反复冲洗后制备悬液,并接种小白鼠乳鼠,分别经3、4代观察,乳鼠表现特异的病状,有的死亡。其脑、肺组织应用IFAT检查EHF病毒抗原阳性。阳性鼠脑悬液再接种Vero E_6细胞亦呈阳性。因而首次证实缓慢细蚤、开皇客蚤能自然感染EHF病毒,其在流行病学中的意义应进一步研究。     

检测DENV-2活病毒含量RTCA标准曲线法的建立

目的利用实时无标记细胞分析系统(real-time cellular analysis,RTCA)建立半数细胞指数时间(CIT50)与病毒滴度(TCID50)相关性的标准曲线,评估RTCA技术在监测2型登革病毒(DENV-2)感染BHK细胞后引发细胞病变效应(CPE)中的可行性和应用价值。方法检测不同培养条件下BHK细胞的CI值变化,筛选合适的初始细胞接种密度及血清浓度;应用RTCA分析已知不同TCID50滴度DENV-2感染BHK细胞的CIT50值,构建CIT50-TCID50标准曲线,计算回归方程;收集DENV-2感染C6/36细胞后第1~4d的培养上清(D1,D2,D3,D4),利用RTCA法和TCID50法分别检测上清中DENV-2滴度,分析CIT50-TCID50标准曲线法的灵敏度和可行性。结果 BHK细胞以初始接种密度为2×104个/孔,血清浓度为2%的培养条件为最佳;不同滴度DENV-2感染后BHK细胞的生长曲线均左移,CIT50与TCID50间呈线性负相关,线性方程为y=-7.007x+75.546(R2=0.9854);不同时间感染C6/36细胞培养上清中病毒含量(logTCID50/mL):RTCA标准曲线法为4.90±0.05(D2)、6.18±0.20(D3)、6.04±0.10(D4);传统TCID50法为0(D1)、6.25±0.49(D3)、5.87±0.35(D4)。结论利用RTCA技术建立的CIT50-TCID50标准曲线法,可用于已知DENV-2在BHK细胞的定量检测,结果较TCID50法更客观,且该法操作便捷且灵敏度高,为评估DENV-2感染性活病毒颗粒的毒力提供了新的技术手段。     

呼肠孤病毒空斑检测方法建立

目的 建立呼肠孤病毒空斑检测方法,为进一步研究该病毒奠定基础。方法 通过Western Blot、免疫荧光检测纳尔逊海湾正呼肠孤病毒Miyazaki病毒株感染L929细胞和BSR细胞情况,病毒RNA水平凝胶电泳验证Miyazaki病毒株核酸成分。建立呼肠孤病毒空斑检测方法,计算病毒滴度。结果 Miyazaki病毒株能成功感染L929细胞和BSR细胞,水平凝胶电泳验证病毒株核酸成分正确,病毒株感染的L929细胞出现空斑,计算得出病毒滴度结果为2.2×10⁸ PFU/mL。结论 呼肠孤病毒空斑检测方法成功建立,该方法可以形成清晰可见的空斑,能稳定地对病毒滴度进行定量测定,为进一步研究呼肠孤病毒提供帮助。     

动物狂犬病病毒街毒株的细胞分离鉴定

目的N2A细胞和BHK-21细胞用于我国动物狂犬病流行街毒株的分离培养。方法狂犬病发病动物脑组织悬液首先脑内接种乳鼠,再采取Nonclon Delta Tube内置盖玻片法从鼠脑中进行病毒分离培养,脑组织悬液接种细胞后96h,利用直接荧光抗体实验检测盖玻片上的病毒感染细胞,判定病毒是否在细胞上增殖;通过测定不同毒株细胞培养上清中的病毒滴度对病毒进行鉴定。结果22份狂犬病病犬、猪和牛的脑组织接种乳鼠后均获得了鼠脑分离毒,将鼠脑毒接种N2A细胞分离获得了22株病毒,接种BHK-21细胞却只分离获得20株病毒。不同病毒株第三代细胞培养上清中的病毒滴度从10-1TCID50/100μL到10-3.6TCID50/100μL。结论N2A细胞对狂犬病病毒街毒株的敏感性高于BHK-21细胞,且不同的病毒株对细胞的适应能力不同。实验分离获得的病毒细胞适应株丰富了我国狂犬病病毒毒种资源,为进一步研究不同病毒分离株的抗原性差异奠定基础。     

从新疆首次分离出20株甲属披膜病毒

1990年3月至1991年8月,用乳鼠及RHK细胞等从新疆西部和北部地区采集的7种20000余只分离到病毒7株,从新疆北部地区采集的蜱7种2300余只分离到病毒13株,共20株。其中从赫坎按蚊 Anopheles hyrcanus分离3株,从尖音库蚊 Culex pipiens pipiens分离2株,从凶小库蚊 Culexmodestus分离2株;从全沟硬蜱Ixodes persulcatus分离11株,从边缘革蜱 Dermacentor marginatus分离2株。电镜检查该病毒为球形有囊膜颗粒,直径为57±1.5nm左右。用分离的20株病毒感染BHK、Vero及C6/36传代细胞,均可在24～72小时内致细胞病变;病毒以脑内、皮下或腹腔途经感染2～4日龄乳小白鼠2～6天规律致死,3周龄小白鼠3～7天规律致死。病毒抵抗5-碘脱氧尿苷(5～Idu),对酸、热、乙醚敏感。酶免疫(ELISA-BHK)检测20株病毒只与甲属披膜病毒有反应。经初步鉴定为甲属披膜病毒(Alpha virus)。     

狂犬病病毒和犬瘟热病毒疫苗株混合感染Vero细胞的实验研究

目的研究狂犬病病毒(RV)及犬瘟热病毒(CDV)共同感染Vero细胞及混合感染对产毒量的影响,比较病毒检测方法的敏感性。方法将单独培养的RV、CDV及RV-CDV混合培养物进行连续10倍稀释后同步接种Vero细胞,37℃5%CO2培养5d,分别以直接免疫荧光(DFA)、RT-PCR及荧光定量RT-PCR方法检测病毒的半数细胞感染量,并对各病毒检测方法进行比较。结果RV和CDV可同时感染Vero细胞,并在其中增殖。CDV单独感染Vero细胞的TCID50为10-5.8/0.05ml,RV和CDV混合感染Vero细胞的TCID50为10-5.5/0.05ml。DFA、RT-PCR和荧光定量RT-PCR阳性的RV-CDV感染最大稀释度分别为10-6、10-5和10-6。结论混合培养对RV和CDV的感染滴度及产毒量影响很小。免疫荧光灶检测与RT-PCR及荧光定量RT-PCR方法检测的敏感性相当。犬瘟热-狂犬病毒联合疫苗的病毒滴度检测可不经中和其中的一个病毒直接将联合疫苗接种Vero细胞,然后分别进行免疫荧光检测。     

1株狂犬病毒减毒株的毒力和分子特性研究

目的 对实验室研发的1株狂犬病减毒株CTN181-3的致病性和基因型特性进行研究。方法 用小鼠脑内、口腔接种法和金黄地鼠口腔接种法测定病毒毒力。将病毒通过乳鼠脑内、豚鼠颌下腺和细胞连续传多代,测定传代后病毒的遗传稳定性。对CTN181-3进行全基因组测序,并与其亲本株进行对比分析。结果 CTN181-3对实验动物毒力高度减弱,对3周龄小鼠无论脑内或口腔接种均无致病性,对2周龄小鼠也表现出脑内低毒力;口腔接种8周龄金黄地鼠亦无发病死亡。遗传稳定性方面,经1~3 d龄乳鼠脑内连续传5代或豚鼠颌下腺传4代,传代增殖后的病毒滴度虽高达7.10 lg PFU/mL或7.63 lg PFU/mL,对小鼠脑内接种仍保留无致病力的弱毒特性;在BSR细胞和Vero细胞上分别传10代,表型特性稳定。全基因组测序结果分析显示CTN181-3株与国内近年来分离株的同源性高于其他疫苗株与国内分离株的同源性。CTN181-3株与其原始的亲本株CTN-1比较,发生8个氨基酸位点的突变,其中G276 L→V和L1496 M→W氨基酸的突变在CTN181株未发生,为CTN181-3株所特有。因此,这2个位点的氨基酸突变应该是CTN181-3株比CTN181株毒力更弱、遗传稳定性更高的分子基础。结论 CTN181-3株的神经毒力高度减弱,毒力稳定,是一种很有应用前景的动物用狂犬病候选疫苗株。     

乙型脑炎病毒野毒株SA14包膜蛋白K279M突变对其神经毒力的影响

目的构建乙型脑炎病毒野毒株SA14包膜蛋白K279M突变株感染性克隆并拯救病毒,探索K279M突变对其毒力的影响。方法应用重叠延伸PCR和分子克隆技术构建含有K279M突变的乙脑突变病毒全长cDNA质粒,经酶切鉴定后,以其为模板体外转录获得RNA,电转染BHK21细胞,得到恢复病毒JEV SA14 (K279M)。分别用野毒株JEV SA14和JEV SA14 (K279M)接种BHK21细胞和脑内接种昆明鼠,比较突变株与野毒株的蚀斑大小、生长特点以及对昆明鼠的神经毒力。结果酶切鉴定表明,突变病毒感染性克隆成功构建。病毒蚀斑试验显示,JEV SA14 (K279M)的蚀斑直径约为2～3 mm,大于野毒株蚀斑直径的1～2 mm。突变株病毒LD50为0.21PFU,大于野毒株病毒LD_(50)的0.05PFU。结论乙脑包膜蛋白K279M突变增大了病毒的蚀斑,但降低了病毒的脑内神经毒力。     

1966年新疆巴楚县出血热病研究报告 Ⅱ.新疆巴楚出血热病原学的初步研究

1966年4~5月从新疆巴楚县阿克沙克马拉勒地区的出血热病人急性期血液和死亡病例的肝、脾、肾及淋巴结等脏器组织标本和牧场中采集的亚洲璃眼蜱成虫标本接种4日龄以内的新生小鼠(乳鼠),结果共分离出14株病毒。标本经脑内和腹腔联合接种乳鼠后6~10天之间出现典型症状,包括:惊跳、弓背、痉挛、继之出现迟纯、软弱、平衡失调、侧卧、拒乳、皮肤苍白而死亡。在乳鼠中连续传代3次后,潜伏期缩短为5天,发病规律,可稳定传代。乳鼠脑组织中的病毒滴度可达6.78logLD50/0.01mL。新生金地鼠在接种急性期病人血液标本后可出现症状和死亡。新生大白鼠用从病人分离的病毒进行脑内和腹腔接种后出现典型症状并死亡。豚鼠在腹腔接种病人急性期血液标本后出现轻度症状。取其心血接种乳鼠后可使乳鼠发病。在有症状和无症状的豚鼠血清中均可测出较高滴度的补体结合抗体。1岁龄绵羊在皮下接种10%感染乳鼠脑悬液后仅出现轻度不适,但用此绵羊的血液接种乳鼠可使乳鼠发病死亡。绵羊的病毒血症至少可持续6天。通过绵羊的病毒在乳鼠中的潜伏期缩短为4天,滴度升高,10-6稀释度可使乳鼠全部死亡。病毒可通过蔡氏滤器EK滤板。可被低浓度甲醛灭活。1/10氯仿、20%乙醚在室...     

活动性病毒性心肌病的冠状动脉病变

本文主要研究柯萨奇B_4病毒和脑心肌炎病毒(EMC病毒)对冠状动脉和主动脉的损害。作者用含柯萨奇B_4病毒(滴度为10~(-3)～10~(-5)TCID50)的猴肾细胞培养液或含EMC病毒(滴度为10~(-6)TCID50)的L-细胞培养物0.025～0.1毫升接种于小白鼠腹腔。接种后24小时至10天内杀死小白鼠,立即采取标本,用光学和电子显微镜观察。作者发现,柯萨奇B_4病毒和EMC病毒不仅引起心肌病,而且使心肌毛细血管、冠状动脉、主动脉     

Isolation and phylogenetic analysis of Batai virus, Germany

A molecular survey including 16,057 mosquitoes captured in Southwest Germany during the summer of 2009 showed the presence of Batai virus (BATV) in Anopheles maculipennis sensu lato. Until this survey, there was no evidence for circulation of BATV in Germany. Analysis of partial S, M, and L segments showed that the sequences from all three segments were most closely related to BATV, indicating that the virus has not undergone reassortment. Phylogenetic analysis revealed a close relationship of the isolated BATV strain from Germany with strains from Slovakia, Ukraine, and Russia.     

Batai Orthobunyavirus: An Emerging Mosquito-Borne Virus in Europe

Batai virus (BATV) is a zoonotic orthobunyavirus transmitted by a wide range of mosquito vectors. The virus is distributed throughout Asia and parts of Africa and has been sporadically detected in several European countries. There is increasing evidence that BATV is emerging in Europe as a potential threat to both animal and human health, having been detected in mosquitoes, mammals, birds and humans. In recent years, serological surveillance in cattle, sheep and goats has suggested an antibody prevalence of up to 46% in European livestock, although human serological prevalence remains generally low. However, the recent and continued spread of invasive mosquito species into Europe may facilitate the establishment of competent populations of mosquitoes leading to increased BATV transmission. Migratory birds may also potentially facilitate the emergence of BATV in geographical locations where it was previously undetected. Although BATV has the potential to cause disease in humans and livestock, our understanding of the impact in wild animal populations is extremely limited. Therefore, there is a need for increased surveillance for BATV in mosquitoes, livestock, wild mammals and birds in Europe to understand the true impact of this virus.     

狂犬病毒CTN-1株在Vero细胞上的适应传代

以我国狂犬病固定毒人二倍体细胞适应株 (CTN - 1)作Vero细胞适应传代研究。显示CTN - 1株能较好适应Vero细胞 ,经 5代培养病毒滴度可达 7 5logLD5 0 /ml,且在 5～ 2 0代以内均稳定在 7 0logLD5 0 /ml左右 ,符合WHO对生产用毒种滴度要求     

Serological and Molecular Investigation of Batai Virus Infections in Ruminants from the State of Saxony-Anhalt,Germany, 2018

Arthropod-borne Batai virus (BATV) is an Orthobunyavirus widely distributed throughout European livestock and has, in the past, been linked to febrile diseases in humans. In Germany, BATV was found in mosquitoes and in one captive harbor seal, and antibodies were recently detected in various ruminant species. We have, therefore, conducted a follow-up study in ruminants from Saxony-Anhalt, the most affected region in Eastern Germany. A total of 325 blood samples from apparently healthy sheep, goats, and cattle were tested using a BATV-specific qRT-PCR and SNT. Even though viral RNA was not detected, the presence of antibodies was confirmed in the sera of all three species: sheep (16.5%), goats (18.3%), and cattle (41.4%). Sera were further analyzed by a glycoprotein Gc-based indirect ELISA to evaluate Gc-derived antibodies as a basis for a new serological test for BATV infections. Interestingly, the presence of neutralizing antibodies was not directly linked to the presence of BATV Gc antibodies. Overall, our results illustrate the high frequency of BATV infections in ruminants in Eastern Germany.     

Vero细胞口服狂犬病疫苗的研究

本试验采用ＣＴＮ－１株经Ｖｅｒｏ细胞传１５代，病毒滴度均在７．０～８．０ｌｏｇＬＤ５０／ｍｌ之间。给８只犬分别口服８．１ｌｏｇＬＤ５０的疫苗３０天的中和抗体几何平均效价为１：１１０，中和抗体的平均国际单位为２．５９ＩＵ／ｍｌ，阳转率为１００％；给４只犬分别口服７．４３ｌｏｇＬＤ５０的疫苗，阳转率为５０％，中和抗体的平均国际单位为２．４８ＩＵ／ｍｌ。选用３５－６０日龄的乳犬１６只口服１０个剂量的疫苗，并在口服后５、１０、２０天、３个月各杀３只犬取脑及唾液腺分别在ＢＨＫ２１细胞上盲传２代，同时在昆明种小白鼠乳鼠脑内盲传２代，进行病毒分离，均为阴性，余下犬观察１２个月均正常。总之该口服狂犬病疫苗对犬具有较好的安全性及口服免疫原性。     

Oral infection and transmission of La Crosse virus by an enzootic strain of Aedes triseriatus feeding on chipmunks with a range of viremia levels

The susceptibility of Aedes triseriatus to oral infection with La Crosse (LAC) virus resulting from feeding on chipmunks with viremia titers of 0.6 to 4.6 log10SMICLD50/0.025 ml was determined. Results indicated that viremia titers must exceed 3.2 log10SMICLD50/0.025 ml before a significant proportion (greater than or equal to 50%) of mosquitoes are infected and capable of transmitting LAC virus. Mosquitoes which fed on chipmunk blood-LAC virus mixtures through a membrane feeder had significantly lower infection rates at virus titers of 1.8 to 4.4 log10SMICLD50/0.025 ml and transmission was also significantly reduced. Application of these data to LAC viremia titers measured in chipmunks in an earlier study indicate that viremias sufficiently high to ensure transmission by the mosquitoes becoming orally infected average only about 1 day per infective bite delivered to the susceptible portion of the amplifier population. Oral infection and transmission rates were also determined for Ae. triseriatus feeding on chipmunk blood containing LAC virus neutralizing (N) antibodies and for Ae. triseriatus feeding on deer blood containing Jamestown Canyon (JC) virus N antibodies. Infection rates were similar to those observed in mosquitoes imbibing blood free of N antibody at the virus titers tested, but, oral transmission was reduced in females feeding on chipmunk blood-LAC virus mixtures containing LAC N antibodies and there was no transmission by females feeding on deer blood-LAC virus mixtures containing JC N antibodies. These data suggest that high LAC antibody prevalences in chipmunk populations and high LAC or JC antibody prevalences in deer populations may be antagonistic to horizontal LAC virus transmission.     

Experimental coxsackie B virus myocarditis in mice: 18 month histopathological and virological study

In a series of experimental studies to test the hypothesis that idiopathic cardiomyopathy in man represents a sequela of virus myocarditis, coxsackie B 1, 3 or 5 virus was inoculated into ICR mice with two different amounts, that is, a small amount (0.1 ml of 10(5.5) TCID50/ml) and a large amount (0.1 ml of 10(7.5) TCID50/ml). Histopathological and immunofluorescent studies of the heart, analysis of the antibody titers in sera and evaluation of the virus concentration in various organs including the heart were carried out in an acute (up to the 21st day) and chronic phase (up to the 18th month) of the experiment. A small amount of coxsackie B 1 or 5 virus did not cause myocarditis, while a large amount of either virus rarely induced mild myocarditis. A small amount of coxsackie B 3 virus frequently caused mild myocarditis without obvious residual pathologic changes of the heart, while a large amount of the same virus always caused acute and severe myocarditis. In these animals, acute myocardial changes are almost in agreement with those in previous investigations except for capillary thrombi. The virus was isolated from the heart with higher titers than from other organs and identified in some cardiocytes by immunofluorescent study until the 14th day. Neutralizing antibody in sera appeared on the 7th day and remained for several months. Approximately two thirds of these mice left no significant myocardial lesions, whereas about one third of them which probably had extensive myocardial lesions in the acute phase developed significant myocardial fibrosis with calcification in the chronic phase. These lesions appeared to become larger after the 6th month. In and around the fibrotic lesions, atrophy, hypertrophy and/or disarray of myocardial fibers were observed. These hearts did not show hypertrophy or dilatation but their histologic findings resembled those seen in some cases of congestive cardiomyopathy except for severe calcification in the myocardium.     

Obtention of rabies antigen through BHK21 cells adhered to microcarriers

Four rabies antigen batches were produced from virus suspensions resulting from BHK21 cells adhered to microcarriers (Cytodex 1), inoculated and cultured in a bioreactor. In parallel the methodology of production of rabies virus through cultures of BHK21 cells in monolayers in bottles was used. The results obtained showed that infecting titles were 10(6.69) DL50/mL and 10(7.28) DL50/mL for suspensions cultured in bottles and in the bioreactor, respectively. The viral suspension volumes collected were on average 11,900 per batch from the bioreactor and 800mL per bottle. Ten horses were immunized with the antigen produced in the bioreactor. The means of antirabies antibody titers found were 240 and 212 IU/mL after the initial and the first booster doses, respectively. Rabies antigen with satisfactory infecting titers can be obtained on a large scale by culturing in a bioreactor inoculated BHK21 cells adhered to microcarriers.     

The role of interferon in the resistance of C57BL/6 mice to various doses of herpes simplex virus type 1

C57BL/6 (B6) mice are relatively resistant to infection with herpes simplex virus type 1 (HSV-1). Paradoxically, B6 mice were resistant to 250 LD50 (50% lethal dose) of HSV-1 but were killed by 25 or 2.5 LD50 of HSV-1 injected intraperitoneally. At the injection site 250 LD50 of virus induced high titers of interferon (IFN) (greater than 1,000 international units) that reached maximal levels 2-4 hr after inoculation, whereas 25 or 2.5 LD50 of HSV-1 generated only borderline levels of IFN (15-40 international units). Simultaneous inoculation of 250 LD50 of HSV-1 and antiserum to mouse IFN (MuIFN) rendered B6 mice susceptible to infection; however, treatment with antiserum to MuIFN did not increase susceptibility to 1 LD50 of virus. Injections of MuIFN given 2 hr before and 2 hr after inoculation with virus protected B6 mice against 25 LD50 of virus. Thus, endogenous IFN induced after injection with 250 LD50 of HSV-1 protects B6 mice, whereas low doses of virus kill the animals because they do not generate a detectable IFN response at the infection site.     

纳米银对流感病毒H3N2的抑制作用

目的探讨纳米银（NaAg）对流感病毒H3N2的抑制作用。方法采用血球凝集试验、MTT分析法、血球吸附试验和鸡胚培养法,探讨NaAg对流感病毒H3N2的抑制作用。结果NaAg／H3N2组和H3N2对照组血球凝集试验效价分别为〈1：4和1：1024（P〈0．001）;NaAg溶液在MD-25细胞上最大无毒浓度为25μg／ml;H3N2在MD-25细胞上的细胞半数感染浓度（TCID50）为10^3.5／0．1ml。等体积的50μg/mlNaAg与40TCID50流感病毒H3N2溶液室温充分混和作用2h后感染MD-25细胞,细胞存活率为（96．77±2．07）％;20TCID。流感病毒H3N2感染MD-25细胞的细胞存活率为（33．09±1．48）％（P〈0．001）。结论NaAg对流感病毒H3N2具有明显的抑制作用。