流行性乙型脑炎病毒的变异V．活疫苗弱毒株的生物学特性

继以前的工作,我们选择SA14-A弱毒变异株中的12-1-7号蚀斑病毒继续进行减毒和蚀斑纯化,获得了二株高度减毒而且毒力不易回升的弱毒株,其中一株5—3株的免疫原性较好,经人体免疫观察证明是安全有效的。5—3株的主要生物学特性如下： 1．在地鼠肾细胞内繁殖引起明显的细胞病变作用,病毒滴度一般可达10-6—10-7(TCID50／0．2毫升)。在琼脂覆盖下的鸡胚单层细胞内形成小于0．1厘米大小的点状蚀斑,蚀斑内细胞未完全破坏死亡。 2．对3周龄小白鼠和2—3公斤恒河猴脑内接种不引起发病和死亡,脑内病理改变较原强毒株显著减轻而且局限。 3．对1-3日龄乳小白鼠,无论脑内或皮下注射均有致病力,但致病力显著减弱,引起乳鼠死亡的毒力(LD50),脑内在3.0对数以下,皮下在2．0对数以下。 4．对3周龄小白鼠皮下注射,病毒只能在周围脏器内轻度繁殖随即消亡,而不能侵入脑内繁殖,脑组织内分离不到病毒,亦不引起病理改变。 5．在组织培养细胞内连续传10代,或在小白鼠脑内传3—5代,其残余毒力无明显回升。 6．对小白鼠皮下一次免疫后表现有较强的保护力。对豚鼠皮下免疫一次后表现有明显的中和抗体产生,阳转率为70—80％。1967年以来对5—3株进行了人群逐步扩大接种观察,结果未发现活疫苗引起的发热 反应和其他异常反应,人体抗体的阳转率为85—91％,人群流行病学效果保护率一般为80—90％,证明活疫苗是安全和有较好的免疫效果。但是5—3株尚存在着病毒滴度下降快,疫苗不稳定,需冷藏保存等缺点,因而往往影响疫苗大面积人体使用效果,需要进一步改进提高。     

云南森林脑炎病毒的动物敏感性研究

本文对分离自云南的森林脑炎病毒进行了动物敏感性研究,实验证明云南森林脑炎病毒对小白鼠有较强的致病性,三日龄乳鼠无论经脑内、腹腔、皮下接种均能致病、死亡,但毒力较国内森林脑炎病毒标准株低;三周龄小白鼠经鼻腔接种亦能发病致死。对乳大白鼠、幼年豚鼠和金黄色地鼠能引起发病或死亡,病毒抗原定位主要在脑组织。病理检查表明感染的各种动物脑组织均有明显病变。此外,对鸡胚敏感,能引起BHK_(21)、Vero、Vero-E_6等传代细胞及人胚肾、乳猪肾原代细胞的CPE_0结果表明了云南森林脑炎病毒对细胞、动物的致病性与国内森林脑炎病毒标准株相似,仅毒力稍低。     

流行性乙型脑炎病毒的变異III．通过地鼠腎细胞后对小白鼠及恒河猴的毒力和免疫力

1．乙型脑炎病毒SA14毒株通过地鼠腎单层细刚胞连续传递20代后,对小白鼠的脑内毒力发生显著变异,由原来的10-5—10-6下降至10-1—10-2.5／0．03ml之间,与地鼠腎细胞的釉胞致病变毒力(TCID50)此鞍,二者相差4．00—5．07log,而皮下,肤腔毒力则巳丧失。 2．变异株对恒河猴的脑腔致病力亦有明显的减弱,以大量病毒脑腔感染猴子后,虽有脑炎症状出现,但发病溉伏期和病程均有延长,而感染的4只猴子中仅有一只死亡。 3．变异株对小白鼠具有较强的免疫力作用,在豚鼠、猴手及小白鼠体内均能产生一定的特异性中和抗体。     

流行性乙型脑炎病毒的变异 Ⅳ.SA14-A株蝕斑病毒的致病力和免疫力

预防流行性乙型脑炎疾病的综合措施中,应用疫苗对人羣进行自动免疫,是控制该病的重要方法之一。由于乙型脑炎死毒疫苗存在着反应重或免疫效果较差的缺点,近年来我们着手研究乙型脑炎活疫苗的工作。本文为继以往的工作,将SA14-A減弱株,继续通过地鼠肾细胞100代的病毒培养液中,挑选出3株对小白鼠脑內感染几乎不致死而对恆河猴完全不致死的毒株,较原有的SA14-A減弱株的毒力又有进一步的减弱,而且这些毒株经小白鼠及豚鼠免疫后仍然表现出良好的免疫性。因此,我们认为将这些毒株再做适当的减毒并保持其一定的毒力稳定性后,可以供乙型脑炎活疫苗做为毒株用。     

流行性乙型脑炎病毒的变異II．不同毒株通过鸡胚组织培养后的毒力变異

1．流行性乙型脑炎不同毒栋通过鸡胚悬浮组织块连续传代50一100代后对小白鼠的神经外毒力逐渐下降,但其表现程度不完全一致,原来神经外毒力较低的SA4毒株则下降比较多,直至完全丧失。脑腔毒力的变化在不同毒株之同的差异更为明显;P3及Lm株经培养200代左右其毒力始格保持恒定,而SA4株毒力(LD50)则自181代以后开始下降1一2log。 2．对小白鼠脑腔毒力下降的SA4株对恒河猴脑腔致病力亦有一定的减弱,主耍表现在猴子于感染病毒后的发病溉伏期及病程比原毒株延长约一倍。 3．通过鸡胚组织块连续传代后的病毒对小白鼠的致病力和免疫力之间有一定的关系,SA4株当其脑腔毒力开始下降,腹腔毒力完全丧失后,则几乎完全失去了对小白鼠的 免疫性,而P3及Lm株的神经外毒力虽有下降,但仍然保持一定的致病力,因而仍然具有一定的免疫力。     

森林脑炎减毒活疫苗的研究I．毒株的选育

为了提高病毒在神经外组织的繁殖能力和增强其免疫原性,将Co乳鼠变异株于小白鼠、睬鼠和金黄地鼠进行了连续传代适应。选育出的减毒株其对嗜神经性毒力显著减弱,当脑腔接种于地鼠和恒河猴时不能引起致病或死亡,同时在中枢神经系统亦检查不到严重的组织学病变。减毒株保留对小白鼠脑腔的残余毒力和原株的抗原性。从对小白鼠的毒力上可认为本系减毒株和其它所报告的减毒株相似。     

狂犬病病毒CTNCEC25株的感染力和致病力特征分析

研究狂犬病病毒CTNCEC25株的增殖能力、病毒的感染力及致病力等表型特征,为将该毒株应用于疫苗生产提供基础信息。将CTNCEC25株及其母株CTN-1株分别接种非洲绿猴肾细胞(Vero细胞)、原代鸡胚成纤维细胞(Primary chicken embryo cell,CEC)和小鼠神经瘤母细胞(Mouse neuroblastoma N2acells,N2a),观察其细胞病变特性及其复制规律;以脑内注射的方式接种动物,观察其对乳鼠、小鼠、豚鼠及家兔等动物的致病力;同时还将CTNCEC25株在CEC上连续传20代,在乳鼠脑内传3代,观察其增殖能力、病毒感染力及致病力的稳定性。研究结果表明,与母株CTN-1株相比,CTNCEC25株在Vero细胞和N2a细胞上的增殖并没有显著差异,但在CEC上,CTNCEC25株具有明显优势,其72h滴度可达107.5~7.6 FFU/mL,而CTN-1株滴度只有105.8 FFU/mL;此外CTNCEC25株在N2a细胞、Vero细胞及CEC上均可产生明显的细胞病变。脑内致病力实验结果表明,CTNCEC25株虽然对1~3日龄的乳鼠仍然具有较强的脑内致病性,但对成年小鼠的脑内致病力大大减弱,对豚鼠和家兔不致病。经细胞和乳鼠脑内连续传代,CTNCEC25株在CEC上增殖稳定,其减弱的毒力并未回升,无弱毒返祖现象。综上所述,本研究初步证实CTNCEC25株是一株高度减毒、不易返祖的弱毒株,其在CEC上可稳定、快速增殖,病毒滴度高(107.0 FFU/mL),是一株安全性较高的狂犬病疫苗候选毒株。     

乙型脑炎14-2株冻干活疫苗的生产研究

乙型脑炎病毒14-2株经地鼠肾细胞连续传23代,各代次的病毒滴度和回传乳鼠后的毒力都较稳定,与5-3株无差异。各代次的神经毒力和中枢神经外毒力也与5-3株相同。脑内感染12～14克小白鼠均不致死,皮下感染也不致病。用地鼠肾细胞经36℃培养4天,病毒增殖达到高峰,滴度为8.0～8.5logTCID50/0.2ml。病毒培养液的pH值为7.4～7.6时,病毒增殖高峰可持续3天。以1％明胶、5％蔗糖为保护剂,在冻干后无真空、不充氮的条件下。14—2株活疫苗在37℃可保存10天,室温(16～31℃)可保存4个月,5～8℃可保存一年,病毒滴度均无明显下降.冻干后充氮或不充氮病毒的滴度及稳定性看不出差异。冻干14—2株活疫苗融化后,用Eagle’s液稀释,置22～23℃8小时,滴度不变,若以生理盐水稀释,则可保持2～4小时;以蒸馏水稀释只能稳定2小时。     

不同狂犬病毒株在地鼠肾细胞上培养的病毒滴度与免疫原性比较

三个狂犬病毒株,分别经地鼠肾细胞培养,将其抗原含量,病毒滴度和免疫原性进行比较。结果显示,三个病毒株的抗原含量有差异。但差异显著性,CTN-V10M3的毒力最高,CTN-BHK3的毒力最低,aG株的毒力虽然居中,但免疫原性最好,它仍是最适于地鼠肾细胞上培养的毒株。     

地鼠肾细胞培养的CTN株狂犬病新疫苗研究

应用细胞毒种代替豚鼠脑毒种制备狂犬病地鼠肾细胞纯化疫苗。将狂犬病毒CTN株在原代地鼠肾细胞（PHKC）传代适应,用病毒培养液上清作为生产用毒种,结果通过在PHKC传10多代,适应后病毒滴度达到了7.0LogLD50/ml,并应用适应株（CTN－LS－HK）细胞毒种制备三批疫苗,其效力在6.11-6.55IU/ml,高于用aG株豚鼠脑毒种制备的三批疫苗效力（3.77-5.85IU/ml)。     

不同代次毒种乙脑减毒活疫苗的滴度和安全性实验结果比较

为比较不同代次的乙脑毒种在疫苗制备过程中对疫苗质量的影响,特制备不同代次的工作种子SA14 14 2PHK7、PHK8、PHK9,检定合格后,分别用这几批毒种制备乙脑减毒活疫苗,检定和比较疫苗的滴度和各项安全性指标。实验表明SA14 -14 -2PHK7、PHK8和PHK9三个代次的乙脑毒种制备的乙脑疫苗平均滴度为 6. 43lgPFU/ml;乳鼠传代返祖试验均值为 1. 1lgLD50 /0. 03ml;致病力均为阴性。证实乙脑毒种SA14 -14 -2 10代以内的生物学特性是稳定的,对疫苗的影响无显著差异。10代以内的乙脑毒种可安全的用于疫苗生产。     

野猪瘁死症病原的分离鉴定

采集病死野猪的脾脏和血清,用特异抗猪瘟病毒抗体进行琼脂扩散试验检测发现有明显的沉淀线出现,证明野猪感染了猪瘟病毒,猪的致病性实验表明并非猪瘟强毒感染。采集病死野猪的心、肝、脾、肺、心血,经镜检、细菌分离培养、纯培养、生化试验和细菌G C mol%含量测定等检验,证实致病细菌有多杀巴氏杆菌,其G C mol%含量为39.7。毒力测试发现巴氏杆菌具有较强的致病性,LD50=10-1.57/0.5 ml。化脓放线杆菌的分离和毒力表明,化脓放线杆菌也参与致病作用。由此推测本次致死野猪的病原体为多杀巴氏杆菌并发或继发猪瘟病毒、化脓放线杆菌的三重感染所致。纸片扩散法(K-B法)药敏试验表明,两株细菌对头孢哌酮、头孢唑啉、丁胺卡拉霉素等高度敏感,为临床治疗和有效预防该瘁死症奠定基础。     

不同代次牛肾原代细胞培养轮状病毒的比较研究

口服轮状病毒活疫苗(LLR株)生产用细胞基质为新生小牛肾原代细胞。原始的初代细胞(P0)产量小,一对牛肾平均生产7瓶细胞。将原始的初代细胞传代可使细胞产量显著增加,传代后(P2代)细胞产量可由7瓶增加为96~112瓶,细胞核型检查传至P5代的细胞染色体数目与初代细胞一致。细胞培养物均一性提高。P0代与P2代细胞病毒培养物滴度分别在6.2±1.5和6.5±0.5lgCCID50/ml,使用P2代细胞培养病毒,产量增加10~15倍。提高了疫苗生产的可控性和质量,生产规模显著放大,经济效益明显。     

Immunization of pigs with the attenuated S- strain of Japanese encephalitis virus.

The attenuated S- strain of Japanese encephalitis virus was produced from a wild strain of this virus by serial cultivation in primary bovine kidney cell cultures at 30 degrees C. Pigs were inoculated with it and examined for ability to produce antibody and protect themselves from infection with a wild strain used for challenge. In pigs inoculated with a single dose of 10(6.5) approximately 10(7.5) TCID50 of the S- strain, the neutralizing antibody titer or hemagglutination-inhibiting antibody (HI) titer increased to 10 approximately 320. An antibody titer exceeding 10 was maintained for 2 approximately 9 weeks. In pigs inoculated twice with 10(6.5) approximately 10(7.0) TCID50 of the S- strain, HI titer increased to 80 approximately 640. In many of these pigs, HI titers of 80 approximately 160 persisted for more than 6 weeks. Pigs inoculated once or twice with 10(7.0) approximately 10(7.5) TCID50 of the S- strain were challenged by inoculation with 10(4.5) approximately 10(5.5) TCID50 of a wild strain and examined for the occurrence of viremia. As a result, an ability to protect from infection was demonstrated in pigs which showed an antibody titer surpassing 10 at the time of challenge. Pregnant sows inoculated with 10(7.0) TCID50 of the S- strain were challenged by inoculation with 10(7.0) TCID50 of a wild strain. Neither death nor infection occurred to any fetus harbored by them. From these results, it is concluded that the S- strain can be used as live virus vaccine for porcine practice.     

Rapid quality control of a live attenuated Peste des petits ruminants (PPR) vaccine by monoclonal antibody based sandwich ELISA.

Peste des petits ruminants (PPR) is a highly contagious and economically important viral disease of goats and sheep. A homologous Vero cell-based attenuated PPR vaccine developed in our laboratory and used extensively throughout the country, is available for control of PPR. The presently used quality control test, titration in Vero cells for PPR virus titre in vaccine batches, takes at least 6-8days to determine the quality and dose of vaccine. In this study, 74 freeze-dried PPR vaccine batches were tested simultaneously by both virus titration and PPR sandwich ELISA (S-ELISA) to correlate the titre of the vaccine virus with reactivity in S-ELISA. It was found that the vaccine batches with titre more than 10(3)TCID(50)/ml gave positive results in S-ELISA and correlated well with the virus titre of the freeze-dried vaccines. The correlation coefficient between the virus titration and S-ELISA reactivity was estimated as 0.96, indicating a high correlation between the two parameters based on 74 batches of freeze-dried PPR vaccine. The vaccine batches with titres of 3.0, 4.3, 4.5, 5.0, 6.5 and 7.0 had shown a positive reaction when tested in two-fold dilutions in S-ELISA at 1, 5, 6, 7, 8 and 9log2 titres, respectively. The test vaccine batches were found to be negative in S-ELISA when the titre of the vaccine was less than 10(3)TCID50/ml, suggesting that the vaccine could not be passed for field use. It is concluded that S-ELISA could be a preliminary tool useful for the quality control of PPR vaccine as it is rapid and easy to perform when compared to virus titration.     

中国首次分离的辛德毕斯病毒XJ-160病毒株感染性全基因组cDNA克隆的构建与分析

报告了中国首次分离的辛德毕斯病毒XJ-160株的感染性全基因组cDNA克隆的构建与鉴定。利用RT—PCR方法获得覆盖病毒全长基因组的cDNA片段,以低拷贝质粒pBR322作为骨架,将基因组cDNA置于SP6RNA聚合酶启动子之后,基因组3’末端带有35个连续的A,通过DNA重组技术组装成病毒基因组全长cDNA克隆。该克隆可在大肠杆菌DH5a中稳定扩增。经体外转录,RNA转录体转染BHK-21细胞,细胞发生病变,恢复病毒滴度达到10^7～10^8PFU／ml。全基因组cDNA克隆构建过程中引入的沉默突变(8453位核苷酸由C变为T)产生XbaⅠ酶切位点作为遗传标记,在子代恢复病毒的基因组中稳定存在。从细胞病变的特征、BHK-21细胞的空斑形态、病毒的抗原性、病毒在细胞中的生长动力学特征以及对乳鼠的致病性等方面比较,恢复病毒和亲本病毒XJ-160没有显著区别,提示获得了具有感染性的XJ-160病毒全长cDNA克隆。该病毒感染性全基因组cDNA克隆可以作为反向遗传学系统,为进一步研究病毒复制和致病机制,以及开发相应的载体表达系统提供分子生物学工具。     

Kinetics of ectromelia virus (mousepox) transmission and clinical response in C57BL/6j, BALB/cByj and AKR/J inbred mice

Research was undertaken to answer basic questions on susceptibility, clinical response and transmission of ectromelia virus in selected strains of inbred mice. C57BL/6J and AKR/J were found to be markedly more resistant to a virulent strain of ectromelia virus (isolated during the 1979-80 outbreak at the National Institutes of Health) than C57LJ, BALB/cByJ, DBA/2J, A.By/SNJ and C3H/HeJ when infected by footpad inoculation. In C57BL/6J and AKR/J the LD50 was about 7 logs higher than the ID50. With one exception, C57LJ, the LD50 and ID50 titers in the other strains were about equal. In C57LJ the LD50 titer was intermediate. Following intragastric inoculation, virus was isolated from feces of C57BL/6J mice for as long as 46 days and up to 29 days from BALB/cByJ mice. Transmission to cage mates from intragastrically infected C57BL/6J and BALB/cByJ occurred up to 36 and 30 days respectively after infection. Virus was isolated from the spleen in 2 of 5 BALB/cByJ mice and 1 of 7 C57BL/6J mice tested 95 days after gastric inoculation. Following footpad inoculation, BALB/cByJ mice consistently transmitted virus to cage mates before death at 10-12 days. C57BL/6J mice transmitted between days 8 and 17, but not beyond. Virus was maintained in C57BL/6J mice by exposure to infected cage mates for seven passages, which was the most attempted. Clinical signs in infected C57BL/6J mice were usually subtle or inapparent.     

Infectivity, speed of kill, and productivity of a baculovirus expressing the itch mite toxin txp-1 in second and fourth instar larvae of Trichoplusia ni

A cDNA clone of the gene coding for the paralytic neurotoxin (tox34) from the female straw itch mite, Pyemotes tritici, was created by RT-PCR and inserted into the genome of the Autographa californica nucleopolyhedrovirus (AcMNPV) under the control of the AcMNPV p10 promoter. This recombinant virus, AcTOX34.4, caused a rigid paralysis in infected larvae. The infectivity of AcTOX34.4 was compared to the wild-type parent strain, AcMNPV-C6, in second and fourth instar larvae of the cabbage looper, Trichoplusia ni. There were no significant differences in LD(50) values between the recombinant virus and its wild-type parent strain but, as expected, the LD(50) was lower for second instar larvae. The mean time to death and yield of occlusion bodies were measured in second and fourth instar T. ni larvae at a high (100% mortality) and low (<50% mortality) doses of the virus. The mean time to death of recombinant infected larvae was reduced by 50-60% compared to larvae infected with the wild-type strain, depending on virus dose and instar, with these larvae becoming paralysed after approximately 60 h and dying 10-20 h later. This is among the fastest speeds of kill recorded for recombinant baculoviruses. Fourth instar larvae were found to succumb to the recombinant virus more quickly than the second instar larvae. The increase in the speed of kill of the recombinant virus was accompanied by a large reduction of approximately 95% in the yield of progeny virus. The yield of virus showed a highly significant relationship with time to death, but this relationship was complex and varied between the different viruses, concentrations, and instars. The yield per unit weight of the larvae was found to be constant at a low virus dose and increased over time at a high virus dose, irrespective of instar and virus. It is predicted that these changes in the performance of the recombinant virus would act toward reducing its fitness, leading to it being outcompeted by the wild type in field situations.     

Study on persistent infection of Japanese encephalitis virus Beijing-1 strain in serum-free Sf9 cell cultures

Sf9 cells have obvious advantages for the conventional production technology of vaccine. They are useful tools for high concentration and large-scale cultures. Sf9 cells were grown to maximal concentration, 8 x 10(6) cells/ml in a 500ml spinner flask, with a doubling time at the exponentially growing phase of 24.5 hours, using serum-free media. To explore the ability of Sf9 cells to be infected by the Japanese encephalitis (JE) virus Beijing-1 strain, Sf9 cells were infected with the virus. By 4-5 days post-infection, 10-15% of the Sf9 cells showed cytopathic effect (CPE), from granularity to the formation of syncytia and multinucleated giant cells continuously observed over a period of 35 days. Positive fluorescent reactions were detected in 30-40% of cells infected with the JE virus Beijing-1 strain, and the uninfected Sf9 cells were completely negative. Virus particles, propagated in Sf9 and Vero cells, were concentrated by sedimentation on 40% trehalose cushions by ultracentrifugation, and showed identical patterns of viral morphogenesis. Complete virus particles, 40 to 50 nm in diameter, were observed, and JE virus envelope (E) proteins, at 53 kDa, were found in the western blot analysis to the anti-JE virus E protein monoclonal antibody and reacted as a magenta band in the same position to the glycoprotein staining. To evaluate whether the infectious virus was produced in Sf9 cells inoculated with the JE virus Beijing-1 stain, Sf9 cells were inoculated with the virus, and sample harvested every 5 days. The titers of the JE virus Beijing-1 strain rose from 1.0 x 10(5) to 1.5 x 10(6) pfu/ml. The infected Sf9 cells could be sub-cultured in serum-free medium, with no change in the plaque sizes formed by the JE virus Beijing-1 strain in the plaque assay. It is suggested that the ability of the JE virus Beijing-1 strain to infect Sf9 cells in serum-free media will provide a useful insect cell system, where the JE virus replication, cytopathogenicity and vaccine immunogen can be studied.     

用8质粒病毒拯救系统产生H9N2/WSN重组A型流行性感冒病毒

把禽流行性感冒(流感)病毒A／Chicken／Shanghai／F／98(H9N2)的血凝素(HA)和神经氨酸酶(NA)基因cDNA克隆至polⅠ-pol Ⅱ双向转录和表达载体pHW2000,用这两种质粒与8质粒病毒拯救系统中流感病毒A／WSN／33(H1N1)6个内部基因cDNA的质粒组合(6 2重排),共转染COS-1细胞,产生了能在鸡胚中高滴度增殖的H9N2／、WSN重组病毒。用A／WSN／33的8个基因cDNA质粒作对照,也产生了转染子病毒。经过EID50测定和MDCK感染实验,新基因型H9N2／WSN病毒感染鸡胚的能力强(EID50为10^-11／0．2m1),而且对鸡胚的毒力弱,在不加胰酶的情况下不使MDCK细胞产牛病变。经电镜观察,两个转染子病毒的形态与野生型流感病毒相似。反向遗传操作技术的建立,为对禽流感病毒基因功能和疫苗构建等方面的研究提供了新的手段。