汉坦病毒在Vero细胞上的适应传代及疫苗候选株的 …

目的 选育出在Ｖｅｒｏ细胞上繁殖力强、免疫原性和抗原性好的汉滩病及汉城病毒候选株。方法 国内外分离的１６株汉坦病毒（ＨＶ）在Ｖｅｒｏ细胞上进行连续终末稀释传代,研究各株的繁殖特性及传代后病毒滴度及抗原量的变化。结果 经过５次终末稀释传代,选育出Ｌ９９（汉城病毒）和８４ＦＬｉ（汉滩病毒）２株滴度高且稳定的病毒,在Ｖｅｒｏ细胞上具有良好和适应性,７～１０ｄ毒力接近峰值。ＥＬＩＳＡ测抗原量高峰则在１０～     

日本脑炎病毒和乙型肝炎病毒抗原真核表达载体的体外共表达

作者构建了日本脑炎病毒（Ｊａｐａｎｅｓｅｅｎｃｅｐｈａｌｉｔｉｓｖｉｒｕｓ，ＪＥＶ）前膜蛋白囊膜蛋白（ｐｒＭＥ）和乙型肝炎病毒（ｈｅｐａｔｉｔｉｓＢｖｉｒｕｓ，ＨＢＶ）前Ｓ蛋白（ｐｒｅＳ）的真核表达载体，分别命名为ｐｃＤＮＡ３Ｅ和ｐＳＧ５ｐｒｅＳ。等量混合这两种质粒，转染ＣＯＳ７细胞进行瞬间表达，应用单克隆抗本ＥＬＩＳＡ法检测特异性抗原。结果表明，单独及混合转染的细胞培养上清中均有特异性抗原表达。本项研究为进一步发展新型日本脑炎病毒和乙型肝炎病毒核酸疫苗打下了基础，也初步显示了核酸疫苗技术在联合疫苗研制中的应用前景     

Sf9昆虫细胞作为流行性乙型脑炎疫苗生产用细胞基质的研究

对Ｓｆ９细胞作为制备乙脑疫苗生产用细胞基质的可能性进行了探讨。用抗胸腺细胞血清处理过的新生小白鼠，检查证明Ｓｆ９昆虫细胞无致肿瘤原性。将在有血清培养液中乙型脑炎病毒（ＪＥＶ）Ｐ３株持续感染的Ｓｆ９细胞，逐步适应到无血清培养液中，此感染细胞持续感染的特性不变。以悬浮培养的方式，无论在有或无血清培养液中培养感染Ｐ３株病毒的Ｓｆ９细胞，其病变细胞数目比例较静止培养均明显增多，病毒滴度形成高峰时间缩短。将感染的Ｓｆ９细胞，经液氮保存后复苏可连续传代，其感染特性未改变。用有血清培养液培养感染细胞第６、１１、２６周和无血清培养液培养的感染细胞上清制备实验性ＪＥＶ疫苗，动物实验证明其效力均能达到疫苗生产规程的要求。Ｓｆ９细胞有望成为大规模悬浮培养制备ＪＥＶ疫苗的细胞基质。     

虎杖金银花联用抗Ⅱ型疱疹病毒的药效分析

Ｈｅｒｐｅｓｓｉｍｐｌｅｘｖｉｒｕｓｔｙｐｅ２（ＨＳＶ２）是外阴疱疹及宫颈癌等多种疾病的病因之一。阿昔洛韦（ａｃｙｃｌｏｖｉｒ,ＡＣＶ）治疗ＨＳＶ２感染易产生耐药毒株;ｃｉｄｏｆｏｖｉｒ肾毒性很大;ＨＳＶ疫苗效果不佳。因此,研制抗病毒新药是相当重要的任务。据报道,１０％虎杖水煎液对ＨＳＶ１、ＨＳＶ２、甲型流感病毒京科６８１株等多种病毒有抑制作用。金银花能延缓呼吸道合胞病毒的ＣＰＥ。为了获得虎杖金银花抗ＨＳＶ制剂的最佳配方比,我们用空斑减数实验,以ＡＣＶ为阳性对照,观察了虎杖金银花单用…     

小盾纤恙螨体内肾综合征出血热病毒增殖的初步研究

为进一步观察小盾纤恙螨感染肾综合征出血热病毒（ＨＦＲＳＶ）后的体内增殖，直接取饲养的小盾纤恙螨幼虫和若虫，每２０天为一批制成无菌滤液，接种Ｖｅｒｏ－Ｅ６细胞测定ＴＣＩＤ５０／ｍｌ滴度，动态观察ＨＦＲＳＶ在螨体内的增殖。结果表明：在小盾纤恙螨幼虫期除采集至测定６０天批未测出ＨＦＲＳＶ滴度和未分离到ＨＦＲＳＶ外，其余１２批均在不同时间内测出ＨＦＲＳＶ滴度和分离到ＨＦＲＳＶ，滴度均在１０－１～１０－５之间。３批若虫亦有２批测出ＨＦＲＳＶ滴度和分离到ＨＦＲＳＶ，滴度为１０－５。所分离的ＨＦＲＳＶ用ＰＣＲ扩增技术亦检测出ＨＦＲＳＶＲＮＡ。这为小盾纤恙螨作为ＨＦＲＳ的传播媒介提供了进一步的证据。     

Sf9昆虫细胞作为流行性乙型脑炎疫苗生产用细胞基…

对Ｓｆ９细胞作为制备乙脑疫苗生产用细胞基质的可能性进行了探讨。用抗胸腺细胞血清处理过的新生小白鼠，检查证明Ｓｆ９昆虫细胞无致肿瘤原性。将在有血清培养液中乙型脑炎病毒（ＪＥＶ）Ｐ３株持续感染的Ｓｆ９细胞，逐步适应到无血清培养液中，此感染细胞持续感染的特性不变。以悬浮培养的方式，无论在有或无血清培养液中培养感染Ｐ３株病毒的Ｓｆ９细胞，其病变细胞数目比例较静止培养均明显增多，病毒滴度形成高峰时间缩短。将     

应用痘苗病毒载体表达猴轮状病毒VP4抗原基因

把编码猴轮状病毒（Ｒｈｅｓｕｓｒｏｔａｖｉｒｕｓ，ＲＲＶ）Ｖｐ４抗原的第４基因片段插入到痘苗病毒表达载体ｐＪＳＡ１１７５的Ｐ７．５启动子下游，构建成在痘苗病毒Ｐ７．５启动子调控下表达猴轮状病毒Ｖｐ４抗原基因的重组质粒ＰＪＳＡ１１７５－ＶＰ４。应用磷酸钙沉淀技术将ＰＪＳＡ１１７５－ＶＰ４ＤＮＡ转入ＴＫ－１４３细胞，在ＢＵＤＲ和Ｘ－ｇａｌ存在下筛选蓝色蚀斑。经３代以上纯化和病毒增殖，获重组病毒Ｒ－ＶＪＳＡ１１７５－Ｖｐ４。蚀斑滴定其满度达到１５×１０１１ＰＦＵ／Ｌ。经核酸杂交试验证明所获得的重组痘苗病毒带有猴轮状病毒Ｖｐ４抗原基因。用重组病毒感染ＴＫ－１４３细胞（或Ｖｅｒｏ细胞），在感染后４８ｈ，用酶免疫法（ＥＩＡ）检测受染细胞上清液和细胞裂解液中表达的猴轮状病毒Ｖｐ４抗原基因均呈阳性反应。本试验为本研究室轮状病毒基因工程疫苗的一部分，为深入了解轮状病毒基因结构及其功能在方法学上奠定了必要的基础。     

柯萨奇病毒A16型YY157株在Vero细胞中培养与增殖特性的研究

目的 探索柯萨奇病毒A16型（CVA16） YY157株在非洲绿猴肾（Vero）细胞中培养与增殖的合适条件.方法 把CVA16 YY157株接种于Vero细胞适应性传代,挑斑纯化后,在不同条件下培养并比较其对病毒滴度的影响.结果 CVA16 YY157株在Vero细胞中培养能导致细胞病变（CPE）,可形成蚀斑.将此CVA16病毒以0.01 ～0.1MOI接种于Vero细胞,并在低于2％牛血清浓度的培养基,35℃培养60 h,CPE可达90％以上;此条件下收获培养液上清,可得到较高滴度的病毒.结论 初步建立了CVA16 YY157株在Vero细胞中培养与增殖的方法,为其下一步的大规模培养及疫苗制备奠定了基础.     

乙型肝炎病毒表面抗原S基因在人5型重组腺病毒中…

从美国Ｗｙｅｔｈ公司引进ｐＡｄ５△Ｅ３载体，将乙型肝炎病毒表面抗原基因（ＨＢＶＳ）ＰｒｅＳ２＋Ｓ，腺病毒晚期表达盒＋ＨＢＶＳ基因插入Ｅ３区，与ＥｃｏＲＩ消化的Ａｄ５ＤＮＡＡ片段共转染细胞获得重组腺病毒，相应命名为ｒＡｄ５Ｓ，ｒＡｄ５ＭＳ，ｒＡｄ５Ｓ２Ｓ。对所获得的重组病毒进行聚合酶链反应（ＰＣＲ）分析，证实重组病毒中含有目的基因，用ＥＬＩＳＡ分析ｒＡｄ５ＭＳ的表达量，其病变细胞和上清液滴度均为１：     

单纯疱疹病毒1型诱导的Hela细胞凋亡及钙浓度的变化

一方面认为宿主细胞可利用细胞凋亡来清除病毒感染细胞 ;另一方面病毒可以抑制细胞凋亡 ,以利于自身增殖需要。而病毒诱导细胞凋亡还是抑制凋亡 ,取决于多方面因素。Ｌｅｐｐａｒｄｉ研究表明野生株ＨＳＶ 1感染Ｖｅｒｏ细胞并不引起Ｖｅｒｏ细胞凋亡 ,但缺乏调节性基因α4的Ｈ     

Preparation of Rocky Mountain spotted fever vaccine suitable for human immunization.

Rocky Mountain spotted fever vaccine was produced from rickettsiae grown in chicken embryo cells in roller bottle cultures. The rickettsiae were concentrated and purified by passage through a sucrose gradient and inactivated with formalin. This vaccine satisfactorily passed preinactivation and final container testing and is believed to be superior to the presently available yolk sac vaccine.     

Growth characteristics of Japanese encephalitis virus in chick embryo cells with special reference to the applicability of the culture to the production of inactivated vaccine

Summary The mouse-adapted Nakayama strain of JE virus was adapted by serial passage to growth in primary chick embryo (CE) cells. It was clone-purified three times. The resulting virus was able to induce a clear CPE in CE cells. It was inoculated to CE cells cultured in bottles and in a spinner culture system and the optimal conditions to give the highest yield were examined. Under the conditions adopted, a basal medium containing no serum or serumalbumin could be used as maintenance medium without lowering the efficiency for yielding a high titer of virus. Also, virus concentrations in culture fluids obtained in the bottle and spinner cultures were about equal. Virus obtained from the spinner culture was concentrated by ethanol precipitation and inactivated by beta-propiolactone treatment in order to produce a vaccine. Three lots of vaccine so prepared were tested for their potencies by the mouse protection test with promising results.     

Pressure-inactivated yellow fever 17DD virus: implications for vaccine development

The successful Yellow Fever (YF) vaccine consists of the live attenuated 17D-204 or 17DD viruses. Despite its excellent record of efficacy and safety, serious adverse events have been recorded and influenced extensive vaccination in endemic areas. Therefore, alternative strategies should be considered, which may include inactivated whole virus. High hydrostatic pressure has been described as a method for viral inactivation and vaccine development. The present study evaluated whether high hydrostatic pressure would inactivate the YF 17DD virus. YF 17DD virus was grown in Vero cells in roller bottle cultures and subjected to 310 MPa for 3 h at 4 °C. This treatment abolished YF infectivity and eliminated the ability of the virus to cause disease in mice. Pressure-inactivated virus elicited low level of neutralizing antibody titers although exhibited complete protection against an otherwise lethal challenge with 17DD virus in the murine model. The data warrant further development of pressure-inactivated vaccine against YF.     

Japanese encephalitis vaccine in travelers

Extensive vaccination against Japanese encephalitis (JE) has been carried out in many Asian countries for the past 20 years and is also increasingly recommended for travelers to endemic areas. Concerns have been raised regarding potential neurological and allergic side effects of the currently available JE vaccine, which is manufactured from mouse brain. A new purified, inactivated JE virus vaccine (IC51) has been developed, which is manufactured in a Vero cell culture substrate. Studies show that the vaccine is both safe and immunogenic and the product will be licensed very soon for use in many industrialized countries. Once a highly immunogenic and safe product is available, wider use of JE vaccine in travelers will be prudent. Currently, vaccination is restricted to travelers with an increased risk of acquiring JE. Individuals at increased risk have been defined quite arbitrarily as travelers with increased behavioral contact to JE-transmitting mosquitoes, in particular, during stays in rural areas and during the transmission season. However, the possibility of an infection with JE virus can never be ruled out when traveling to endemic areas and infection can prove disastrous for the individual concerned. Since a safe product will be available very soon, guidelines and recommendations will have to be reconsidered.     

肾综合征出血热纯化疫苗候选毒株在Vero细胞上的适应传代及其免疫原性研究

目的 将肾综合征出血热纯化疫苗候选毒株适应于Vero细胞，并对其抗原性和免疫原性进行研究。方法 将汉滩病毒H8207株和汉城病毒Y86013株在Vero细胞上进行连续终末稀释传代，采用间接免疫荧光法、酶联免疫吸附试验和空斑减少中和试验，研究传代后毒株的繁殖特性、病毒滴度、抗原量及免疫原性。结果 经过5次终末稀释传代，两株病毒在Vero细胞上均显示出良好的适应性，从第六代开始病毒滴度稳定在7．0Lg TCID50/m1以上，第八代两毒株抗原量均已达到1：64，H8207株抗原量继续升高，最高时达1：256。用两株病毒不同培养代次上清液制备的单价原液灭活疫苗免疫家兔二针，免疫血清对同型毒株的中和效价均达到1：10。结论 两株病毒已适应于Vero细胞，且具有病毒滴度高和免疫原性良好的特性，适合用作Vero细胞肾综合征出血热灭活纯化疫苗的候选毒株。     

T-cell activation and induction of antibodies and memory T cells by immunization with inactivated Japanese encephalitis vaccine

Mouse brain-derived inactivated Japanese encephalitis (JE) vaccine is the only currently internationally accepted vaccine against JE virus. We analyzed cellular and humoral immune responses to the JE vaccine in healthy adults in order to understand the protective immunity induced by this vaccine. Immunization with the JE vaccine induced T-cell activation in vivo, demonstrated by increase in the plasma levels of interleukin (IL)-2 and soluble CD8. JE virus-specific antibodies determined in radioimmunoprecipitation (RIP), hemagglutination inhibition (HI), and neutralization assays were also induced by immunization with the JE vaccine. JE virus-specific memory T cells were detected 60 days after immunization. These results suggest that protective immunity induced by the inactivated JE vaccine includes JE virus-specific T cells as well as antibodies with multiple biological activities.     

Formalin-inactivated,aluminum phosphate gel-adsorbed vaccine of bovine ephemeral fever virus

Summary An inactivated vaccine prepared from virus grown in hamster lung cells produced specific neutralizing antibody by intramuscular inoculation in cattle as well as in mice. Antibody response of cattle after one dose of vaccine was poor both in antibody level and seroconversion rate. However, a second dose given at intervals of 3 or more weeks induced a rapid and high-titered antibody formation in almost all animals. The antibody levels thus attained declined rather rapidly in several months, yet a booster dose given one year later provoked a rapid antibody response. Considering this pattern of antibody response together with the seasonal incidence of the disease it seems adequate that the initial immunization with two doses and the booster inoculation be made in late spring or early summer, immediately before the epizootic season. The vaccination prevented the disease in calves challenged with virulent virus. The vaccine exerted little side effects in cattle. Cows vaccinated during pregnancy gave birth to healthy calves in term. No adverse effects of vaccination on milk production was shown. All these findings indicate efficacy and safety of the vaccine, although the final evaluation rests on largescale field trials.     

Tissue culture inactivated vaccine for the prevention of Japanese encephalitis: experimental and laboratory process and control layout

A process flow diagram was elaborated for production and control of tissue culture inactivated vaccine (TCIV) against Japanese encephalitis (JE). The vaccine was prepared on the basis of the earlier patented JE virus strain (K3). Experimental laboratory JE TCIV series were obtained; their safety and high immunogenicity were tested on animals. Regulations (an instruction) for preparing and controlling JE TCIV have been worked out, which have been approved by the Academic Council of the D. I. Ivanovsky Institute of Virology.     

Immunisation with gamma globulin to murray valley encephalitis virus and with an inactivated Japanese encephalitis virus vaccine as prophylaxis against australian encephalitis: evaluation in a mouse model

In northwestern Australia, the flavivirus Murray Valley encephalitis (MVE) poses a significant health risk to infants in some aboriginal communities, particularly during each wet season. While there are too few cases to warrant the development of a vaccine against MVE, a safe, effective prophylaxis for these children is still urgently required. The use of passive transfer of human gamma globulin to MVE or immunisation with a vaccine to the closely related Japanese encephalitis (JE) virus were investigated as potential strategies. When 40 microg of IgG was purified from MVE-immune human sera and transferred to 3-week-old mice, the animals were protected from lethal IP inoculation with MVE virus while still producing a detectable immune response to the virus. Similarly, sera from adult mice infected sublethally with MVE or JE virus provided significant protection against MVE infection. However, sera from mice sublethally infected with the related Kunjin or immunised with the inactivated JE vaccine (Biken) provided no protection against MVE challenge. In fact, mice immunised passively with the latter appeared to succumb to MVE challenge more rapidly than mice that received serum from unimmunised animals, suggesting that antibody to the vaccine had accelerated the progression of disease. These preliminary trials in mice indicate that passive immunisation with human gamma globulin has the greatest potential as a strategy for MVE prophylaxis, whilst the apparent enhancement of MVE by antibodies to the JE vaccine requires further investigation, with particular reference to current vaccination programs in areas of Australia and Papua New Guinea, where both JE and MVE occur.     

Ethylenimine-inactivated rabies vaccine of tissue culture origin.

The replication of seven rabies virus strains (CVS, HEP, PV, ERA, WIRAB, CPZ and BOLIVAR) in BHK cells and the inactivation dynamics of these strains by beta-propiolactone, acetylethylenimine, and ethylenimine were studied to find the most immunogenic strain and the most economic and stable inactivating agent for the production of an inactivated tissue culture rabies vaccine for animal use. The seven strains reached the peak of virus production 3 to 5 days after inoculation of the cell culture; PV yielded the highest virus titer (10(9) plaque-forming units/ml). The infectivity of virus suspensions containing 10(7) to 10(8) plaque-forming units/0.1 ml was inactivated by beta-propiolactone in 0.5 h, acetylethylenimine in 3.0 h, and ethylenimine in 1.0 h. Most of the vaccine lots prepared with the different strains and inactivating agents passed a modified National Institutes of Health potency test. The vaccines prepared with the PV strain had consistently higher antigenic values (equal or better than four) than the other six strains. This difference was highly significant (F6,12=59.8), whereas there were no statistically significant differences among the antigenic values of the vaccine lots prepared with the three inactivating agents. Batches of lyophilized and liquid vaccine stored at 4 C maintained potency for over 1 year. Ten dogs vaccinated with a vaccine prepared with the PV strain and inactivated with ethylenimine developed a good antibody response and resisted challenge 60 days after vaccination, while seven of eight nonvaccinated controls died of rabies. This information indicates that an inactivated, stable, economic, and easy-to-prepare rabies vaccine can be produced in BHK cells by using the PV strain and ethylenimine as an inactivating agent.