A型流感病毒持续感染细胞系来源的IVpi-189病毒生物学性状的初步研究

为明确E61-24-P15 A型重组流感病毒的第189代传代子病毒(IVpi-189)是否具备流感病毒温度敏感减毒活疫苗候选株的特点,将IVpi-189病毒感染MDCK细胞,并于不同培养温度条件下培养,观察其致细胞病变效应,病毒合成、释放情况,以及不同温度条件下病毒存活时间。结果显示32℃培养温度下,IVpi-189病毒具有等同于亲代野生病毒株的诱导细胞病变能力,而当培养温度上调至38℃,IVpi-189病毒致细胞病变效果出现缓慢且程度明显减轻。空斑形成单位实验发现IVpi-189病毒在38℃培养条件下增殖能力明显下降,其原因与病毒灭活速度及子病毒释放无关,但与感染细胞病毒合成能力下降有关。上述实验结果初步证实流感病毒持续感染细胞系来源的IVpi-189病毒具有温度敏感减毒活疫苗的生物学特性,在许可培养温度条件下具有良好的增殖能力,而在非许可培养温度下,病毒增殖活性受到明显抑制。本研究为流感病毒减毒活疫苗的开发研制提供实验佐证。     

Batai and Ngari viruses: M segment reassortment and association with severe febrile disease outbreaks in East Africa

Ngari virus is an orthobunyavirus recently recognized as a reassortant between Bunyamwera virus and an as yet unidentified M segment donor. Analysis of M segment sequences of Batai and Ilesha viruses revealed 95% deduced amino acid identity between Batai virus and Ngari virus. These findings suggest Batai virus as the donor of Ngari virus M segment sequence. Analysis of Batai virus-related African isolates identified UgMP-6830, isolated from mosquitoes in Uganda, as an isolate of Batai virus. KV-141, isolated during a febrile disease outbreak in Sudan, was identified as another isolate of Ngari virus, emphasizing a role of this reassortant virus in severe human illness throughout East Africa.     

Yaba-like disease virus: an alternative replicating poxvirus vector for cancer gene therapy

Vaccinia virus is being investigated as a replicating vector for tumor-directed gene therapy. However, the majority of cancer patients have preformed immunologic reactivity against vaccinia virus, as a result of smallpox vaccination, which may limit its use as a vector. The Yaba-like disease (YLD) virus was investigated here as an alternative, replicating poxvirus for cancer gene therapy. We have demonstrated that the YLD virus does not cross-react with vaccinia virus antibodies, and it replicates efficiently in human tumor cells. YLD virus can be expanded and purified to high titer in CV-1 cells under conditions utilized for vaccinia virus. The YLD virus RNA polymerase was able to express genes regulated by a synthetic promoter designed for use in orthopoxviruses. We sequenced the YLD virus TK gene and created a shuttle plasmid, which allowed the recombination of the green fluorescent protein (GFP) gene into the YLD virus. In a murine model of ovarian cancer, up to 38% of cells in the tumor expressed the GFP transgene 12 days after intraperitoneal virus delivery. YLD virus has favorable characteristics as a vector for cancer gene therapy, and this potential should be explored further.     

A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus

CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus).     

流感病毒在无动物细胞的细菌共生培养基里的培养研究

流行性感冒病毒流行时,在一些病人的上呼吸道里,常可以见到流感病毒和一些细菌(如甲型链球菌、葡萄球菌、流感杆菌等)共存,以往的实验也证明它们的关系比较密切。此试验发现:流感病毒在仅有甲型链球菌生长的培养基中有自我复制的现象,共生培养的病毒经过一系列的病毒学实验方法,证明确实是流感病毒。流感病毒在共生培养基里与它在鸡胚里的生长曲线基本相同,共生培养基里,只要细菌生长,流感病毒就能生长,在不同的温度(15℃、22℃、37℃)条件下,流感病毒和细菌的生长趋势出现密切的平行关系。甲型链球菌以外的其它细菌,如乙型链球菌、肠球菌、葡萄球菌、酵母菌、革兰氏阳性杆菌等菌株也能与流感病毒共生,只有个别的菌株不共生。共生培养的流感病毒能在较低的温度(22℃)环境下保持它的活性达2个月之久;在8℃的环境里,流感病毒也能共生繁殖,经过长期低温共生培养,其致病性减弱;流感病毒和其它两种病毒能在同一共生培养基里共生繁殖。实验研究中还简要讨论了共生的机理和实际应用等问题。     

Chikungunya Virus Vaccine Prepared by Tween-Ether Extraction

Chikungunya virus vaccines prepared by Tween 80 and ether inactivation of virus grown in green monkey kidney cell cultures were shown to be as immunogenic as comparable Formalin-inactivated vaccines. Both types of vaccine stimulated hemagglutination-inhibiting, complement-fixing, and neutralizing antibody and afforded protection to mice against a live virus challenge. It was shown after Tween-ether treatment of chikungunya virus that the infectivity of the virus was lost and the hemagglutinin titer was increased. By characterization of the resultant hemagglutinin by sucrose and cesium chloride density gradient centrifugation, it was found that the extracted particle was smaller in size and greater in density than the parent virus particle. Removal of lipid may account for the alterations in the physical characteristics of the infectious virus particle. Conditions for treatment of chikungunya virus with Tween and ether were found that preserved high titers of hemagglutinin as well as the immunogenicity of the virus preparations.     

基于痘苗病毒载体的恶性肿瘤基因治疗研究进展

基因治疗一直是肿瘤生物治疗的重要策略,而以溶瘤痘苗病毒为载体的肿瘤治疗近年来受到较多关注。该文总结了目前用于恶性肿瘤治疗的痘苗病毒和基于痘苗病毒载体的基因治疗研究进展及其在各个领域的成果。     

Glycoprotein K Specified by Herpes Simplex Virus Type 1 Is Expressed on Virions as a Golgi Complex-Dependent Glycosylated Species and Functions in Virion Entry

To facilitate detection of glycoprotein K (gK) specified by herpes simplex virus, a 12-amino-acid epitope tag was inserted within gK domain III. Recombinant virus gKprotC-DIII, expressing the tagged gK, was isolated. This virus formed wild-type plaques and replicated as efficiently as the wild-type KOS virus in Vero cells. Anti-protein C MAb detected high-mannose and Golgi complex-dependent glycosylated gK within cells as well as on purified virions. The gK-null virus DeltagK (gK(-/-)) entered Vero cells substantially more slowly than the wild-type KOS (gK(+/+)), while DeltagK virus grown in complementing VK302 cells (gK(-/+)) entered with entry kinetics similar to those of the KOS virus.     

Comparison of 6/94 Virus and Sendai Virus RNA by RNA-RNA Hybridization

The genomic RNA of 6/94 virus, an agent isolated from the brains of multiple sclerosis patients, was studied for sequence homology by RNA-RNA hybridization with closely related Sendai virus and another paramyxovirus virus, Newcastle disease virus. It was found that the genomic RNA of 6/94 virus hybridizes equally as well to the virus-specific 18S RNA found in Sendai-infected cells as that of Sendai virus.     

Bovine Diarrhea Virus

Marked cytopathic changes were induced by challenge with Newcastle disease (ND) virus in bovine testicle or kidney cell cultures which were previously infected with non-cytopathogenic strains of bovine diarrhea (BD) virus. No cytopathic changes were induced by ND virus in similar cells not infected with BD virus. The development of cytopathic effect was shown to be associated with enhancement of ND virus replication. This exalting effect of BD virus appears to be dependent on infectivity, since the effect was inhibited when infection of the cells with BD virus was blocked by specific antiserum. Various factors involved in the phenomenon were investigated and an in vitro method (END) for the assay of BD virus and its antibodies was developed. The use of this method eliminates the difficulties in recognizing non-cytopathogenic strains of BD virus which hampered systematic investigations of the nature and behavior of BD virus as well as of the natural history and pathogenesis of the infection in cattle.     

Salivary Excretion of Coxsackie B-1 Virus in Rabbits

Coxsackie B-1 virus was injected into the ear vein of albino doe rabbits. Saliva and blood samples were taken before the injection of virus and at specific times thereafter. Virus was recovered in the whole saliva when the blood titer was approximately 10(4) TCID(50) per 0.1 ml or greater. The virus could be detected in the saliva as early as 2 min after the initiation of the viremia. The recovered virus was shown to be the same as the injected virus by serological identification of the recovered virus with neutralizing antibody for Coxsackie B-1 virus. These results suggest that virus may be transmitted to other animals in the saliva of animals who are in the viremic phase of infection without infection of the oropharyngeal tissues.     

Transcription of 70S RNA by DNA polymerases from mammalian RNA viruses.

DNA polymerases purified by the same procedure from four mammalian RNA viruses, simian sarcoma virus type 1, gibbon ape lymphoma virus, Mason-Pfizer monkey virus, and Rauscher murine leukemia virus are capable of transcribing heteropolymeric regions of viral 70S RNA without any other primer. In this reconstituted system the enzymes from simian sarcoma virus type 1, Mason-Pfizer monkey virus, and Rauscher murine leukemia virus transcribe viral 70S RNA almost as efficiently as the DNA polymerase from the avian myeloblastosis virus, but gibbon ape lymphoma virus DNA polymerase is approximately three-to fivefold less efficient. Although there is a substantial difference among the sizes of these DNA polymerases (160,000 daltons for the avian myeloblastosis virus enzyme, 110,000 daltons for the Mason-Pfizer monkey virus enzyme, and 70,000 daltons for the mammalian type C viral polymerases), the ability to transcribe viral 70S RNA is a characteristic common to these enzymes.     

Rabies virus infection of cultured rat sensory neurons.

The axonal transport of rabies virus (challenge virus strain of fixed virus) was studied in differentiated rat embryonic dorsal root ganglion cells. In addition, we observed the attachment of rabies virus to neuronal extensions and virus production by infected neurons. A compartmentalized cell culture system was used, allowing infection and manipulation of neuronal extensions without exposing the neural soma to the virus. The cultures consisted of 60% large neuronal cells whose extensions exhibited neurofilament structures. Rabies virus demonstrated high binding affinity to unmyelinated neurites, as suggested by assays of virus adsorption and immunofluorescence studies. The rate of axoplasmic transport of virus was 12 to 24 mm/day, including the time required for internalization of the virus into neurites. The virus transport could be blocked by cytochalasin B, vinblastine, and colchicine, none of which negatively affected the production of virus in cells once the infection was established. It was concluded that, for the retrograde transfer of rabies virus by neurites from the periphery to the neuronal soma, the integrity of tubulin- and actin-containing structures is essential. The rat sensory neurons were characterized as permissive, moderately susceptible, but low producers of rabies virus. These neurons were capable of harboring rabies virus for long periods of time and able to release virus into the culture medium without showing any morphological alterations. The involvement of sensory neurons in rabies virus pathogenesis, both in viral transport and as a site for persistent viral infection, is discussed.     

核型多角体病毒与侧沟茧蜂对斜纹夜蛾幼虫的协同作用

研究了斜纹夜蛾幼虫体内的斜纹夜蛾侧沟茧蜂存活率、发育历期、寄主感染病毒时间、病毒浓度之间的关系,并测定了斜纹夜蛾侧沟茧蜂的传毒效率．结果表明,病毒对寄主体内寄生蜂历期无明显影响,寄生在幼虫体内的寄生蜂能在寄主病死前完成发育,存活比例因寄主感染病毒的时间和浓度而异．斜纹夜蛾被寄生后接种病毒(SINPV),距离寄生时间越长,饲毒浓度越低,寄生蜂完成发育的比例越大,但饲毒时间是主要影响因素．从感病幼虫体内发育成的侧沟茧蜂或曾经在感病寄主上产过卵的寄生蜂,以及通过人工方式使产卵器被病毒污染的寄生蜂,均能携带一定数量的病毒．通过产卵活动,侧沟茧蜂成蜂能在寄主幼虫个体间传递病毒．当寄生蜂在感病的寄主幼虫上产卵带毒后,平均可传递病毒给2．14头幼虫;发育于感病幼虫体内的寄生蜂,平均可传递病毒给2．45头幼虫．通过用病毒液浸茧或用混有病毒的蜂蜜饲喂成蜂等方式使产卵器污染病毒的寄生蜂,传毒效率随饲毒浓度增加而提高,平均可传递病毒1．45头和0．94头幼虫     

Growth characteristics of bovine herpesvirus 1 (infectious bovine rhinotracheitis) in human diploid cell strain WI-38.

IBR virus was found to replicate in WI-38 cells. At a high input multiplicity the virus yield was comparable to that obtained in bovine cells, but comparable degree of CPE took longer to achieve. At a low input multiplicity of IBR virus, such as may be encountered in virus contaminated bovine serum, virus yield was only about 1% of that in bovine cells, with 50% of the cells showing CPE, followed by cell regrowth. Infectious virus was not recoverable from the regrown cells by 5 weeks after initial infection, and these regrown cells were susceptible to reinfection with IBR virus. Aging of WI-38 cells in cultures for as little as 1 week reduced IBR virus yield to 90% less than the yield from the same lot of cells inoculated 7 days earlier.     

AN ATTEMPT TO INHIBIT THE MULTIPLICATION OF TOBACCO MOSAIC VIRUS IN TISSUE CULTURE BY ITS ANTISERUM

Extracts from tobacco tissue cultures infected with tobacco mosaic virus grown in medium containing antiserum to the virus had only one-half to one-sixteenth as much virus as extracts from tissues grown in medium without the antiserum. When tissues grown with antiserum were thoroughly washed before they were extracted, the extracts contained as much virus as extracts of tissues grown without antiserum. The antiserum did not affect virus multiplication, but antibodies in the tissues may have precipitated virus either in the cells or when the tissues were macerated.     

针对多种强致病性病毒的基因芯片检测方法的建立

为了制备灵敏的可检测多种烈性病毒性病原体的基因芯片,本研究设计了针对21种烈性病毒性病原体的基因芯片检测探针,每种5条,长50 bp.并以甲病毒属的基孔肯亚病毒和黄病毒属的黄热病毒细胞培养物为检测模型,摸索了合适的病毒基因处理与扩增方法.将提取的病毒RNA先用DNase Ⅰ处理,以去除掉其中的DNA分子,然后利用病毒属特异性引物进行反转录,以引导病毒基因组的合成,从而尽可能地减少宿主细胞基因成分的干扰.进行随机PCR扩增后将扩增产物与基因芯片进行杂交,分别出现了4条基孔肯亚病毒探针信号和5条黄热病毒的探针信号,说明所设计的检测探针具有较好的特异性,可用于这2种病毒的特异性检测.这种病毒基因样品的处理和扩增方法也为此基因芯片的临床应用奠定了基础.     

Top 10 plant viruses in molecular plant pathology

Many scientists, if not all, feel that their particular plant virus should appear in any list of the most important plant viruses. However, to our knowledge, no such list exists. The aim of this review was to survey all plant virologists with an association with Molecular Plant Pathology and ask them to nominate which plant viruses they would place in a 'Top 10' based on scientific/economic importance. The survey generated more than 250 votes from the international community, and allowed the generation of a Top 10 plant virus list for Molecular Plant Pathology. The Top 10 list includes, in rank order, (1) Tobacco mosaic virus, (2) Tomato spotted wilt virus, (3) Tomato yellow leaf curl virus, (4) Cucumber mosaic virus, (5) Potato virus Y, (6) Cauliflower mosaic virus, (7) African cassava mosaic virus, (8) Plum pox virus, (9) Brome mosaic virus and (10) Potato virus X, with honourable mentions for viruses just missing out on the Top 10, including Citrus tristeza virus, Barley yellow dwarf virus, Potato leafroll virus and Tomato bushy stunt virus. This review article presents a short review on each virus of the Top 10 list and its importance, with the intent of initiating discussion and debate amongst the plant virology community, as well as laying down a benchmark, as it will be interesting to see in future years how perceptions change and which viruses enter and leave the Top 10.