Myeloproliferative virus, a cloned murine sarcoma virus with spleen focus-forming properties in adult mice.

Myeloproliferative virus, derived from Moloney sarcoma virus, causes erythroleukemia and myeloid leukemia in adult mice. This virus is also capable of fibroblast transformation in vitro. The virus consists of two separable biological entities which have been cloned. The helper virus component caused no visible changes in adult mice, whereas the defective virus induced both spleen focus formation and a large increase in erythroid precursor cells but retained the sarcoma virus property of transforming fibroblasts in vitro. Thus, myeloproliferative virus is the first murine sarcoma virus which induces erythroleukemia in adult animals.     

RNAi的抗病毒研究进展

RNA干扰(RNA interference,RNAi)是真核生物中的特异核苷酸序列产生的基因沉默现象,被认为有抑制病毒复制的功能。最近的研究表明,通过诱导RNAi可以抑制多种病毒的复制,包括人类免疫缺陷病毒Ⅰ型,乙型肝炎病毒,丙型肝炎病毒,登革热病毒,脊髓灰质炎病毒,流感病毒,口蹄疫病毒和重症急性呼吸综合征病毒等。总结了目前运用RNA干扰技术抑制病毒复制的研究进展,展望基于RNAi技术的抗病毒治疗的可能性。     

Identification of a herpesvirus isolated from cytomegalovirus-transformed human cells.

Human cells transformed by cytomegalovirus and transplanted to athymic nude mice yielded a cytopathic virus, Hershey Medical Center virus, following prolonged in vitro passage of the tumor cells. The virus is a double-enveloped herpesvirus, is sensitive to ether, and is inhibited by iododeoxyuridine. No significant antigenic relationship to herpes simplex virus was detected using herpes simplex virus-immune sera in neutralization and immunofluorescence tests, but indirect immunofluorescence tests revealed cytomegalovirus-related antigenicity. Further immunological tests revealed that Hershey Medical Center virus is antigenically indistinguishable from infectious bovine rhinotracheitis virus. Thus, it appears that Hershey Medical Center virus is infectious bovine rhinotracheitis virus, which presumably appeared in the cell culture as a contaminant from fetal calf serum.     

Glycoprotein gIII of pseudorabies virus is multifunctional.

One of the major glycoproteins of pseudorabies virus, gIII, is nonessential for growth in cell culture. Mutants defective in gIII, however, consistently yield lower titers of infectious virus (3- to 20-fold) than does wild-type virus. The interactions of gIII- mutants with their host cells were compared with those of wild-type virus in an attempt to uncover the functions of gIII. We show that gIII plays a major role in the stable adsorption of the virus to its host cell; in the absence of gIII, the rate of adsorption is reduced and adsorption is easily reversed by washing. Thus, adsorption of pseudorabies virus can be said to occur in at least the following two ways: (i) a gIII-mediated rapid adsorption or (ii) a slower and more labile adsorption that is independent of gIII. After virions have been complexed with monoclonal antibodies against gIII (but not some monoclonal antibodies against other glycoproteins), both modes of adsorption were inhibited. Glycoprotein gIII affects virus stability and virus release, as well as adsorption. The effect on virus release is marked when the virus is defective in additional functions. Thus, although we found no obvious difference in the release of virus from gIII- or wild-type virus-infected rabbit kidney cells, release of a gIII-/gI- double mutant from the cells occurred less readily than did release of a gI- mutant. The gIII-/gI- and gIII- mutants, however, adsorbed to cells at a similar rate, indicating that the effects of gIII on adsorption and virus release constitute separate functions. The Bartha vaccine strain of pseudorabies virus has a defective gIII gene and is released poorly from rabbit kidney cells. After the resident Bartha gIII gene was replaced by the gIII gene of wild-type virus, virus release was enhanced considerably. Since inactivation of gIII in wild-type pseudorabies virus did not significantly affect virus release, the Bartha strain must be defective in another function which, in conjunction with gIII, significantly affects virus release. These results indicate again that gIII affects virus release in conjunction with other functions. Also, although the Bartha strain was functionally defective in virus release, it adsorbed to cells as well as wild-type virus did, showing that the effects of gIII on virus adsorption and release constitute separate functions. We conclude that gIII is a multifunctional glycoprotein.     

Effect of Ionic Strength on the Binding of Sindbis Virus to Chick Cells

Sindbis virus can adsorb to chicken embryo fibroblasts in two different ways. "Loosely" bound virus can be washed off the cell with buffers of ionic strength 0.2 or greater, whereas "tightly" bound virus remains attached under these conditions. When Sindbis virus is adsorbed to chick cells at 4 C from a buffer of ionic strength 0.17, 40 to 50% of the adsorbed virus is loosely bound, the remainder tightly bound. Infection of chick cells by Sindbis virus has only small effects on the total amount of virus that can be bound to the cells. However, the amount of Sindbis virus that can be tightly bound declines rapidly beginning at 2 to 3 h after infection. By 7 h after infection, the amount of virus that can be tightly bound is only 10 to 20% of the amount bound to uninfected cells. The adsorption (and penetration) of virus at 37 C is most efficient at an ionic strength of 0.15 to 0.17; at this ionic strength most of the adsorbed virus is tightly bound. At higher ionic strengths the virus adsorbs poorly. At lower ionic strengths most of the virus is loosely bound. A second enveloped virus, vesicular stomatitis virus, has been studied for the purposes of comparison; its adsorption behavior differs from that of Sindbis virus.     

Ngari virus is a Bunyamwera virus reassortant that can be associated with large outbreaks of hemorrhagic fever in Africa

Two isolates of a virus of the genus Orthobunyavirus (family Bunyaviridae) were obtained from hemorrhagic fever cases during a large disease outbreak in East Africa in 1997 and 1998. Sequence analysis of regions of the three genomic RNA segments of the virus (provisionally referred to as Garissa virus) suggested that it was a genetic reassortant virus with S and L segments derived from Bunyamwera virus but an M segment from an unidentified virus of the genus Orthobunyavirus. While high genetic diversity (52%) was revealed by analysis of virus M segment nucleotide sequences obtained from 21 members of the genus Orthobunyavirus, the Garissa and Ngari virus M segments were almost identical. Surprisingly, the Ngari virus L and S segments showed high sequence identity with those of Bunyamwera virus, showing that Garissa virus is an isolate of Ngari virus, which in turn is a Bunyamwera virus reassortant. Ngari virus should be considered when investigating hemorrhagic fever outbreaks throughout sub-Saharan Africa.     

Nucleotide sequence analysis and expression from recombinant vectors demonstrate that the attachment protein G of bovine respiratory syncytial virus is distinct from that of human respiratory syncytial virus

Bovine respiratory syncytial (BRS) virus causes a severe lower respiratory tract disease in calves similar to the disease in children caused by human respiratory syncytial (HRS) virus. While there is antigenic cross-reactivity among the other major viral structural proteins, the major glycoprotein, G, of BRS virus and that of HRS virus are antigenically distinct. The G glycoprotein has been implicated as the attachment protein for HRS virus. We have carried out a molecular comparison of the glycoprotein G of BRS virus with the HRS virus counterparts. cDNA clones corresponding to the BRS virus G glycoprotein mRNA were isolated and analyzed by dideoxynucleotide sequencing. The BRS virus G mRNA contained 838 nucleotides exclusive of poly(A) and had a major open reading frame coding for a polypeptide of 257 amino acid residues. The deduced amino acid sequence of the BRS virus G polypeptide showed only 29 to 30% amino acid identity with the G protein of either the subgroup A or B HRS virus. However, despite this low level of identity, there were strong similarities in the predicted hydropathy profiles of the BRS virus and HRS virus G proteins. A cDNA molecule containing the complete BRS virus G major open reading frame was inserted into the thymidine kinase gene of vaccinia virus by homologous recombination, and a recombinant virus containing the BRS virus G protein gene was isolated. This recombinant virus expressed the BRS virus G protein, as demonstrated by Western immunoblot analysis and immunofluorescence of infected cells. The BRS virus G protein expressed from the recombinant vector was transported to and expressed on the surface of infected cells. Antisera to the BRS virus G protein made by using the recombinant vector to immunize animals recognized the BRS virus attachment protein but not the HRS virus G protein and vice versa, confirming the lack of antigenic cross-reactivity between the BRS and HRS virus attachment proteins. On the basis of the data presented here, we conclude that BRS virus should be classified within the genus Pneumovirus in a group separate from HRS virus and that it is no more closely related to HRS virus subgroup A than it is to HRS virus subgroup B.     

Role of Interferon in the Propagation of MM Virus in L Cells

MM virus propagated in mouse brain replicates to low titers in L cells without production of cytopathic effect (CPE). After growing the virus in BHK-21 cells, however, the virus replicates to high titers in L cells with complete CPE. It was found that suspensions of MM virus propagated in L cells directly from the mouse brain contained much more interferon than did suspensions of virus which had first been grown in BHK-21 cells. Mouse brain suspensions of the virus were also found to contain high interferon titers. Treatment of L cells with actinomycin D before infection with mouse brain-grown virus resulted in full virus replication with CPE. BHK-21 cell-grown virus diluted in L cell interferon behaved like mouse brain-grown virus in L cells. It is concluded that the presence of interferon in the inoculum is largely responsible for the suppression of MM virus replication in L cells.     

Nonrelatedness between symptoms and cucumber virus 4 content in different cucumber cultivars

The virus replication capacity (determined by density gradient centrifugation and spectrophotometric virus assay) and severity of symptoms were compared in ten cucumber(Cucumis sativus L.) cultivars infected by cucumber virus 4. Except the expected resistant susceptible and symptomless virus carrier plants a new paradoxial group of cultivars was found. This group is characterized by low virus reproduction rate but simultaneously associated with severe symptoms and is responsible for difficulties in the virus transmission and purification. It is concluded that the critical evaluation of the resistance against viruses of TMV-group must include symptomatic and quantitative virus assays, as there need not be a direct relationship between virus symptoms and the virus increase.     

Nucleotide sequence of Chinese rape mosaic virus (oilseed rape mosaic virus), a crucifer tobamovirus infectious on Arabidopsis thaliana

The complete nucleotide sequence of Chinese rape mosaic virus has been determined. The virus is a member of the tobamovirus genus of plant virus and is able to infect Arabidopsis thaliana (L.) Heynh systemically. The analysis of the sequence shows a gene array that seems to be characteristic of crucifer tobamoviruses and which is slightly different from the one most frequently found in tobamoviruses. Based on gene organization and on comparisons of sequence homologies between members of the tobamoviruses, a clustering of crucifer tobamoviruses is proposed that groups the presently known crucifer tobamovirus into two viruses with two strains each. A name change of Chinese rape mosaic virus to oilseed rape mosaic virus is proposed.Abbreviations 2-ME 2-mercaptoethanol - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate - UTR untranslated region - MP movement protein - CP capsid protein - CRMV Chinese rape mosaic virus - TVCV turnip vein clearing virus - PaMMV paprika mild mottle virus - PMMV-I pepper mild mottle virus (Italian isolate) - PMMV-S pepper mild mottle virus (Spanish isolate) - ToMV tomato mosaic virus - TMV tobacco mosaic virus - TMGMV tobacco mild green mosaic virus - ORSV odontoglossum ringspot virus - SHMV sunn hemp mosaic virus - CGMMV cucumber green mottle mosaic virus - ORMV oilseed rape mosaic virus     

H7N9禽流感病毒与其他流感病毒对雪貂致病性与传播力的比较

目的针对2013年3月中国爆发的人感染H7N9禽流感病毒,在雪貂体内进行致病性及传播力的研究,并与甲型H1N1流感病毒、H5N1禽流感病毒进行比较。方法对新发H7N9毒株、甲型H1N1流感病毒、H5N1禽流感病毒感染雪貂后的临床症状、体征,呼吸道排毒情况,组织病理学变化等进行评价和比较,并对H7N9毒株在雪貂群体中的传播力进行研究。结果雪貂模型的临床症状、死亡率、病毒传播以及组织病理学分析显示:H7N9病毒的致病性低于H5N1,与2009年起源于北美的甲型H1N1流感病毒相当。新发H7N9禽流感病毒可以在雪貂的呼吸道、心脏、肝脏以及嗅球进行复制。值得注意的是H7N9禽流感可以通过飞沫在雪貂间进行低水平的传播,并且在传播过程中,病毒基因组内有多个位点的氨基酸发生了替换。结论 H7N9禽流感病毒对雪貂的致病性较H5N1禽流感病毒低,与甲型H1N1流感病毒对雪貂的致病性相当,H7N9禽流感病毒可在雪貂间进行传播。     

Integrin β1 mediates vaccinia virus entry through activation of PI3K/Akt signaling

Vaccinia virus has a broad range of infectivity in many cell lines and animals. Although it is known that the vaccinia mature virus binds to cell surface glycosaminoglycans and extracellular matrix proteins, whether additional cellular receptors are required for virus entry remains unclear. Our previous studies showed that the vaccinia mature virus enters through lipid rafts, suggesting the involvement of raft-associated cellular proteins. Here we demonstrate that one lipid raft-associated protein, integrin β1, is important for vaccinia mature virus entry into HeLa cells. Vaccinia virus associates with integrin β1 in lipid rafts on the cell surface, and the knockdown of integrin β1 in HeLa cells reduces vaccinia mature virus entry. Additionally, vaccinia mature virus infection is reduced in a mouse cell line, GD25, that is deficient in integrin β1 expression. Vaccinia mature virus infection triggers the activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling, and the treatment of cells with inhibitors to block P13K activation reduces virus entry in an integrin β1-dependent manner, suggesting that integrin β1-mediates PI3K/Akt activation induced by vaccinia virus and that this signaling pathway is essential for virus endocytosis. The inhibition of integrin β1-mediated cell adhesion results in a reduction of vaccinia virus entry and the disruption of focal adhesion and PI3K/Akt activation. In summary, our results show that the binding of vaccinia mature virus to cells mimics the outside-in activation process of integrin functions to facilitate vaccinia virus entry into HeLa cells.     

Vaccinia virus envelope H3L protein binds to cell surface heparan sulfate and is important for intracellular mature virion morphogenesis and virus infection in vitro and in vivo

An immunodominant antigen, p35, is expressed on the envelope of intracellular mature virions (IMV) of vaccinia virus. p35 is encoded by the viral late gene H3L, but its role in the virus life cycle is not known. This report demonstrates that soluble H3L protein binds to heparan sulfate on the cell surface and competes with the binding of vaccinia virus, indicating a role for H3L protein in IMV adsorption to mammalian cells. A mutant virus defective in expression of H3L (H3L(-)) was constructed; the mutant virus has a small plaque phenotype and 10-fold lower IMV and extracellular enveloped virion titers than the wild-type virus. Virion morphogenesis is severely blocked and intermediate viral structures such as viral factories and crescents accumulate in cells infected with the H3L(-) mutant virus. IMV from the H3L(-) mutant virus are somewhat altered and less infectious than wild-type virions. However, cells infected by the mutant virus form multinucleated syncytia after low pH treatment, suggesting that H3L protein is not required for cell fusion. Mice inoculated intranasally with wild-type virus show high mortality and severe weight loss, whereas mice infected with H3L(-) mutant virus survive and recover faster, indicating that inactivation of the H3L gene attenuates virus virulence in vivo. In summary, these data indicate that H3L protein mediates vaccinia virus adsorption to cell surface heparan sulfate and is important for vaccinia virus infection in vitro and in vivo. In addition, H3L protein plays a role in virion assembly.     

Are Animal Tumor Viruses Always Virus-Like?

Three tumors initiated by well characterized viruses, but in which virus is not detectable by ordinary virological techniques, are discussed. The question of the possible state of the virus within these seemingly non-infectious tumors is considered, largely from the standpoint of findings with the rabbit papilloma virus. This agent in its natural host, the cottontail rabbit, is infective, can be seen as virus bodies with the electron microscope, and can be visualized with fluorescent antibody only in the upper keratinizing cells of individual papillomas. At the growing bases of such papillomas, where neoplasia is in active progress, no infective virus is demonstrable and viral bodies cannot be visualized by either the electron microscope or fluorescent antibody. A hypothesis is presented that rabbit papilloma virus exists in cottontail papillomas in two forms—one, the complete mature virus, composed of nucleic acid and protein, and the other, immature virus, composed of naked viral nucleic acid without its protein coating. The function of the mature papilloma virus is to initiate tumor formation,—that of the immature virus, to maintain neoplasia. In the non-infective domestic rabbit papilloma, the viral nucleic acid and protein fail to combine to form mature infective virus and, as in the cottontail papilloma, neoplasia is maintained by the activity of the viral nucleic acid alone.     

综述: 病毒与宿主细胞的糖基化修饰及相关功能

病毒的复制和对宿主的入侵与自身结构蛋白的糖基化修饰密切相关．对于宿主而言,在病毒感染宿主和宿主抗病毒的过程中,宿主的糖基化过程一方面可抑制病毒的复制和入侵,另一方面可促进病毒对宿主的感染,抑制宿主糖苷酶可抑制病毒的复制．从病毒方面来看,由于病毒自身缺乏糖基化修饰系统,病毒的糖基化过程是借宿主细胞内的合成系统对自身进行糖基化修饰．病毒的糖基化过程对病毒蛋白的折叠与稳定、病毒的感染和入侵、参与识别宿主细胞受体和参与病毒的免疫逃逸等过程起着重要的作用．随着糖基化研究技术的发展,以糖基化为基础的功能应用也越来越深入：如新型病毒疫苗和新型抗病毒药物的研制,以糖蛋白质组学研究为基础的质谱技术和生物信息学方法的发展,以及利用糖基化对病毒性疾病的诊断和治疗等,这些均为糖基化深入研究发展奠定了基础．本文就病毒与宿主细胞糖基化过程、相关功能以及研究应用等进展作一综述．     

Transfection of RNA from Organ Samples of Infected Animals Represents a Highly Sensitive Method for Virus Detection and Recovery of Classical Swine Fever Virus

Translation and replication of positive stranded RNA viruses are directly initiated in the cellular cytoplasm after uncoating of the viral genome. Accordingly, infectious virus can be generated by transfection of RNA genomes into susceptible cells. In the present study, efficiency of conventional virus isolation after inoculation of cells with infectious sample material was compared to virus recovery after transfection of total RNA derived from organ samples of pigs infected with Classical swine fever virus (CSFV). Compared to the conventional method of virus isolation applied in three different porcine cell lines used in routine diagnosis of CSF, RNA transfection showed a similar efficiency for virus rescue. For two samples, recovery of infectious virus was only possible by RNA transfection, but not by the classical approach of virus isolation. Therefore, RNA transfection represents a valuable alternative to conventional virus isolation in particular when virus isolation is not possible, sample material is not suitable for virus isolation or when infectious material is not available. To estimate the potential risk of RNA prepared from sample material for infection of pigs, five domestic pigs were oronasally inoculated with RNA that was tested positive for virus rescue after RNA transfection. This exposure did not result in viral infection or clinical disease of the animals. In consequence, shipment of CSFV RNA can be regarded as a safe alternative to transportation of infectious virus and thereby facilitates the exchange of virus isolates among authorized laboratories with appropriate containment facilities.     

狂犬病毒aG株在Vero细胞上传代适应研究

为研制有效、安全和稳定的Vero细胞狂犬病疫苗提供实验室基础资料。采用狂犬病固定毒4aG株在Vero细胞上进行传代适应,同时对该毒株在Vero细胞上的增殖条件,病毒液的回收方法进行研究。结果显示,狂犬病固定毒4aG株在Vero细胞上多次传代后获得一株Vero细胞适应株(4aG-V株),该毒株的毒力可达8.50 logLD50/ml,且具有很好的抗原性及免疫原性。结果表明,感染Vero细胞最适种毒比例为1∶103,病毒滴度随着时间的延长而增强到第12天后逐渐减弱,且采用低温冻融破碎法回收的病毒液滴度优于直接收液法。4aG-V株在Vero细胞上维持时间长,可连续收液,有望其生产高滴度的狂犬病毒液。     

Distinct domains of the influenza a virus M2 protein cytoplasmic tail mediate binding to the M1 protein and facilitate infectious virus production

The cytoplasmic tail of the influenza A virus M2 protein is highly conserved among influenza A virus isolates. The cytoplasmic tail appears to be dispensable with respect to the ion channel activity associated with the protein but important for virus morphology and the production of infectious virus particles. Using reverse genetics and transcomplementation assays, we demonstrate that the M2 protein cytoplasmic tail is a crucial mediator of infectious virus production. Truncations of the M2 cytoplasmic tail result in a drastic decrease in infectious virus titers, a reduction in the amount of packaged viral RNA, a decrease in budding events, and a reduction in budding efficiency. The M1 protein binds to the M2 cytoplasmic tail, but the M1 binding site is distinct from the sequences that affect infectious virus particle formation. Influenza A virus strains A/Udorn/72 and A/WSN/33 differ in their requirements for M2 cytoplasmic tail sequences, and this requirement maps to the M1 protein. We conclude that the M2 protein is required for the formation of infectious virus particles, implicating the protein as important for influenza A virus assembly in addition to its well-documented role during virus entry and uncoating.