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.     

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。     

Isolation of a new rhabdovirus in Australia related to Tibrogargan virus

A virus isolated from the blood of a healthy steer and designated DPP53 was shown to have rhabdovirus morphology. Although DPP53 virus was antigenically related to Tibrogargan virus by reciprocal immunofluorescence and neutralization tests, the viruses were distinguishable by neutralization tests. DPP53 virus contained RNA and was sensitive to both ether and chloroform. The geographical distribution of neutralizing antibody to DPP53 virus in Australian cattle corresponded to the distribution of Culicoides brevitarsis indicating that this virus may be arthropod-borne with this midge as a possible vector. Antibody to DPP53 virus was detected in serum from cattle, buffalo, dogs and one horse, but not in serum from deer, pigs, humans or wallabies. Highest virus titres were obtained by growth in Vero and BHK21 cell cultures, but the virus could also be grown in Aedes albopictus cell cultures. Higher virus titres were obtained when the multiplicity of infection was low. The name advanced for DPP53 virus is 'Coastal Plains' virus.     

Nucleocapsid-glycoprotein interactions required for assembly of alphaviruses.

We have studied interactions between nucleocapsids and glycoproteins required for budding of alphaviruses, using Ross River virus-Sindbis virus chimeras in which the nucleocapsid protein is derived from one virus and the envelope glycoproteins are derived from the second virus. A virus containing the Ross River virus genome in which the capsid protein had been replaced with that from Sindbis virus was almost nonviable. Nucleocapsids formed in normal numbers in the infected cell, but very little virus was released from the cell. There are 11 amino acid differences between Ross River virus and Sindbis virus in their 33-residue E2 cytoplasmic domains. Site-specific mutagenesis was used to change 9 of these 11 amino acids in the chimera from the Ross River virus to the Sindbis virus sequence in an attempt to adapt the E2 of the chimera to the nucleocapsid. The resulting mutant chimera grew 4 orders of magnitude better than the parental chimeric virus. This finding provides direct evidence for a sequence-specific interaction between the nucleocapsid and the E2 cytoplasmic domain during virus budding. The mutated chimeric virus readily gave rise to large-plaque variants that grew almost as well as Ross River virus, suggesting that additional single amino acid substitutions in the structural proteins can further enhance the interactions between the disparate capsid and the glycoproteins. Unexpectedly, change of E2 residue 394 from lysine (Ross River virus) to glutamic acid (Sindbis virus) was deleterious for the chimera, suggesting that in addition to its role in nucleocapsid-E2 interactions, the N-terminal part of the E2 cytoplasmic domain may be involved in glycoprotein-glycoprotein interactions required to assemble the glycoprotein spikes. The reciprocal chimera, Sindbis virus containing the Ross River virus capsid, also grew poorly. Suppressor mutations arose readily in this chimera, producing a virus that grew moderately well and that formed larger plaques.     

Characterization of bovine respiratory syncytial virus proteins and mRNAs and generation of cDNA clones to the viral mRNAs.

We have characterized the proteins and mRNAs of bovine respiratory syncytial (BRS) virus strain 391-2 and constructed cDNA clones corresponding to 9 of the 10 BRS virus mRNAs. The proteins of BRS virus-infected cells were compared with the proteins from human respiratory syncytial (HRS) virus-infected cells. Nine proteins specific to BRS virus-infected cells, corresponding to nine HRS virus proteins, were identified. Only a BRS virus polymerase protein remains to be identified. The BRS virus G glycoprotein showed major antigenic differences from the HRS virus G glycoprotein by immunoprecipitation and Western (immuno-) blot analysis, whereas the BRS virus F, N, M, and P proteins showed antigenic cross-reactivity with their HRS virus counterparts. Analysis of RNAs from BRS virus-infected cells showed virus-specific RNAs which had electrophoretic mobilities similar to those of mRNAs of HRS virus but which hybridized poorly or not at all with HRS virus-specific probes in Northern (RNA) blot analysis. To analyze the BRS virus RNAs further, cDNA clones to the BRS virus mRNAs were generated. Nine separate groups of clones were identified and shown to correspond to nine BRS virus mRNAs by Northern blot analysis. A 10th BRS virus large mRNA was identified by analogy with the HRS virus polymerase mRNA. These data show that like HRS virus, BRS virus has 10 genes coding for 10 mRNAs.     

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     

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.     

Discrimination by gel-diffusion serology of digitaria striate, maize sterile stunt and other rhabdoviruses of Poaceae

Eight rhabdoviruses from grass and cereal hosts and their antisera were used to examine virus relationships by gel-diffusion serology. Nucleocapsid (Nc) preparations from digitaria striate virus (DSV) and maize sterile stunt virus (MSSV) both contained a major protein of c. 52 OOO daltons, and antisera prepared to these readily discriminated related planthopper-transmitted rhabdoviruses. MSSV showed a moderately close relationship to barley yellow striate mosaic virus (BYSMV) when an antiserum prepared to whole virus was used, but the Nc antiserum showed clearer discrimination. Worthern cereal mosaic virus and DSV showed a distant relationship to BYSMV and MSSV. There was no serological relationship between any of these viruses and cereal chlorotic mottle virus, cynodon chlorotic streak virus, festuca leaf streak virus or maize mosaic virus.     

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.     

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).     

Sexual transmission of hepatitis C virus and its relation with hepatitis B virus and HIV.

OBJECTIVE--To determine the extent of transmission of hepatitis C virus in sexual partners of intravenous drug misusers and to examine the relation between the prevalences of HIV, hepatitis B virus, and hepatitis C virus infections in homosexual men and intravenous drug misusers and their sexual partners. DESIGN--Serum samples collected between 1984 and 1988 were tested for hepatitis B virus markers and antibodies against hepatitis C virus by enzyme linked immunosorbent assay (ELISA) and for HIV antibody by enzyme immune analysis and western blotting. SETTING--Large referral university hospital with an external AIDS clinic in the metropolitan area of Barcelona, Spain. SUBJECTS--243 Intravenous drug misusers, 143 of their regular heterosexual partners, and 105 homosexual men. MAIN OUTCOME MEASURES--Prevalences of hepatitis C virus, hepatitis B virus, and HIV infections. RESULTS--In all, 178 of the 243 (73%) intravenous drug misusers, 16 out of 143 (11%) of their partners, and 17 of the 105 (16%) homosexual men had antibodies against hepatitis C virus. The presence of hepatitis C virus infection was unrelated to sex, age, the presence of HIV or hepatitis B virus infections, or the Centers for Disease Control stage of HIV. In sexual partners of intravenous drug misusers there were strong correlations between the presence of hepatitis C virus infection and that of HIV (p = 0.001) and hepatitis B virus (p = 0.013) infections. CONCLUSIONS--Intravenous drug misusers have a high risk of acquiring hepatitis C virus, hepatitis B virus, and HIV infections, but the presence of hepatitis C virus infection seems to be unrelated to the presence of the other two viruses. Homosexual men have a high prevalence of HIV and hepatitis B virus infections with a low prevalence of hepatitis C virus infection, the presence of which is not related to that of the other two infections. Conversely, heterosexual partners of intravenous drug misusers have low prevalences of the three virus infections, but the presence of hepatitis C virus infection correlates significantly with the presence of HIV and hepatitis B infections. The rate of sexual transmission of hepatitis C virus seems to be low, even in partners of people known to be seropositive for this virus.     

Detection of multiple viruses in queens of the honey bee Apis mellifera L

Individual honey bee Apis mellifera L. queens were examined for the presence of six honey bee viruses including acute bee paralysis virus, chronic bee paralysis virus, black queen cell virus, deformed wing virus, Kashmir bee virus, and sacbrood virus. All viruses, except ABPV, were detected in the samples. Among queens examined for virus infections, 93% had multiple virus infections. The detection of viruses in queens raises the possibility of a vertical transmission pathway wherein infected queens can pass virus through their eggs to their offspring.     

Virus of Pekin ducks with structural and biological relatedness to human hepatitis B virus.

A virus found in the sera of Pekin ducks appears to be a new member of the human hepatitis B-like family of viruses. This virus had a diameter of 40 nm and an appearance in the electron microscope similar to that of human hepatitis B virus. The DNA genome of the virus was circular and partially single stranded, and an endogenous DNA polymerase associated with the virus was capable of converting the genome to a double-stranded circle with a size of ca. 3,000 base pairs. An analysis for viral DNA in the organs of infected birds indicated preferential localization in the liver, implicating this organ as the site of virus replication. In all of these aspects, the virus bears a striking resemblance to human hepatitis B virus and appears to be a new member of this family, which also includes ground squirrel hepatitis virus and woodchuck hepatitis virus.     

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.     

Distribution of the Receptor Sites for Sindbis Virus on the Surface of Chicken and BHK Cells

Sindbis virus was adsorbed to chicken cells or to BHK cells, and the distribution of virus over the surface of the cell was examined by electron microscopy of surface replicas. The distribution of virus particles on the cell was used to indicate the position of virus receptors at the cell surface. When purified Sindbis virus was adsorbed at 37 C to cells prefixed with glutaraldehyde, the virus particles were evenly distributed over the surface of most cells. There was a large variability from cell to cell, however, in the number of virus particles adsorbed, and regions with different concentrations of virus particles were sometimes observed on the same cell. The concentration of virus receptors observed varied from 20 to 160/mum(2) of cell surface, and, thus, the total number of virus receptors per chicken cell is on the order of 10(5). When virus was adsorbed to unfixed cells at 4 C, the virus particles were clustered into aggregates varying from a few particles to large crystalline arrays (the latter seen only in chicken cells). These conditions are apparently conducive to virus aggregation, and this, coupled with free lateral diffusion of the virus-receptor complex in the cell membrane at 4 C, leads to the observed clustering.     

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。     

Development,standardization and assessment of PCR systems for purity testing of avian viral vaccines

The European Pharmacopoeia (Ph. Eur.) requires avian viral vaccines to be free of adventitious agents. Purity testing is an essential quality requirement of immunological veterinary medicinal products (IVMPs) and testing for extraneous agents includes monitoring for many different viruses. Conventional virus detection methods include serology or virus culture, however, molecular tests have become a valid alternative testing method.Nucleic acid testing (NAT) is fast, highly sensitive and has a higher degree of discrimination than conventional approaches. These advantages have led to the development and standardization of polymerase chain reaction (PCR) assays for the detection of avian leucosis virus, avian orthoreovirus, infectious bursal disease virus, infectious bronchitis virus, Newcastle disease virus, infectious laryngotracheitis virus, influenza A virus, Marek's disease virus, turkey rhinotracheitis virus, egg drop syndrome virus, chicken anaemia virus, avian adenovirus and avian encephalomyelitis virus. This paper reviews the development, standardization and assessment of PCR for extraneous agent testing in IVMPs with examples from an Official Medicines Control Laboratory (OMCL).     

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.     

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.     

Neutralizing antibodies to visna lentivirus: mechanism of action and possible role in virus persistence.

Lentiviruses are nononcogenic retroviruses that cause persistent infections and slowly progressive diseases. Visna virus, a lentivirus of sheep, persists in cells of the macrophage lineage despite the presence of neutralizing antibodies in the animal. These antibodies are measured by prevention of virus replication in sheep fibroblast cell cultures. In this study we have compared the antiviral properties of the antibodies in sheep fibroblast and macrophage cell cultures, the latter being more relevant to infection in the animal. Using infectivity assays, binding of radiolabeled virus to cell membranes, cellular processing of labeled virus into acid-precipitable and acid-soluble components, and in situ hybridization of viral nucleic acid, we show that the antibodies prevented virus replication in both fibroblasts and macrophages. However, the site of neutralization differed between the two cell types. In fibroblasts, the site of virus neutralization was at the cell membrane, when the antibodies prevented virus attachment. In macrophages, virus incubated with the antibodies was phagocytized rapidly, followed by uncoating of the virions. However, virus RNA was not transcribed. Despite this ability of the antibodies to abort virus replication in macrophages, the kinetics of binding of the antibodies to the virus was much slower than the binding of virus to the macrophages. Therefore, persistent virus replication in immune sheep may be the result of virus spreading from macrophage to macrophage before the agent can be neutralized by antibodies in the plasma.