Efficient isolation of wild strains of canine distemper virus in Vero cells expressing canine SLAM (CD150) and their adaptability to marmoset B95a cells

We have previously shown that canine signaling lymphocyte activation molecule (SLAM; also known as CD150) acts as a cellular receptor for canine distemper virus (CDV). In this study, we established Vero cells stably expressing canine SLAM (Vero.DogSLAMtag cells). Viruses were isolated in Vero.DogSLAMtag cells one day after inoculation with spleen samples from five out of seven dogs with distemper. By contrast, virus isolation with reportedly sensitive marmoset B95a cells was only successful from three diseased animals at 7 to 10 days after inoculation, and no virus was recovered from any dogs when Vero cells were used for isolation. The CDV strain isolated in Vero.DogSLAMtag cells did not cause cytopathic effects in B95a and human SLAM-expressing Vero cells, whereas the strain isolated in B95a cells from the same dog did so in canine or human SLAM-expressing Vero cells as well as B95a cells. There were two amino acid differences in the hemagglutinin sequence between these strains. Cell fusion analysis after expression of envelope proteins and vesicular stomatitis virus pseudotype assay showed that their hemagglutinins were responsible for the difference in cell tropism between them. Site-directed mutagenesis indicated that glutamic acid to lysine substitution at position 530 of the hemagglutinin was required for the adaptation to the usage of marmoset SLAM. Our results indicate that Vero cells stably expressing canine SLAM are highly sensitive to CDV in clinical specimens and that only a single amino acid substitution in the hemagglutinin can allow the virus to adapt to marmoset SLAM.     

In Vitro Studies with Modoc Virus in Vero Cells: Plaque Assay and Kinetics of Growth, Neutralization, and Thermal Inactivation

A sensitive and quantitative assay system is described for plaquing Modoc virus in Vero cells. Neutralizing antibodies to Modoc virus could be detected by using this in vitro system by their interference with viral plaque formation. Virus was readily neutralized within 30 min at 37 C by a 1:10 dilution of hyperimmune hamster serum. The rate of neutralization and the total amount of virus neutralized was not altered significantly by the addition of 20 U of guinea pig complement to the hyperimmune hamster serum. A study of the growth of Modoc virus in Vero cells is also presented. After an initial latent period of 20 h, viral titer increased exponentially for 20 h. By 83 h after infection, 8,000 plaque-forming units of virus were detected per cell. The stability of viral infectivity in phosphate-buffered saline at pH 7.4 was evaluated. No reduction in viral titer was detected after 3 days at 7 or 22 C. A continuous decrease in infectivity at 37 C was observed, however, throughout the observation period.     

Neurovirulence in mice of neural cell-adapted canine distemper virus

Neurovirulence of the Onderstepoort strain of canine distemper virus (CDV) adapted to human neural cell lines was determined by the intracerebral inoculation of DDD mice at 3 and 5 weeks of age. Intensity of neurovirulence was estimated by histopathological changes in the central nervous system and clinical symptoms. The original virus propagated in Vero cells induced leptomeningoencephalitis, whereas neuroblastoma-adapted virus induced nerve cell degeneration and mild encephalitis with relatively low morbidity and fatality. In contrast, the viruses adapted to glioblastoma and oligodendroglioma caused high morbidity and fatality. The latter two viruses induced necrotizing encephalopathy including edema and hyperemia. In addition, the glioblastoma-adapted virus induced formation of giant cells. The oligodendroglioma-adapted virus caused demyelination and spongy state associated with degeneration of glial cells and axons. These observations are discussed in regard to a possible correlation between the neurovirulence of CDV in mice and its tropism for neural cells in vitro.     

Characteristics of fusion of respiratory syncytial virus with HEp-2 cells as measured by R18 fluorescence dequenching assay.

The characteristics of fusion of respiratory syncytial virus (RSV) with HEp-2 cells were studied by the R18 fluorescence dequenching assay of membrane fusion. A gradual increase in fluorescence intensity indicative of virion-cell fusion was observed when R18-labeled RSV was incubated with HEp-2 cells. Approximately 35% dequenching of the probe fluorescence was observed in 1 h at 37 degrees C. Fusion showed a temperature dependence, with significant dequenching occurring above 18 degrees C. The dequenching was also dependent on the relative concentration of target membrane. Thus, increasing the concentration of target membrane resulted in increased levels of dequenching. In addition, viral glycoproteins were shown to be involved in this interaction, since dequenching was significantly reduced by pretreatment of labeled virus at 70 degrees C for 5 min or by trypsinization of R18-labeled virions prior to incubation with HEp-2 cells at 37 degrees C. The fusion of RSV with HEp-2 cells was unaffected over a pH range of 5.5 to 8.5, with some increase seen at lower pH values. Treatment of HEp-2 cells with ammonium chloride (20 and 10 mM), a lysosomotropic agent, during early stages of infection did not inhibit syncytium formation or progeny virion production by RSV. At the same concentrations of ammonium chloride, the production of vesicular stomatitis virus was reduced approximately 4 log10 units. These results suggest that fusion of the virus with the cell surface plasma membrane is the principal route of entry.     

Nectin4 is an epithelial cell receptor for canine distemper virus and involved in neurovirulence

Canine distemper virus (CDV) uses signaling lymphocyte activation molecule (SLAM), expressed on immune cells, as a receptor. However, epithelial and neural cells are also affected by CDV in vivo. Wild-type CDV strains showed efficient replication with syncytia in Vero cells expressing dog nectin4, and the infection was blocked by an anti-nectin4 antibody. In dogs with distemper, CDV antigen was preferentially detected in nectin4-positive neurons and epithelial cells, suggesting that nectin4 is an epithelial cell receptor for CDV and also involved in its neurovirulence.     

CD9, a tetraspan transmembrane protein, renders cells susceptible to canine distemper virus.

Canine distemper virus (CDV), a lymphotropic and neurotropic negative-stranded RNA virus of the Morbillivirus genus, causes a life-threatening disease in several carnivores, including domestic dogs. To identify the cellular receptor(s) involved in the uptake of CDV by susceptible cells, we isolated a monoclonal antibody (MAb K41) which binds to the cell surface and inhibits the CDV infection of several cell lines from various species. Pretreatment of cells with MAb K41 reduces the number of infectious centers and the size of the syncytia. Using affinity chromatography with MAb K41, we purified from HeLa and Vero cell extracts a 26-kDa protein which contained the amino acid sequence TKDEPQRETLK of human CD9, a member of the tetraspan transmembrane or transmembrane 4 superfamily of cell surface proteins. Transfection of NIH 3T3 or MDBK cells with a CD9 expression plasmid rendered these cells permissive for viral infection and raised virus production by a factor of 10 to 100. The mechanism involved is still unclear, since we were unable to detect direct binding of CDV to CD9 by using immunoprecipitation and a virus overlay protein binding assay. These findings indicate that human CD9 and its homologs in other species are necessary factors for the uptake of CDV by target cells, the formation of syncytia, and the production of progeny virus.     

犬瘟热病毒CDV-3株感染性克隆的构建与鉴定

以国内商品化水貂犬瘟热病毒疫苗所用毒株CDV-3为模板,构建犬瘟热病毒感染性c DNA克隆,为犬瘟热病毒新型疫苗研制、致病机理研究提供理论基础。设计13对引物对其全基因组序列测定,分析单一酶切位点,将CDV-3的全长分5个片段进行RT-PCR扩增。经酶切拼接,将5个片段顺次插入到酶切位点改造后的真核载体pc DNA3.2的多克隆酶切位点处,同时在F1首端和F5末端分别加入锤头状核酶和丁型肝炎核酶序列,获得CDV-3株的全长c DNA质粒(pcDNA3.2-CDV-3)。构建表达CDV-3 N、P、L蛋白的3个辅助质粒。利用转染试剂Lipofectamine~(TM) 2000将全长质粒和3个辅助质粒共转染293T细胞,3 d后,将上清接种到Vero细胞。观察犬瘟热病毒典型合胞体病变,对重组病毒进行免疫荧光鉴定和标签鉴定。最后,比较wtCDV-3和rCDV-3的生长特性。全长质粒和辅助质粒的酶切鉴定和序列测序均正确。拯救的重组病毒能在Vero细胞上形成典型的合胞体病变,经RT-PCR、间接免疫荧光和电镜观察鉴定,证明成功拯救出重组病毒rCDV-3株。rCDV-3的病毒滴度最高达到10~(7.667) TCID_(50)/mL,比wtCDV-3的滴度10~(6.667) TCID_(50)/mL高出约10倍。rCDV-3感染Vero细胞后,迅速大量增殖,于感染后36 h达到最高病毒滴度。而wt CDV-3增殖平缓,感染后72 h时病毒含量达到最大。文中建立的高效CDV-3株反向遗传操作平台,为犬瘟热病毒新型疫苗研制和致病机理研究奠定基础。     

Poliovirus and polio antibody assay in HEp-2 and Vero cell cultures

HEp-2 cell cultures were about three to 30 times more sensitive for poliovirus titration than Vero cells. Attenuated strains induced a complete cytopathic effect in HEp-2 but not in Vero cells. For polio antibody titration, HEp-2 and Vero cells were equally suitable. A high degree of sensitivity and reproducibility of virus neutralization was achieved in tests utilizing a low virus dose and serum-virus incubation overnight at 36 degrees C. Staining of infected trays with crystal violet obviated reading of viral CPE under the microscope and expedited the evaluation of larger-scale tests.     

Vaccinia virus recombinants expressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge.

cDNA clones of the genes encoding either the hemagglutinin (HA) or fusion (F) proteins of the Edmonston strain of measles virus (MV) were expressed in vaccinia virus recombinants. Immunofluorescence analysis detected both proteins on the plasma membranes of unfixed cells as well as internally in fixed cells. Immunoprecipitation of metabolically radiolabeled infected-cell extracts by using specific sera demonstrated a 76-kDa HA polypeptide and gene products of 60, 44, and 23 kDa which correspond to a MV F precursor and cleavage products F0, F1, and F2, respectively. Neither recombinant induced cell fusion of Vero cells when inoculated individually, but efficient cell fusion was readily observed upon coinfection of cells with both recombinants. Inoculation of dogs with the vaccinia virus-MV F recombinant (VV-MVF) did not give rise to detectable MV-neutralizing antibody. Inoculation of dogs with the vaccinia virus-MV HA recombinant (VV-MVHA) or coinoculation with both recombinants (VV-MVF and VV-MVHA) induced significant MV-neutralizing titers that were increased following a booster inoculation. Inoculation of dogs with the vaccinia virus recombinants or with MV failed to induce canine distemper virus (CDV)-neutralizing antibodies. Upon challenge with a lethal dose of virulent CDV, signs of infection were observed in dogs inoculated with (VV-MVF). No symptoms of disease were observed in dogs that had been vaccinated with VV-MVHA or with VV-MVHA and VV-MVF and then challenged with CDV. All dogs vaccinated with the recombinant viruses as well as those inoculated with MV or a vaccine strain of CDV survived CDV challenge.     

Herpes simplex virus type 1 glycoprotein K is not essential for infectious virus production in actively replicating cells but is required for efficient envelopment and translocation of infectious virions from the cytoplasm to the extracellular space.

We characterized the glycoprotein K (gK)-null herpes simplex virus type 1 [HSV-1] (KOS) delta gK and compared it to the gK-null virus HSV-1 F-gKbeta (L. Hutchinson et al., J. Virol. 69:5401-5413, 1995). delta gK and F-gKbeta mutant viruses produced small plaques on Vero cell monolayers at 48 h postinfection. F-gKbeta caused extensive fusion of 143TK cells that was sensitive to melittin, a specific inhibitor of gK-induced cell fusion, while delta gK virus did not fuse 143TK cells. A recombinant plasmid containing the truncated gK gene specified by F-gKbeta failed to rescue the ICP27-null virus KOS (d27-1), while a plasmid with the delta gK deletion rescued the d27-1 virus efficiently. delta gK virus yield was approximately 100,000-fold lower in stationary cells than in actively replicating Vero cells. The plaquing efficiencies of delta gK and F-gKbeta virus stocks on VK302 cells were similar, while the plaquing efficiency of F-gKbeta virus stocks on Vero cells was reduced nearly 10,000-fold in comparison to that of delta gK virus. Mutant delta gK and F-gKbeta infectious virions accumulated within Vero and HEp-2 cells but failed to translocate to extracellular spaces. delta gK capsids accumulated in the nuclei of Vero but not HEp-2 cells. Enveloped delta gK virions were visualized in the cytoplasms of both Vero and HEp-2 cells, and viral capsids were found in the cytoplasm of HEp-2 cells within vesicles. Glycoproteins B, C, D, and H were expressed on the surface of delta gK-infected Vero cells in amounts similar to those for KOS-infected Vero cells. These results indicate that gK is involved in nucleocapsid envelopment, and more importantly in the translocation of infectious virions from the cytoplasm to the extracellular spaces, and that actively replicating cells can partially compensate for the envelopment but not for the cellular egress deficiency of the delta gK virus. Comparison of delta gK and F-gKbeta viruses suggests that the inefficient viral replication and plaquing efficiency of F-gKbeta virus in Vero cells and its syncytial phenotype in 143TK- cells are most likely due to expression of a truncated gK.     

Establishment of a persistent mutant of canine distemper virus

We produced a B95a lymphoid cell line persistently infected with canine distemper virus (CDV), in which virus-specific antigens were present in nearly 100% of cells without causing cytopathic effect. The virus recovered from this cell line was able to infect fresh B95a cells persistently, indicating that a persistent CDV was established.     

Effect of visible light on canine distemper virus

Nemo, George J. (The Catholic University of America, Washington, D.C.), and Ernest C. Cutchins. Effect of visible light on canine distemper virus. J. Bacteriol. 91:798-802. 1966.-Canine distemper virus (CDV) was inactivated by visible light. The virus was light-sensitive in fluid suspension (in vitro) as well as during intracellular replication (in vivo). The addition of calf serum or glutathione reduced the extent of inactivation. CDV was less sensitive when suspended in distilled water or in the amino acid or Earle's salts components of the minimal essential medium of Eagle than when suspended in the vitamin component of the minimal essential medium of Eagle or in riboflavine (0.1 mg per liter). These findings indicate that, whereas some ingredient of the medium may enhance light sensitivity, its presence is not necessary for light inactivation of CDV. It is proposed that some substance derived from the host cell and intimately associated with the virus particle serves to render CDV light-sensitive.     

Isolation and characterization of canine distemper virus-specific RNA

Ten species of virus-specific RNA were detected in Vero cells infected with the FXNO strain of canine distemper virus (CDV). The largest RNA was the genome-sized RNA and the nine smaller species were polyadenylated RNAs. Similar results were obtained for nine other strains of CDV. The molecular weights of these ten RNAs were determined to be 4.61 X 10(6), 2.46 X 10(6), 1.52 X 10(6), 1.32 X 10(6), 1.19 X 10(6), 1.07 X 10(6), 0.77 X 10(6), 0.65 X 10(6), 0.58 X 10(6), and 0.48 X 10(6). By in vitro translation of the polyadenylated RNAs in a rabbit reticulocyte lysate system, three different proteins which probably correspond to H, NP, and M were synthesized from the fraction containing RNAs 7, 8, 9, and 10.     

Vero cells infected with the Lederle strain of canine distemper virus have increased Fas receptor signaling expression at 15 h post-infection

We evaluated the expression of the Fas receptor gene in Vero cells infected with the Lederle vaccine strain of canine distemper virus using RT-PCR. Vero cells were plated, and after being grown for 24 h in MEM with 5% FBS, 80-90% confluent monolayer cultures were infected with the virus. The cells were harvested at 3, 6, 9, and 15 h post-infection. Uninfected Vero cells were used as a control. Total RNA was isolated from Vero cells using 1 mL Trizol(?) LS, and RT was performed using 2 μg total RNA. Primer pairs for RT-PCR amplification for the canine distemper virus nucleocapsid gene, the S26 reference gene, and the Vero rFas gene were used to analyze expression in Vero cells. RT-PCR results revealed virus activity at 3, 6, 9, and 15 h in the virus-infected Vero cells. The S26 housekeeping gene was amplified in virus infected and control samples. However, expression of the cell death receptor Fas was detected in Vero cells only at 15 h post-infection. We suggest that the Lederle vaccine induces apoptosis by Fas receptor signaling, possibly through caspase-8 signaling rather than through mitochondrial signaling in the infected cells.     

Evidence for post-translational glycosylation of a nonglycosylated precursor protein of herpes simplex virus type 1.

Incubation of herpes simplex virus type 1-infected Vero and HEp-2 cells at a reduced temperature (34 degrees C) enhanced the detection of the nonglycosylated precursors (pgB97 and pgC75) to the gB and gC glycoproteins in the cytoplasmic and nuclear fractions. Relative to the fully glycosylated and high-mannose forms detected, the nonglycosylated precursors were the predominant components associated with the nuclear fraction of infected cells. Furthermore, addition of protease inhibitors to the fractionation buffers did not affect the distribution or abundance of the nonglycosylated precursors, suggesting that the presence of pgB97 and pgC75 was not the result of proteolysis. When infected Vero or HEp-2 cells were harvested at various times postinfection, the nonglycosylated precursors were detected after the initial appearance of the high mannose components (pgB110 and pgC105). In Vero cells, pgB97 and pgC75 were detected simultaneously at 8 h postinfection, whereas detection was not apparent in HEp-2 cells until 20 h postinfection. Conditions which favored detection of appreciable amounts of nonglycosylated precursors provided an unique approach to probe possible post-translational modifications in the absence of inhibitors of glycosylation. In nuclear fractions isolated from cycloheximide-treated HEp-2 or Vero cells, numerous discrete gC-immunoreactive bands migrating with decreased electrophoretic mobility relative to the nonglycosylated precursor pgC75 were observed. This series of one to four additional bands was eliminated by digestion with endoglycosidase H, and the appearance of these bands was blocked by the addition of tunicamycin. Collectively, the data suggest that high-mannose core oligosaccharides may be added to the nonglycosylated precursor of the gC glycoprotein of herpes simplex virus type 1 in a post-translational fashion.     

犬瘟热病毒小熊猫株反向遗传系统的构建及其鉴定

以驯化致弱的犬瘟热病毒小熊猫株(Canine distemper virus,CDV)为模板,构建犬痘热病毒感染性cDNA克隆.对其全基因组序列测定后,用RT-PCR的方法获得组成全长基因的7个片段,通过酶切、拼接将7段CDVcDNA序列插入到真核表达载体pCI的MCS上,构建犬瘟热病毒小熊猫株的全长cDNA质粒(pCI-CDV-LP),同时分别克隆CDV小熊猫株N、P、L蛋白ORF构建三个辅助质粒.酶切鉴定和序列测定表明,pCI载体中插入的核酶及CDV cDNA序列正确无误,使用转染试剂Lipofectamine TM 2000将全长质粒和三个辅助质粒共转染中国仓鼠肾细胞(BSR),经RT-PCR、间接免疫荧光和病毒感染VERO-SLAM细胞试验鉴定,成功拯救出CDV小熊猫株,显示CDV小熊猫株反向遗传系统构建完成,为犬瘟热病毒致病机理及免疫研究奠定基础.     

Infectious entry pathway of influenza virus in a canine kidney cell line

The entry of fowl plague virus, and avian influenza A virus, into Madin- Darby canine kidney (MDCK) cells was examined both biochemically and morphologically. At low multiplicity and 0 degrees C, viruses bound to the cell surface but were not internalized. Binding was not greatly dependent on the pH of the medium and reached an equilibrium level in 60-90 min. Over 90% of the bound viruses were removed by neuraminidase but not by proteases. When cells with prebound virus were warmed to 37 degrees C, part of the virus became resistant to removal b neuraminidase, with a half-time of 10-15 min. After a brief lag period, degraded viral material was released into the medium. The neuraminidase- resistant virus was capable of infecting the cells and probably did so by an intracellular route, since ammonium chloride, a lysosomotropic agent, blocked both the infection and the degradation of viral protein. When the entry process was observed by electron microscopy, viruses were seen bound primarily to microvilli on the cell surface at 0 degrees C and, after warming at 37 degrees C, were endocytosed in coated pits, coated vesicles, and large smooth-surfaced vacuoles. Viruses were also present in smooth-surfaced invaginations and small smooth-surfaced vesicles at both temperatures. At physiological pH, no fusion of the virus with the plasma membrane was observed. When prebound virus was incubated at a pH of 5.5 or below for 1 min at 37 degrees C, fusion was, however, detected by ferritin immunolabeling. t low multiplicity, 90% of the prebound virus became neuraminidase- resistant and was presumably fused after only 30 s at low pH. These experiments suggest that fowl plague virus enters MDCK cells by endocytosis in coated pits and coated vesicles and is transported to the lysosome where the low pH initiates a fusion reaction ultimately resulting in the transfer of the genome into the cytoplasm. The entry pathway of fowl plague virus thus resembles tht earlier described for Semliki Forest virus.     

Role of receptor-binding activity of the viral hemagglutinin molecule in the presentation of influenza virus antigens to helper T cells.

The concentration of antigen required to stimulate influenza virus-specific helper T cells was observed to be dependent upon the antigenic form bearing the relevant determinant: intact, nonreplicative virus was needed only in picomolar amounts, while denatured proteins, protein fragments, or synthetic peptides were required in micromolar concentrations for a threshold level of stimulation. Antigenic efficiency of intact virus was found to result from the attachment of virus to sialic acid residues on the surface of the antigen-presenting cell since spikeless viral particles lacking the hemagglutinin molecule were much less efficient antigens for helper T cells and continuous presence of hemagglutination-inhibiting antihemagglutinin antibodies reduced efficiency of stimulation by intact virus approximately 100-fold for both hemagglutinin and internal virion proteins. Influenza virus associated rapidly with antigen-presenting cells; less than 10 min at 20 degrees C was sufficient to introduce virus for a maximal level of T-cell stimulation. This rapid attachment was blocked by antibodies to the hemagglutinin or by pretreatment of the antigen-presenting cells with neuraminidase to remove the cellular virus receptor. Following viral adsorption by antigen-presenting cells, a lag period of 30 min at 37 degrees C was required for the expression of helper T-cell determinants. One early event identified was the movement of the virus to a neuraminidase-insensitive compartment, which can occur at 10 degrees C, but which was not equivalent to expression of helper T-cell determinants. Preincubation of cells with virus at 10 degrees C for 4 h reduced the lag period of helper T-cell determinant expression to 15 min when these cells were shifted to 37 degrees C, suggesting that transition of the virus to a neuraminidase-resistant state is a required step in presentation of T-cell antigenic determinants.