Histone acetylation in CV-1 cells infected with simian virus 40.

In CV-1 cells infected with simian virus 40 (SV40) the histone acetylation of host cell chromatin was compared to that of intracellular 70 S minichromosomes. The degree of acetylation in the histones of cell chromatin did not substantially increase after infection; the 70 S minichromosomes contained histones that were not as highly acetylated as those contained in virions, but more acetylated than those associated with the infected cell chromatin. The rate of acetylation, as measured by [³H]acetate uptake, appeared to be nearly equal for the histones associated with viral and cellular DNA; the acetyl groups incorporated by the 70 S histones, however, were retained in larger proportion compared to those incorporated by the host histones.     

Electron microscopic demonstration of Crimean hemorrhagic fever virus in CV-1 cells.

Crimean hemorrhagic fever (CHF) virus was detected by electron microscopy in ultrathin sections of CV-1 cells. The virus particles appeared spherical or oval and occurred both intra- and extracellularly. Based on their size and density of their contents, two types of virions, probably representing different phases of the reproduction cycle of CHF virus, could be distinguished.     

Cloning and sequencing of the mumps virus fusion protein gene

The fusion protein (F) gene of mumps virus was cloned from a cDNA library constructed from infected cell mRNA. The F-specific plasmids were identified by hybridization to a degenerate oligonucleotide probe whose sequence was deduced from the N-terminal amino acid sequence of the F₂ protein. The complete nucleotide sequence of the F gene was determined. The gene is 1786 nucleotides long and encodes one long open reading frame of 538 amino acids. The F protein has a 19-amino acid signal peptide cleaved between Cys and Val residues. The cleavage site for activation of the F₀ protein into themature F_1,2 is ArgArgHisLysArg. A stretch of 30 hydrophobic amino acids near the C-terminus of the protein is followed by serveral charged amino acids and appears to serve as the anchoring domain for the protein in the lipid bilayer. The F gene of mumps virus is highly related to the F gene of the paramyxovirus SV-5.     

CV-1 cells release proteins that facilitate infection by simian virus 40 and echovirus 6

Summary Uninfected simian cells (CV-1) produce two different proteins, one of which enhances the infectivity of echovirus 6 and the other enhances the infectivity of SV 40 in less susceptible cells. The enhancers are released by metabolizing cells into the culture medium. The SV 40 enhancer protein is larger and less acidic than the echovirus enhancer. The SV 40 enhancer protein can be dissociated into 2 subunits with apparent molecular weights of 42,000 and 24,000. The echovirus enhancer protein consists of 2 subunits with apparent molecular weights of 28,000 and 31,000. Electrophoretically purified enhancer proteins interact with virions and retain their biological activities and viral specificities. The SV 40 enhancer stimulates expression of SV 40-T antigen under conditions in which untreated virus does not initiate expression of T antigen. This result and the observation that the enhancers do not increase the infectivity of the nucleic acids of their respective viruses suggest that the enhancers act either at the stage of penetration or uncoating.With 5 Figures     

Replication of mumps virus in murine cells

Summary Mumps virus replication was examined in various culture cells derived from mice. Eight of 16 lymphoid cell lines and 4 of 13 non-lymphoid cell lines supported the replication of Vero cell-adapted Enders strain (EY) of mumps virus. EY strain replicated more efficiently in lymphoid cell lines than in non-lymphoid ones. T cell preference, however, was not observed in this study.The growth kinetics of EY strain in high yield cell lines such as EL-4, L1210 and NS-18 cells were similar to that in Vero cells, while in low yield cell lines such as DK, C243 and 203GL cells the growth patterns varied respectively.Nineteen kinds of primary culture cells of murine origin all proved not to be susceptible for EY strain, even when spleen cells were stimulated with lectins or allogeneic cells.Seven other strains of mumps virus were examined for their ability to replicate in EL-4, L1210 and L929 cells. Four and 6 strains replicated in EL-4 cells and L1210 cells respectively, although the growth patterns and yields varied in each virus-cell combinations. On the other hand, none of 7 strains showed sufficient replication in L929 cells.With 2 Figures     

Purification and amino-terminal protein sequence analysis of the mumps virus fusion protein

The fusion (F) protein of mumps virus was purified by immunoaffinity chromatography using an anti-F monoclonal antibody. The F protein was reduced and alkylated, and the F1 and F2 chains were isolated by high-pressure size exclusion chromatography. Twenty-three amino acid residues from the amino terminus of each chain were identified following automated Edman degradation. The amino-terminal sequence of the F1 chain was homologous to previously reported F1 sequences from three other paramyxoviruses (simian virus 5, Newcastle disease virus, and Sendai virus). Secondary structure predictions suggest an alpha-helical conformation for the mumps virus F1 amino-terminal sequence. A helical wheel model of the paramyxovirus F1 NH2 terminus is presented which defines conserved and variable arcs of the helix and provides a spatial representation of this critical functional domain of the paramyxovirus fusion protein.     

Structural polypeptides of mumps virus.

The structural polypeptides of egg grown mumps virus were analysed by SDS-polyacrylamide-slab-gel electrophoresis. Mumps virions contained eight major polypeptides with mol. wt. of 75, 73, 71, 61, 47, 44, 42 and 40 X 10(3). The 75 K and 61 K polypeptides were glycosylated. In virions treated with pronase and trypsin, the 75 K glycoprotein was removed more readily from the virus than the 61 K glycoprotein. The gradual removal of the 75 K glycoprotein was paralleled by a decrease of haemagglutinating activity. The large glycoprotein was cleaved into a 40 K glycoprotein by trypsin treatment. Pronase and trypsin treatment also removed the smallest 40 K non-glycosylated polypeptide. Thus this polypeptide appears to be located on the outside of the virion and probably represents a cleavage product of the large glycoprotein. Treatment of virions with 2% Triton-X 100 under alkaline conditions in the absence or presence of 2 M-KCl solubilized the two glycoproteins and a fraction of the 71 and 44 K polypeptides, but not the 73 and 47 K polypeptides. The two smallest polypeptides were solubilized by treatment with 2% Triton X-100 in the presence of 2 M-KCl. Since the 40 K polypeptide was interpreted to represent a cleavage product of the large surface glycoprotein the 42 K polypeptide was proposed to represent the membrane protein of mumps virus. The 44 K polypeptide co-migrated with Vero cell actin. The nature of the 47 K polypeptide could not be determined, but it is probably located in the central part of the virus. The 73 K polypeptide and in some experiments also the 71 K polypeptide were found in purified nucleocapsid preparations. It is concluded that mumps virus has a general polypeptide composition similar to other paramyxoviruses. However, the molecular weights of the different polypeptides of mumps virus differ markedly from the corresponding polypeptides in Newcastle disease virus and Sendai virus.     

Experiences with the adaptation of crimean hemorrhagic fever virus to the CV-1 monkey cell line.

Of several cell lines tested, the CV-1 monkey cell line proved to be the most suitable system for propagation of Crimean hemorrhagic fever (CHF) virus. Two CHF strains could be adapted to this cell line by serial passaging. The viral progeny from these passages was specifically cytocidal and produced plaques in CV-1 cells, which could be used in serological diagnosis of CHF. After adaptation of CHF virus to CV-1 cells, the rate of virus synthesis was more rapid, but the yield of virus produced by these cells did not substantially increase. Data reported on cultivation of CHF-Congo viruses are discussed in comparison with the present findings.     

Studies on the defectiveness of adeno-associated virus (AAV). II. Effect of growth in CV-1 cells on the replication of adeno-associated virus.

The mechanisms by which helper viruses can promote AAV macromolecular synthesis have been investigated. The third system used was a continuous line of African green monkey kidney cells (CV-1) which is permissive for AAV when a simian adenovirus helper is used. When HSV was used as a helper, no AAV DNA, structural proteins, or IF antigens were detected, indicating the absence of HSV helper activity. HSV production and plaqueing in CV-1 cells were found to be comparable to those seen in Hep-2 cells (an epidermoid cancer line) and Vero cells (another line of African green monkey kidney cells). In both of these cell lines, HSV expresses its AAV helper functions. It was also observed that AAV had no effect on HSV replication in either Vero or CV-1 cells. Comparison of HSV protein production in CV-1 and Hep-2 cells demonstrated the absence of HSV proteins ICPO and ICP46 in the CV-1 line following the release of a cycloheximide block.     

Antibody responses to mumps virus proteins in natural mumps infection and after vaccination with live and inactivated mumps virus vaccines

Paired sera from 20 patients with acute mumps infection, 16 from persons vaccinated with live attenuated mumps virus vaccine, and 12 from persons vaccinated with formalin-inactivated virus vaccine were studied for mumps antibodies by single radial hemolysis (SRH), hemagglutination inhibition (HI), and by enzyme immunoassays (EIA) specific for whole virus, envelope glycoprotein, and nucleocapsid antibodies. Mumps patients had diagnostic rises in serum mumps antibodies in 90–100% of the cases depending on the method of assay. Vaccination resulted in seroconversion in 75–88% (live vaccine) and in 92% (inactivated vaccine) of the cases as detected by SRH or EIAs, whereas HI detected seroconversion only in 38% and 58% of the cases, respectively. Immunoprecipitation analyses revealed that all sera from mumps patients and nearly all postvaccination sera had antibodies against the main structural proteins of mumps virus. By immunoblotting, antibodies against denatured hemagglutinin-neuraminidase (HN) and fusion protein (F) were detected in 15–25 % of mumps patients and persons vaccinated with live vaccine, whereas most postvaccination sera from those vaccinated with inactivated vaccine had HN (92%) and F (83%) protein antibodies, suggesting that antibodies against the denatured form of proteins are formed.     

CV-1 and MRC-5 mixed cells for simultaneous detection of herpes simplex viruses and varicella zoster virus in skin lesions.

BACKGROUND: Culture for varicella zoster virus (VZV) is relatively insensitive. Herpes simplex viruses (HSV) culture methods, which rely on primary rabbit kidney (pRK), mink lung (Mv1Lu) or the ELVIS HSV culture system fail to detect VZV. Culture of atypical vesicular skin lesions should be able to detect both HSV and VZV. OBJECTIVES: In this study, we evaluated the sensitivity of a newly developed mixture of CV-1/MRC-5 cells for the concurrent detection of both HSV and VZV. STUDY DESIGN: The CV-1/MRC-5 mixed cells were compared with pRK cells and Mv1Lu cells for the detection of HSV and to MRC-5 and A-549 cells for the detection of VZV. Fresh clinical samples submitted for HSV culture, VZV culture, and/or direct immunofluorescent assay (DFA) as well as frozen clinical samples previously positive for VZV were used for these comparisons. RESULTS: This preliminary study suggest that CV-1/MRC-5 mixed cells are as sensitive as pRK and Mv1Lu cells for the detection of HSV and appear to be more sensitive than MRC-5 and A-549 cells for the detection of VZV. Although the sample size is small, pre-CPE staining with VZV specific monoclonal antibody (Mab) at day 2 post-inoculation may provide a rapid detection of VZV with these mixed cells, but not with MRC-5 or A549 cells. In addition, culture of VZV in mixed cells from fresh clinical specimens appears to be as sensitive as antigen detection by DFA. Finally, 1% of specimens from skin lesions submitted for HSV culture grew VZV, highlighting the importance of culturing for both VZV and HSV, particularly in the case of atypical lesions. CONCLUSION: CV-1/MRC-5 mixed cells are highly sensitive for the simultaneous culture of HSV and VZV. The ability to detect either HSV or VZV from skin lesions is important for patient management.     

Expression of mumps virus glycoproteins in mammalian cells from cloned cDNAs: both F and HN proteins are required for cell fusion.

Two recombinant plasmids were constructed by inserting the cDNAs of either the fusion (F) or the hemagglutinin-neuraminidase (HN) protein genes of mumps virus into the pcDL-SR alpha expression vector. Both the F and the HN proteins expressed in COS7 cells transfected with their respective recombinant plasmids were indistinguishable in terms of electrophoretic mobility from their counterparts synthesized in mumps virus-infected cells. The F protein was cleaved and expressed on the cell surface, but uncleaved forms were also detected. The expressed HN protein was transported to the cell surface and adsorbed guinea pig erythrocytes. Syncytium formation was induced when COS7 cells were transfected with both recombinant plasmid DNAs together, but not with the recombinant plasmid only carrying the F gene. This observation indicates that cell fusion mediated by mumps virus requires both the F and the HN glycoproteins.     

The mumps virus V protein is unstable in virus infected cells

Summary The mumps virus (MuV) V protein was characterized in virus infected cells by the use of antipeptide sera. In radioimmune precipitation assay (RIPA), the sera reacted with the V protein and also immunoprecipitated the nucleocapsid (NP) and phospho (P) proteins. However, by depletion RIPA (in which either the NP and P proteins or the V protein were removed) and Western immunoblotting, it was demonstrated that the V protein was not associated with the NP and P proteins, but that the anti-V sera cross-reacted with the NP protein. Pulse-chase experiments demonstrated that the V protein was gradually decreased during the chase period and could not be detected by antibodies raised against peptides representing three different regions of the protein at the end of the chase, while the NP and P proteins were relatively stable during the chase period. These results suggest that the V protein is unstable and degraded gradually in virus infected cells.     

The development of CV-1 cells resistant to SV 40

Summary CV-1 cells which survived SV 40 infection developed resistance to SV 40 and changes in growth potential and morphology. The changes in growth and resistance to SV 40 were not permanent in the majority of the surviving cells. The number of surviving colonies decreased exponentially over a five week period. During this time, secondary changes in cell morphology took place. Stable cell strains emerged. Although these strains continued to release small quantities of SV 40, an increase in the number of infected cells (containing V antigen) could not be produced by superinfection. An isolated clone of the resistant cells remained free of detectable virus for two months but then spontaneously released virus. Treatment with anti-SV 40 serum reduced the concentration of extracellular virus but not that of intracellular virus. The fraction of cells in which T antigen fluorescence could be found varied from 10–50% but never reached 100%, even after long term cultivation.The results are consistent with the concept that both the T antigen positive cells and those that continue to release virus arise from T antigen negative, abortively transformed cells in which the viral genome is unstably suppressed.Part of these results has been reported at the Third Heidelberg Symposium, Aktuelle Probleme aus dem Gebiet der Cancerologie III, September, 1970.     

Membrane fusion activity, oligomerization, and assembly of the rabies virus glycoprotein.

The spike glycoprotein (G protein) of rabies virus (CVS strain) expressed in HeLa cells from cloned cDNA mediated membrane fusion after exposure to pHs of 6.1 or below. Chemical crosslinking showed that the rabies G protein, like the vesicular stomatitis virus (VSV) G protein, could be crosslinked to dimers and trimers, indicating that rabies G protein is a trimer. However, unlike the VSV G protein, rabies G protein trimers were not stable to sedimentation in sucrose gradients, even at a mildly acidic pH which stabilizes the VSV G protein trimers. In addition, we report that the expressed rabies virus G protein was functional because it could assemble into VSV particles (tsO45) lacking VSV G protein and rescue infectivity. These VSV (rabies) pseudotypes were neutralized only by an antibody to the rabies G protein. We also examined the properties of a hybrid protein containing the extracellular domain of the rabies virus glycoprotein and the transmembrane and cytoplasmic domains of the VSV G protein. This protein was transported to the cell surface and could be crosslinked to form dimers and trimers, but had little or no detectable membrane fusion activity. The lack of fusion activity was paradoxical because the hybrid protein could rescue VSV infectivity, although the titers were lower than those obtained with the wild-type rabies G protein.     

Characterisation of mumps virus genotype C among patients with mumps in India

Measles, mumps and rubella (MMR) vaccine failure had been reported globally and here, we report that it occurs in India now. MMR vaccinated people have developed acute mumps accompanied by anti-mumps immunoglobulin M. Genotypic characterisation revealed that the circulating mumps strain was genotype C, which is distinct from the vaccine strain of genotype N (L-Zagreb). This is the first report in India to suggest that genotype C is responsible for the present mumps infection. Thus, the present MMR vaccine must be revamped and optimised for its efficacy to prevent any future mumps epidemics.     

Scanning electron microscopy of CV-1 and HeLa cells infected with herpes simplex viruses types 1 and 2

Production of virus particles in CV-1 or HeLa BU-25 cells was investigated after their infection with several strains of herpes simplex virus (HSV) types 1 and 2, especially in relation to the association of virus particles with cells, the ratio of plaque forming units (PFU) to the whole number of virus particles and the morphological characteristics of cytopathic effect (CPE). Growth curves of the viruses differed according to the combination of cells and infecting virus strains. At 20 hr p.i., the number of cell-associated or cell-free viruses ranged from 5 X 10(8) to 5 X 10(9) PFU/35 mm dish or from 5 X 10(2) to 1.5 X 10(4) PFU/cell irrelevant of virus serotype or the morphology of CPE. In the case of CV-1 cells, the ratios of the number of the virus particles to PFU ranged from 100 to 640 and/or 18 to 110, respectively, depending on the CPE of rounding type or of fusion type. In case of CPE of fusion type, a higher rate of infectious particles was observed.     

The multiplication of influenza virus in enucleated BHK cells fused with chicken erythrocytes.

Chicken erythrocyte nuclei that were introduced by cell fusion into enucleated BHK cells supported the production of influenza virus nucleoprotein but not haemagglutinin or neuraminidase antigens.     

Antigenic and genetic characterization of the fusion (F) protein of mumps virus strains

Summary.  Twelve strains of mumps virus, belonging to the A, C and D genotypes of the small hydrophobic (SH) protein gene, were investigated by nucleotide sequencing of the fusion protein gene. The nucleotide sequences and deduced amino acid sequences were aligned and compared with previously reported sequences of the gene. In addition an antigenic comparison between the F protein of different strains of the A, C and D genotypes was performed with ten monoclonal antibodies directed against the F protein of genotype A. Phylogenetic analysis of the coding region of the F gene showed the expected clustering of the different genotypes, as previously determined from the SH protein gene. Comparison of the 538 long amino acid sequence of the protein showed that only a small number of amino acids differed between the viral strains. The A genotype differed from B, C and D whereas the latter showed fewer consistent amino acid differences between themselves. Nine of the ten monoclonal antibodies reacted with the C and D genotypes and one failed to react with these genotypes. It is concluded that the structure and antigenicity of the F protein is well conserved both intra- and intergenotypically over long periods of time. Received September 7, 1999 Accepted November 17, 1999