Enhanced plating efficiency of herpesvirus DNA on herpes simplex virus type 1 DNA-transformed cells.

Partial resistance of hamster cells transformed by sheared herpes simplex virus (HSV) DNA to superinfection by intact HSV could be overcome by transfection with viral DNA's. HSV DNA plated more efficiently on HSV type 1 DNA-transformed hamster cells than on spontaneously transformed hamster or rabbit cells or on hamster cells transformed by other DNA viruses.

A critical view of cell substrates with regard to cell transformation and cancer.

The basic heuristic method in experimental medicine of proceeding from animals to human beings to facilitate the understanding of the etiology of a disease, is not completely relevant to viral oncogenesis in particular. The situation does not diminish scientific interest in tumor viruses because the study of these oncogens opens the way to identification of cancer genes and the understanding of their functions, expression and regulation in transformed cells. The results of these investigations are essential in comprehending animal tumor biology and from this, within limits, extrapolating to human neoplasia. However, the results of epidemiological, cellular and molecular approaches to human cancer all suggest a multiple and complex origin of this disease. In light of our current knowledge of cell transformation and cancer, the nature of permanent heteroploid cell lines is discussed in relation to their use as substrates for production of biologicals.

Quantitation of cell DNA in the evaluation of heteroploid cells as substrate for the preparation of killed viral vaccines.

To evaluate the risk of using heteroploid cell lines as substrates for viral vaccine production, the presence of cell DNA in poliovirus suspensions was examined. The time course of [3H]-thymidine-labeled HeLa cell DNA release during lytic infection with type 1 poliovirus was investigated. More DNA was found in filtered supernatants of poliovirus-inoculated cultures than in control cell supernatants. DNA concentration increased with time, paralleling virus release, but did not exceed 1.5% of the total DNA content of the culture. Only about 10% of this cell DNA was resistant to DNase treatment. By both ion-exchange chromatography and rate-zonal centrifugation it was possible to remove practically all cell DNA contaminating filtered poliovirus suspensions. Results obtained in this study permitted quantitative evaluation of cell substrate DNA present in poliovirus suspensions during successive steps of killed poliovirus vaccine preparation. Based on the sensitivity of our method, the amount of residual DNA was estimated at less than 0.02 pg per dose of purified vaccine.

Presence and quantification of cell substrate DNA in inactivated poliovirus vaccine.

In order to follow the fate of cell substrate DNA in inactivated poliovirus vaccine (IPV), experiments simulating different steps in IPV preparation were performed. For this purpose, 3H-thymidine-labeled HeLa cell DNA released during lytic infection with Mahoney type 1 poliovirus strain was used as tracer. Low speed centrifugation (1500 xg for 15 min.) of crude virus suspension removed on the average 98.5% of the total labeled cell substrate DNA. Repeated freezing and thawing of the crude virus suspension before centrifugation did not increase the residual 1.5% radioactivity found in the supernatant. This level of contamination was not significantly influenced by filtration of the supernatant through a 0.22 micrometer pore size membrane filter. DEAE-Sepharose CL-6B chromatography of poliovirus suspensions containing large amounts of 3H-thymidine labeled HeLa cell extracts completely removed the contaminating substrate DNA. These data showed that IPV free of cell substrate DNA can easily be obtained by a usual purification procedure.

[Structural polypeptide VP1 in poliovirus type 1 induced by neutralizing antibodies]. / Le polypeptide structural VP1 du poliovirus type 1 induit des anticorps neutralisants.

Anti-type 1 (Mahoney) poliovirus neutralizing antibodies were obtained by immunizing rabbits with structural polypeptide VP1. This polypeptide was isolated from the virion by PAGE-SDS electrophoresis and extracted from the gel by electroelution. Anti VP2 and anti VP3 sera prepared in the same was did not display any significant antipolio neutralizing activity.

Cell substrate and risk in killed poliomyelitis vaccine.

By using 3H-T labeled HeLa cell DNA it was possible to evaluate the quantity and the state of substrate DNA present in crude and purified killed poliovaccine. The results showed that 1.7 x 10(5) pg/ml of DNA is present in crude vaccine while in purified vaccine this product can not be detected (the limit of the detection method was 1.1 x 10(2) pg/ml). The theoretical problems of the risk of the formation of poliovirus pseudotypes and the potential contamination of the vaccine with a type C retrovirus are examined.

Enhanced efficiency of transfection of HSV DNA in HSV-1 DNA-transformed hamster cells.

The efficiency of transfection, i.e., the number of plaques per microgram of DNA, was compared for herpes simplex virus (HSV) type 1 and type 2, and herpesvirus eidolon (antigenically unrelated to HSV) DNAs in HSV DNA-transformed cells (EH/A44) and control hamster cells (EHT). The efficiency of transfection of HSV-1 and HSV-2 DNAs was significantly higher in EH/A44 cells than in EHT cells, showing that an early step in HSV infection was involved in the partial resistance of HSV DNA-transformed cells to superinfection by various intact herpesviruses.

Purification of poliovirus obtained from human diploid cells by means of DEAE-Sepharose.

Mahoney, MEF and Saukett poliovirus strains were grown on human diploid cells (MRC 5) and concentrated on Amicon filter. Concentrated viral suspensions containing 3H-uridine labelled virus were mixed with 14C-amino acid labelled cell extracts containing calf serum and passed through a DEAE-Sepharose column. Fractions eluted at various ionic strengths were analyzed for infectivity, radio-activity and serum content by counter immunoelectrophoresis in the presence of a rabbit anti-calf serum. A peak containing 40--60% of the input infectivity was easily obtained by elution with 0.01 M phosphate buffer at pH 7. The virus peak did not contain 14C or calf serum and its purity was controlled by PAGE-SDS electrophoresis and electron microscopy. This technique may be useful in the large-scale purification of viruses used in the preparation of inactivated vaccines.

Comparison of occurrence of antibodies to human cytomegalovirus as demonstrated by immunofluorescence and indirect hemagglutination techniques.

Indirect immunofluorescence and hemagglutination study of reactivity to human cytomegalovirus immediate early, early, and late antigens revealed that antibody responses to these antigens varies greatly and that hemagglutination is positive when sera contain only antibody to one or both early antigens.

Immunofluorescence technique for detection of human cytomegalovirus (HCMV) antibodies against HCMV-induced late antigens with elimination of immunoglobulin G-receptor staining.

An immunofluorescence technique is described which permits the elimination of nonspecific cytoplasmic staining from late antigen preparations by using in situ a buffer containing Nonidet P-40.

Human cytomegalovirus-induced immediate early antigens: analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis after immunoprecipitation.

Immediate early antigen (IEA) induced in human lung fibroblasts by human cytomegalovirus was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after immunoprecipitation with IEA-positive human sera. Two polypeptides of 76,000 daltons (76K) and 82K were detectable within 90 min after infection. Polypeptides of similar molecular weight were also found in immunoprecipitates of human cytomegalovirus-infected cells nonpermissive for virus replication. IEA is located within the nucleus, although some of the 76K material appears to be located on the outer nuclear membrane. Raising salt concentrations in the extraction buffer increased antigen extraction. The contribution of these IEA polypeptides to IEA nuclear fluorescent staining is discussed.

Comparison of HSV1 DNA-induced and spontaneous transformation of hamster cells.

Primary cultures of hamster embryo fibroblasts were transfected by purified, fragmented herpes simplex virus type 1 (HSV1) strain A44 DNA in the presence of carrier DNA. Control cultures were transfected with carrier DNA only. A cell line transformed by HSV1 DNA (EH/A44 cell line) was established after serial passages, without senescent crisis. A control cell line (EHT cell line), spontaneously transformed in vitro, was established after a 13th passage crisis. Both cell lines originated from the same primary culture thereby allowing the comparison of virus DNA induced and spontaneous transformation. Various parameters were studies for both cell lines and their clones: cell growth in vitro, presence of viral antigens, permissivity to superinfection and oncogenicity.

Late transient expression of human hepatitis B virus genes in monkey cells.

The expression of human hepatitis B virus (HBV) surface (HBS) and e (HBe) antigens has been studied comparatively in monkey and mouse cell lines co-transfected with HBV DNA and the dominant selective marker aminoglycoside 3'-phosphotransferase gene. We have found that the kinetics and stability of expression of the HBS gene varies with the cell lines used. Only a late transient expression of both HBS and HBe is observed between 1 and 5 weeks after transfection in monkey kidney Vero cells transfected with the complete HBV genome, while a permanent expression of HBS and HBe is obtained in mouse cells. HBS and HBe are excreted into the cell culture medium. HBe is expressed in cells transfected with the complete HBV genome, but not with isolated HBS gene. In clones of Vero cells transformed with the HBS gene, HBV sequences were rearranged or lost.

Cloning of the active thymidine kinase gene of herpes simplex virus type 1 in Escherichia coli K-12.

A herpes simplex virus DNA fragment that is produced by digestion with BamHI endonuclease and carries the thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene has been cloned in Escherichia coli. A recombinat plasmid, pFG5, has been analyzed extensively and a detailed restriction map is presented. pFG5 DNA efficiently transforms TK- mouse L cells. The TK coding sequence in the cloned fragment has been localized and a smaller recombinant plasmid, pAG0, also carrying an active TK gene, has been constructed to serve as a more convenient vector for transfer, into TK- cells, of genes previously cloned in E. coli.

Cloning of the herpes simplex virus type 1 thymidine kinase gene in E. coli K 12: a selective marker for gene transfer into animal cells.

The Herpes Simplex Virus type 1 (HSV 1) thymidine kinase (TK) gene, carried by a 3.6 kb Bam H1 DNA fragment, was isolated and ligated to Bam H1 cleaved bacterial plasmid pBR 322. The TK gene, cloned in E. coli, is functional when it is transferred back into Eukaryotic cells, and can thus be used as a selective marker for gene transfer.

Detection by monoclonal antibodies of an antigenic determinant critical for poliovirus neutralization present on VP1 and on heat-inactivated virions.

Hybridoma cell lines were established against poliovirus type 1 (Mahoney) heat-denatured virions (C particles). Each anti-C monoclonal antibody (McAb) immunoprecipitated specifically one of the individualized poliovirus capsid polypeptides VP1, VP2, or VP3. One of the anti-C McAb (C-3), reacting with VP1, neutralized homologous virus and immunoprecipitated infectious D particles. Its properties have been compared to those of a neutralizing anti-D McAb (D-Ic). In contrast with the C-3 antigenic site, the D-Ic epitope was not present on C particles nor on individualized structural polypeptide. This demonstrates that C-3 and D-Ic epitopes represent two independent antigenic determinants, both critical for poliovirus neutralization.

Natural variation of poliovirus neutralization epitopes.

Poliovirus-neutralizing monoclonal antibodies were prepared against type 1, type 2, and type 3 wild laboratory (Mahoney, MEF1, and Saukett) and Sabin vaccine strains. Fifty-five poliovirus laboratory strains and field isolates were assayed by neutralization index test with a panel of homotypic monoclonal antibodies. A total of 27 monoclonal antibodies were used. Two categories of neutralization epitopes were found, i.e., cross-reacting (K), which is present on almost all strains of the same serotype, and strain-specific (V, variable), either wild (VW) or Sabin (VS). Several distinct neutralization epitopes were defined for each of the three poliovirus serotypes in almost every category. The study of antigenic variation of the Sabin type 1 vaccine virus during replication in human intestine showed that the VS neutralization epitope may be lost and even replaced by the VW epitopes of the parental Mahoney virus. A late isolate from a vaccine-fed infant recovered the complete neutralization epitope pattern of the Mahoney strain. Upon in vivo virus replication, a different kind of antigenic variation was also detected in which an epitope lost its function in virus neutralization but kept its antigenic conformation unaltered. Neutralization epitope analysis demonstrated that the presence of VS epitopes on a field isolate suggests the Sabin origin of the strain when the isolate displays the same epitope pattern as the original Sabin virus, or confirms it when the VS epitope(s) is mutually exclusive of VW epitopes. The lack of VS epitopes on a field isolate does not rule out its being of Sabin origin.

Construction of a dominant selective marker useful for gene transfer studies in animal cells.

Biochemically transformed clones of murine, simian and human cells were obtained after transfection with a new dominant selective marker. This marker is a hybrid gene which was constructed with a bacterial neomycin-kanamycin resistance gene coding region and the transcription signals of the herpes simplex virus thymidine kinase gene.