Limited proteolysis of herpes simplex virus glycoproteins that occurs during their extraction from vero cells.

Herpes simplex virus glycoproteins extracted from infected Vero cells can be smaller in apparent size than the same viral products extracted from infected HEp-2 cells. Here we show that the differences in size result primarily from limited proteolysis, during or after their extraction, of the viral glycoproteins made in Vero cells. In the absence of appropriate protease inhibitors, both mature and immature forms of four different glycoproteins specified by herpes simplex virus type 2 were significantly smaller (based on electrophoretic mobilities in acrylamide gels) when extracted from Vero cells than when extracted from HEp-2 cells. Inclusion of certain protease inhibitors in the extraction buffer, however, permitted isolation of immature forms from Vero cells that were indistinguishable in size from the immature forms extracted from HEp-2 cells. Under these conditions, the mature forms of glycoproteins B and E were also indistinguishable by electrophoretic sizing from those made in HEp-2 cells, whereas the mature forms of glycoproteins D and F were smaller, indicating the possibility of differences between Vero and HEp-2 cells in the post-translational processing of glycoproteins D and F.     

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.     

A microplate method for isolation of viruses from infants and children with acute respiratory infections

Between December 1984 and December 1986, a microplate technique was adopted for isolation of viruses from infants and children with acute respiratory infections. By using two kinds of tissue culture microplates, i.e., the HHVM plate, containing human embryonic fibroblast (HEF), HEp-2, Vero and MDCK cells, and the MK plate which contains secondary monkey kidney cells, 1,080 field viruses were isolated from 1,061 (24.9%) out of 4,254 throat swabs. Of these 1,080 isolates, 1,003 (92.9%) were recovered in the HHVM plates and the remaining 77 (7.1%) in the MK plates. With the HHVM plate, influenza A and B viruses were cultivated in MDCK, RS virus in HEp-2, parainfluenza and mumps viruses in Vero, adenoviruses in both HEF and HEp-2, polioviruses in HEF, HEp-2 and Vero, coxsackie B viruses in both HEp-2 and Vero, rhino and echo viruses in HEF, herpes simplex virus in both HEF and HEp-2, and cytomegalovirus in HEF, although MK were more sensitive than Vero to parainfluenza and coxsackie B viruses. There was no difference in the rate of isolation of viruses between the microplate and ordinary tube methods. Cross contamination in the microplates was negligible for routine work.     

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.     

A new plaque system for canine distemper: characteristics of the green strain of canine distemper virus.

A Vero cell adapted Green strain of canine distemper virus (CDV) was tested for its plaque-forming capacity in different cell lines. Plaque formation was observed in HEp-2, BS-C-1, and HeLa cells but not in Vero or dog kidney cells even though replication and cytopathology were observed in the latter cell types. In the cells in which the virus was capable of producing plaques, the plaques were observed within 24 h post infection and continued to increase in size with subsequent cellular destruction such that by 72 h postinfection the size of the plaques approached 0.5 mm. With the use of the plaquing technique, it was possible to demonstrate the thermal lability of the virus as well as the kinetics of adsorption. Thus, it was shown that the half-life of the virus was 125 min at 25 degrees C, 75 min at 35 degrees C, and 65 min at 37 degrees C. The rate of adsorption of CDV to HEp-2 cells was 17.2% in 30 min at 37 degrees C and continued slowly for 4 h before completion. Application of this rapid plaque-forming assay to plaque-reduction tests for CDV antibody and for CDV-infected cells by the infectious center assay are described.     

Redistribution of microtubules and Golgi apparatus in herpes simplex virus-infected cells and their role in viral exocytosis.

Earlier studies have shown that the Golgi apparatus was fragmented and dispersed in herpes simplex virus 1-infected Vero and HEp-2 cells but not in human 143TK- cells, that the fragmentation and dispersal required viral functions expressed concurrently with or after the onset of DNA synthesis (G. Campadelli-Fiume, R. Brandimarti, C. Di Lazzaro, P. L. Ward, B. Roizman, and M. R. Torrisi, Proc. Natl. Acad. Sci. USA 90:2798-2802, 1993), and that in 143TK- cells, but not Vero or HEp-2 cells, infected with viral mutants lacking the UL20 gene virions were glycosylated and transported to extracellular space (J. D. Baines, P. L. Ward, G. Campadelli-Fiume, and B. Roizman, J. Virol. 65:6414-6424, 1991; E. Avitabile, P. L. Ward, C. Di Lazzaro, M. R. Torrisi, B. Roizman, and G. Campadelli-Fiume, J. Virol. 68:7397-7405, 1994). Experiments designed to elucidate the role of the microtubules and of intact or fragmented Golgi apparatus in the exocytosis of virions showed the following. (i) In all cell lines tested (Vero, 143TK-, BHK, and Hep-2) microtubules underwent fragmentation particularly evident at the cell periphery and then reorganized into bundles which circumvent the nucleus. This event was not affected by inhibitors of viral DNA synthesis. We conclude that redistribution of microtubules may be required but is not sufficient for the fragmentation and dispersal of the Golgi apparatus. (ii) In all infected cell lines tested, nocodazole caused fragmentation and dispersal of the Golgi and a far more extensive depolymerization of the microtubules than was seen in untreated, infected Vero or HEp-2 cells. Taxol precluded the depolymerization of the microtubules and fragmentation of the Golgi in both infected cell lines. Neither nocodazole nor taxol affected the exocytosis of infectious virus from Vero, HEp-2, or 143TK- cells infected with wild-type virus. We conclude that the effects of nocodazole or of taxol are dominant over the effects of viral infection in the cell lines tested and that viral exocytosis is independent of the organization of microtubules or of the integrity of the Golgi apparatus. Lastly, the data suggest that herpes simplex viruses have evolved an exocytic pathway for which the UL20 protein is a component required in some cells but not others and in which this protein does not merely compensate for the fragmentation and dispersal of the Golgi apparatus.     

Differential cell membrane labilizing effect of adenovirus type 5 and 12 observed during productive and abortive infection.

Culture fluid of human epitheloid (HEp-2) cells was examined for extracellular lactate dehydrogenase activity as an indicator of cell damage during a 48 h period in which virus replication and changes in cell morphology occurred. Uninfected and adenovirus type 5-infected cells had the same levels of extracellular enzyme activity both before and after the appearance of morphological changes in cells due to virus infection, whereas adenovirus type 12-infected cells showed increased extracellular enzyme activity. Cells infected with either adenovirus type 5 or type 12 had the same total cellular and extracellular lactate dehydrogenase activity. Hydrocortisone, a membrane stabilizing agent, prevented abnormal leakage of lactate dehydrogenase from adenovirus type 12-infected cells, but had no effect on virus replication or total enzyme activity of infected cells. After inoculation of monkey kidney (Vero) cells the yield of progeny adenovirus type 5 virions was greatly reduced and there was no production of adenovirus type 12 virions. The pattern of extracellular lactate dehydrogenase activity of uninfected and adenovirus type 5- and type 12-infected Vero cells was like that with HEp-2 cells. Therefore, production of adenovirus type 12 virions is not necessary for the virus-cell interaction causing cell membrane labilization.     

Cytotoxic activity of Serratia marcescens clinical isolates

Twenty Serratia marcescens isolates from clinical specimens were examined for their cytotoxic activity on four cell lines (HEp-2, Vero, CHO, J774). Most of the isolates were found to be cytotoxic to CHO (70%), Vero (75%) and HEp-2 cells (90%). CHO cells were the most sensitive to cell-free supernatants, followed by HEp-2 and Vero cells. Two strains produced cytotonic toxins which caused elongation of CHO cells. Moreover, twelve isolates (60%) revealed cytotoxic potential to macrophage cell line J774. The results indicate that these bacteria may destroy phagocytes and epithelial cells, which may lead to spread within the host.     

Herpes simplex virus 1 mutant deleted in the alpha 22 gene: growth and gene expression in permissive and restrictive cells and establishment of latency in mice.

R325-beta TK+, a herpes simplex virus 1 mutant carrying a 500-base-pair deletion in the alpha 22 gene and the wild-type (beta) thymidine kinase (TK) gene, was previously shown to grow efficiently in HEp-2 and Vero cell lines. We report that in rodent cell lines exemplified by the Rat-1 line, plating efficiency was reduced and growth was multiplicity dependent. A similar multiplicity dependence for growth and lack of virus spread at low multiplicity was seen in resting, confluent human embryonic lung (HEL) cells. The shutoff of synthesis of beta proteins was delayed and the duration of synthesis of gamma proteins was extended in R325-beta TK+-infected HEL cells relative to cells infected with the wild-type parent, but no significant differences were seen in the total accumulation of viral DNA. To quantify the effect on late (gamma 2) gene expression, a recombinant carrying the deletion in the alpha 22 gene and a gamma 2-TK gene (R325-gamma 2 TK) was constructed and compared with a wild-type virus (R3112) carrying a chimeric gamma 2-TK gene. In Vero cells, the gamma 2-TK gene of R325-gamma 2TK was expressed earlier than and at the same level as the gamma 2-TK gene of R3112. In the confluent resting HEL cells, the expression of the gamma 2-TK gene of the alpha 22- virus was grossly reduced relative to that of the alpha 22+ virus. Electron microscopic studies indicated that the number of intranuclear capsids of R325-beta TK+ virus was reduced relative to that of the parent virus in resting confluent HEL cells, but the number of DNA-containing capsids was higher. Notwithstanding the grossly reduced neurovirulence on intracerebral inoculation in mice, R325-beta TK+ virus was able to establish latency in mice. We conclude that (i) the alpha 22 gene affects late (gamma 2) gene expression, and (ii) a host cell factor complements that function of the alpha 22 gene to a greater extent in HEp-2 and Vero cells than in confluent, resting HEL cells.     

Lactate dehydrogenase release assay from Vero cells to distinguish verotoxin producing Escherichia coli from non-verotoxin producing strains

The Vero cell assay presently used for virulence testing of verotoxigenic Escherichia coli (VTEC) requires at least 48-96 h where cytotoxicity effects are examined under a microscope. Here, a complimentary rapid assay was developed that measures endogenous lactate dehydrogenase (LDH) release from Vero or HEp-2 cells as an indicator of cytotoxicity. Toxin preparations from 24 VTEC strains induced 36-89% LDH from Vero cells and 15-62% LDH from HEp-2 cells in 12-16 h. A verotoxin-positive but enterohemolysin negative strain also showed a similar cytotoxicity effect. In contrast, three VT-negative strains caused only 13-16% LDH from Vero cells and 1-7% LDH from HEp-2 cells. Five presumptive E. coli isolates from naturally contaminated food and clinical sources did not induce significant LDH release from either cell lines. PCR analysis confirmed the presence of vt1 or vt2 genes in E. coli showing positive LDH values. Similarly, RiboPrinter analysis confirmed and identified the test strains as E. coli except for two meat isolates, which were identified as Hafnia alvei. Cytopathic effects of toxin preparations from VTEC revealed severe lysis, vacuole formation and death in Vero cells and multiple vacuoles and cell elongation in HEp-2 cells. The colorimetric cytotoxicity assay described here can provide quantitative data for determining the virulence potential of verotoxigenic E. coli in 12-16 h.     

Growth, plaque assay and immunofluorescent studies on Tataguine virus in cell culture.

The growth characteristics of Tataguine virus were studied in Cercopithecus monkey kidney (Vero); rhesus monkey kidney (LLC-MK2), baby hamster kidney (BHK-21); porcine kidney (PK-15), mouse fibroblasts (L-929) and Aedes albopictus cell monolayers. The virus replicated without producing any cytopathology in Vero, BHK-21 and Aedes albopictus: but not in the other three cell culture systems. Two or three subsequent serial blind passages in those cultures supporting the growth of the virus did not produce any appreciable increase in virus titre. Immunofluorescent staining of inoculated Vero cells demonstrated the presence of Tataguine virus antigen in the cytoplasm of infected cells. Plaques 1--1.5 mm in diameter were produced only in Vero cell culture. In neutralization tests performed on Tataguine virus, immune mouse and hamster sera, higher antibody titres were obtained by plaque reduction than mouse protection tests.     

Molecular Genetics of Herpes Simplex Virus II. Mapping of the Major Viral Glycoproteins and of the Genetic Loci Specifying the Social Behavior of Infected Cells

We have mapped the location in herpes simplex virus (HSV) DNA of (i) three mutations at different loci (syn loci) which alter the social behavior of infected cells from clumping of rounded cells to polykaryocytosis, (ii) a mutation which determines the accumulation of one major glycoprotein [VP8.0(C(2))], and (iii) the sequences encoding four major virus glycoproteins [VP8.0(C(2)), VP7(B(2)), VP8.5(A), and VP19E(D(2))]. The experimental design and results were as follows. (i) Analysis of HSV-1 x HSV-2 recombinants showed that the sequences encoding the VP19E(D(2)) glycoprotein map in the S component, whereas the sequences encoding the other three major glycoproteins are in two locations in the L component of HSV DNA. The templates specifying the HSV-1 and HSV-2 glycoprotein VP8.0(C(2)) appear not to be colinear; we isolated recombinants specifying glycoproteins comigrating in sodium dodecyl sulfate-polyacrylamide gels with VP8.0(C(2)) of both HSV-1 and HSV-2. (ii) Marker rescue of a ts mutant defective in accumulation of glycoprotein VP7(B(2)) showed that the mutation maps within a region containing the sequences encoding that glycoprotein. (iii) Marker transfer experiments involving transfection of rabbit skin cells with donor HSV-1(F) DNA and fragments from several donor strains causing fusion of Vero or both Vero and HEp-2 cells revealed the existence of three syn loci specifying the social behavior of cells and one locus (Cr) determining the accumulation of glycoprotein VP8.0(C(2)). The Cr locus maps to the right of the template specifying VP8.0(C(2)) glycoprotein. Loci syn 1 and syn 2 map at or near the Cr locus but can be segregated from it. Locus syn 3 maps at or near the template specifying glycoproteins VP7(B(2)) and VP8.5(A). The expression of mutations in the syn 1 and syn 3 loci appear to be cell type dependent, in that recombinants with these mutations fuse Vero cells but not HEp-2 cells. Recipients of the syn 2 locus or of both syn 2 and syn 1 loci fuse both Vero and HEp-2 cells.     

Tumor localizing agents: the transport of meso-tetra(p-sulfophenyl) porphine by Vero and HEp-2 cells in vitro.

The mechanism of transport of the tumor localizing agent, meso-tetra(p-sulfophenyl)porphine (TPPS4), was investigated in Vero and HEp-2 cells in vitro. Vero cells proved to be basically impermeable to the porphyrin, but a slow transport was observed. The uptake was linear with time and appeared to be carrier mediated, as it was strongly inhibited by cyanide or low temperature and demonstrated saturation kinetics. Transport in HEp-2 cells was more rapid and non-linear, reaching a plateau after about 2 h. Analysis of this uptake over a 20-fold range of porphyrin concentration revealed it to be biphasic. A low affinity, high capacity component appeared to be unsaturable and was unaffected by low temperature or metabolic inhibitors. This system is probably one of a passive diffusion. The high affinity, low capacity phase is probably carrier mediated. The tumor cells appear to be "leaky" to the porphyrin, with respect to the Vero cells. This may explain part of the localizing ability of TPPS4.     

Response of various cell lines to Escherichia coli toxic products.

Seven cell lines were compared with Y-1 and Vero cells for morphological response to E. coli toxic products. FL and WI-38, like Y-1 and Vero, responded to heat-labile enterotoxin. FL, HEp-2, HeLa, and BGM, like Vero but not Y-1, responded to heat-labile cytotoxin. FL was comparable to Vero in sensitivity to these two toxic products.     

In Vitro Selection of Variants of Herpes Simplex Virus Type 1 Which Differ in Cytopathic Changes

To analyze the mechanisms for in vitro emergence of the syncytial variants of herpes simplex virus type 1 (HSV-1), several cell lines were infected with a mixture of equal amounts of two HSV-1 variants, one syncytial and the other non-syncytial, and changes in their relative abundance were monitored during passage. With a combination of two variants of the Miyama strain of HSV-1, the syncytial variant became dominant during passage in Vero, RK-13 and FL cells. On the other hand, the ratios of the two variants remained around 1:1 during the passage in HEp-2, MGC and HEL cells. In another set of variants of the SKO strain of HSV-1, the outcomes were different from those of the Miyama strain in the FL, MGC and HEp-2 cells. The ratios of the two variants remained around 1:1 during passage in FL cells, while the non-syncytial variant became dominant during passage in MGC and HEp-2 cells. In addition, we examined the effects of a complement and interferon-β (IFN-β) on the outcome of the selection. As a result, the complement slowed the selection of a syncytial variant, whereas IFN-β facilitated it.     

In vitro potency assay for yellow fever vaccines: comparison of three vero cell lines sources.

Quality control of Yellow Fever vaccines performed by Control Authorities prior to marketing vaccines batches requires in vitro potency assays. The two currently available methods are the plaque formation assay and the cytopathic effect assay based on the use of porcine kidney PS cells or monkey kidney Vero cells. Among several sources of variation in virus titration, the cell systems are considered as important issues and Quality Assurance strongly recommends working with cell banks from certified suppliers. The aim of our study was to compare the behaviour and the sensitivity of three Vero cell sources obtained from ATCC, WHO and EP used at different passage levels in a plaque formation test. The conclusion of this work was that the yellow fever live attenuated virus titration, adapted in Vero cell lines appeared as a reliable method applicable for routine in vitro potency assay. The comparison of Vero cell lines, originated from three different sources, showed that they could be equally used as substrates by laboratories having the basic facility of cell culture, without influence on the final viral titre.     

狂犬病毒蚀斑方法在病毒研究和疫苗研制中的应用

本文采用狂犬病毒CTN-1和4aC株,经Vero细胞传代适应后,以Vero细胞为培养基质,建立了狂犬病毒蚀斑试验和蚀斑减少试验的方法。目前已将此方法应用于病毒鉴定、病毒克隆、病毒滴定以及抗狂犬血清的检测,并取得了较好的结果。     

Accumulation of herpes simplex virus type 1 early and leaky-late proteins correlates with apoptosis prevention in infected human HEp-2 cells

We previously reported that a recombinant ICP27-null virus stimulated, but did not prevent, apoptosis in human HEp-2 cells during infection (M. Aubert and J. A. Blaho, J. Virol. 73:2803-2813, 1999). In the present study, we used a panel of 15 recombinant ICP27 mutant viruses to determine which features of herpes simplex virus type 1 (HSV-1) replication are required for the apoptosis-inhibitory activity. Each virus was defined experimentally as either apoptotic, partially apoptotic, or nonapoptotic based on infected HEp-2 cell morphologies, percentages of infected cells with condensed chromatin, and patterns of specific cellular death factor processing. Viruses d27-1, d1-5, d1-2, M11, M15, M16, n504R, n406R, n263R, and n59R are apoptotic or partially apoptotic in HEp-2 cells and severely defective for growth in Vero cells. Viruses d2-3, d3-4, d4-5, d5-6, and d6-7 are nonapoptotic, demonstrating that ICP27 contains a large amino-terminal region, including its RGG box RNA binding domain, which is not essential for apoptosis prevention. Accumulations of viral TK, VP16, and gD but not gC, ICP22, or ICP4 proteins correlated with prevention of apoptosis during the replication of these viruses. Of the nonapoptotic viruses, d4-5 did not produce gC, indicating that accumulation of true late gene products is not necessary for the prevention process. Analyses of viral DNA synthesis in HEp-2 cells indicated that apoptosis prevention by HSV-1 requires that the infection proceeds to the stage in which viral DNA replication takes place. Infections performed in the presence of the drug phosphonoacetic acid confirmed that the process of viral DNA synthesis and the accumulation of true late (gamma(2)) proteins are not required for apoptosis prevention. Based on our results, we conclude that the accumulation of HSV-1 early (beta) and leaky-late (gamma(1)) proteins correlates with the prevention of apoptosis in infected HEp-2 cells.