Growth In Vero Cells Research Articles

Application of a serum-free medium for the growth of Vero cells and the production of reovirus.

Two strains of reovirus (serotype 1 Lang/TIL and serotype 3 Dearing/T3D) were propagated in Vero cells grown in stationary or agitated cultures in a serum-free medium, M-VSFM. Solid microcarriers (Cytodex-1) were used to support cell growth in agitated cultures with a normal doubling time of 25 h. Cell yields of 1 x 10(6) cells/mL were obtained from an inoculum of 2 x 10(5) cells/mL in 4 days in microcarrier cultures. The growth profile and cell yield was not significantly different from serum-supplemented cultures. The virus titer increased by 3-4 orders of magnitude over a culture period of 150 h. The maximum virus titer in stationary cultures reached >1 x 10(9) pfu/mL for both strains of reovirus in M-VSFM. M-VSFM also supported high viral yields in microcarrier cultures. Both the specific productivity and final viral yield was higher in M-VSFM than serum-supplemented cultures. The high viral productivity suggests that this is a suitable system for the production of reovirus as an oncolytic agent for human therapeutic use.

Metal content and biochemical analyses of a recombinant collagenase PrtV from Vibrio parahaemolyticus.

PrtV is an extracellular metalloprotease of Vibrio parahaemolyticus and regarded as a collagenase. Inductively coupled plasma-optical emission spectrometry analysis indicated that the recombinant PrtV contains 1 mol of zinc per mol of the native enzyme. On the basis of a kinetic study using 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA, the specific substrate for bacterial collagenase) as a substrate, it was suggested that metal ions may play a significant role in the binding and catalytic steps of the substrate. PrtV hydrolyzed type I, II, III, and IV collagens; however, it did not hydrolyze type V. In addition, the hydrolysis of native proteins and synthetic substrates revealed that PrtV possesses higher activity toward collagen and collagen-like sequences. The result of the thermal stability study indicated that PrtV was thermostable up to 40 C; at 50 C, stability gradually decreased. In addition, PrtV showed higher storage stability at -20 and 4 C, respectively, than at 25 C. Compared with collagenases from Clostridium histolyticum and Vibrio alginolyticus, PrtV was immunologically different and had no significant effect on the growth of CHO, HeLa, and Vero cells. Taken together, the results of the studies described in this paper advance our knowledge concerning the metal content and biochemical properties of PrtV.

A simplified objective method for quantification of peste des petits ruminants virus or neutralizing antibody

A simplified and standardized assay based on haemagglutination of infected culture supernatants was developed to detect peste des petits ruminants (PPR) virus growth in vero cells and to quantify PPR neutralizing antibody. Virus titres estimated by visual reading of cytopathic effects as a criterion were compared with those estimated by haemagglutination of infected supernatants and no statistically significant differences were seen between them. During quantification of PPR antibodies, the titres based on haemagglutination of supernatants on day 5 post-infection had a high sensitivity, specificity and agreement in qualititative comparison with those determined by visual examination of cytopathic effects. In quantitative comparison, the correlation coefficient between serum neutralization titres estimated by visual examination of cytopathic effects or haemagglutination of supernatants was 0.96, when haemagglutination was done on day 5 post-infection. The virus and serum neutralization titres can thus be estimated objectively using the haemagglutination of supernatants as criterion to measure endpoints. The haemagglutination–inhibition titres also correlated well with serum neutralization titres with a coefficient of 0.78. Thus the haemagglutination–inhibition test appears to be a suitable alternative to the serum neutralization test for quantification of PPR neutralizing antibody.

Biological characteristics of genetic variants of Urabe AM9 mumps vaccine virus

The Urabe AM9 mumps vaccine is composed of a mixture of variants distinguishable by a difference at nucleotide (nt) 1081 of the hemagglutinin-neuraminidase (HN) gene (Brown, E.G., Dimock, K., Wright, K.E., 1996. The Urabe AM9 mumps vaccine is a mixture of viruses differing at amino acid (aa) 335 of the hemagglutinin-neuraminidase gene with one form associated with disease. J. Infect. Dis. 174, 619–622.). Further genetic and biological variation was detected in plaque purified viruses from the Urabe AM9 vaccine by examining the HN gene sequence, plaque morphology, cytopathic effects and growth in Vero cells, and temperature sensitivity (ts). Infection of Vero cells with plaque purified viruses with a G at nt 1081 of the HN gene produced large, clear plaques, caused significant CPE early after infection but yielded lower titres of virus than other purified viruses. None of these viruses were ts. In contrast, half of the plaque purified viruses with an A at nt 1081 were sensitive to a temperature of 39.5°C. These viruses produced small plaques, caused significant CPE and grew to low titres. Two ts viruses possessed a unique aa substitution at aa 468 of HN. The remaining A 1081 viruses were not ts, produced large plaques but little CPE, and grew to titres 10-fold higher than the G 1081 viruses. Isolates of Urabe AM9 associated with post-vaccination illness were similar to these non-ts A 1081 viruses, but could be further sub-divided into two groups on the basis of a difference at aa 464 of HN. The post-vaccination isolates may represent insufficiently attenuated components of the vaccine, while the G 1081 and ts subset of A 1081 viruses may be more fully attenuated.

V and C Proteins of Measles Virus Function as Virulence Factors in Vivo

The measles virus (MV) P gene encodes three proteins: the P protein and two nonstructural proteins, C and V. Because the functions of both the C and V protein are unknown, we used MV C (C−) and V (V−) deletion recombinants generated by the MV reverse genetics system (F. Radecke, P. Spielhofer, H. Schnieder, K. Kaelin, M. Huber, C. Dotsch, G. Christiansen, and M. A. Billeter 1995. EMBO J. 14, 5773–5784). Compared to parental vaccine strain, Edmonston (Ed) MV, both had normal growth and cytopathic effects in Vero cells and showed similar growth kinetics in human neuroblastoma SK-N-MC cells and in primary mouse neurons expressing the MV receptor, CD46. However, in vivo, using YAC-CD46 transgenic mice as a model for MV induced CNS disease (M. B. A. Oldstone, H. Lewicki, D. Thomas, A. Tishon, S. Dales, J. Patterson, M. Manchester, D. Homann, D. Naniche, and A. Holz 1999. Cell 98, 629–640), C− and V− viruses differed markedly from wt Ed(V+C+) virus. Newborn mice inoculated with as little as 103 PFU of Ed strain became ill and died after 10–15 days. In contrast, those inoculated with 103 or 104 PFU of MV C− or MV V− showed significantly fewer and milder clinical symptoms and had a lower mortality. A total of 105 PFU V− virus were required to kill most YAC-CD46 mice, and less than half (44%) were killed with a corresponding dose of MV C−. Immunohistochemical staining for MV antigens showed similar extents of spread for MV C− and MV Ed but restricted spread for MV V− throughout the brain. Viral load and transcription were markedly reduced for V− but not for C−. Multiple cytokines and chemokines were equivalently upregulated for all three viruses. Therefore, MV C and V proteins encode virulence functions in vivo and likely operate via separate mechanisms.

Effect of a soluble pseudo-receptor on verotoxin 2-induced toxicity

To neutralize the toxicity of verotoxin (VT) produced by Escherichia coli type O-157, a soluble pseudo-receptor (Lyso Gb3) was synthesized with the deacylated form of the natural receptor, globotrisylceramide (Galalpha 1-4Galbeta1-4-glucosylceramide; Gb3). In this study, we evaluated the characteristics and pharmacological effects of Lyso Gb3, using VT2. It was confirmed that Lyso Gb3 specifically recognized VT2. Lyso Gb3 itself had little influence on the in-vitro growth of Vero cells, but markedly augmented VT2-induced cytotoxicity. In addition, the VT2-induced killing of mice was not decreased, but was, rather, increased by Lyso Gb3. These results indicate that the soluble pseudo-receptor Lyso Gb3 recognized VT2. However, it did not reduce, but, rather, enhanced, VT2-induced toxicity in the presence of the natural receptor, although Lyso Gb3 alone had no toxicity.

A simplified and standardized neutralization enzyme immunoassay for the quantification of measles neutralizing antibody

A simplified and standardized neutralization enzyme immunoassay (Nt-EIA) was developed to detect measles virus growth in Vero cells and to quantify measles neutralizing antibody. Heat-inactivated sera were diluted serially 4-fold and tested in duplicate. The 50% reduction point (50%RP) of virus growth was calculated using the Reed–Muench formula and the neutralizing antibody titre of test sera was converted into mIU/ml by comparing their 50%RP with that of the international standard serum. The optimal virus input and incubation time were found to be 50–100 plaque forming unit (PFU)/well and 64–72 h, respectively. The simplified Nt-EIA had a good reproducibility with only 3.7–4.2% of duplicate tests having a ratio >4 in an evaluation of intra assay variation and the coefficients of variance were 2–9% in an evaluation of inter assay variation. In addition, the simplified Nt-EIA had a high sensitivity(98.6%), specificity (100%) and agreement (98.8%) in qualitative comparison with plaque reduction neutralization test (PRNT). In quantitative comparison, the correlation coefficient between Nt-EIA and PRNT was 0.83 without log transformation or 0.77 after log transformation and 90% of 61 positive sera had a ratio <4 between antibody titre tested by the two methods. The simplified Nt-EIA is thus a suitable alternative to the PRNT for the quantification of measles neutralizing antibody.

Attachment, spreading and growth of VERO cells on microcarriers for the optimization of large scale cultures

The VERO cell attachment, spreading and growth were measured as a function of the substrate and temperature used for cell cultivation, the presence of fetal calf serum (FCS) in the medium and the initial cell inoculum used for cultivation on MCs. The data show that the cell attachment kinetics were comparable at RT or 37 °C, a higher rate of cell attachment occurred to MCs and the presence of FCS inhibited the cell attachment to glass or plastic but not to MCs. The cell spreading, in general higher at 37 °C, was dependent on the presence of FCS, comparable on glass or plastic substrate and lower on MCs. The spread of VERO cells over MCs was fully dependent on the presence of FCS and decreases progressively with a delayed addition of FCS into the medium. The cell detachment by trypsin was slower from MCs and the cells recovered showed lower viability and reattachment. Better results of detachment, viability and reattachment were obtained by treatment with the trypsin at pH of 8 instead of 7. The lower was the number of cells/MC for the initial inoculum, the higher was the percent of unoccupied MCs (with 1 cell/MC we had 35.6% of unoccupied MCs), which were shown to remain uncovered during the whole period of culture. With an initial inoculum of 4, 6 and 8 VERO cells/MC, respectively 46%, 76% and 83% of the MCs were totally covered by cells after 7 days, the cultures showing at this time, respectively, 5.1 × 105, 8.8 × 105 and 1.8 × 106 cells/ml, which represented a biomass production of respectively 8.5x, 9.7x and 15.5x. When compared to 175 cm2 T-flasks, using the same amount of medium, a VERO cell culture on 2 mg/ml of MCs offers about 10 times more available surface for cell growth and allowed the obtention of 7 times more cells. The optimization procedures concerning initial steps of VERO cell cultures, such as the attachment, spreading and growth as a function of parameters like initial cell inoculum and medium supplementation are of special interest mainly due to the perspective of a large use of VERO cell cultures for human viral vaccine production.

Influence of hydrodynamic shear stress on microcarrier-attached cell growth: Cell line dependency and surfactant protection

Animal cell injuries due to fluid-mechanical forces generated by hydrodynamic mixing in bioreactors could become more detrimental for microcarrier systems. In this work, the effects of cell protective surfactants such as pluronic F68 (F68) and polyvinyl alcohol (PVA) were investigated on microcarrier-grown cells under various hydrodynamic stress conditions. Experimental results indicated that adding 0.2% F68 or 0.2% PVA in media enhanced Vero Cell growth against fluid-mechanical forces, but not CHO-K1 and BHK-21 cells. Although affecting the specific growth rate of Vero cells, hydrodynamic shear forces only slightly influenced CHO-K1 and BHK-21 cells. The cellular sensitivity against fluid-mechanical forces for these three cell lines had the following order: Vero cells > CHO-K1 cells > BHK-21 cells. It seems that, the hydrodynamic effects on microcarrier-grown cells and the effectiveness of surfactant protection are heavily dependent on the culture cell type.

Measles virus attenuation associated with transcriptional impediment and a few amino acid changes in the polymerase and accessory proteins.

Measles virus (MV) isolated in B95a cells, a marmoset B-cell line, retains full pathogenicity for cynomolgus monkeys, while its derivative obtained by adaptation to the growth in Vero cells, a monkey kidney cell line, loses the pathogenic potential (F. Kobune, H. Sakata, and A. Sugiura, J. Virol. 64:700-705, 1990). Here, we show with a pair of strains, a fresh isolate (9301B) in B95a cells and its Vero cell-adapted form (9301V), that the in vivo attenuation parallels the decrease of replication and syncytium-inducing capabilities in the original B95a cells and that these in vitro phenotypes are attributable to impediment of transcription, which is already obvious at the level of primary transcription catalyzed by the virion-associated RNA polymerase. On the other hand, cell fusion assays detected no functional difference between the glycoproteins of the two viruses. Essentially the same transcriptional impediment with reduced syncytium induction following Vero cell adaptation was found with two other pairs of strains that had been similarly prepared. Nucleotide sequence comparison between the 9301B and 9301V viruses revealed that a few (at most five) amino acid changes, which sporadically took place in the polymerase (L and P proteins) and/or accessory V and C proteins, were responsible for the in vitro and in vivo attenuation through adaptation to growth in Vero cells.

ALTERATIONS IN THE GROWTH AND ADHESION PATTERN OF VERO CELLS INDUCED BY NUTRITIONAL STRESS CONDITIONS

The pattern of growth, adhesion and protein synthesis in Vero cells submitted to nutritional stress conditions was investigated. The control cells presented a characteristic pattern, with monolayer growth, while the stressed cells presented multilayered growth, with aggregate or spheroid formation which detached on the flask surface and continued their growth in another region. In the soft agar assay, with reduced amount of nutrients, only the stressed cells presented growth, indicating physical and nutritional independence. A 44-kDa protein was observed in stressed cells and was absent in non-stressed cells. The adhesion index and fibronectin synthesis and distribution were altered in stressed cells. After confluence, control cells presented fibronectin accumulation in lateral cell–cell contact regions, while this fibronectin accumulation pattern was not observed in stressed cells. These alterations may be responsible for the multilayered growth and decreased adhesion index observed in stressed cells which were transformed by nutritional stress conditions.

Cell metabolism and medium perfusion in VERO cell cultures on microcarriers in a bioreactor

Parameters of VERO cell growth and metabolism were studied in cultures performed on microcarriers (MCs) using a bioreactor with a working capacity of 3.7 l. Kinetic studies of VERO cell growth in batch, semi-batch and perfusion cultures using concentrations of 2 and 10 mg/ml of MCs showed that a high concentration of MCs (10 mg/ml) and the use of medium perfusion allowed the attainment of higher final yields of VERO cells (6 × 106 cells/ml after 10 days of culture). Perfusion also allowed better use of MCs as indicated by the observation of about 100% of MCs totally covered by cells and the appearance of multilayered cells on 64% of MCs after 13 days of VERO cell culture with 2 mg/ml of MCs. Concerning the concentration of nutrients in the cultures, the medium perfusion was able to sustain suitable levels of galactose and glutamine, which quickly decreased after 4 days in batch cultures. The air inlet in the batch cultures was capable of eliminating the NH4+ which accumulated in the medium culture. Lactate accumulated during the first days of culture but then was utilized by the cells and decreased along the culture time. The optimization of VERO cell cultures on microcarriers as indicated by the concentration of MCs, medium perfusion and air inlet is discussed.

Construction and Characterization of a Replication-Defective Herpes Simplex Virus 2 ICP8 Mutant Strain and Its Use in Immunization Studies in a Guinea Pig Model of Genital Disease

A replication-defective mutant of herpes simplex virus 2 (HSV-2) was engineered by replacing the ICP8 gene of HSV-2 strain 186 with an ICP8–lacZ fusion gene from the herpes simplex virus 1 (HSV-1) HD-2 mutant strain. The resulting virus, HSV-2 5BlacZ, is defective for growth in Vero cells but is capable of growth in a cell line that expresses HSV-1 ICP8. In Vero cells, the mutant virus is defective for DNA synthesis but is able to express many viral proteins at levels similar to those of wild-type virus, including several of the late kinetic class. SDS–PAGE and Western blot analysis demonstrated the expression of glycoproteins B and D by 5BlacZ in Vero cells. Initial studies have shown that immunization with 5BlacZ protects guinea pigs from intravaginal HSV-2 challenge. Immunized animals had less severe genital skin disease and reduced replication of the challenge virus in the genital tract during primary infection and reduced episodes of recurrent disease. Thus, HSV-2 ICP8 shows gene regulatory properties similar to those of HSV-1 ICP8, and this HSV-2 ICP8 mutant virus shows a phenotype similar to those of HSV-1 ICP8 mutant strains. Replication-defective mutants of HSV-2 offer a potential vaccine approach for immune intervention against HSV-2 genital disease and latent infection.

The Product of a 1.9-kb mRNA Which Overlaps the HSV-1 Alkaline Nuclease Gene (UL12) Cannot Relieve the Growth Defects of a Null Mutant

Alkaline nuclease, a relatively abundant viral phosphoprotein in herpes simplex virus type 1 (HSV-1)- or HSV-2-infected cells, is encoded by a 2.3-kb mRNA (R. H. Costa, K. G. Draper, L. Banks, K. L. Powell, G. Cohen, R. Eisenberg, and E. K. Wagner, 1983.J. Virol.48, 591–603). This mRNA is a member of a family of five unspliced 3′-coterminal messages. Costaet al.proposed that another member of this family of mRNAs (1.9-kb) may encode an N-terminally truncated protein which shares its carboxy-terminus with the alkaline nuclease protein. We previously described the isolation of AN-1, a deletion/insertion mutant of the alkaline nuclease gene (S. K. Weller, R. M. Seghatoleslami, L. Shao, D. Rowse, and E. P. Carmichael, 1990.J. Gen. Virol.71, 2941–2952). The deletion in AN-1 would be predicted to abolish gene products of both the 2.3- and the 1.9-kb mRNAs. To investigate whether the putative truncated version of alkaline nuclease encoded by the 1.9-kb mRNA has enzymatic activity and plays a role in the viral life cycle, a viral mutant (AN-F1) was constructed which is predicted to abolish the gene product of the 2.3-kb mRNA (full-length alkaline nuclease) but leave intact the putative product of the 1.9-kb mRNA. Using a highly sensitive polyclonal antiserum raised against a bacterially expressed full-length alkaline nuclease, we observed a 60-kDa protein in KOS- and AN-F1-infected cells but not in AN-1-infected cells. This suggests that the 60-kDa protein is likely to be expressed from the 1.9-kb mRNA; the open reading frame is now designated UL12.5. Despite the presence of the 60-kDa band, AN-F1 failed to exhibit any alkaline exonuclease activity. This result suggests that the truncated polypeptide (UL12.5) is not enzymatically active, has low levels of activity, or possesses enzymatic activity which is not detected because of the low abundance of the polypeptide. AN-1 and AN-F1 are both severely restricted with respect to growth in Vero cells, as viral yields are 100- to 1000-fold lower than those of wild-type virus. We previously reported that the major defect in AN-1 is in the ability of DNA-containing capsids which form in the nucleus to mature into the cytoplasm (L. Shao, L. M. Rapp, and S. K. Weller, 1993.Virology196, 146–162); AN-F1 exhibits the same defect. These results indicate that although the 1.9-kb mRNA encodes a 60-kDa protein presumably from the UL12.5 open reading frame, this polypeptide cannot substitute for the full-length UL12 product.

Rapid Recovery of Rinderpest Virus in Vero Cells from Fresh Infected Tissues by Fusion and Co-culture Methods

Abstract Singh, R.K, Bhat, P.N. and Bhat, P.P. 1995. Rapid recovery of rinderpest virus in vero cells from fresh infected tissues by fusion and co-culture methods. J. AppL Anim. Res., 7: 145–151. Two virulent rinderpest virus isolates (Hisar and Bangalore) and one caprinised strain known as goat tissue virus (GTV) were adapted to in vitro growth in vero cells by fusion and co- culture methods. Both these methods were found to be efficient for rapid recovery of rinderpest virus but the co-culture technique was simpler in comparison to the fusion method where more stringent fusion conditions were required. The co-culture technique can easily be adopted for use in any virology laboratory engaged in routine virological work.

Impairment of multicycle influenza virus growth in Vero (WHO) cells by loss of trypsin activity.

We demonstrated that influenza virus replication in Vero (WHO) cells, a subline of African green monkey kidney cells, is impaired by rapid inactivation of trypsin in the culture fluids. Trypsin inactivation was caused by a factor secreted by Vero cells into the media. Repeated addition of trypsin to the culture medium of influenza virus-infected Vero cells restores the multicycle growth pattern of influenza A virus strains, allowing high yields to be obtained at a low multiplicity of infection. These findings may permit efficient use of Vero (WHO) cells in the production of influenza vaccines.

Inhibition of human cytomegalovirus in culture by alkenyl guanine analogs of the thiazolo[4,5-d]pyrimidine ring system

A series of alkyl and alkenyl guanine analogs containing a thiazolo[4,5-d]pyrimidine ring system were prepared by reaction of the appropriate alkyl halide with the sodium salt of the heterocycle. In preliminary antiviral efficacy evaluations against laboratory strains of both human cytomegalovirus (HCMV) and herpes simplex virus types 1 and 2, it was determined that two of the compounds (T70072 and T01132) were more active and less toxic in stationary-phase cell monolayers than were the other derivatives tested. T01132 and T70072, which have 2-pentenyl and 3-methyl-2-butenyl moieties attached to position 3 of the 5-aminothiazolo[4,5-d]pyrimidine-2,7-dione, respectively, were then more extensively evaluated for anti-HCMV activity. The concentrations of T01132 and T70072 required to inhibit HCMV by 50% in plaque reduction assays were approximately 0.5 and 6.8 microM, respectively. These two compounds inhibited the growth of KB, MRC-5, or Vero cells at concentrations of 75 to 150 microM, depending upon the cell line. In bone marrow progenitor cells T01132 was slightly less toxic than ganciclovir (DHPG). The 50% inhibitory concentrations of T01132 against clinical isolates and DHPG-resistant strains of HCMV were approximately the same as those obtained for laboratory strains of HCMV (approximately 0.5 microM). When tested in combination with DHPG, the resultant antiviral activity was determined to be additive but not synergistic. Experiments performed using variations of the viral multiplicity of infection (MOI) demonstrated that T01132 was more active than DHPG at a low MOI (0.002 or 0.02). However, when a higher MOI (0.2 or 2.0) was used, DHPG was more efficacious than T01132. In experiments in which drug was added at various times post-viral infection, T01132 was most effective when added within the first 24 h post-HCMV infection while DHPG was able to protect cells in this assay system when added up to 48 h postinfection, indicating that T01132 is exerting its antiviral effect on events leading up to and possibly including viral DNA synthesis. The data presented in this report suggest that the antiviral activity of alkenyl-substituted thiazolopyrimidine derivatives may represent a mechanism of action against herpesviruses alternative to that of classical nucleoside analogs such as acyclovir or DHPG.

A comparison of monoclonal antibody productivity in different hollow fiber bioreactors

Cell culture in hollow fiber bioreactors has been used as a method for large-scale production of monoclonal antibodies, viruses, cell-associated proteins and cancer antigens. We have examined an important variable in culturing cells in hollow fiber bioreactors: bioreactor composition. Eight different bioreactor designs which varied in nominal molecular weight cutoff, surface area, fiber material and ultra filtration rate were compared in large-scale hollow fiber cultures. A standard protocol utilizing the hybridoma 3C11 (ATCC HB 8511) or African green monkey kidney cells (Vero cells, ATCC CRL 1587) was designed so that the only variable examined was the hollow fiber bioreactor in use. The results suggest that surface area has little effect on antibody productivity, while fiber composition and ultrafiltration rate may play an important role. Vero cell growth was affected by both fiber composition and ultrafiltration rate.

Transinhibition of herpes simplex virus replication by an inducible cell-resident gene encoding a dysfunctional VP19c capsid protein

This study demonstrates that cells expressing a dysfunctional analog of a herpes simplex virus (HSV) capsid protein inhibits HSV replication. Vero cell lines expressing HSV-1 capsid protein VP19c/β-galactosidase fusion proteins were constructed and tested for their kinetics of expression, intracellular location, and ability to interfere with HSV replication. Two chimeric genes were constructed for these studies. The larger chimeric gene encodes the amino terminal 327 amino acids (aa) of VP19c fused to the carboxy terminal 1026 aa of β-galactosidase, and the shorter chimeric gene encodes VP19c aa 1–30 and 302–327 fused to the carboxy-terminal 1026 aa of β-galactosidase. Cell lines V32G-1 and V32G-2 containing the larger and the shorter chimeric genes, respectively, were isolated after cotransfection with plasmid pSV2-neo DNA, cell selection, and limiting-dilution cloning. The chimeric VP19c/β-galactosidase genes resident in V32G-1 and V32G-2 cell lines were induced by early gene products of superinfecting wild-type HSV-1 and HSV-2, but were not constitutively expressed. The hybrid proteins expressed in infected V32G-1 and V32G-2 cells both colocalized with infected cell protein 8 (ICP8) into virus-replicative compartments in the cell nuclei. HSV-1 and HSV-2 growth in V32G-1 cells (which express the larger chimeric gene) was significantly reduced compared to growth in V32G-2 and control Vero cells. The data suggest that the larger VP19c/β-galactosidase hybrid protein interferes with virus capsid assembly or morphogenesis in a competitive manner. Results also demonstrate that a small portion of VP19c containing the predicted endoplasmic reticulum signal sequence for this capsid protein (aa 1–30) promotes incorporation of the VP19c/β-galactosidase fusion proteins into nuclear viral replication compartments.

Effect of High Glucose on Cellular Proliferation and Lipid Peroxidation in Cultured Vero Cells

Nephropathy is common among the diabetic population. However, the molecular mechanisms by which hyperglycemia can cause alteration in kidney structure and function is not known. In this study, we examined the effect of high glucose levels on cellular growth and membrane lipid peroxidation in Vero cells, an African green monkey kidney cell line. Cell growth was assessed by a tetrazolium salt reduction test (MTT); and lipid peroxidation was measured by thiobarbituric acid reactivity. Our results show that elevated levels of glucose (25, 50 and 100 mM) can cause up to 50% reduction in cell growth in cultured Vero cells as compared with controls (8 mM). Also, we observed a significant increase in the amount of oxidative damage in Vero cells cultured with elevated glucose concentrations. This study demonstrates that hyperglycemia can cause increased lipid peroxidation and affect the cellular proliferation in a monkey kidney cell line.