Susceptibility of continuous lines of monkey kidney cells to influenza and parainfluenza viruses in the presence of trypsin

LLC-MK2, GMK AH-1, BSC-1, and Vero cells were compared in titrations of recent isolates and laboratory strains of influenza A and B and parainfluenza types 1, 2, and 3 viruses. About the same titres, as determined by haemadsorption in cell cultures, were obtained in LLC-MK2, GMK AH-1, and BSC-1 cells when trypsin had been added to the medium, whereas the Vero cells were less sensitive to the influenza virus strains tested. Virus titres were usually low in the absence of trypsin. A laboratory strain of parainfluenza 2 virus reached about the same titres in medium without as in medium with trypsin, possibly owing to prior adaptation by passages in Vero cells. Comparative titrations of influenza A, and parainfluenza 1 and 3 viruses suggested the same susceptibility of LLC-MK2 cells with trypsin as of primary monkey kidney cells. Re-isolation experiments from 38 clinical specimens showed LLC-MK2 cells to be as efficient as primary monkey kidney cells for isolation of influenza and parainfluenza viruses, whereas the susceptibility of the other cell lines to clinical material has not yet been tested on a larger scale. It is concluded that a continuous line of monkey kidney cell culture may be acceptable as an alternative to primary monkey kidney cells for the isolation of influenza and parainfluenza viruses from patients.     

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.     

Activation of metastatic potential in african green monkey kidney cell lines by prolongedin vitro culture

Summary Studies on the tumorigenicity of Vero kidney cells ofCercopithecus aethiops monkey origin were extended to various passage levels of BSC-1 aneuploid cells and to low passage CV-1 diploid cells (derived also fromC. aethiops monkey kidney). It was found that BSC-1 cells—like Vero cells—showed increased tumorigenicity with increasing passage level in antitymocyte globulins (ATG) treated newborn rats and in nude mice. Cells passaged over 250 times in cultures formed invasive adenocarcinomas in newborn rats. Their malignant tumor growth was further demonstrated around the 500 passage level when tumor metastases were detected in the lungs of four of the 14 inoculated rats. Vero cells induced such lung metastases in rats already at passage 227. CV-1 diploid cells at low passage level produced small nodules of epithelioid cells in newborn rats at 6th day after inoculation that had disappeared by the 21st day, and caused no local invasion nor lung metastasis. In vitro tumorigenicity tests on BSC-1 and CV-1 cells, using chick embryo skin, human muscle and colony formation in agarose, confirmed the animal test results. The results of this study indicate that BSC-1 and Vero cell lines at low and high passage levels may prove to be useful tools to study the molecular basis of malignancy. Editor's Statement In this paper Contreras et al. document the increased malignancy of commonly-used monkey cell lines upon long-term culture. These observations have implications for the use of these cell lines in studies of cancer cell biology, as well as the use of these lines for the production of biologicals. David W. Barnes     

Alteration of the In Vitro Host Range of Rabies Virus after Serial Chick Embryo Cell Passage Using Alkaline Maintenance Medium

HEP Flury strain of rabies virus maintained by 7-day chicken egg passage (parent line) and the same strain serially passaged in primary chick embryo (CE) cells using alkaline maintenance medium (AM line) were inoculated to cells of various species. Growth was negative in primary mouse embryo, L and HeLa cells, and positive in primary hamster kidney and BHK21 cells with both lines. An all-or-none difference between the two lines was observed in primary monkey kidney and Vero cells. The parent line did not multiply in these monkey cells, whereas the AM line grew to high titers. In the case of Vero cells a unique cytopathic effect (CPE) was induced by the AM line. After five consecutive passages in Vero cells, the CPE-inducing agent was identified as rabies virus by a neutralization test. It was infective to intracerebrally inoculated suckling mice but not to adult mice, and its Vero cell-infective titer determined by CPE induction was about 1 log lower than the baby mouse-infective and CE plaque-forming titers. In contrast to the AM line, HEP Flury strain receiving 150 CE cell passages under neutral maintenance medium and three other strains receiving similar CE cell passages all failed to grow in Vero cells.     

Simian virus 40 host range/helper function mutations cause multiple defects in viral late gene expression.

Simian virus 40 (SV40) deletion mutants dlA2459 and dlA2475 express T antigens that lack the normal carboxy terminus. These mutants are called host range/helper function (hr/hf) mutants because they form plaques at 37 degrees C on BSC-1 and Vero monkey kidney cell lines but not on CV-1p monkey kidney cells. Wild-type SV40 can provide a helper function to permit growth of human adenoviruses in monkey kidney cells; the hr/hf mutants cannot. Progeny yields of hr/hf mutants are also cold sensitive in all cell lines tested. Patterns of viral macromolecular synthesis in three cell lines (Vero, BSC-1, and CV-1) at three temperatures (40, 37, and 32 degrees C) were examined to determine the nature of the growth defect of hr/hf mutants. Mutant viral DNA replication was similar to that of the wild type in all three cell lines, indicating that the mutations affect late events in the viral lytic cycle. In mutant-infected Vero cells, in which viral yields were highest, late mRNA levels were similar to those observed during wild-type infection. Levels of viral late mRNA from mutant-infected CV-1 and BSC-1 cells at 32 and 37 degrees C were reduced relative to those of wild-type-infected cells. The steady-state level of the major viral capsid protein, VP1, in mutant-infected CV-1 cells was reduced to the same extent as was late mRNA. The synthesis of agnoprotein could not be detected in mutant-infected CV-1 cells but was readily detected in CV-1 cells infected by wild-type SV40. Primer extension analyses indicated that most late mRNAs from mutant-infected CV-1 cells utilize start sites downstream from the major wild-type cap site (nucleotide 325) and the agnoprotein initiation codon (nucleotide 335). These results indicate that deletion of the carboxyl-terminal domain of T antigen affects viral late mRNA production, both quantitatively and qualitatively. The agnoprotein is detected late in the wild-type SV40 lytic cycle and is thought to play a role in the assembly or maturation of virions. Reduced hr/hf progeny yields could result from decreased capsid protein synthesis and, in the absence of detectable levels of agnoprotein, from inefficient use of available capsid proteins.     

Exogenous ACE2 expression allows refractory cell lines to support severe acute respiratory syndrome coronavirus replication

Of 30 cell lines and primary cells examined, productive severe acute respiratory syndrome coronavirus (Urbani strain) (SARS-CoV) infection after low-multiplicity inoculation was detected in only six: three African green monkey kidney epithelial cell lines (Vero, Vero E6, and MA104), a human colon epithelial line (CaCo-2), a porcine kidney epithelial line [PK(15)], and mink lung epithelial cells (Mv 1 Lu). SARS-CoV produced a lytic infection in Vero, Vero E6, and MA104 cells, but there was no visible cytopathic effect in Caco-2, Mv 1 Lu, or PK(15) cells. Multistep growth kinetics were identical in Vero E6 and MA104 cells, with maximum titer reached 24 h postinoculation (hpi). Virus titer was maximal 96 hpi in CaCo-2 cells, and virus was continually produced from infected CaCo-2 cells for at least 6 weeks after infection. CaCo-2 was the only human cell type of 13 tested that supported efficient SARS-CoV replication. Expression of the SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2), resulted in SARS-CoV replication in all refractory cell lines examined. Titers achieved were variable and dependent upon the method of ACE2 expression.     

CD163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses

Direct functional screening of a cDNA expression library derived from primary porcine alveolar macrophages (PAM) revealed that CD163 is capable of conferring a porcine reproductive and respiratory syndrome virus (PRRSV)-permissive phenotype when introduced into nonpermissive cells. Transient-transfection experiments showed that full-length CD163 cDNAs from PAM, human U937 cells (histiocytic lymphoma), African green monkey kidney cells (MARC-145 and Vero), primary mouse peritoneal macrophages, and canine DH82 (histocytosis) cells encode functional virus receptors. In contrast, CD163 splice variants without the C-terminal transmembrane anchor domain do not provide PRRSV receptor function. We established several stable cell lines expressing CD163 cDNAs from pig, human, and monkey, using porcine kidney (PK 032495), feline kidney (NLFK), or baby hamster kidney (BHK-21) as the parental cell lines. These stable cell lines were susceptible to PRRSV infection and yielded high titers of progeny virus. Cell lines were phenotypically stable over 80 cell passages, and PRRSV could be serially passed at least 60 times, yielding in excess of 10(5) 50% tissue culture infective doses/ml.     

Release of simian virus 40 virions from epithelial cells is polarized and occurs without cell lysis.

We have investigated the process of release of simian virus 40 (SV40) virions from several monkey kidney cell lines. High levels of virus release were observed prior to any significantly cytopathic effects in all cell lines examined, indicating that SV40 utilizes a mechanism for escape from the host cell which does not involve cell lysis. We demonstrate that SV40 release was polarized in two epithelial cell types (Vero C1008 and primary African green monkey kidney cells) grown on permeable supports; release of virus occurs almost exclusively at apical surfaces. In contrast, equivalent amounts of SV40 virions were recovered from apical and basal culture fluids of nonpolarized CV-1 cells. SV40 virions were observed in large numbers on apical surfaces of epithelial cells and in cytoplasmic smooth membrane vesicles. The sodium ionophore monensin, an inhibitor of vesicular transport, was found to inhibit SV40 release without altering viral protein synthesis or infectious virus production.     

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.     

Polyvinyl formal surface promotes continuous growth of Vero cells in protein-free medium.

The effect of polyvinyl formal (PVF) culture surface on the growth of 10 mammalian continuous cell lines, including Swiss 3T6, NCTC clone 929 L, BHK-21 clone 13, CHO-K1, PK 15, A 431, HeLa, MDCK, LLC-MK2 and Vero in protein-free 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 supplemented with trace elements and L-ascorbic acid 2-phosphate, was investigated. Most of the cell lines showed only some initial proliferation on PVF similar to the polystyrene (PS) surface of commercially available culture flasks. In contrast, proliferation of monkey kidney cell line Vero was by far greater on PVF than on PS or poly-D-lysine treated culture surface. In addition, Vero cells on PVF could be subcultured in the protein-free medium without any significant decrease of growth rate in successive passages. These results showed that PVF provides a culture surface which selectively promotes continuous growth of Vero cells in protein-free, chemically defined medium.     

African green monkey kidney (Vero) cells provide an alternative host cell system for influenza A and B viruses.

The preparation of live, attenuated human influenza virus vaccines and of large quantities of inactivated vaccines after the emergence or reemergence of a pandemic influenza virus will require an alternative host cell system, because embryonated chicken eggs will likely be insufficient and suboptimal. Preliminary studies indicated that an African green monkey kidney cell line (Vero) is a suitable system for the primary isolation and cultivation of influenza A viruses (E. A. Govorkova, N. V. Kaverin, L. V. Gubareva, B. Meignier, and R. G. Webster, J. Infect. Dis. 172:250-253, 1995). We now demonstrate for the first time that Vero cells are suitable for isolation and productive replication of influenza B viruses and determine the biological and genetic properties of both influenza A and B viruses in Vero cells; additionally, we characterize the receptors on Vero cells compared with those on Madin-Darby canine kidney (MDCK) cells. Sequence analysis indicated that the hemagglutinin of Vero cell-derived influenza B viruses was identical to that of MDCK-grown counterparts but differed from that of egg-grown viruses at amino acid positions 196 to 198. Fluorescence-activated cell sorting analysis showed that although Vero cells possess predominantly alpha2,3 galactose-linked sialic acid, they are fully susceptible to infection with either human influenza A or B viruses. Moreover, all virus-specific polypeptides were synthesized in the same proportions in Vero cells as in MDCK cells. Electron microscopic and immunofluorescence studies confirmed that infected Vero cells undergo the same morphological changes as do other polarized epithelia] cells. Taken together, these results indicate that Vero cell lines could serve as an alternative host system for the cultivation of influenza A and B viruses, providing adequate quantities of either virus to meet the vaccine requirements imposed by an emerging pandemic.     

Concurrent Replication and Methylation at Mammalian Origins of Replication

Observations made with Escherichia coli have suggested that a lag between replication and methylation regulates initiation of replication. To address the question of whether a similar mechanism operates in mammalian cells, we have determined the temporal relationship between initiation of replication and methylation in mammalian cells both at a comprehensive level and at specific sites. First, newly synthesized DNA containing origins of replication was isolated from primate-transformed and primary cell lines (HeLa cells, primary human fibroblasts, African green monkey kidney fibroblasts [CV-1], and primary African green monkey kidney cells) by the nascent-strand extrusion method followed by sucrose gradient sedimentation. By a modified nearest-neighbor analysis, the levels of cytosine methylation residing in all four possible dinucleotide sequences of both nascent and genomic DNAs were determined. The levels of cytosine methylation observed in the nascent and genomic DNAs were equivalent, suggesting that DNA replication and methylation are concomitant events. Okazaki fragments were also demonstrated to be methylated, suggesting that the rapid kinetics of methylation is a feature of both the leading and the lagging strands of nascent DNA. However, in contrast to previous observations, neither nascent nor genomic DNA contained detectable levels of methylated cytosines at dinucleotide contexts other than CpG (i.e., CpA, CpC, and CpT are not methylated). The nearest-neighbor analysis also shows that cancer cell lines are hypermethylated in both nascent and genomic DNAs relative to the primary cell lines. The extent of methylation in nascent and genomic DNAs at specific sites was determined as well by bisulfite mapping of CpG sites at the lamin B2, c-myc, and β-globin origins of replication. The methylation patterns of genomic and nascent clones are the same, confirming the hypothesis that methylation occurs concurrently with replication. Interestingly, the c-myc origin was found to be unmethylated in all clones tested. These results show that, like genes, different origins of replication exhibit different patterns of methylation. In summary, our results demonstrate tight coordination of DNA methylation and replication, which is consistent with recent observations showing that DNA methyltransferase is associated with proliferating cell nuclear antigen in the replication fork.     

Phenylalanine hydroxylation and tyrosine requirement of cultured cells : Evidence of phenylalanine hydroxylation in mastocytoma cells in culture

Growth of cells in tyrosine-free medium can be used to detect and select cells able to convert phenylalanine to tyrosine. Nine different cell types have been tested: fibroblastic cells derived from human skin, human amniotic fluid cells, continuous human lymphoid cells, rat hepatoma, Vero (monkey kidney), HeLa, mouse neuroblastoma, mouse melanoma, and mouse mastocytoma. Two cell lines, mastocytoma and hepatoma, grew well in tyrosine-free medium, and subsequent enzyme assay showed substantial phenylalanine hydroxylation. This enzyme activity has already been documented in hepatoma [1], but the demonstration of phenylalanine hydroxylation by a cell line of non-hepatic origin is new. Fibroblastic cells survived but did not grow without tyrosine. No conversion of [¹⁴C]phenylalanine to tyrosine was demonstrated in these cells.     

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.     

Tumorigenicity of Vero cells

One of the current criteria for evaluating the acceptability of cell lines for use in vaccine production is lack of tumorigenicity. Vero cells represent an example of a class of cells known as continuous cell lines. They were derived from African green monkey kidney, and their growth properties and culture characteristics have many advantages over other cell substrates for use in vaccine production. We have tested Vero cells for tumorigenicity in nude mice and in a human muscle organ culture system, and found a significant increase in their tumorigenic potential with increasing passage numbers. Cells at passage 232 and higher produced nodules in all nude mice inoculated. Histologically the nodules were well defined, anaplastic tumors, which exhibited some of the characteristics of renal adenocarcinomas. In about 6 to 8 days all of the nodules began to regress. Data were obtained that suggested an immune mechanism was the basis for the regression phenomenon.     

Verotoxin-resistant cell clones are deficient in the glycolipid globotriosylceramide: differential basis of phenotype

Escherichia coli-derived verotoxin is an extremely toxic protein and is highly selective toward certain primate cells. Two susceptible cell lines are the Daudi cell line (human Burkitt lymphoma) and the Vero cell line (Green African monkey kidney). Both of these cell lines contain significant levels of the verotoxin binding glycolipid globotriosylceramide (Gb3) (1 nmol/10(7) cells and 3 nmol/10(6) cells, respectively). A clone was selected from the Vero cell line for resistance to Verotoxin 2, while a mutant from the Daudi cell line was selected for resistance to Verotoxin 1. Both were found to be deficient in globotriosylceramide with a corresponding increase in the precursor glycolipid lactosylceramide. Cell free assay of alpha-galactosyltransferase activity revealed that the Vero cell clone (VRP) contained significantly reduced enzyme activity, whereas in the case of the Daudi mutant (VT20), no significant decrease in activity was noted in vitro. These observations suggest a complex regulation of Gb3 biosynthesis which is considered in relation to P blood group antigen expression.     

Patterns of integration of exogenous DNA sequences transfected into mammalian cells of primate and rodent origin

We studied the cotransfer and cointegration of several genes transfected into four cell lines of primate origin. Mouse thymidine-kinase-negative LM cells, which had been extensively studied previously, were used as a reference. We found that in monkey kidney Vero cells, on average between 3.5 and 6.0 kb of plasmid sequences was integrated per clone, while in the murine LM cell Une, 9–186 kb of exogenous DNA was integrated per clone. Transformed Vero clones which had integrated more than 6 kb of DNA did not integrate larger DNA fragments in a second transformation assay than had the parental Vero cells. We found that the efficiency of gene cointegration is similar in Vero, HeLa and GM4312A cells, the latter being deficient in the repair of UV-induced damage. The human hepatocarcinoma Hep G2 cells integrated on the average 2 kb more exogenous DNA than the three other primate cell Unes, which resulted in a 4–5 times higher efficiency of gene cointegration. Plasmid penetration and persistence in a free state between 24 h and two weeks after transfection was similar in Vero and LM cells. No major post-integration DNA rearrangement could be demonstrated after the isolation of Vero clones. These observations correlate the low efficiency of gene cointegration in some primate cell lines with a genomic recombination step or with rearrangements taking place during early cell divisions following integration     

Isolation and in vitro characterization of a herpesvirus from ground squirrels (Citellus sp).

A viral agent was isolated from ground squirrel primary kidney cultures which presented a spontaneous cell layer alteration. The cultural, morphologic, and physico-chemical characteristics of the agent indicated that it belongs in the herpesvirus group. The isolate presented a narrow in vitro cell host range, growing best in marmoset monkey, owl monkey, rabbit, and hamster kidney cultures and poorly in Vero and dog fetal lung cells. The agent is readily released from infected cells, a fact which indicates that it is a cell-free virus. Good plaque formation was observed only in marmoset monkey kidney monolayers. The virus was not neutralized by antisera against other herpesviruses with the exception of a partial 1-way cross-neutralization between ground squirrel agent and Herpesvirus saguinus antisera. All these characteristics indicate that this virus is likely a new member of the herpesvirus family.     

Evaluation of cell lines and immunofluorescence and plaque assay procedures for quantifying reoviruses in sewage.

Twelve continuous cell lines were tested to determine their sensitivity to reovirus types 1, 2, and 3 isolated from sewage. Madin-Darby bovine kidney (MDBK), rhesus monkey kidney (LLC-MK2), and human embryonic intestinal (intestinal 407) cells were most sensitive, respectively. In a similar study, MDBK cells were more sensitive than LLC-MK2 and Buffalo green monkey kidney (BGM) cells to sewage-isolated, protamine-precipitated reoviruses which had not been serotyped and had no previous cell contact. Sewage-isolated, protamine-precipitated reoviruses were also used in conjunction with MDBK cells in a comparative evaluation of immunofluorescent cell count and plaque assay procedures. The immunofluorescence assay is more sensitive and more rapid than the plaque assay. Reoviruses in excess of 10(4)/liter of raw sewage were detected by the immunofluorescent cell count assay.     

CNF1-induced ubiquitylation and proteasome destruction of activated RhoA is impaired in Smurf1-/- cells

Ubiquitylation of RhoA has emerged as an important aspect of both the virulence of Escherichia coli producing cytotoxic necrotizing factor (CNF) 1 toxin and the establishment of the polarity of eukaryotic cells. Owing to the molecular activity of CNF1, we have investigated the relationship between permanent activation of RhoA catalyzed by CNF1 and subsequent ubiquitylation of RhoA by Smurf1. Using Smurf1-deficient cells and by RNA interference (RNAi)-mediated Smurf1 knockdown, we demonstrate that Smurf1 is a rate-limiting and specific factor of the ubiquitin-mediated proteasomal degradation of activated RhoA. We further show that the cancer cell lines HEp-2, human embryonic kidney 293 and Vero are specifically deficient in ubiquitylation of either activated Rac, Cdc42, or Rho, respectively. In contrast, CNF1 produced the cellular depletion of all three isoforms of Rho proteins in the primary human cell types we have tested. We demonstrate that ectopic expression of Smurf1 in Vero cells, deficient for RhoA ubiquitylation, restores ubiquitylation of the activated forms of RhoA. We conclude here that Smurf1 ubiquitylates activated RhoA and that, in contrast to human primary cell types, some cancer cell lines have a lower ubiquitylation capacity of specific Rho proteins. Thus, both CNF1 and transforming growth factor-beta trigger activated RhoA ubiquitylation through Smurf1 ubiquitin-ligase.