Evaluation of mixed cell types and 5-iodo-2'-deoxyuridine treatment upon plaque assay titers of human enteric viruses

Four continuous cell lines, BGM, L-132, HEL-299, and RD, were compared both when cultured separately and as mixtures for use in plaque assay titrations of human adenovirus 1 and six human enterovirus serotypes. The effect of incubating these cell cultures in media containing 5-iodo-2'deoxyuridine (IDU) prior to inoculation with virus was also studied. The use of mixed-cell cultures revealed cell line-dependent synergistic effects as well as inhibitory effects. These effects were strongly virus dependent. In particular, enterovirus 69 did not form plaques on any of the four cell lines when cultured independently. However, it did form plaques on nearly all of the cell lines when cultured as mixtures. Contrary to this effect, when BGM cells were used in combination with the other cell lines, plaque counts for adenovirus 1 were greatly reduced. The effect of IDU pretreatment was also virus and cell line specific and enabled some viruses to form plaques on cell lines when they otherwise would not. Overall, IDU pretreatment resulted in an approximate twofold increase in plaque titers over those obtained without treatment.     

Plaque Formation by Mumps Virus and Inhibition by Antiserum

Boston and ABC strains of mumps virus produced plaques approximately 1.0 mm in diameter in monolayers of BGM cells. The plaques were circular and either clear or target-like in form. Ricki strain virus produced plaques of similar size and form but, in addition, a red plaque was observed with this agent. The vaccine strain of mumps virus, Jeryl Lynn, produced minute clear plaques approximately 0.3 mm in diameter. Incorporation of diethylaminoethyl (DEAE)-dextran into the overlay medium did not affect the size difference between Jeryl Lynn plaques and those of the other strains. However plaques of the Jeryl Lynn and Ricki strains were more easily visualized when the overlay medium contained 400 mug/ml of DEAE-dextran. Simultaneous titration by plaque formation and roller tube infectivity showed that these two methods were of equal sensitivity. Virus neutralization by antibody was demonstrated by plaque reduction. Rise in antibody titer was observed in sera from human and animal infection, human vaccination, and rabbit immunization.     

Comparative Sensitivity of Various Cell Culture Systems for Isolation of Viruses from Wastewater and Fecal Samples

In efforts to define the most sensitive cell culture systems for recovery of viruses from wastewaters, 181 samples were inoculated in parallel into tube cultures of various cell types and were plaqued in bottle and petri dish cultures of three types of monkey kidney cells. Polioviruses were recovered most frequently in the RD line of human rhabdomyosarcoma cells, group A coxsackieviruses in RD and human fetal diploid kidney (HFDK) cells, group B coxsackieviruses in the BGM line of African green monkey kidney cells, echoviruses in RD and primary rhesus monkey kidney (RhMK) cells, and reoviruses in RhMK cells. BGM cells were unsatisfactory for recovery of viruses other than polioviruses and group B coxsackieviruses, and a line of fetal rhesus monkey kidney (MFK) was not a satisfactory substitute for primary RhMK. With RhMK cells, comparable numbers of virus isolations were made in tube cultures and in plaque assays conducted in bottle cultures, but with BGM and MFK cells, fewer isolations were made by plaquing than by inoculation of tube cultures. In comparative plaque assays on fecal samples under three different overlays in bottle and plate cultures of RhMK, BGM, and MFK cells, it was found that plaquing in the most sensitive system, RhMK, was less efficient for virus recovery than was inoculation of tube cultures of RhMK or HFDK cells. Overall, plaque assays performed in petri dishes in a CO₂ incubator yielded fewer virus isolates than did parallel plaque assays performed in closed bottle cultures. Other limitations of plaque assays for recovery of human enteric viruses are discussed.     

Comparative sensitivity of various cell culture systems for isolation of viruses from wastewater and fecal samples.

In efforts to define the most sensitive cell culture systems for recovery of viruses from wastewaters, 181 samples were inoculated in parallel into tube cultures of various cell types and were plaqued in bottle and petri dish cultures of three types of monkey kidney cells. Polioviruses were recovered most frequently in the RD line of human rhabdomyosarcoma cells, group A coxsackieviruses in RD and human fetal diploid kidney (HFDK) cells, group B coxsackieviruses in the BGM line of African green monkey kidney cells, echoviruses in RD and primary rhesus monkey kidney (RhMK) cells, and reoviruses in RhMK cells. BGM cells were unsatisfactory for recovery of viruses other than polioviruses and group B coxsackieviruses, and a line of fetal rhesus monkey kidney (MFK) was not a satisfactory substitute for primary RhMK. With RhMK cells, comparable numbers of virus isolations were made in tube cultures and in plaque assays conducted in bottle cultures, but with BGM and MFK cells, fewer isolations were made by plaquing than by inoculation of tube cultures. In comparative plaque assays on fecal samples under three different overlays in bottle and plate cultures of RhMK, BGM, and MFK cells, it was found that plaquing in the most sensitive system, RhMK, was less efficient for virus recovery than was inoculation of tube cultures of RhMK or HFDK cells. Overall, plaque assays performed in petri dishes in a CO(2) incubator yielded fewer virus isolates than did parallel plaque assays performed in closed bottle cultures. Other limitations of plaque assays for recovery of human enteric viruses are discussed.     

Detection of biologically active adenovirions unable to plaque in human cells

Butel, Janet S. (Baylor University College of Medicine, Houston, Tex.), Joseph L. Melnick, and Fred Rapp. Detection of biologically active adenovirions unable to plaque in human cells. J. Bacteriol. 92:433-438. 1966.-Plaque formation in green monkey kidney (GMK) cells by a defective simian virus 40-adenovirus 7 "hybrid" population (PARA-adenovirus 7) was enhanced by the addition of excess adenovirions. Adenovirus types 2, 7, and 12 were capable of providing enhancement, although none of these viruses gives rise to plaques in simian cells in the absence of PARA (particle aiding replication of adenovirus). Near maximal enhancement of the PARA plaque titer on simian cells was obtained with input multiplicities ranging from 0.02 to 0.14 plaque-forming units (PFU) of helper adenovirus per GMK cell. The PFU of helper adenoviruses tested (types 2, 7, and 12) were measured in the most sensitive assay system, human kidney cells. This input corresponded to three to nine helper virus particles per GMK cell. The majority of particles capable of enhancing plaque formation by PARA banded at a density of 1.34 in CsCl. Adenoviruses inactivated by heat or ultraviolet light were not capable of enhancing plaque formation by PARA. Highest titers were obtained when PARA and helper adenovirus were inoculated simultaneously. Inoculation of the helper adenovirus 24 hr prior to the inoculation of PARA resulted in the formation of only 50% as many plaques, and no enhanced plaques developed when the adenovirus preceded PARA by 48 hr. Conversely, the addition of adenovirus 48 hr after the inoculation of PARA initiated 56% as many plaques as simultaneous inoculation; 4% of the enhanced plaques still formed when helper virus was added as late as 5 days after inoculation of PARA. These results suggest that adenovirus particles unable to plaque on human or monkey kidney cells are nevertheless capable of interacting with PARA in simian cells, thereby facilitating replication of both particles.     

Studies on the interaction between coxsackievirus A9 and HeLa cells. I. Plaque-forming ability of coxsackievirus A9 in HeLa cell cultures.

Most of the coxsackievirus A9 (CA 9 virus) including the prototype strain formed plaques in HeLa cell monolayers under agar overlay, although they showed little or no cytopathogenicity under fluid medium. These viruses were isolated or passaged in primary cynomolgus monkey kidney (MK) cell cultures, and the infectivity of any strain in terms of plaque-forming units was much higher in MK cells than in HeLa cells, even after plaque purification of the virus in HeLa cell cultures. CA 9 virus contained in the original throat swabs as well as some clones obtained by plaque purification in MK cells failed to form plaques in HeLa cells, but virus preparations obtained after several undiluted passages through MK cells included plaque-formers in HeLa cells, suggesting that such plaque (HeLa)-forming viruses may have developed at a certain rate during multiplication of the original non-plaque (HeLa)-forming virus population in MK cells. Out of four lines of HeLa cells examined, two, including a clonal line S3, failed to support plaque formation by CA 9 virus.     

Sensitivity of Herpes Simplex Virus, Vaccinia Virus, and Adenoviruses to Deoxyribonucleic Acid Inhibitors and Thiosemicarbazones in a Plaque Suppression Test

Herpes simplex and vaccinia viruses and adenovirus types 1, 2, 5, and 7 were tested by plaque suppression methods for sensitivity to halogenated deoxyuridines (5-iodo-, 5-bromo-, 5-chloro-, and 5-fluoro-), cytosine arabinoside, isatin-beta-thiosemicarbazone, and N-methylisatin-beta-thiosemicarbazone. After incubation for 12 days in HeLa cell cultures, vaccinia virus plaques were still readily suppressed by deoxyribonucleic acid (DNA) inhibitors and thiosemicarbazones. Herpes simplex virus plaques were likewise suppressed by at least three DNA inhibitors. Adenovirus plaques were not suppressed by DNA inhibitors or thiosemicarbazones. 5-Fluoro-2'-deoxyuridine could not be shown to have any antiviral activity, but it did produce a substantial lethal action on the cells.     

Identification of the Simian Virus 40 Which Replicates When Simian Virus 40-Transformed Human Cells Are Fused with Simian Virus 40-Transformed Mouse Cells or Superinfected with Simian Virus 40 Deoxyribonucleic Acid

Simian virus 40 (SV40) was rescued from heterokaryons of transformed mouse and transformed human cells. To determine whether the rescued SV40 was progeny of the SV40 genome resident in the transformed mouse cells, the transformed human cells, or both, rescue experiments were performed with mouse lines transformed by plaque morphology mutants of SV40. The transformed mouse lines that were used yielded fuzzy, small-clear, or large-clear plaques after fusion with CV-1 (African green monkey kidney) cells. The transformed human lines that were used did not release SV40 spontaneously or after fusion with CV-1 cells. From each mouse-human fusion mixture, only the SV40 resident in the transformed mouse cells was recovered. Fusion mixtures of CV-1 and transformed mouse cells yielded much more SV40 than those from transformed human and transformed mouse cells. The rate of SV40 formation was also greater from monkey-mouse than from human-mouse heterokaryons. Deoxyribonucleic acid (DNA) from SV40 strains which form fuzzy, largeclear, or small-clear plaques on CV-1 cells was also used to infect monkey (CV-1 and Vero), normal human, and transformed human cell lines. The rate of virion formation and the final SV40 yields were much higher from monkey than from normal or transformed human cells. Only virus with the plaque type of the infecting DNA was found in extracts from the infected cells. Two uncloned sublines of transformed human cells [W18 Va2(P363) and WI38 Va13A] released SV40 spontaneously. Virus yields were not appreciably enhanced by fusion with CV-1 cells. However, clonal lines of W18 Va2(P363) did not release SV40 spontaneously or after fusion with CV-1 cells. In contrast, several clonal lines of WI38 Va13A cells did continue to shed SV40 spontaneously.     

Simian rotavirus SA11 replication in cell cultures.

Understanding the basic virology of rotavirus infections has been hampered by the fastidiousness of most isolates and by the lack of a rapid quantitative assay method. The growth characteristics of the simian rotavirus SA11 were studied because it grows to high titers in tissue culture and infectivity can be quantitated by plaque assay. SA11 replication was analyzed in a variety of primary cell cultures or continuous cell lines derived from both homologous and heterologous hosts. Viral replication was observed in each of the cell cultured examined. The individual cell cultures demonstrated marked variability in their susceptibility to rotavirus infection. The highest titers were obtained with MA104, BSC-1, CV-1, and BGM cells. Observable cytopathic effect was found to correlate with the percentage of infected cells in the culture. This study presents growth curves of the simian rotavirus in a variety of cell cultures.     

Occurrence of adenovirus and other enteric viruses in limited-contact freshwater recreational areas and bathing waters

Aims: The goal of this study was to characterize enteric virus concentrations and their infectivity in a variety of limited‐contact recreation and bathing waters, including Great Lakes beaches, inland lakes, rivers, and an effluent‐dominated urban waterway. Additionally, we evaluated associations between point sources of human faecal pollution and enterovirus and adenovirus presence and concentrations. Methods and Results: Quantitative polymerase chain reaction (qPCR) and two cell culture lines were used to identify and quantify viruses in water samples. The presence of human adenoviruses F was strongly associated with effluent‐dominated waters (odds ratio 6·1, confidence interval 2·3, 15·7), as was adenovirus concentration; though, neither enterovirus presence nor concentration was associated with an effluent source. Samples with high concentrations of qPCR targets all tested positive by cell culture on both cell lines, although qPCR target concentrations were not correlated with culture values. Conclusions: Adenovirus was strongly associated with point sources of human faecal pollution while enterovirus was not, indicating that adenovirus measured by qPCR is a better target than enterovirus for identifying wastewater discharges in recreational freshwaters. Significance and Impact of the Study: The development of monitoring for enteric human viral pathogens at recreational waters should include adenovirus testing. Further research is needed to interpret the results of qPCR testing in relationship to the presence of infectious viruses using cell culture.     

Adenovirus type 5 precursor terminal protein-expressing 293 and HeLa cell lines.

HeLa and 293 cell lines that express biologically active adenovirus type 5 precursor terminal protein (pTP) have been made. The amount of pTP synthesized in these cell lines ranges from barely detectable to greater than that observed in cells infected with the wild-type virus. The pTP-expressing cell lines permit the growth of a temperature-sensitive terminal protein mutant virus sub100r at the nonpermissive temperature. A higher percentage of the stably transfected 293 cell lines expressed terminal protein, and generally at considerably higher levels, than did the HeLa cell lines. While 293 cells appeared to tolerate pTP better than did HeLa cells, high-level pTP expression in 293 cells led to a significantly reduced growth rate. The 293-pTP cell lines produce infectious virus after transfection with purified viral DNA and form plaques when overlaid with Noble agar after infection at low multiplicity. These cell lines offer promise for the production of adenoviruses lacking pTP expression and therefore completely defective for replication.     

Role of antibodies and host cells in plaque enhancement of Murray Valley encephalitis virus.

Chicken antisera to Murray Valley encephalitis (MVE) virus, when incubated with virus and assayed for plaques on chicken embryo (CE) monolayers, neutralized MVE virus at high concentrations of antibody, but caused increases in plaque counts at low concentrations of antibody. Plaque enhancement did not occur when the same virus-antibody mixtures were assayed on a continuous line of rhesus monkey kidney cells (LLC-MK2), nor when the anti-MVE antibody was of mammalian origin and the assay system was CE monolayers. Correspondingly, chicken anti-MVE did not enhance the plaque formation of MVE virus in a stable line of mouse macrophages, P-388D1, whereas rabbit and mouse anti-MVE did enhance plaque formation. This enhancing activity was associated with noncytophilic immunoglobulin G (IgG). The Fc terminus of the IgG molecule was required, as no plaque enhancement occurred with chicken anti-MVE Fab. These data indicate that there is a requirement for taxonomic complementarity between Fc termini and Fc receptors in the above systems. CE cell monolayers were found to contain approximately 2% of Fc receptor-bearing cells among the fibroblast-like cells. Fc receptor-bearing cells in CE monolayers were isolated and found to be of the mononuclear phagocytic lineage. These mononuclear phagocytes, which originate in lymphoid tissues and blood associated with CE tissue fragments, are integrated into primary CE monolayers and form infectious centers in the presence of virus and low dilutions of antibody.     

Construction and Preliminary Characterization of a Library of “Lethal” Preterminal Protein Mutant Adenoviruses

Adenoviruses containing lethal in-frame insertion mutant alleles of the preterminal protein (pTP) gene were constructed with cell lines that express pTP. Thirty in-frame insertion mutant alleles, including 26 alleles previously characterized as lethal and 4 newly constructed mutant alleles, were introduced into the viral chromosome in place of the wild-type pTP gene. The viruses were tested for ability to form plaques at 37 degrees C in HeLa-pTP cells and at 32 degrees C and 39.5 degrees C in HeLa cells. Two of the newly constructed viruses exhibited temperature sensitivity for plaque formation, one virus did not form plaques in the absence of complementation, seven additional mutants exhibited a greater than 10-fold reduction in plaque formation in the absence of complementation, and another eight mutants exhibited stronger phenotypes than did previously characterized in-frame insertion mutants in the plaque assay. These mutant viruses offer promise for analysis of pTP functions.     

A New Candidate Type of Human Enterovirus (Drilon Strain) Isolated in the Philippines

A cytopathogenic agent was isolated in monkey kidney (MK) cell cultures from the stool specimen of a 3-month-old Filipina hospitalized with lower respiratory disease. The agent was designated the Drilon strain. It was characterized as an enterovirus on the basis of electron microscopic morphology, nucleic acid type (RNA), resistance to ether and acid (pH 3.0) treatments, stabilization by molar MgCl₂ against heat inactivation, and buoyant density in CsCl. The strain caused mild febrile illness in experimentally inoculated cynomolgus monkeys, but not in suckling mice. In addition to its effect in primary MK cells, the virus was cytopathogenic in primary and secondary human amnion or embryonic lung cell cultures and in WI-38 or HEp-2 cell lines, but not in primary bovine kidney, primary porcine kidney, primary embryonic mouse or primary embryonic chick cell cultures. The Drilon strain was not neutralized by reference antisera against the known enterovirus serotypes, and the antiserum prepared with the Drilon strain did not neutralize any of the recognized prototype enterovirus strains. Although the patient's sera were not available, antibodies against the Drilon strain were prevalent in normal Filipinos and Indonesians, but not in Japanese people. The Drilon strain fulfilled the criteria of human enterovirus and is considered a candidate for designation as a new type.     

Studies on the interaction between coxsackievirus A9 and HeLa cells. II. Mode of growth of coxsackievirus A9 in HeLa cell cultures and the effect of sulfated polysaccharide on plaque formation.

For the purpose of clarifying the mechanism of plaque formation in HeLa cell cultures by coxsackievirus A9, which does not show definite CPE in fluid cultures, we investigated the growth pattern of the virus in HeLa cells, comparing plaque (HeLA)-forming and non-plaque (HeLa)-forming viruses. It was revealed that the yield of both viruses per cell was nearly the same, but non- plaque (HeLa)-forming virus was far less efficient in infecting HeLa cells. Dextran sulfate was effective in releasing more virus from cells, when HeLa cell cultures were infected with plaque (HeLa)-forming virus, but not in cultures infected with non-plaque (HeLa)-forming virus. From these experimental results, the mechanism by which plaques are formed in HeLa cell cultures by coxsackievirus A9 was discussed.     

Comparative Studies on Large- and Small-Plaque-Forming Clones of Newcastle Disease Virus (Miyadera Strain)

The Miyadera strain of Newcastle disease virus (NDV) consisted predominantly of virus particles forming small plaques on monolayers of chick embryo fibroblasts (CEF), and contained small amounts of virus particles forming large plaques. These large- and small-plaque-forming clones of this virus (NDV-L and NDV-S) were isolated. The small size of the NDV-S plaques did not appear to be due to an agar inhibitor. NDV-L produced a much higher yield of infective virus particles in CEF and they were released more completely from the infected cells than were those produced by NDV-S. The yield of infective virus of NDV-L per cell from cultures of CEF was comparable to the yield from the allantoic cells. The infectivity/hemagglutinin ratio for NDV-L from CEF was as high as the ratio for virus from the allantoic cells, but the ratio for NDV-S from CEF was lower. NDV-S demonstrated an autointerference phenomenon in CEF when infected at high multiplicities, but NDV-L did not. Contrary to virus multiplication, NDV-S exhibited a more rapid and marked cytopathic effect on monolayers of CEF than NDV-L. In the allantoic cavity of eggs NDV-S produced slightly higher virus yields than NDV-L. No correlation existed between plaque size of the two viruses and the capacity to induce interferon synthesis or the susceptibility to the action of interferon. The properties of both distinctive plaque isolates were stable on egg passage.     

Optimization of the BGM cell line culture and viral assay procedures for monitoring viruses in the environment.

An in-depth study of the continuous cell line designated BGM is described herein, and recommendations are made for standardizing cell culture and viral assay procedures. Based on data gathered from a survey of 58 laboratories using this cell line, a research plan was developed that included the study of growth media, sera, NaHCO3 levels, culture bottles, cell concentration, overlay media, agar, virus infection conditions, and cell-dissociating agents. Additionally, a comparative virus isolation study with BGM cells and nine other cell types was conducted with 37 sewage samples collected from nine different geographic areas. The results of the study indicated that the BGM cell line is superior for virus isolation when compared with the other cell types and that certain media and additives tend to increase BGM cell sensitivity to a specific group of viruses. A standardized procedure for cultivation of BGM cells is described which provides a more effective enterovirus assay system.     

Optimization of the BGM cell line culture and viral assay procedures for monitoring viruses in the environment

An in-depth study of the continuous cell line designated BGM is described herein, and recommendations are made for standardizing cell culture and viral assay procedures. Based on data gathered from a survey of 58 laboratories using this cell line, a research plan was developed that included the study of growth media, sera, NaHCO3 levels, culture bottles, cell concentration, overlay media, agar, virus infection conditions, and cell-dissociating agents. Additionally, a comparative virus isolation study with BGM cells and nine other cell types was conducted with 37 sewage samples collected from nine different geographic areas. The results of the study indicated that the BGM cell line is superior for virus isolation when compared with the other cell types and that certain media and additives tend to increase BGM cell sensitivity to a specific group of viruses. A standardized procedure for cultivation of BGM cells is described which provides a more effective enterovirus assay system.     

Influence of inoculum size, incubation temperature, and cell culture density on virus detection in environmental samples

The influence of inoculum size and cell culture density on virus titer by cytopathic effect or plaque assay was studied using poliovirus type 1 and BGM (Buffalo green monkey) cells as a model for this evaluation. With a plaque assay system, a linear relationship was observed for an inoculum size of up 1 mL/25 cm2; a marked decrease in the number of plaques was observed when over 1 mL of sample was inoculated on this surface area. Cell culture density also affected virus titer; maximal titers were observed when cells were seeded at 25 000 to 75 000 cells/mL and incubated for 6 days before infection with the virus. Viral density, evaluated as most-probable-number and measured by cytopathic effect under liquid overlay, revealed that the viral titer was similar up to 1 mL inoculum and increased only when over 1 mL was inoculated. Cell density had no significant effect on the viral titer measured by the most-probable-number method and cytopathic effect. Inactivation of inoculum due to an incubation temperature of 37 degrees C for a short period was shown to be minimal for poliovirus type 1, reovirus type 2, coxsackievirus B-5, and the simian rotavirus SA-11. Longer inactivation time led to a 2 logs reduction of the infectious titer of coxsackievirus B-5 (in 48 h) while the other viruses showed a significant reduction in titer only after 96 h.     

Double-layer plaque assay for quantification of enteroviruses

We describe here a double-layer plaque assay for the quantification of enteroviruses, combining a monolayer plaque assay and a suspended-cell plaque assay. The double-layer assay provides significantly greater counts than other methods of virus quantification of both suspensions of pure culture viruses and naturally occurring viruses. The counts obtained by this method are approximately one order of magnitude greater than those obtained with the more commonly used method, the monolayer plaque assay. We conclude that the methods available for quantifying viruses rank in efficiency as follows: double-layer plaque assay >or=suspended-cell plaque assay > counting cytopathogenic virus adsorbed to cellulose nitrate membrane filters >or= most probable number of cytopathogenic units > monolayer plaque assay. Moreover, the double-layer plaque assay allows the use of two different cell lines in the two layers. Using the human colonic carcinoma cell line CaCo2 facilitates the recovery of a greater number and diversity of naturally occurring enteroviruses in water than the monolayer agar method. In addition, the pretreatment of cells with 5-iodo-2'-deoxyuridine (IDU) prior to the quantification of enteroviruses by the double-layer plaque assay provides significantly higher recoveries than the use of IDU does with the other methods of quantification.