Higher Virus Yields Research Articles

Cell killing by simian virus 40: variation in the pattern of lysosomal enzyme release, cellular enzyme release, and cell death during productive infection of normal and simian virus 40-transformed simian cell lines

Simian virus 40 (SV40) growth on rhesus kidney cells and on the T-22 line of SV40-transformed green monkey kidney (GMK) cells is largely limited by the low plating efficiency of SV40 on these cells. In addition, a fraction of the rhesus kidney and T-22 cells are resistant to infection by SV40. Nevertheless, 72-h viral yields per infected rhesus kidney and T-22 cell are nearly equivalent to that obtained on normal GMK cells and are independent of the multiplicity of infection. Despite the production of high viral yields, infected rhesus kidney and T-22 cells are killed slowly by SV40. Monolayers of these cells are also refractory to plaque formation by SV40. SV40 induces the release of lysosomal N-acetyl-beta-glucosaminidase into the cytoplasmic fractions of rhesus kidney and T-22 cells to an extent equal to that observed during infection of rapidly killed normal GMK cells. In contrast, damage to the plasma membrane, as indicated by the release of the cellular enzymes lactic dehydrogenase and glutamic oxaloacetic transaminase into the overlay media, occurred to a much greater extent in the normal GMK cells than in the rhesus kidney or T-22 cells. Neither a lysosomal hydrolase mechanism nor viral release appear to be responsible for this phenomenon. The different rates and extents of the SV40 cytocidal process on these cells do not result from the differences in the viral plating efficiency on them.

Possible Interference in the Isolation of IPN Virus from Carrier Fish

This report describes an instance of possible homologous viral interference in the isolation of IPN virus from carrier fish. Brook trout (Salvelinus fontinalis), suspected of being carriers of IPN virus, were examined for the presence of the virus by inoculating monolayer cultures of RTG-2 cells with different dilutions of tissue homogenates of the suspected fish. Cultures of RTG-2 cells infected with concentrated tissue homogenates exhibited little, if any, cytopathic effect (CPE) and yielded little infectious IPN virus at 72 h after infection. However, cell cultures infected with a relatively high dilution of tissue homogenate exhibited extensive CPE and resulted in high yields of infectious virus.

Environmental factors for the growth of animal cells.

Although cells can grow throughout a wide range of osmotic pressures, any additional salt beyond that required to meet the minimal level exerts an adverse effect, as evidenced by delay in onset of logarithmic growth, slower growth rate, decreased maximal population, and accelerated death. The toxicity of K+ is directly related to the osmotic pressure of the culture. Cells appear to ingest most of the nutrients from the medium within the 1st day after being inoculated into it and do not remove significant amounts of nutrients for the next 7 to 8 days of active growth. Dissolved oxygen and medium oxidation-reduction potential (ORP) do not always vary identically and, of the two parameters, ORP is the more critical. If the ORP of the culture is not held constant during growth, enzymatic activity fluctuates during the culture growth cycle. However, if the ORP is held constant, then enzymatic activity and isoenzyme distribution patterns remain constant during the growth cycle. For L cells, the optimum ORP is 100 mv. The ORP of the medium controls the rate and extent of growth of cells. If the ORP is lowered, cells can be inoculated into media and held at 37°C for at least 5 days with no detrimental effect; then, when the ORP is raised, the cells grow as well as if they had just been inoculated into fresh media. By controlling the osmotic pressure, media composition, and environmental parameters, high yields of cells and virus may be consistently obtained.

Production of sindbis, influenza, and vesicular stomatitis viruses in chicken embryo and rat embryo cell suspensions.

AbstractStudies were carried out on the production of Sindbis, influenza and vesicular stomatitis viruses in suspensions of chicken embryo and rat embryo cells. The yield of Sindbis virus in chicken embryo cell suspensions was independent of the multiplicity of infection over the range 0.0001 to 0.01 although reduction in multiplicity caused a delay in virus production. With influenza virus the use of higher multiplicities gave increased virus yields possibly due to the very slow production at low multiplicities. In both monolayer and suspension cultures of chicken embryo cells addition of serum or use of media richer than minimum essential medium (Eagle) had little effect on Sindbis virus production, but if the glucose were omitted the virus yield was markedly reduced. In cell suspensions, a marked reduction in virus yield occurred if infection were delayed more than 24 hr after cell preparation whereas in monolayers the delay of infection allowed cell propagation and hence a higher yield of virus. It was also shown that vesicular stomatitis virus can be produced in chicken embryo cell suspensions, and that rat embryo primary cell suspensions can be used to prepare both Sindbis and vesicular stomatitis viruses. Sindbis virus obtained from chicken embryo cell suspensions was purified by polyethylene glycol precipitation and sucrose density gradient centrifugation and shown to contain only those proteins previously identified as viral, without any contamination from chicken cell proteins. The relative ease and economics of virus production by cell suspension and monolayer methods is compared.

Effect of proteolytic enzymes on the infectivity of a number of herpesviruses.

The characteristics of cytomegaloviruses (CMV) that have been used to distinguish them from the rest of the herpes group include species specificity, slower growth, lower yield of virus, and close association with cellular material. In previous work, however, we have shown that a murine strain of CMV (MCMV) can grow in strains of cells derived from species other than mice [1]. Furthermore, the high virus yield of MCMV and the lack of close association with cellular material would appear to place the murine strain in the standard group of herpesviruses, as opposed to the CMV subgroup. In this study, the sensitivity of a number of herpes group viruses to the proteolytic enzymes trypsin and chymotrypsin were tested; there were marked differences in sensitivity between MCMV and herpes simplex virus type 1 (HSV-1) on the one hand and a number of strains of CMV derived from humans (HCMV) on the other.

East African strains of cowpea aphid-borne mosaic virus.

SUMMARYCowpea aphid‐borne mosaic virus (CAMV) was isolated for the first time in East Africa where three distinct strains, type, veinbanding and mild, were differentiated by host range and serology. The three strains infected 17/38, 18/37 and 10/35 legume species, and 11/21, 7/21 and 3/19 non‐legume species, respectively.The viruses were propagated in cowpea and assayed in Chenopodium amaranticolor. Isolates of all three strains had similar in vitro properties: dilution end point between 10‐3 and 10‐4; thermal inactivation point between 56 and 58 °C; longevity in vitro between 2 and 3 days. Infectivity of sap from frozen leaves was high after 4 wk but much less after 7 wk; infectivity was largely precipitated by 50% acetone but inactivated by 50% ethanol. High yields of virus were consistently obtained from cowpea by extracting systemically infected leaves in 0.5 m sodium citrate containing 1% mercaptoethanol (pH 8.1), and clarifying with 8.5 ml n‐butanol/100 ml sap. Virus preparations contained numerous unaggregated and aggregated virus particles c. 750 nm long and contained components with sedimentation coefficients (s°20, w) of 150S and 175S (presumably unaggregated and aggregated particles, respectively). CAMV is serologically distantly related to bean common mosaic virus, but not to bean yellow mosaic or eight other morphologically similar viruses. It is a typical but distinct member of the potato virus Y group.

Inhibition of replication of lactic dehydrogenase virus by actinomycin.

Lactic dehydrogenase virus replicated rapidly in monolayers of primary mouse embryo cells and reached a titer of 10(8) mean infective dose per ml within 18 h after infection. Despite the high virus yield, cytopathology was not observed. Examination of the tissue culture media failed to reveal any evidence of interferon, but the virus was found to be as sensitive to mouse interferon as vesicular stomatitis virus. Incubation of mouse embryo cells with actinomycin D markedly inhibited viral replication, whereas cytosine-beta-d-arabinofuranoside and 5-fluorodeoxyuridine had no effect on replication. These findings indicate that new DNA synthesis is not required but suggest that the intact function of cellular DNA may be required for lactic dehydrogenase virus replication.

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.

The RNAs of infective and incomplete influenza virions grown in MDBK and HeLa cells

The MDBK line of bovine kidney cells inoculated with the WSN strain of influenza virus at high or low multiplicity produce a high yield of infective virus containing at least five distinct pieces of RNA. There is a significant decrease in infectivity and PFU HA ratio only after three or four serial undiluted passages of virus in MDBK cells, but when this decrease occurs, it is accompanied by a decrease in the largest piece of viral RNA and an increase in the amount of the small, heterogeneous RNA. HeLa cells inoculated with the WSN strain of influenza virus grown in MDBK cells produce infective virus containing at least 5 pieces of RNA similar to those in virus obtained from chick embryo and MDBK cells; however, HeLa cells inoculated with virus grown in the chick embryo produce noninfective virus with a deficiency of the largest piece of viral RNA and an increase in the amount of small, heterogeneous RNA. These results confirm the partial host cell dependence of incomplete virus formation, the loss of the largest piece of viral RNA, and the increase in the amount of small RNA in incomplete virus. They also indicate that HeLa cells can produce infective influenza virus if the inoculum is relatively free of incomplete virus, and suggest that the mechanism involved in the abortive infection of HeLa cells is similar to that involved in the von Magnus type of incomplete virus formation in other cells.

Restricted replication of frog virus 3 in selected variants of BHK cells.

Synthesis and maturation of frog virus 3 deoxyribonucleic acid (DNA) in BHK cells and selected variants, in chick fibroblasts, and in minnow cells were compared. Wide ranges in rates of DNA synthesis and assembly of virions were found. At least three variants of BHK cells can be obtained: (i) fully permissive, characterized by rapid DNA synthesis and assembly to give a high yield of infective virus; (ii) semipermissive, in which viral DNA is synthesized slowly for extended periods of time, and the yield of infective virus is poor; and (iii) nonpermissive, in which virus adsorbs to cells and arrests host functions but viral DNA is not synthesized. Kinetics of appearance of virions and procedures for their separation from cell extracts are described.

Plasma membrane lipids and parainfluenza virus assembly

The lipids of bovine kidney (MDBK) and hamster kidney (HaK) cells, their plasma membranes, and parainfluenza (SV5) virions grown in these cells have been analyzed and compared with previous results with rhesus monkey kidney (MK) and baby hamster kidney (BHK21-F) cells. Features common to all plasma membranes include a high content of cholesterol, sphingomyelin, and saturated fatty acids. The fatty acid composition of cells and plasma membranes depends to a large extent on the fatty acids in the medium. However, each of the membranes has a distinct and specific phospholipid pattern and molar ratio of cholesterol to phospholipid. With few exceptions, the lipids of virions closely resemble those of the plasma membrane of the cell in which the virus was grown. The results indicate that, in general, the virions acquire the lipid pattern of the plasma membrane of the host cell. However, a lipid pattern characterized by a high phosphatidylethanolamine and relatively lower phosphatidylcholine content may be optimal for the formation of SV5 virions. These conditions are met in the MK membrane, and this cell produces a very high yield of SV5 virions. MDBK membranes have a slightly different lipid pattern, but do contain much phosphatidylethanolamine, and MDBK cells produce a moderately high yield of virus with a lipid composition similar to that of virus grown in MK cells. This suggests that, within narrow limits, some selective rearrangement of membrane lipids incorporated into virions is possible in some cells. Hamster cell membranes have a very different set of lipids, which are incorporated essentially unchanged into virions, and these cells produce a very low yield of virus. The data suggest a correlation between membrane lipid composition and virus yield, and possibly other biological properties of the plasma membranes.

Further Studies on the Differences in the Interaction of Simian Virus 40 with African Green Monkey Kidney and Human Diploid Cells

Infection of human diploid cells with Simian virus 40 (SV 40) leads to an abortive virus cycle characterized by little virus production, little or no cell destruction, no inhibition of cell replicating capacity, low efficiency of induction of tumour antigen (T-antigen) and eventual transformation of the morphological, chromosomal, and growth characteristics of the cells (Koprowski et al. 1962; Shein & Enders, 1962; Rabson et al. 1962; Weinstein & Moorhead, 1965). In marked contrast, in stationary-phase primary African green monkey kidney (GMK) cells, SV 40 infection leads to high yields of virus, highly efficient induction of T-antigen and extensive cell destruction (Carp & Gilden, 1966). The determination of the cause of these marked differences might clarify the mechanism of the transformation process. Previous results indicated that the virus adsorbed and entered into eclipse phase with equal efficiency for the two cell types (Carp & Gilden, 1966).

Replication of influenza virus in a continuous cell line: High yield of infective virus from cells inoculated at high multiplicity

Replication of influenza virus in a continuous cell line: High yield of infective virus from cells inoculated at high multiplicity

Electron microscopic studies on bovine ephemeral fever virus.

In an effort to raise the titre of Ephemeral fever virus (EFV) in BHK-21 cells, cell cultures were treated with 0.2 μg/ml “cortisol”. Relatively high yields of virus resulted in the first electron micrographs of EFV which is cone-shaped, having a height of 176 mμ, and a basal diameter of 88 mμ. The cone apparently consists of a spiral of 10 turns with a pitch of 16.6 mμ. From these figures the total length of the spiral is approximately 2.2 μ. The electron-dense spiral is presumably nucleoprotein in nature. Suggestions are made with respect to the classification of the virus.

Development of measles virus in vitro

The sequence of development of measles virus in cells cultivated in vitro was studied by electron microscopic techniques. The observations were correlated with virus growth measured by infectivity assay. Viral nucleoprotein tubules were first detected in the cytoplasm of infected cells, and shortly thereafter similar tubules were detected in the nucleus. In any one cell tubules were usually present either in the nucleus or in the cytoplasm, and rarely in both. No evidence suggesting that tubules migrated from one site to the other was obtained. At later stages in the infectious cycle, cytoplasmic tubules were extruded by a budding process at the cell surface, resulting in the formation of enveloped particles, interpreted as measles virions. Cytoplasmic tubule formation was associated with active budding and high yields of virus, whereas when intranuclear formation was predominant, little or no budding was detected and viral yields were low. The distribution of lengths of isolated nucleocapsids in negatively stained preparations showed a peak at 1.4-1.6 μ. When allowance was made for stretching of the nucleocapsid helix, this figure was reduced to 1.1 μ. The number of helical turns per nucleocapsid peaked sharply at a value of 204. On the assumption that the conformation of the measles nucleocapsid is similar to that of tobacco mosaic virus, the length and molecular weight of measles virus RNA were calculated and found to be 6.4μ and 6.2 × 10 6 daltons, respectively.

Virus enhancement with a growth product of Streptomyces scabies. II. Increased release of coxsackievirus B5 from HEp-2 monolayers treated with scaben

A single-cycle infection of scaben-treated HEp-2 monolayers with coxsackievirus B5 showed no increase in total virus yield but an enhancement of virus release which was correlated with an earlier cytopathic effect. These findings were further substantiated in multiple-cycle infections where enhanced release shortened the period necessary to infect all the cells in a monolayer, thus, in effect, producing higher yields of virus in a shorter time. Larger plaques were obtained with both coxsackievirus B5 m and m+ variants when scaben was added to the agar overlay. The mechanism of action of scaben is not clear but we suspect that it alters the cell membrane and thus enhances virus release.

Enumeration of VSV particles and a demonstration of their growth kinetics by electron microscopy.

SummaryGrowth curve studies indicate that the m.o.i. plays a major role in the replication and resultant quantity of VSV. Consistent high yields of the virus can be obtained when the m.o.i. is kept at approximately 1. Natural aggregation studies using the count technique indicate that the virus is extruded from the cells in loosely connected chains. The agar sedimentation procedure for counting virus particles can be readily adapted to vesicular stomatitis virus with high efficiency.The author is most grateful for the excellent technical assistance of Mary-Martin Bratcher.

On the role of the response of the cell membrane in determining virus virulence. Contrasting effects of the parainfluenza virus SV5 in two cell types.

The simian myxovirus SV5 multiplies in a continuous line of baby hamster kidney (BHK21-F) cells causing extensive cell fusion, followed by cell death. After inoculation of 15 PFU/cell, the latent period was 7 hr, the doubling time approximately 60 min, and the yield 7 PFU per cell. Giant cell formation began about 6 hr after infection and rapidly progressed to the formation by 14 to 18 hr of a single syncytium which disintegrated by 24 to 36 hr. In contrast, SV5 multiplies in primary rhesus monkey kidney cells for long periods of time producing high yields of virus with little cytopathic effect. High multiplicities of SV5 induced cell fusion in BHK21-F cells within 1 hr in the absence of virus multiplication but had no visible effect on monkey kidney cells. Time-lapse photomicrography has demonstrated that giant cells form by fusion of infected cells, and that some polykaryocytes divide. During aberrant division of polykaryocytes giant nuclei are formed from the nuclear material of several parent nuclei. The cytoplasmic development of viral antigens as demonstrated by immunofluorescence is similar in BHK21-F and monkey kidney cells. Synthesis of cellular DNA, RNA, and protein in monkey kidney cells is not shut off by SV5-infection, and in BHK21-F cells synthesis of these macromolecules is not inhibited until after extensive cell fusion has occurred 12 to 15 hr after infection. Persistently infected BHK21-F and monkey kidney cells have been serially carried through 11 and 28 cell passages, respectively. The results suggest that whether SV5 acts as a moderate virus, as in monkey kidney cells, or a virulent virus, as in BHK21-F cells, depends on the response of the cell membrane to the virus.

Interrelationship of Adenovirus (Type 4) and Papovavirus (SV-40) in Monkey Kidney Cell Cultures

Summary Our experimental data indicate: a) Pure Type 4 adenovirus will not produce infectious progeny in monkey kidney cell culture; b) pure Type 4 adenovirus will reproduce in HEK and HEp-2 cells and give high yields of infectious virus; c) the incomplete form of adenovirus developed in monkey kidney tissue is associated with a lack of formation of intranuclear RNA; d) a dual infection of Type 4 adenovirus and SV-40 in primary monkey kidney cell culture allows the adenovirus to mature in an apparently normal manner; e) simultaneous massive infection of monkey kidney cell cultures with SV-40 is required for maximum growth of Type 4 adenovirus.

Multiplication of a myxovirus (SV5) with minimal cytopathic effects and without interference

SV5 virus, a simian myxovirus, multiplies to high titer in rhesus monkey kidney cells, but causes only minimal cytopathic effects. It forms plaques with high efficiency, the plaques consisting of intact cells with diminished neutral red staining.After inoculation of SV5 virus at a multiplicity of 40–70 PFU/cell, the latent period was 6–7 hours, and the doubling time during the period of exponential increase in infective virus was approximately 50 minutes. In 24 hours, yields of 500–1500 PFU/cell were obtained. Cells continued to produce high yields of infective virus for periods up to 30 days.Inoculation of SV5 virus at high multiplicity did not result in autointerference or incomplete virus formation. Multiplication of SV5 virus in monkey kidney cells for 48 hours did not interfere with subsequent infection and plaque formation by a second virus, and did not significantly reduce the yield of a superinfecting virus.