Yield Of Virus Particles Research Articles

Modeling multipartite virus evolution: the genome formula facilitates rapid adaptation to heterogeneous environments†.

Multipartite viruses have two or more genome segments, and package different segments into different particle types. Although multipartition is thought to have a cost for virus transmission, its benefits are not clear. Recent experimental work has shown that the equilibrium frequency of viral genome segments, the setpoint genome formula (SGF), can be unbalanced and host-species dependent. These observations have reinvigorated the hypothesis that changes in genome-segment frequencies can lead to changes in virus-gene expression that might be adaptive. Here we explore this hypothesis by developing models of bipartite virus infection, leading to a threefold contribution. First, we show that the SGF depends on the cellular multiplicity of infection (MOI), when the requirements for infection clash with optimizing the SGF for virus-particle yield per cell. Second, we find that convergence on the SGF is very rapid, often occurring within a few cellular rounds of infection. Low and intermediate MOIs lead to faster convergence on the SGF. For low MOIs, this effect occurs because of the requirements for infection, whereas for intermediate MOIs this effect is also due to the high levels of variation generated in the genome formula (GF). Third, we explored the conditions under which a bipartite virus could outcompete a monopartite one. As the heterogeneity between environments and specificity of gene-expression requirements for each environment increased, the bipartite virus was more likely to outcompete the monopartite virus. Under some conditions, changes in the GF helped to exclude the monopartite competitor, highlighting the versatility of the GF. Our results show the inextricable relationship between MOI and the SGF, and suggest that under some conditions, the cost of multipartition can be outweighed by its benefits for the rapid tuning of viral gene expression.

Hydrophobic-interaction chromatography for purification of influenza A and B virus

Options for hydrophobic-interaction chromatography (HIC) for purification of cell culture-derived influenza A and B virus have been assessed using a 96-well plate format using a semi-high-throughput approach. Follow-up experiments at preparative scale were used to characterize dynamic binding capacity, viral hemagglutinin protein (HA protein) recovery, and the influence of influenza virus (IV) strains on yield and contamination levels. Virus recoveries of up to 96% with a residual DNA level of about 1.3% were achieved.To achieve DNA contamination levels required for manufacturing of influenza vaccines for human use, a purification train comprising clarification, inactivation, concentration, column-based anion-exchange chromatography (AEC), and HIC was used in a final set-up. AEC using strong quaternary ammonium ligands was applied as an orthogonal method for DNA depletion by adsorption. Subsequently, HIC (with polypropylene glycol as functional group) was used to reversibly bind virus particles for capture and to remove residual contaminating DNA and proteins (flow-through). This two-step chromatographic process, which requires neither a buffer exchange step nor nuclease treatment had a total virus particle yield for IV A/PR/8/34 (H1N1) of 92%. The protein and the DNA contamination level could be reduced to 42% and at least 1.0%, respectively. With 17.2 μg total protein and 2.0 ng DNA per monovalent dose, this purity level complies with the limits of the European Pharmacopeia for cell culture-derived human vaccines. Overall, the presented downstream process represents a valuable alternative to existing virus purification schemes. Furthermore, it utilizes only off-the-shelf materials and is a simple as well as an economic process for production of cell culture-derived viruses and viral vectors.

HPV-16 E1, E2 and E6 each complement the Ad5 helper gene set, increasing rAAV2 and wt AAV2 production.

Adeno-associated virus type 2 (AAV) is a popular vector for human gene therapy, because of its safety record and ability to express genes long term. Yet large scale recombinant (r)AAV production remains problematic due to low particle yield. The adenovirus (Ad) and herpes (simplex) virus (HSV) helper genes for AAV have been widely used and studied, but the helper genes of human papillomavirus (HPV) have not. HPV-16 E1, E2 and E6 help wild type (wt) AAV productive infection in differentiating keratinocytes, however HEK293 cells are the standard cell line used for generating rAAV. Here we demonstrate that the three HPV genes were unable to stimulate significant rAAV replication in HEK293 cells when used alone. However, when used in conjunction (complementation) with the standard Ad5 helper gene set, E1, E2 and E6 were each capable of significantly boosting rAAV DNA replication and virus particle yield. Moreover, wt AAV DNA replication and virion yield were also significantly boosted by each HPV gene along with wt Ad5 virus co-infection. Mild to moderate changes in rep- and cap–encoded protein levels were evident in the presence of the E1, E2 and E6 genes. Higher wt AAV DNA replication was not matched by similar increases in the levels of rep-encoded protein. Moreover, while rep mRNA was up-regulated, cap mRNA was up-regulated more. Higher virus yields did correlate most consistently with increased Rep52, VP3 and VP-related 21/31 kDa species. The observed boost in wt and rAAV production by HPV genes was not unexpected, as the Ad and HPV helper gene sets do not seem to recapitulate each other. These results raise the possibility of generating improved helper gene sets derived from both the Ad and HPV helper gene sets.

Stochastic population balance modeling of influenza virus replication in vaccine production processes

A distributed population balance model of influenza A virus replication in adherent Madin-Darby canine kidney cells has been developed to reproduce and interpret flow cytometry data for virus propagation in microcarrier culture. The population of cells is differentiated into uninfected, infected and degraded cells. As an internal coordinate the number of intracellular viral components is considered. The main focus of the model is to link the time course of intracellular virus protein accumulation monitored by flow cytometry with the total yield of virus particles measured by the hemagglutination assay. The model allows simulating the extracellular virus dynamics for multiplicities of infection in the range 0.025–3.0. Shape of predicted histograms is in general agreement with distributions obtained by flow cytometry. Differences in time course at about 12–14 and 20 h post infection indicate that additional assumptions on intracellular virus dynamics are required to fully explain experimental data. Furthermore, prerequisites for virus replication, like receptor binding sites, the number of endosomes or the demand for free amino acids and nucleotides for virus synthesis can be estimated and compared with cellular resources available. Simulation results suggest that intracellular pools of free amino acids as well as early cell death due to influenza virus-induced apoptosis can limit virus yields. It is expected that based on a better understanding of the infectivity status of cells and the spreading of viruses in population of cells in bioreactors strategies on design and optimization of vaccine production processes can be developed.

Role of the TSG101 Gene in Epstein-Barr Virus Late Gene Transcription

Rta, an Epstein-Barr virus (EBV)-encoded immediate-early protein, governs the reactivation of the viral lytic program by transactivating a cascade of lytic gene expression. Cellular transcription factors such as Sp1, ATF2, E2F, and Akt have been demonstrated to mediate Rta transactivation of lytic genes. We report herein that Rta associates with another potent transcription factor, tumor susceptibility gene 101 (TSG101), to promote the activation of EBV late genes. Results from an EBV cDNA array reveal that depletion of TSG101 by siRNA potently inhibits the transcription of five Rta-responsive EBV late genes, BcLF1, BDLF3, BILF2, BLLF1, and BLRF2. Depletion of TSG101 impairs the Rta transactivation of these late promoters severely. Moreover, a concordant augmentation of Rta transactivating activity is observed when TSG101 is overexpressed following ectopic transfection. Mechanistically, Rta interaction with TSG101 causes the latter to accumulate principally in the nuclei, wherein the proteins colocalize and are recruited to the viral promoters. Of note, TSG101 is crucial for the efficient binding of Rta to these late promoters. As a result, cells with defective TSG101 fail to express late viral proteins, leading to a decrease in the yield of virus particles. Thus, the contribution of TSG101 to Rta-mediated late gene activation is of great importance for completion of the EBV productive lytic cycle. These observations consolidate a role for TSG101 in the replication of EBV, a DNA virus, that differs from what is observed for RNA viruses, where TSG101 aids mainly in the endosomal sorting of enveloped late viral proteins for assembly at the plasma membrane.

Inhibition by prostaglandin PGA1 on the multiplication of influenza virus is a dose-dependent effect

Cyclopentenone prostaglandins (PGs), are strong inhibitors of the multiplicative cycle of a wide variety of enveloped RNA and DNA viruses. Their antiviral activity is generally associated with alterations in the synthesis or maturation of specific virus proteins. In this report, we describe the effect of cyclopentenone PGA1 on the replication of influenza A virus Ulster 73 in LLC-MK2 cells. PGA1 was found to inhibit viral replication in a dose-dependent fashion and virus particle yield was reduced at a PGA1 concentration, which did not suppress protein synthesis in mock-infected cells. The kinetic of late viral protein synthesis was delayed in PGA1-treated cells till 10 h post-infection; after that period, viral polypeptide synthesis appeared to be similar in PGA1-treated as well as untreated cells both infected by Ulster 73 virus. This finding suggests that PGA1 might interfere with one or more events in the viral multiplicative cycle such as protein synthesis and assembly, correct insertion of virus polypeptides into the cell membrane and, or maturation of Ulster 73 virion particles. In particular, inhibition of viral replication in LLC-MK2 cells by PGA1 is accompanied by the induction of a cellular polypeptide of 70K molecular weight. We identified this cell protein as a heat shock protein (HSP) related to the inducible isoform of HSP 70, a polypeptide of 72K molecular weight. Induction of this polypeptide by PGA1 was found to be dose-dependent and a substantial accumulation could be seen at a PGA1 concentration that did not inhibit cell protein synthesis in uninfected cells. HSP 70 synthesis started after the beginning of PGA1 treatment and remained at the same level for at least 10 h, leading us to hypothesize that the delay of production of late Ulster 73 proteins could be the consequence of HSP 70 synthesis. These results suggest that HSP 70 could play a role in the antiviral activity of cyclopentenone PGA1 in LLC-MK2 cells.

Comparative susceptibility of two different hosts to genotypic variants of the Anticarsia gemmatalis nuclear polyhedrosis virus

The susceptibility of third instar larvae ofAnticarsia gemmatalis (Lepidoptera: Noctuidae) and Diatraea saccharalis (Lepidoptera: Pyralidae) to ten distinct plaque purified genotypic variants of a selected isolate of the Anticarsia gemmatalis multiple-embedded nuclear polyhedrosis virus (AgMNPV), was compared. Despite the fact that this isolate, AgMNPV-Ds20, represents a wild strain of the AgMNPV selected for higher virulence to D. saccharalis, an alternate host, most of the variants are much more virulent to the original host Anticarsia than to Diatraea. Bioassays have shown an over one hundred-fold variation in LD 50 values ranging from 1700 polyhedron inclusion bodies (PIBs) to more than 200 000 PIBs/larva. The PIB production in infected larvae increased with the pathogenicity of the variant to the host, showing an average ten-fold reduction in Diatraea when compared to Anticarsia for the same variant. The virus particle yield ranged from 6 x 10 7 to more than 10 9 PIBs/g of infected larvae in Diatraea and from 8x10 8 to more than 10 10 PIBs/g of infected Anticarsia larvae. The data show a clear difference of the pathogenicity of the genotypic variants of AgMNPV in vivo both between the original and alternate host and between the individual variants for the same host. These differences found in vivo indicate that monitoring of shifts in variant frequency of wild and laboratory-propagated viral isolates in these highly heterogeneous populations would help ensure the efficacy of biological control programs.

Induction of programmed cell death (apoptosis) by influenza virus infection in tissue culture cells.

The process of cell death caused by influenza virus infection in cultured MDCK and HeLa cells was analysed. This infection gave rise to nuclear fragmentation and chromatin condensation accompanied by chromosomal DNA fragmentation into oligonucleosomes. Chromosomal DNA fragmentation progressed concomitantly with cell lysis of MDCK cells and HeLa cells, producing high and low yields of virus particles, respectively, indicating that the extent of cell lysis was not proportional to the virus production. The endonuclease inhibitor zinc blocked DNA fragmentation in MDCK cells. Cycloheximide inhibited DNA fragmentation as well as cell lysis. Inhibition occurred when the drug was added to the medium within 2 h after infection but not efficiently at 4 h or later. Infection induced the Fas Ag gene, which encodes a possible apoptosis-mediating molecule, in the early infectious stage followed by the expression of Fas Ag on the cell surface. These results suggested that influenza virus infection causes apoptotic death of cultured cells, and their fate might be determined at an early stage of the infection by induction of an apoptotic gene.

Analysis of deletions and thermosensitive mutations in Rous sarcoma virus gag protein p10.

Rous sarcoma virus protein p10 is a gag component of the virion present in stoichiometric amount but of unknown function. To characterize this protein, a series of mutants of p10 with linker insertions or deletions was generated by site-directed mutagenesis of a cloned proviral DNA. The deletions and two of the linkers insertions, which disrupted proline pairs, reduced the yield of virus particles upon transfection. These two linker insertion mutants were moreover thermosensitive for this phenotype, producing fewer virus particles at 41 degrees C than at 36 degrees C. Examination of the intracellular viral proteins demonstrated that for all mutants, the amount of gag precursor was similar to the wild-type level. Moreover, the amount of mature gag CA that could be detected by this analysis was similar between each of the mutants and the wild type. This finding suggests that the transport of gag to the membrane and the initial stages of maturation were not affected by the mutations. The virus particles contained normal amounts of active reverse transcriptase, showing that the gag-pol polyprotein was incorporated and cleaved properly. Viral RNA was quantitatively and qualitatively similar in mutant and wild-type virions. However, the infectivity of the mutants virions differed; one of the thermosensitive linker insertions that had no effect on particle production at 36 degrees C was nevertheless noninfectious at that temperature. Together, these data suggest that the p10 protein is involved in a late steps of virus maturation, possibly budding, and perhaps also in an early event of viral infection.

An improved procedure for the purification of apple chlorotic leaf spot virus

A reproducible procedure is described for the purification of apple chlorotic leaf spot virus (CLSV) from Chenopodium quinoa. Two cycles of sucrose density-gradient centrifugation in a magnesium-containing buffer were used. A relatively high yield of virus particles was obtained with little degradation observed. The average yield was 2.3 mg 100 g−1 fresh leaf tissue. The modal lengths of purified virus particles and particles from leaf sap were 692 nm and 704 nm, respectively.Key words: Chenopodium quinoa, sucrose density gradient, magnesium-containing buffer, intact virus

The effect of cicloxolone sodium on the replication in cultured cells of adenovirus type 5, reovirus type 3, poliovirus type 1, two bunyaviruses and Semliki Forest virus.

The effect of cicloxolone sodium (CCX) on the replication of typical representatives of different virus families [adenovirus type 5 (Ad-5), reovirus type 3 (Reo-3), Bunyamwera and Germiston viruses, poliovirus type 1 (Polio-1) and Semliki Forest virus (SFV)] in tissue culture was investigated. The Golgi apparatus inhibitor monensin (Mon) and CCX were shown to have analogous effects on some aspects of virus replication. Although the Mon-like effect of CCX played no role in the antiviral activity against Ad-5, Reo-3 or Polio-1, it could entirely account for the antiviral activity against the Bunyamwera and Germiston viruses, for which inhibition of glycoprotein processing was responsible for the antiviral activity. In the case of SFV, the Mon-like activity of CCX caused cytoplasmic assembly of fully infectious SFV within vacuoles and thus impaired virus release without altering total infectious virus yield. Fewer Ad-5 and Reo-3 progeny were produced in the presence of the drug. CCX had a dose-dependent biphasic effect on the particle:p.f.u. ratio of the Reo-3 yield. At low CCX concentration (less than 50 microM) the virus yield contained poor quality, non-infectious virus, but at higher CCX concentration (greater than or equal to 100 microM) low quality virus could no longer be successfully assembled. We conclude that the antiviral effect can be manifested in three ways: (i) by a reduction in the virus particle yield produced; (ii) by a loss of quality (relative infectivity); (iii) by a virucidal effect of the drug. We have previously defined three CCX sensitivity classes. Mechanisms (i), (ii) and (iii) operate against viruses belonging to class CCXs-1 [herpes simplex virus (HSV) type 1, HSV-2 and vesicular stomatitis virus], but essentially only (i) and (ii) affect Reo-3 (CCXs-2), whereas (i) and possibly (iii) affect Ad-5 (CCXs-2). In the case of SFV (CCXs-3) none of these mechanisms operate, but relocation of assembled virus is found.

Purification and particle properties of groundnut rosette assistor virus and production of a specific antiserum*

SUMMARYPurified preparations of the luteovirus, groundnut rosette assistor virus (GRAV), were made by treatment of groundnut leaf extracts with cellulase, followed by sucrose density gradient centrifugation. Yields of virus particles were about 0·5‐1·0 mg/kg leaf material. The preparations contained isometric particles c. 28 nm in diameter with a sedimentation coefficient (s20, w) of 115 S, a buoyant density in Cs2SO4 of 1·34 g/cm3, and A260/A280 of 1·86. The particles contained a single species of nucleic acid (presumably RNA), of mol. wt 2·09 × 106and with no detectable polyadenylate sequence, and a single protein species, of mol. wt 24 × 103. An antiserum produced in a rabbit had a titre of 1/256 in gel diffusion tests and detected GRAV in leaf extracts by ELISA. GRAV particles reacted in F(ab')2‐ELISA and immunosorbent electron microscopy (ISEM) tests with antisera to bean leaf roll, potato leafroll and tobacco necrotic dwarf luteoviruses, but did not react with antisera to carrot red leaf luteovirus.

The effect of triterpenoid compounds on uninfected and herpes simplex virus-infected cells in culture. III. Ultrastructural study of virion maturation.

Electron microscope studies been made of mock-infected or herpes simplex virus (HSV) type 1 (strain 17)- or HSV-2(strain HG52)-infected Flow 2002 cells grown in the absence or presence of various concentrations of cicloxolone sodium (CCX). Fifty non-serial, thin sections of mock-infected or HSV-infected cells, which contained a portion of the nucleus, were examined by transmission electron microscopy. With increasing drug concentration, counts of capsid structures both in the nucleus and cytoplasm showed a decrease in the number of virions per cell section, an increase in the ratio of nuclear to cytoplasmic virus, a relative reduction in the percentage of cytoplasmic enveloped virus particles, but no effect on the ratio of empty to core-containing capsid structures. The high particle to p.f.u. ratio induced by CCX treatment is thus not explained through failure to assemble morphologically mature core-containing capsids. In part it can be explained by non-envelopment, but in addition, specific effects on other virion proteins (tegument and envelope) must be involved. The extracellular virus particle yield was unaffected, indicating that CCX treatment enhances the egress of HSV. In the presence of CCX the encapsidation of HSV DNA into DNase-resistant structures was unaffected.

Replication of Red Clover Necrotic Mosaic Virus RNA in Cowpea Protoplasts: RNA 1 Replicates Independently of RNA 2

SUMMARY Inoculation of cowpea mesophyll protoplasts with unseparated RNA 1 and RNA 2 from red clover necrotic mosaic virus in the presence of polyethylene glycol resulted in virus RNA replication, the synthesis of virus capsid polypeptide and the formation of virus particles; 75 to 85% of the viable protoplasts became infected and the yield of virus particles or virus RNA after 72 h incubation corresponded to about 3 × 106 virus genomes per infected protoplast. In contrast, no replication could be detected when protoplasts were inoculated with RNA 2 alone. However, inoculation of protoplasts with RNA 1 alone resulted in its replication and the formation of virus particles, with a yield similar to that obtained after inoculation with both RNAs. Since infection of plants requires both RNA 1 and RNA 2 to be present, the demonstration of the independent replication of RNA 1 in single cells strengthens the hypothesis that RNA 2 plays a role in the cell-to-cell transmission of the virus.

Possible role of ADP-ribosylation of adenovirus core proteins in virus infection.

We have investigated the role of poly(ADP)-ribosylation of adenoviral proteins in virus infection. Viral core proteins V and the precursor to protein VII were shown to be in vivo and in vitro acceptors of ADP-ribose. In vivo ADP-ribosylation was restricted to viral proteins as the histones were not labeled during the late phase of infection. The ADP-ribosylated core proteins were assembled into mature virus particles. In vitro ADP-ribosylation of adenoviral core proteins performed with purified poly(ADP-ribose) polymerase led to relaxation of the chromatin structure of both ts1 and wild type pyridine cores and pentonless particles and triggered the complete dissociation of wild type particles. A critical role for poly(ADP)-ribosylation in virus infection was confirmed by measuring the effect of the inhibitors 3-aminobenzamide and nicotinamide on virus particle yield and infectivity. Both inhibitors depressed particle yield by up to 9-fold, but infectivity was reduced by up to 10(4)-fold. These results suggest that ADP-ribosylation of adenovirus core proteins may have a role in virus decapsidation.

Purification of particles of subterranean clover red leaf virus using an industrial-grade cellulase

Particles of two isolates of subterranean clover red leaf virus were purified by a method in which infected plant tissue was digested with an industrial-grade cellulase, Celluclast ® 2.0 L type X. The yields of virus particles using this enzyme were comparable with those obtained using either of two laboratory-grade cellulases, Cellulase type 1 (Sigma) and Driselase®. However, the specific infectivity or aphid transmissibility of the particles purified using Celluclast ® was 10–100 times greater than those of preparations obtained using laboratory-grade cellulases or no enzyme. The main advantage of using Celluclast® is that at present in Australia its cost is only ca. 1% of laboratory-grade cellulases.

Structure of poliovirus replicative intermediate RNA: Electron microscope analysis of RNA cross-linked in vivo with psoralen derivative

The structure of the poliovirus replicative intermediate RNA was examined by electron microscopy after cross-linking in vivo with 4′-aminomethyl-4,5′,8-trimethylpsoralen. After purification from infected cells, undenatured RI † † Abbreviations used: RI, replicative intermediate; AMT, 4′-aminomethyl-4,5′,8-trimethylpsoralen; RF, double-stranded poliovirus RNA. appeared as a double-stranded backbone of genome length, with an average of three (and occasionally up to eight) nascent, single-stranded tails. After denaturation, however, only single strands of heterogeneous length were visualized, indicating that the RI in the cell contains little or no duplex structure, and thus nascent chains are only transiently hydrogen-bonded to their template over short regions. The double-stranded backbone of undenatured RI, observed previously by others and in these experiments, is due to collapse of complementary chains during the deproteinization and purification procedures. The effectiveness of the in vivo cross-linking procedure was demonstrated by the complete inhibition of viral RNA synthesis in treated cells and by direct binding of [ 3H]AMT to RI molecules in vivo. Mature polio virions are impermeable to AMT; however, growth of virus in cells incubated with AMT in the dark resulted in normal yields of virus particles containing RNA genomes, whose infectivity could be subsequently photo-inactivated. The frequency of AMT-induced cross-linking was determined by analyses of double-stranded poliovirus RNA (RF). Cross-linking in vitro followed by spreading for electron microscopy under denaturing conditions yielded bubbled duplex structures with a minimum of one interstrand cross-link per 80 base-pairs. RF cross-linked in vivo also showed extensive cross-linking, decreased about fivefold from the in vitro cross-linked value. Thus, the failure to detect cross-linked RI under these conditions indicates that extensive base-pairing does not exist in vivo.

Purification of bovine papilloma virus by gel filtration on sephacryl ® S-1000 superfine

Bovine papilloma virus was purified from crude extracts of bovine warts by gel filtration in Sephacryl ® S-1000 Superfine. Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis and electron microscopy shows that the virus preparation is as pure as that obtained by alternative techniques. The yield of virus particles is in the range 55–80%. The main advantage of using the gel filtration technique is that it is much less time-consuming than currently used centrifugation procedures.

The effect of cytochalasin D and monensin on enveloped vaccinia virus release

Both monensin and cytochalasin D reduced the production of infectious cell associated virus and infectious extracellular virus with the latter clearly being the more sensitive. The difference in yields were even more clearly seen if the yield of virus particles was monitored instead of yield of infectious virus. Addition of 1 microgram/ml cytochalasin D or 1 microM monensin to the growth medium of vaccinia virus-infected cells inhibited the appearance of extracellular enveloped vaccinia virus (EEV) in the growth medium without affecting the production of intracellular naked vaccinia virus (INV) particles. Although EEV was not released into the medium of cytochalasin D treated cells, EEV was, nevertheless, detectable in CsCl gradients of cell associated virus. Monensin treatment did not affect the synthesis of vaccinia glycoproteins but did significantly reduce the transport of these glycoproteins to the cell surface and also reduced the secretion of proteins. Monensin had the additional effect of causing the appearance of numerous large vacuoles in the cytoplasm. Vaccinia is normally wrapped by cytoplasmic membranes in preparation for release. The monensin induced conversion of cytoplasmic membranes to large vacuoles is presumably the basis for the block in virus wrapping and subsequent release. Cytochalasin D did not alter any of the steps in protein synthesis, transport or secretion. Electronmicroscopic studies confirmed the existence of EEV on the surface of infected cells treated with cytochalasin D. This drug which specifically affects cellular microfilament organisation thus imposes a block on the final release of EEV from the cell surface.

The Effect of Horse Serum on the Quality of Vaccinia Virus Grown in L Cell Cultures

Summary Vaccinia virus, inoculated at a multiplicity of 10 particles per L cell in closed tube cultures with media containing the serum of certain individual horses has produced progeny notably poor in plaque-forming efficiency (quality). The total virus particle yield from these cultures has been high and independent of the particular serum used. These differences were greatly reduced when the sera were heated at 56°C for 30 min. Possible reasons for these effects include indirect influence of a heat-labile serum component upon progeny virus quality through the agency of the cell.