Growth characteristics and intraspecies host specificity of a large virus infecting the dinoflagellate Heterocapsa circularisquama

The growth characteristics and intraspecies host specificity of Heterocapsa circularisquama virus (HcV), a large icosahedral virus specifically infecting the bivalve-killing dinoflagellate H. circularisquama, were examined. Exponentially growing host cells were more sensitive to HcV than those in the stationary phase, and host cells were more susceptible to HcV infection in the culture when a higher percent of the culture was replaced with fresh medium each day, suggesting an intimate relationship between virus sensitivity and the physiological condition of the host cells. HcV was infective over a wide range of temperatures, 15 to 30 degrees C, and the latent period and burst size were estimated at 40 to 56 h and 1,800 to 2,440 infective particles, respectively. Transmission electron microscopy revealed that capsid formation began within 16 h postinfection, and mature virus particles appeared within 24 h postinfection at 20 degrees C. Compared to Heterosigma akashiwo virus, HcV was more widely infectious to H. circularisquama strains that had been independently isolated in the western part of Japan, and only 5.3% of the host-virus combinations (53 host and 10 viral strains) showed resistance to viral infection. The present results are helpful in understanding the ecology of algal host-virus systems in nature.     

High-titer replication of nondefective Sendai virus in MDBK cells.

Egg-grown Sendai virus was adapted to growth in a bovine kidney cell line (MDBK cells) by serial passage under defined conditions. The adapted virus contained only 50S RNA and was highly infectious for MDBK cells. Infection of these cells with a high multiplicity of adapted virus resulted in a yield of 10(8) MDBK-infectious units/ml by 18 h, accompanied by severe cytopathic changes in the host. Cell fusion did not occur. Examination of the proteins of the adapted virus revealed that despite the high infectivity of this virus for MDBK cells the virions contained considerable quantities of Fo, the precursor to the F glycoprotein that is responsible for cell fusion and high infectivity in other systems.     

Growth Characteristics and Intraspecies Host Specificity of a Large Virus Infecting the Dinoflagellate Heterocapsa circularisquama

The growth characteristics and intraspecies host specificity of Heterocapsa circularisquama virus (HcV), a large icosahedral virus specifically infecting the bivalve-killing dinoflagellate H. circularisquama, were examined. Exponentially growing host cells were more sensitive to HcV than those in the stationary phase, and host cells were more susceptible to HcV infection in the culture when a higher percent of the culture was replaced with fresh medium each day, suggesting an intimate relationship between virus sensitivity and the physiological condition of the host cells. HcV was infective over a wide range of temperatures, 15 to 30°C, and the latent period and burst size were estimated at 40 to 56 h and 1,800 to 2,440 infective particles, respectively. Transmission electron microscopy revealed that capsid formation began within 16 h postinfection, and mature virus particles appeared within 24 h postinfection at 20°C. Compared to Heterosigma akashiwo virus, HcV was more widely infectious to H. circularisquama strains that had been independently isolated in the western part of Japan, and only 5.3% of the host-virus combinations (53 host and 10 viral strains) showed resistance to viral infection. The present results are helpful in understanding the ecology of algal host-virus systems in nature.     

Formation of a host range mutant of the lymphotropic strain of minute virus of mice during persistent infection in mouse L cells

Minute virus of mice (i), the lymphotropic strain of minute virus of mice, established a persistent infection in normally restrictive L cells. The carrier state, which lasted 150 days, exhibited three clearly distinguishable stages. During the early stage (days 1 to 10 postinfection), small amounts of virus were formed. A "crisis" then developed that lasted 50 to 60 days and was characterized by massive cell lysis and high titers of virus. This was followed by a 70- to 80-day period in which small but stable quantities of virus were produced. Virus shed by the carrier culture during the latter phase had acquired an altered host range, namely, it had lost its ability to replicate in T-lymphocyte cell lines and had adapted to growth in L cells. Virus isolated at this time from a single plaque in L cells, designated hr301, was shown to possess similar host range properties. No differences, however, could be found between the DNAs of minute virus of mice (i) and of hr301 by restriction enzyme analysis, suggesting that the mutation that affected the viral host range did not involve an extensive region of the viral genome.     

Optimization of reovirus production from mouse L-929 cells in suspension culture

Reovirus serotype 3 Dearing (T3D) has shown potential as a novel cancer therapy. To support the increasing demand for reovirus, a two-stage perfusion mode scheme is proposed for cell growth and reovirus production. Mouse L-929 cells were used as the host for reovirus infection due to their ability to grow well in suspension culture. Several L-929 cell growth and reovirus infection characteristics were investigated and optimized in spinner flask batch cultures. For the growth of L-929 cells, a balanced nutrient-fortification of SMEM medium increased the maximum cell density by 30%, compared to normal SMEM; however, ammonia and lactate accumulations were found to inhibit further cell growth. For the production of reovirus, approximately 90% increase in viral yield resulted when the infection temperature was reduced from 37 to 33 degrees C. Infectious reovirus particles were shown to be stable in conditioned medium at 37 and 33 degrees C. The final virus titer was dependent on the multiplicity of infection (MOI) and the host cell density at the time of infection. A combination of an MOI of 0.1 pfu/cell and an initial host cell density of 1.0 x 10(6) cells/mL in fortified medium resulted in a maximum virus titer of (4.59 +/- 0.16) x 10(9) pfu/mL and a specific yield of (2.34 +/- 0.08) x 10(3) pfu/cell. At an optimal harvest time of the infection process, 99% of the virus was associated with the cellular debris. Finally, the presence of 5.0 mM ammonia in the culture medium was shown to seriously inhibit the reovirus yield, whereas lactate concentrations up to 20 mM had no effect.     

Sindbis virus glycoproteins: effect of the host cell on the oligosaccharides.

Sindbis virus was grown in four different host cells and the carbohydrate portions of the glycoproteins were analyzed. Sindbis virus grown in BHK-21 cells has more sialic acid and galactose than Sindbis virus grown in chicken embryo cells. In other respects the carbohydrates from virus grown in these two hosts are very similar. Sindbis virus grown either in chick cells transformed by Rous sarcoma virus or in BHK cells transformed by polyoma virus was also examined. In comparisons of virus from normal and transformed cells, differences in the amount of sialic acid were observed; but otherwise the carbohydrate structures appeared basically similar. The growth conditions used for the host cell also affected the degree of completion of the carbohydrate chains of the viral glycoproteins.     

Reciprocal productive and restrictive virus-cell interactions of immunosuppressive and prototype strains of minute virus of mice

Viral and cellular factors responsible for parvovirus target cell specificity have been examined for two serologically indistinguishable strains of the minute virus of mice which infect mouse cells of dissimilar differentiated phenotype. Both the prototype strain and the immunosuppressive strain grow in and form plaques on monolayers of simian virus 40-transformed human fibroblasts, a finding that has allowed the comparison of several aspects of their virus-host cell interactions. Although closely related by antigenic and genomic criteria, both the prototype strain and the immunosuppressive strain are restricted for lytic growth in each other's murine host cell, that is, in T cells and fibroblasts, respectively. The host range of each virus variant appears to be specified by a genetic determinant that is stably inherited in the absence of selection. In the restrictive virus-host interaction lytic growth is limited to a small or, in some cases, undetectable subset of the host cell population, and the majority of the infected cells remain viable, continuing to grow at the normal rate without expressing viral antigens. The susceptible host cell phenotype is dominant in T lymphocyte x fibroblast fusion hybrids, implying that different cell types express different developmentally regulated virus helper functions that can only be exploited by the virus variant that carries the appropriate strain-specific determinant.     

Growth of an enveloped mycoplasmavirus and establishment of a carrier state.

The growth of an enveloped DNA-containing mycoplasmavirus (MVL2 obtained from R.N. Gourlay) has studied, by using the indicator host Acholeplasma laidlawii strain JA1. From virus one-step growth curves, artificial lysis experiments, and infected cell growth curves, it was found that virus infection is nonlytic. Newly infected cells grow slower and are osmotically more stable than uninfected cells. However, 4 to 6 h after infection, the cells reach a carrier state in which cell growth rate and osmotic fragility are indistinguishable from uninfected cells. Carrier cultures contain free virus. Every carrier culture cell gives rise to either a clone of carrier cells or a clone of MVL2-resistant cells.     

Formation of Viral Ribonucleic Acid and Virus in Cells that are Permissive or Nonpermissive for Murine Encephalomyelitis Virus (GDVII)

GDVII virus growth in BHK-21 cells, a permissive host for the virus, resembled productive infections with other picornaviruses. Virus yields ranged from 100 to 600 plaque-forming units (PFU)/cell. Virus replication in HeLa cells, a nonpermissive host for GDVII virus, was characterized by virus yields of only 0.1 to 5 PFU/cell. Similar low yields of virus have been obtained from HeLa cells at all multiplicities of input up to 6,000 per cell. The progeny particles from HeLa cells were, like the infecting particles, restricted in the HeLa cell host. Despite the great difference in final yields of virus from BHK-21 and HeLa cells, the times when maximal yields were reached were similar. GDVII virus stock grown in BHK-21 cells was designated HeLa(-). A variant of GDVII virus which is capable of extensive growth in HeLa cells was obtained. This variant, designated HeLa(+) GDVII virus, was passaged serially in HeLa cells. Virus yields of 50 to 150 infective virus particles per cell were obtained from infection of HeLa cells with HeLa(+) GDVII virus. The major species of HeLa(+) virus-specific ribonucleic acid (RNA) produced was single stranded and sedimented with an S value of 35S. The rate of accumulation of HeLa(+) virus-specific RNA in HeLa cell cultures was about four times that of HeLa(-) RNA. The amount of virus-specific HeLa(+) RNA formed in HeLa cells was several-fold greater than that of HeLa(-) RNA. With HeLa(-) parent GDVII virus undergoing productive replication in BHK-21 cells or abortive replication in HeLa cells, the major species of virus-specific RNA produced was single stranded and sedimented with an approximate S value of 35S. The amount of HeLa(-) virus-specific RNA extracted from BHK-21 cells was several-fold greater than the amount obtained from HeLa cells.     

Glucose uptake and lactic acid production of adenovirus type 5-infected HEp-2 cells cultured under exponential growth and stationary phase conditions

Exponentially growing HEp-2 cells have a higher rate of glucose uptake and lactic acid production than stationary phase cells. Infection of cells with adenovirus type 5 stimulates glycolysis irrespective of the original rate of the host cells. Therefore, infected cells cultured under exponential growth conditions have a higher rate of glycolysis than infected stationary phase cells. The rate of host cell glycolysis does not influence the time required for virus replication, the yield of infectious virus particles produced, or the time of appearance and progression of virus-induced cytopathology.     

Human cytomegalovirus UL38 protein blocks apoptosis

Apoptosis is an innate cellular defense response to viral infection. The slow-replicating human cytomegalovirus (HCMV) blocks premature death of host cells prior to completion of the infection cycle. In this study, we report that the HCMV UL38 gene encodes a cell death inhibitory protein. A mutant virus lacking the pUL38 coding sequence, ADdlUL38, grew poorly in human fibroblasts, failed to accumulate viral DNA to wild-type levels, and induced excessive death of infected cells. Cells expressing pUL38 were resistant to cell death upon infection and effectively supported the growth of ADdlUL38. Cells infected with the pUL38-deficient virus showed morphological changes characteristic of apoptosis, including cell shrinkage, membrane blebbing, vesicle release, and chromatin condensation and fragmentation. The proteolytic cleavage of two key enzymes involved in apoptosis, namely, caspase 3 and poly(ADP-ribose) polymerase, was activated upon ADdlUL38 infection, and the cleavage was blocked in cells expressing pUL38. The pan-caspase inhibitor Z-VAD-FMK largely restored the growth of ADdlUL38 in normal fibroblasts, indicating that the defective growth of the mutant virus mainly resulted from premature death of host cells. Furthermore, cells expressing pUL38 were resistant to cell death induced by a mutant adenovirus lacking the antiapoptotic E1B-19K protein or by thapsigargin, which disrupts calcium homeostasis in the endoplasmic reticulum. Taken together, these results indicate that the HCMV protein pUL38 suppresses apoptosis, blocking premature death of host cells to facilitate efficient virus replication.     

Requirement of cell nucleus for Sindbis virus replication in cultured Aedes albopictus cells.

The ability of Sindbis virus to grow in enucleated BHK-21 (vertebrate) and Aedes albopictus (invertebrate) cells was tested to determine the dependence of this virus upon nuclear function in these two phylogenetically unrelated hosts. Although both cell types could be demonstrated to produce viable cytoplasts (enucleated cells) which produced virus-specific antigen subsequent to infection. BHK cytoplasts produced a significant number of progeny virions, whereas mosquito cytoplasts did not. The production of vesicular stomatitis virus in mosquito cells was not significantly reduced by enucleation. That such a host function was not essential for vesicular stomatitis virus growth in insect cells is supported by the observation that the production of this virus by mosquito cells is not actinomycin D sensitive. This result agrees with a previously published report in which it was shown that Sindbis virus maturation in invertebrate cells is inhibited by actinomycin D, indicating a possible requirement for host cell nuclear function (Scheefers-Borchel et al., Virology, 110:292-301, 1981).     

Simian virus 40-permissive cell interactions: selection and characterization of spontaneously arising monkey cells that are resistant to simian virus 40 infection.

A fraction of permissive cells survive simian virus 40 (SV40) infection. The frequency of such surviving cells depends only upon the concentration of infecting virus, both parental and progeny, to which the cells are exposed during the course of selection. Surviving clones, which can be freed of virus by cloning in the presence of SV40 antiserum, are indistinguishable from parental cells in their growth of characteristics and display no SV40 antigen; thus they are not transformed. Most surviving clones are less than 10% as susceptible as parental cells to SV40 infection; 5 to 10% are less than 1% as susceptible. None of these SV40-resistant clones is absolutely resistant to SV40 infection. Analysis of 16 independently arising resistant clones indicates that they all block SV40 infection at an early stage after adsorption and eclipse but before full uncoating. Viral mutants have been isolated that partially overcome the block to infection in these cells; these host range viruses plaque on resistant lines fivefold more efficiently than wild-type SV40 and have a characteristic plaque morphology. Fluctuation analysis indicates that resistant cells arise spontaneously during the growth of normally susceptible permissive cells. Thus, SV40-resistant cells are selected for, not induced by, SV40 infection.     

Sulfolobus tengchongensis spindle-shaped virus STSV1: virus-host interactions and genomic features

A virus infecting the hyperthermophilic archaeon Sulfolobus tengchongensis has been isolated from a field sample from Tengchong, China, and characterized. The virus, denoted STSV1 (Sulfolobus tengchongensis spindle-shaped virus 1), has the morphology of a spindle (230 by 107 nm) with a tail of variable length (68 nm on average) at one end and is the largest of the known spindle-shaped viruses. After infecting its host, the virus multiplied rapidly to high titers (>10(10) PFU/ml). Replication of the virus retarded host growth but did not cause lysis of the host cells. STSV1 did not integrate into the host chromosome and existed in a carrier state. The STSV1 DNA was modified in an unusual fashion, presumably by virally encoded modification systems. STSV1 harbors a double-stranded DNA genome of 75,294 bp, which shares no significant sequence similarity to those of fuselloviruses. The viral genome contains a total of 74 open reading frames (ORFs), among which 14 have a putative function. Five ORFs encode viral structural proteins, including a putative coat protein of high abundance. The products of the other nine ORFs are probably involved in polysaccharide biosynthesis, nucleotide metabolism, and DNA modification. The viral genome divides into two nearly equal halves of opposite gene orientation. This observation as well as a GC-skew analysis point to the presence of a putative viral origin of replication in the 1.4-kb intergenic region between ORF1 and ORF74. Both morphological and genomic features identify STSV1 as a novel virus infecting the genus Sulfolobus.     

Bacterial Growth Rate and Marine Virus–Host Dynamics

Abstract The dynamics of a marine virus–host system were investigated at different steady state growth rates in chemostat cultures and the data were analyzed using a simple model. The virus–host interactions showed strong dependence on host cell growth rate. The duration of the infection cycle and the virus burst size were found to depend on bacterial growth rate, and the rate of cell lysis and virus production were positively correlated with steady state growth rate in the cultures (r ² > 0.96, p < 0.05). At bacterial growth rates of 0.02 to 0.10 h⁻¹ in the chemostats the virus burst size increased from 12 ± 4 to 56 ± 4, and the latent period decreased from 2.0 to 1.7 h. Resistant clones of the host strain were present in the cultures from the beginning of the experiment and replaced the sensitive host cells following viral lysis in the cultures. Regrowth of resistant cells correlated significantly (r ²= 1.000, p < 0.02) with the lysis rate of sensitive cells, indicating that release of viral lysates stimulated growth of the non-infected, resistant cells. The constructed model was suitable for simulating the observed dynamics of the sensitive host cells, viruses and resistant clones in the cultures. The model was therefore used in an attempt to predict the dynamics of this virus–host interaction in a natural marine environment during a certain set of growth conditions. The simulation indicated that a steady state relationship between the specific viruses and sensitive and resistant bacterial clones may occur at densities that are reasonable to assume for natural environments. The study demonstrates that basic characterization and modeling of specific virus–host interactions may improve our understanding of the behavior of bacteria and viruses in natural systems. Received: 12 November 1999; Accepted: 2 May 2000; Online Publication: 11 August 2000     

Interactions of minute virus of mice and adenovirus with host nucleoli.

Biochemical evidence is presented that both minute virus of mice (MVM) and adenovirus interact with the nucleolus during lytic growth and that MVM can also target specific changes involving nucleolar components in adenovirus-infected cells. These virus-nucleolus interactions were studied by analysis of intranuclear compartmentalization of both viral DNAs and host nucleolar proteins: (i) MVM in mouse cells (its normal host) replicates its DNA in the host nucleoli; (ii) specific nucleolar proteins as well as small nuclear ribonucleoprotein antigens are recompartmentalized to multiple intranuclear foci in adenovirus-infected HeLa cells; and (iii) when adenovirus helps MVM DNA replication in a nonpermissive human cell (HeLa), the MVM DNA is also recompartmentalized for synthesis. The data suggest mechanisms for disruption of nucleolar function common to oncogenic or oncolytic virus lytic growth and cell transformation.     

Virustat, a device for continuous production of viruses

Methods for continuous production of viruses, and operation of the virustat, an apparatus in which such production was accomplished, were studied. Continuous production requires a separate continuous host growth chamber, such as the chemostat, and a multiunit virus growth chamber into which the virus-inoculated host cells are led. Successful continuous output of MS-2 and ϕX174 viruses, the latter in lysates, over periods of several days and at titers approximating those of batch lysates, was observed. Design problems include chamber sizes and flow rates, growth of resistant mutants within both virus and host growth chambers, clogging by lysis debris, and the phenomenon of self-inoculation. The latter represents virus growth in the first section of the chamber in excess of the washout rate, leading to lack of need for virus inoculation after an initial period. Use of the virustat for production and research purposes will require some attention to the formation of resistant bacterial colonies at pockets and surface sites of limited washout. With the virustat as a continuous virus production device, continuous purification methods are desirable. Research use of the virustat in continuous mutagenic population studies would require suppression of self-inoculation by use of many sections in the chamber, and improved servo control of host populations at low concentrations.     

Specific binding sites for a parvovirus, minute virus of mice, on cultured mouse cells.

The early interactions between parvoviruses and host cells have not been extensively described previously. In this study we have characterized some aspects of viral binding to the cell surface and demonstrated the existence of specific cellular receptor sites for minute virus of mice (MVM) on two murine cell lines that are permissive for viral growth. The interaction had a pH optimum of 7.0 to 7.2, and both the rate and extent of the reactions were slightly affected by temperature. Mouse A-9 cells (L-cell derivative) had approximately 5 X 10(5) specific MVM binding sites per cell, and Friend erythroleukemia cells had 1.5 X 10(5) MVM sites per cell. In contrast, the nonpermissive mouse lymphoid cell line L1210 lacked specific viral receptors. Also, cloned lines of A-9 cells resistant to viral infection have been isolated. One of these lines lacked the "specific" virus attachment sites but exhibited low levels of nonsaturable virus binding. Based on these examples, infectivity is correlated with the presence of specific viral receptors on the cell surface.     

Intracellular restriction on the growth of induced subgroup E avian type C viruses in chicken cells.

Subgroup E avian type C viruses produced by bromodeoxyuridine-treated 100 X 7, line 7, or line C chicken cells were restricted in their intracellular growth on K28 chicken cells but not on line 15 chicken cells. Cells from embryos of line 15 chickens bred with K28 chickens did not restrict the growth of the subgroup E induced leukosis viruses (ILVs). This result indicates that the phenotype for the intracellular restriction of the growth of subgroup E ILVs found in K28 cells is recessive. Long-term growth of the subgroup E ILVs in K28 cells resulted in the appearance of subgroup E virus that grew well on K28 cells. No change in growth characteristics was observed for subgroup E ILVs grown in line 15 cells indicating that appearance of nonrestricted virus occurred only during growth of the subgrouo E ILVs on a restrictive host. RAV-0, a subgroup E virus closely related to the ilvs, had the same growth characteristics as the subgroup E ILVs. RAV-60, a subgroup E virus formed by recombination of exogenous avian leukosis virus with endogenous subgroup E virus coat information, grew well on both line 15 and K28 cells.     

Multiplication of Polyoma Virus in Mouse-Hamster Somatic Hybrids: a Hybrid Cell Line Which Produces Viral Particles Containing Predominantly Host Deoxyribonucleic Acid

The multiplication of polyoma virus in a mouse-hamster (3T3 x BHK) somatic hybrid line (10A), which, although permissive for viral multiplication, produces very low amounts of virus, has been studied. In this cell line, the efficiency of productive infection is high, but the yield of infectious virus is on the order of 0.5% of that of 3T3 cells. The amount of viral deoxyribonucleic acid (DNA) synthesized by these cells upon infection is about 5% of that of 3T3 cells. An examination of the virus produced in hybrid 10A revealed that it was only one-tenth as infectious as the virus grown in 3T3. Although the viral DNA synthesized in the infected 10A cells is normal, the DNA extracted from purified virus grown in 10A consists of approximately 10% of normal, supercoiled polyoma DNA molecules and of approximately 90% linear DNA molecules with a sedimentation coefficient of 14 to 16S. These DNA molecules appear to be of cellular origin but contain a limited amount of viral DNA sequences. The host DNA-containing particles are not infectious but appear to possess some biological activity; they give rise to a weak complementation effect, and part of them are able to induce T-antigen synthesis. In addition, the host DNA present in these particles is predominantly that which has been synthesized after infection. The correlation between the block in viral DNA synthesis in this cell line and the abnormal encapsidation of host DNA is discussed.