Growth characteristics of reovirus type 2: The intracellular fate of viral RNA

Summary Tritium-labeled reovirus type 2 was adsorbed rapidly and irreversibly to HeLa cells at 37°C. However, if adsorption was carried out at 4°C and the virus-cell complex subsequently incubated at 37° C, more than 25% of the labeled particles was rapidly eluted in 5 minutes. The adsorbed virus remaining at 37°C was stable and no further elution occurred. The eluted virions were found to be biologically and physically unaltered and the elution phenomenon was determined to be dependent on the multiplicity of virus used.In following the fate of the non-eluted adsorbed virion, labeled viral UNA analyzed in sucrose gradients was found to retain its structural integrity throughout the eclipse period. Loss of radioactivity of the viral genome did not occur until about 12 hours after infection, which was several hours after the first appearance of newly-formed progeny.This investigation was supported by Grant AI04685 from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health.     

Abortive infection of neural cells by herpes simplex virus type 2

Summary The nature of the refractoriness of C6 rat glioma cells to herpes simplex virus type 2 (HSV-2) was examined. Infection of C6 cells with HSV-2 results in low virus yields, not exceeding the input virus. Although virus growth studies suggested a restricted cycle of virus replication, synthesis of HSV-2 DNA and HSV-2-specific antigens could not be detected. In addition, HSV-2 yields in C6 cells were unaffected by interferon, cycloheximide, tunicamycin, actinomycin D and cytosine arabinoside. However, trypsin, but not EDTA, treatment of infected C6 cells at 4 hours postinfection (p.i.) reduced maximal HSV-2 yields at 24 hours p.i. by 61 percent. These data: 1) indicate that HSV-2 fails to replicate in C6 cells and is prohibited from directing the synthesis of virus macromolecules; and 2) suggests that the increment of HSV-2 yields observed during the synthesis phase of the virus growth cycle represents re-envelopment and egress of a portion of the input virus.With 4 Figures     

Avian adeno-associated virus (AAAV) and fowl adenoviruses (FAV): Studies of viral interactions in chicken cell cultures

As the growth kinetics of avian adeno-associated virus (AAAV) in chicken cells demonstrate, the three serotypes of fowl adenovirus (FAV), FAV -1, -5 and -8, provide complete helper activity for the production of infectious AAAV. Under one step conditions, the growth cycle of AAAV in primary chicken kidney cell (CKC) cultures is characterised by an eclipse phase of 8 hours and an exponential increase of the virus infectivity by 4 to 5 logs until 24 hours post-adsorption (p.a.). These growth characteristics do not depend on the serotype of FAV used as helper. In chicken embryo fibroblast (CEF) cultures the eclipse phase is prolonged to 12 hours p.a. and the virus infectivity increases only by 2 logs. In addition, the low efficiency of plating of FAV -1 in this cell system does not allow one step growth curves for AAAV. In CKC and CEF cultures coinfected with FAV and AAAV the multiplication of helper FAV is reduced. The degree of growth inhibition depends on the AAV multiplicity used. Sequential infection of CKC cultures with FAV -1 and AAAV modifies the AAAV growth cycle, i.e. there is a time reduction of the eclipse phase and a decrease of the virus yield. Infectious AAAV was determined by an indirect immunofluorescence assay and infectious FAV by a plaque assay.     

Growth characteristics of reovirus type 2: Ultraviolet light inactivated virion preparations and cell death

Summary Ultraviolet light inactivated preparations of purified reovirus type 2 when added to monolayers of HeLa cells have been found to cause a cytotoxic effect (CTE). At multiplicities of 80–100 infectious units per cell (IU/cell) cellular death due to CTE was initially observed at 3–4 hours post infection and reached maximum 9–10 hours after infection. The cell survival curves suggest that the rate of cell killing was dependent upon the multiplicity of infection and exhibited multiple hit type of kinetics. The CTE was preceded by marked alterations in the synthesis of cellular RNA, DNA, and protein, and not produced by UV-irradiated preparations of uninfected HeLa cell homogenates nor by UV-R2 preparations heated at 56°C for 30 minutes. When UV-R2 preparations were subjected to ultracentrifugation (45,000 r.p.m., 1.5 hours), activity was restricted to the sediment only. In addition, CTE could not be serially transferred and could be prevented by addition of reovirus type 2 antiserum. The data strongly suggest that the toxic factor is an intimate part of the UV-inactivated viral particle.This investigation was supported by USPH grant AI-04685 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.NIH. Trainee 5TI AI243.     

Murine cytomegalovirus: Observations on growth in vitro, cytopathic effect, and inhibition with 5-iododeoxyuridine

Summary Growth characteristics, CPE and inhibition with IUdR have been describedin vitro for the murine cytomegalovirus (Smith strain). The virus produced a CPE only in mouse embryonic fibroblasts. Its one-step growth cycle was 28 hours though the length of the eclipse period and time of onset of CPE depended on the multiplicity of infection. Once formed, new virus was rapidly released from infected cells. Interferon was produced in low titre as a result of the initial adsorption of inoculated virus, and later in much higher titre with development and spread of the infection. In stained cultures, cytopathic changes consisted of intranuclear inclusions, nucleolar hypertrophy, chromatin margination, cytomegalia, occasional amitotic nuclear division and cell lysis. Cytoplasmic inclusions comparable to HCMV infection were not observed. With inputs less than 1.0 PFU/eell, viral DNA first appeared 12 hours after infection, preceding assembly of mature virus by 5–6 hours.A plaque reduction-neutralization test was developed for MCMV using tissue culture adapted virus as the test antigen. Neutralizing antibody was obtained from experimentally infected mice and from rabbits inoculated with ultracentrifuged virus suspensions.IUdR inhibited synthesis of infectious virus, but did not prevent the CPE. However, with IUdR, inclusions were smaller, less well defined, and frequently multiple as compared to controls. They showed green fluorescence with acridine orange, and their nature is not clear. In vitro, MCMV differs in several respects from the human cytomegalovirus and these differences are briefly discussed.Supported by the Otho S. A. Sprague Memorial Institute, the Illinois Division of the American Cancer Society and the National Institutes of Health (2G-129).Special Post-Doctoral Research Fellow, National Cancer Institute (1-F3-CA-30, 434-01).     

Role of lysine in the replication of reovirus

Summary Omission of lysine from the extracellular medium of HeLa cells infected with reovirus type 2 results in the production of virus structures which could be separated by isopycnic sedimentation in CsCl into a top band of empty particles (L-T) and a bottom band (L-B) containing a mixture of defective and complete virus structures. The L-T and L-B particles were found to be similar to the complete virus in the following properties: adsorption characteristics to HeLa cells, RNA base composition, and qualitative distribution of the double-stranded viral RNA segments and structural viral proteins.Polyacrylamide gel electrophoresis analyses of the capsid proteins of L-T and L-B particles indicated a quantitative alteration in the distribution of their protein components. In addition, L-T particles contained markedly reduced amounts of the single-stranded adenine-rich RNA component. The synthesis of viral double-stranded RNA and cytoplasmic proteins were both reduced in lysine-deficient cell cultures. In contrast, the synthesis of single-stranded RNA remained unaltered. Addition of increasing concentrations of lysine to lysine-deficient cultures resulted in increasing yields of infectious virus.This investigation was supported by research grant AI 07647 from the National Institutes of Health, U.S. Public Health Service.Post-doctoral trainee NIH 5 T 1 AI 243.     

Receptor utilization by reovirus type 3: distinct binding sites on thymoma and fibroblast cell lines result in differential compartmentalization of virions.

Reovirus type 3/D infects cells following binding to specific cell-surface receptors. The characteristics of these receptors may play an important role in determining post-binding events critical to the viral life cycle. Some cell lines, i.e. L-cells, appear to bind reovirus type 3/D utilizing sialylated proteins as specific receptors for viral adsorption. Such binding results in productive infection. Other cell lines, i.e. R1.1 thymoma cells, bind reovirus type 3/D in a sialic acid independent manner which does not result in productive infection. Yet, a peptide analogue of the viral binding site is capable of inhibiting binding of reovirus type 3 to both cell types, suggesting the same viral epitope interacts with both cellular receptors. When binding of reovirus is studied by electron microscopy, the virus particles enter the L cells via coated pits, and are later seen in large accumulations in endocytic vesicles near the transGolgi network. In contrast, R1.1 cells appear to divert the reovirus particles to a cell membrane elaboration, with reovirus remaining bound to the cell membrane. At later time points with R1.1 cells, there are no apparent intracellular accumulations. These studies demonstrate that viruses can attach to different cells utilizing distinct receptors, and this may play a role in the ability of the virions to productively infect the cells. The capacity of virus to be adsorbed to cellular receptors which do not lead to internalization may be an important mechanism for the sequestration and clearance of virus. These observations have implications for the tissue tropism demonstrated by reovirus type 3/D and other viruses.     

Growth characteristics of rhinoviruses in human gingival cells

Summary Type M and H rhinoviruses have been shown to replicate in human gingival tissue culture and under selective conditions to produce CPE. Both of these viruses replicate best at approximately 33° C with TCID₅₀/ml of 10⁵ and 10⁴ for rhinoviruses 2060 and 11,757, respectively.In gingival cells the type H rhinovirus (strain 11,757) has an eclipse phase from 1/2 to 6 hours and a maximum intracellular viral titer at 16 hours, whereas the type M rhinovirus (strain 2060) has an eclipse phase from 2 to 8 hours and a maximum intracellular viral titer at 24 hours. A more rapid viral replication of the type H virus was indicated by its rapid attachment to the host cell, short latent period and early virus release.This report was presented in part at the 46th General Meeting of the International Association for Dental Research, San Francisco, California, March 21–24, 1968.This investigation was supported by a General Research Support Grant awarded to the School of Dental Medicine, University of Pittsburgh by the National Institutes of Health, U.S. Public Health Service.     

Charakterisierung einiger Vertreter der Columbia-SK-Virusgruppe mit fluoreszierenden Antikörpern

Zusammenfassung Nach Infektion von L-Zellen mit einem Mengovirus-Stamm (5 bzw. 1 PBE pro Zelle) konnte mit Hilfe fluoreszierender Antikörper bereits 30 Min. p. i. bei einigen Zellen in einem umschriebenen Bereich des perinukleären Zytoplasmas Virusantigen nachgewiesen werden. 7 bis 8 Stdn. p. i. enthielt eine maximale Anzahl von Zellen zahlreiche fluoreszierende Granula im Zytoplasma. Höchste Titer an extra- und intrazellulärem Virus traten nach etwa 8 Stdn. auf. Ein Vermehrungszyklus des Mengovirus beträgt in L-Zellen demnach 8 Stdn. Bei Infektion mit etwa 1 PBE pro Zelle konnten innerhalb von 24 Stdn. drei Vermehrungszyklen beobachtet werden.

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Summary In L cells infected with 5 or 1 PFU/cell of a mengovirus strain, virus antigen could be demonstrated in the perinuclear cytoplasm, in a few cells already 30 min. after infection. After 7–8 hours a maximal number of cells contained numerous fluorescent granules in the cytoplasm. The highest titres of extracellular and intracellular virus were found after about 8 hours. One replication cycle of mengovirus in L cells takes, therefore, 8 hours. On infection with about 1 PFU per cell three replication cycles could be seen within 24 hours.

     

Replication of equine herpesvirus type 3: kinetics of infectious particle formation and virus nucleic acid synthesis

The kinetics of equine herpesvirus type 3 (EHV-3) multiplication and of the synthesis of EHV-3 specific DNA and RNA were investigated. A one-step growth curve of EHV-3 in equine epithelial cells from a transitional cell carcinoma was characterized by: (1) a short eclipse period (4 h); (2) an exponential increase in infectious virus between 5 and 10 h post-inoculation; and (3) a slow, inefficient release of newly formed virus into the extracellular fluid. Two hours after infection of cells with EHV-3, the rates of incorporation of specific precursors into total cell RNA or DNA were reduced to 30% and 10%, respectively, of that seen in uninfected cells. With the aid of DNA-RNA hybridization and caesium chloride isopycnic centrifugation techniques, the rates of synthesis of EHV-3 specific nucleic acids at different stages of the virus replication cycle were determined. Virus RNA and DNA synthesis was detectable 2 h after infection and reached maximum levels at an interval (4 to 7 h post-inoculation) corresponding to that period of the virus replication cycle just preceding the time of maximal synthesis of infectious virus.     

Effect of enhancer on growth cycle of ECHO virus 6 plaque mutant

Summary Comparison of the growth pattern of them ⁺ mutant of ECHO virus 6 in its permissive (human) and restrictive (monkey) hosts indicated that defects in monkey cells were expressed as low efficiency of plaquing, prolonged eclipse period and retarded release of virus. Simultaneous addition of virus and a subcellular fraction obtained from permissive cells (enhancer) removed these growth defects ofm ⁺ virus in monkey cells. Increasing the input multiplicity could correct only part of the defect, -i.e., shorten the eclipse period. Addition of enhancer during the eclipse period (0.5 to 5.0 hours) accelerated release of virus, but did not enhance efficiency of plaquing. Studies of events early in the growth cycle suggested that virus could be enhanced only prior to attachment. Although the mechanism has not been elucidated, the results suggest that enhancer may facilitate entry as well as exit ofm ⁺ virus grown in restrictive cells.Supported by research grant (AI-2396) and training grant (AI-98) from the National Institute of Allergy and Infectious Diseases.Recipient of Research Career Award (AI-1136) from the National Institute of Allergy and Infectious Diseases.     

Experimental reovirus infection in chickens: observations on early viraemia and virus distribution in bone marrow, liver and enteric tissues

The nature of viraemia and tissue distribution of reovirus were studied in the early phase after oral infection of 1-day-old specific-pathogen-free (SPF) White Leghorn chicks with the R2 strain of avian reovirus. A range of tissues collected up to 3 weeks after infection was titrated for their viral content. Virus was present in the plasma, erythrocyte and mononuclear fractions of the blood within 30 hours post-inoculation (p.i.) and was widely distributed in tissues, including the bone marrow by 3 to 5 days p.i. A greater part of the viraemia was associated with plasma, virus in the blood mononuclear fraction being detected only occasionally. There was more infectious virus in the duodenum than the liver and the highest virus titres were found in cloacal swabs taken 1 to 5 days p.i. It was also evident that virus reached the liver within a very short time after infection (<6 hours p.i.) although the source of this early hepatic virus was considered to be residual inoculum absorbed directly into the portal blood. Viraemic virus titres could not be correlated either with duodenal or hepatic virus titre alone.     

Virus development in enucleate cells: echovirus, poliovirus, pseudorabies virus, reovirus, respiratory syncytial virus and Semliki Forest virus.

A group of RNA viruses, echovirus, poliovirus, reovirus, respiratory syncytial virus and Semliki Forest virus have been examined for ability to grow in enucleate African green monkey kidney (BSCi) cells. Semliki Forest virus produced an almost normal yield of virus but poliovirus, echovirus, reovirus and respiratory syncytial virus, although showing clear evidence of virus replication when compared with a nuclear DNA virus (pseudorabies virus) gave much lower yields than those from nucleate cells. Analysis of enucleate cells infected with echovirus and reovirus showed no evidence of a specific block in the synthesis of any virus-specified polypeptide. Infection with vesicular stomatitis virus at intervals after enucleation demonstrated a diminishing ability to support virus growth with increasing time. It is suggested that the yield of virus obtained from an enucleate cell is related to the length of the growth cycle of the virus, the reduced yield obtained with some viruses reflecting the declining ability of the enucleate cell to support virus growth.     

Selection of a mutant S1 gene during reovirus persistent infection of L cells: role in maintenance of the persistent state

LR-7 cells, variant L cells derived from a type 3 reovirus persistently infected (p.i.) carrier culture (R. Ahmed, W. M. Canning, R. S. Kauffman, A. H. Sharpe, J. V. Hallum, and B. N. Fields, Cell 25, 325-332, 1983) were used to define the viral genes critical for maintenance of the persistent state. A cloned viral isolate (L/C virus) derived from the p.i. culture replicated normally in LR-7 cells, while wild-type (wt) viruses of the three reovirus serotypes replicated less efficiently. To identify the viral gene(s) permitting enhanced replication of L/C virus in LR-7 cells, viral reassortants were prepared by mixed infection of L cells with L/C virus and type 1 wt. Study of the one-step growth curves and final yields of large numbers of reassortants in both L cells and LR-7 cells revealed that the presence of the S1 gene from L/C virus was critical for normal viral replication in LR-7 cells. However, this phenotype was suppressed by the simultaneous presence in reassortants of both the M2 and S4 genes from the type 1 wt parent. The critical change in the S1 gene occurred by passage 13 (63 days) after initiation of the carrier culture. Although multiple mutations are present in the viral population from p.i. cultures, certain specific mutations can be identified as critical for maintenance of the persistent state.     

Hormones and the lung II. Immunohistochemical localization of thyroid hormone binding in type II pulmonary epithelial cells clonally-derived from adult rat lung

The type II pulmonary epithelial cell is the recognized site of surfactant synthesis and storage. Results of recent studies indicate that the thyroid hormones, triiodothyronine (T₃) and thyroxine (T₄), may be important regulators of surfactant production and/or release. Direct and indirect immunofluorescence techniques were used in an attempt to demonstrate binding of T³ and T⁴ in monolayer cultures of isolated type II cells. These cultured epithelial cells are clonally-derived from adult rat lung, retain a diploid karyotype through 35 population doublings in vitro, contain granular inclusions (lamellar bodies) in the perinuclear cytoplasm, and synthesize phosphatidylcholine via the CDP-choline pathway. In isolated type II cells, either of two fluorescent patterns was observed: (a) nuclear fluorescence accompanied by a reticular perinuclear network; or (b) diffuse cytoplasmic accumulations with concentrations around perinuclear cytoplasmic inclusions. Ultrastructurally these inclusions had the typical appearance of lamellar bodies. Histochemical studies demonstratedthat these inclusions contained surfactant-associated nonspecific esterases and stained with Nile blue hydrochloride. The positive reactions with these two recognized markers for pulmonary surfactant indicate that these inclusions are indeed lamellar bodies, the putative sites of surfactant synthesis and/or storage. These findings suggest that the type II pulmonary epithelial cell contains specific binding sites for thyroid hormones, and support the hypothesis that thyroid hormones are regulators of surfactant metabolism.     

Characterization and Serotyping of Three Feline Reovirus Isolates

Three feline virus isolates were shown to be members of the reovirus group by their growth characteristics in cell cultures, physicochemical properties, and appearance under an electron microscope. Their close serological relationship to, or identity with, human reovirus type III was revealed by hemagglutination, hemagglutination inhibition, serum neutralization, and gel diffusion tests. One feline isolate (636) was pathogenic for suckling mice.     

Comparative cytopathology of canine distemper and measles viruses in ferret kidney cell cultures

Summary Multinucleated giant cells, cytoplasmic and intranuclear inclusions were characteristic findings in ferret kidney cultures inoculated with canine distemper virus. Variations observed among strains were of a quantitative nature and these did not serve as distinguishing features. Contracted giant cells were observed which were interpreted as being degenerative forms of the multinucleated giant cells. Ferret kidney cell cultures inoculated with measles virus showed the same characteristics.Aided by Grant AI 04372-02 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md.     

The preferential cytotoxicity of reovirus for certain transformed cell lines

Summary The susceptibility of a variety of cell lines of different mammalian origin to cytotoxic (CT) induction by either ultraviolet light-irradiated reovirus type 2 (UVR2) or viable reovirus type 2 plus the protein synthesis inhibitor, cycloheximide, was examined. The following groups of cells were found to be susceptible to CT-induction: certain tumor cells and spontaneously transformed cell lines of human origin and certain virally and spontaneously transformed cell lines of murine origin. The following groups of cells were found to be resistant: normal human diploid cell lines, primary and continuous cell cultures of subhuman primates, primary mouse cells, normal rat kidney cells and baby hamster kidney cells. Susceptibility to CT-induction could not be related to the adsorption of virus to cells, nor to the capacity of the cell to support virus replication.The research was supported by Public Health Service grant CA-13768 from the National Cancer Institute.     

Induction of Interferon in L Cells by Reoviruses

L cell cultures challenged with reoviruses types 1 and 3 produced little to no detectable interferon under conditions which permitted virus replication and under conditions which prevented replication of a temperature-sensitive mutant strain of reovirus type 3 (ts-1). Ultraviolet-irradiated reoviruses and double-stranded ribonucleic acids extracted from purified reovirus type 3 also induced little to no interferon in L cell cultures. Under similar conditions, MM virus proved to be an effective inducer of interferon. Exposure of L cells to interferon prior to challenge with virus (priming) failed to enhance interferon production upon subsequent challenge with reovirus although priming increased the amount of interferon produced following MM virus challenge. L cell cultures that were challenged with reovirus type 3 and subsequently with either MM or Colorado tick fever viruses produced similar titers of interferon as cell cultures that were challenged with either MM or Colorado tick fever virus alone, respectively. These data show that the presence of production of reovirus type 3 double-stranded ribonucleic acid is not sufficient for induction of interferon in L cell cultures and that additional processes which are required for induction of interferon in L cell cultures are not expressed by reovirus type 3.     

Hormones and the lung. II. Immunohistochemical localization of thyroid hormone binding in type II pulmonary epithelial cells clonally-derived from adult rat lung.

The type II pulmonary epithelial cell is the recognized state of surfactant synthesis and storage. Results of recent studies indicate that the thyroid hormones, triiodothyronine (T3) and thyroxine (T4), may be important regulators of surfactant production and/or release. Direct and indirect immunofluorescence techniques were used in an attempt to demonstrate binding of T3 and T4 in monolayer cultures of isolated type II cells. These cultured epithelial cells are clonally-derived from adult rat lung, retain a diploid karyotype through 35 population doublings in vitro, contain granular inclusions (lamellar bodies) in the perinuclear cytoplasm, and synthesize phosphatidylcholine via the CDP-choline pathway. In isolated type II cells, either of two fluorescent patterns was observed: (a) nuclear fluorescence accompanied by a reticular perinuclear network; or (b) diffuse cytoplasmic accumulations with concentrations around perinculear cytoplasmic inclusions. Ultrastructurally these inclusions had the typical appearance of lamellar bodies. Histochemical studies demonstrated that these inclusions contained surfactant-associated nonspecific esterases and stained with Nile blized markers for pulmonary surfactant indicate that these inclusions are indeed lamellar bodies, the putative sites of surfactant synthesis and/or storage. These findings suggest that the type II pulmonary epithelial cell contains specific binding sites for thyroid hormones, and support the hypothesis that thyroid hormones are regulators of surfactant metabolism.