Generation of Defective Interfering Particles of Semliki Forest

Size: px
Start display at page:

Download "Generation of Defective Interfering Particles of Semliki Forest"

Transcription

1 JOURNAL OF VIROLOGY, Apr. 1979, p X/79/ /07$02.00/0 Vol. 30, No. 1 Generation of Defective Interfering Particles of Semliki Forest Virus in a Clone of Aedes albopictus (Mosquito) Cells KELVIN B. LOGAN Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, England Received for publication 17 November 1978 Serial undiluted passage of Semliki Forest virus in a clone of Aedes albopictus cells resulted in a marked decrease in infectious virus yields due to the generation and accumulation of defective interfering particles. Virus from the third passage had a high particle/infectivity ratio and interfered specifically with homologous but not heterologous standard virus replication. Two RNA species of molecular weights 0.78 x 10' and 0.61 x 106 were the major RNA components of purified passage 4 virus. These RNA species were also the predominant virus RNA species detected in cells infected with passage 3 virus. Synthesis of standard virus RNA and virus-specified protein was much reduced in passage 3 virus-infected cells. Interference with standard virus replication and the synthesis of large amounts of defective interfering RNA were also observed in chicken embryo cells infected with passage 3 virus from mosquito cells. Serial high-multiplicity passaging of alphaviruses, such as Semliki Forest virus (SFV) or Sindbis virus, in cultured vertebrate cells leads to the accumulation of defective interfering (DI) particles (2, 13, 15, 26-28). The presence of DI particles in virus stocks is inferred from two observations. First, DI particles interfere specifically with the replication of homologous standard virus in infected cells so that the yields of infectious virus are reduced. Second, the replication of alphavirus DI particles interferes with the synthesis, and alters the pattern, of intracellular viral RNA species. Two major virus-specified, single-stranded RNAs of positive polarity are synthesized in cells infected with standard virus. These are the 42S RNA, which is identical to virion RNA, and 26S RNA, which is the mrna for virus structural proteins (3, 4, 7, 29, 36) and contains the sequences of the 3'-terminal one-third of the 42S RNA (21, 37). On the other hand, infection of cells with virus stocks containing DI particles leads to the synthesis of smaller DI RNA species, whereas 42S RNA synthesis is depressed (3, 13, 15, 35). These smaller DI RNA species are also isolated from purified DI virus (2, 15) and arise by deletion of internal regions of the 42S standard virus genome during replication (16, 31). Alphaviruses are included in the arthropodborne group of viruses (arboviruses) and replicate in both vertebrate and invertebrate cells. However, whereas the infection of cultured vertebrate cells invariably leads to a cytopathic effect and eventual cell destruction, the infection of cultured mosquito cells by alphaviruses gives rise to an inapparent, noncytocidal, persistent infection (9, 24, 32, 34). The role of DI particles in alphavirus replication in mosquito cells is a curiously paradoxical problem (18). Attempts at generating DI particles in cultured cells of the mosquito Aedes albopictus by serial undiluted passage of Sindbis virus have been unsuccessful (19). Furthermore, it has been reported that cultured mosquito cells are unable to support the replication of DI particles of SFV or Sindbis virus isolated by serial passage in chick or BHK cells (11, 19). However, DI virus has recently been isolated from cultured A. albopictus cells after several weeks of a persistent infection by Sindbis virus (12). This result confirmed an earlier observation that persistently infected mosquito cells contain RNA species characteristic of an infection by DI particles of Sindbis virus (18, 33). Cultured A. albopictus cells consist of a mixed population of cells, reflecting their origin from whole mosquito larvae (30). Cloning experiments have shown that the cells vary in both the yields of progeny virus and the development of a cytopathic effect after infection by such alphaviruses as Sindbis (25), Chikungunya (17), and SFV (K. B. Logan, manuscript in preparation). Since the response to standard virus infection by these high virus-producing cell clones is apparently different from that of the mixed cell line, I examined whether these clones also respond differently in terms of their ability to generate and/or replicate DI particles of SFV. The experiments reported here show that serial 38

2 VOL. 30, 1979 DI PARTICLES OF SFV IN MOSQUITO CELLS 39 undiluted virus passage of SFV in a clone of A. albopictus cells leads to the generation and accumulation of DI particles, which are able to interfere with standard virus replication in both mosquito and chick cells. MATERIALS AND METHODS Materials. Actinomycin D was obtained from Merck Sharpe & Dohme Research Laboratories, Rahway, N.J. Agarose (type II, medium EEO) was obtained from Sigma (London) Chemical Co., Ltd., London, U.K. Tri-isopropylnaphthalenesulfonic acid was obtained from Eastman-Kodak, Rochester, N.Y. 4- Aminosalicylic acid was obtained from BDH Chemicals Ltd., Poole, Dorset, England. L-['S]methionine (740 Ci/mmol) and 32Pi (80 to 130 Ci/mg of phosphorus) were obtained from the Radiochemical Centre, Amersham, England. All media, sera, and N-2-hydroxyethyl piperazine-n'-2-ethanesulfonic acid (HEPES) were obtained from Flow Laboratories Ltd., Irvine, Scotland. All other chemicals were of the best grade available commercially. Cells. Primary cultures of chicken embryo cells were prepared and cultured as described previously (23). A. albopictus cells (30) that had been previously adapted to growth in vertebrate cell culture medium (17, 25) were a generous gift from V. Stollar, College of Medicine and Dentistry of New Jersey, Rutgers Medical School, New Brunswick, N.J. G8 cells were clonally derived in this laboratory by S. I. T. Kennedy and were grown in the Glasgow modification of Eagle (10) medium supplemented with nonessential amino acids and 15% heat-inactivated (56 C/45 min) fetal bovine serum. Virus. The wild-type strain of SFV and the AR339 strain of Sindbis virus were plaque purified, and stocks were obtained as described previously (1, 2). A standard preparation of T2 phage was obtained from Miles Laboratories Ltd., Slough, England. Conditions of serial undiluted passage. Monolayer cultures of clone G8 mosquito cells in 150-cm2 Nunclon tissue culture flasks were infected with SFV at a high multiplicity (100 PFU/cell), and extracellular virus was harvested after 20 h at 28 C. The virus suspension was clarified by centrifugation and used, without dilution, to infect fresh cultures of cells. Serial undiluted virus passaging was continued, and the fluids obtained were stored at -70 C. Viral infectivity was assayed by plaque formation on primary chick cells under agar overlay at 33 C. Particle/infectivity ratios. All preparative procedures were performed either on ice or at 4 C. Extracellular virus in 60 ml of clarified tissue culture fluids was concentrated and partially purified by centrifugation at 100,000 x g for 2 h through a 10-ml layer of 15% (wt/vol) sucrose in 50 mm Tris (ph 7.4) containing 100 mm NaCl and 1 mm EDTA (TNE) plus 1% calf serum. Pelleted virus was gently resuspended in a small volume of the same buffer and stored at -70 C. SFV preparations were diluted with distilled water, mixed with a standard preparation of T2 phage and virus particles adsorbed onto Formvar-coated copper grids, and negatively stained with 1% phosphotungstic acid (ph 7.0). Grids were examined in an AEI Corinth 275 electron microscope, and SFV particles were enumerated by comparative counts with T2 phage. Labeling, extraction, and analysis of viral RNA. Confluent cell monolayers were washed once with phosphate-free Eagle minimum essential medium (Glasgow modification) and infected. After adsorption for 1 h at 28 C the inocula were replaced by the same medium containing 5% dialyzed, heat-inactivated fetal calf serum plus 1 jig of actinomycin D per ml. At times specified in the individual experiments, the fluids were replaced by 15 ml of medium containing 1 mci of 'Pi. At the end of the labeling period cell sheets were washed three times with ice-cold phosphate-buffered saline, scraped into 10 ml of phosphate-buffered saline, and collected by centrifugation. Cell pellets were resuspended and dissolved in 50 mm Tris (ph 8.5) containing 1% (wt/vol) tri-isopropylnaphthalenesulfonic acid and 6% (wt/vol) 4-aminosalicylic acid and deproteinized by addition of an equal volume of a phenol-chloroform-isoamyl alcohol mixture prepared as follows: 500 g of phenol (detached crystals), 70 ml of redistied m-cresol, and 0.5 g of8-hydroxyquinoline, saturated with 50 mm Tris, ph 8.5 (50 volumes); chloroform (50 volumes); and isoamyl alcohol (1 volume). After vigorous shaking and centrifugation the aqueous phase was removed and extracted twice more with the phenol mixture, twice with chloroform-octanol (24:1, vol/vol), and finally twice with ether. Residual ether was blown off in a stream of N2, and NaCl was added to a final concentration of 100 mm. Most of the cell DNA was removed after gentle mixing with an equal volume of ethanol for 15 s at room temperature and centrifugation at 1,000 x g for 3 min to remove the precipitate. The remaining nucleic acid was precipitated by addition of a further 1.5 volumes of ethanol and storage overnight at 4 C. The RNA was recovered by centrifugation, dried in a stream of N2, dissolved in one-tenth concentration of TNE containing 0.2% sodium dodecyl sulfate, 0.001% bromophenol blue, and 10% sucrose, and analyzed on 17.5-cm slab gels of 2% agarose prepared in electrophoresis buffer (36 mm Tris [ph 8], 30 mm NaH2PO4, 1 mm EDTA, 0.2% sodium dodecyl sulfate). After prerunning the gel for 1 h, samples were electrophoresed for 16 h at a 30-V fixed potential. Gels were fixed in an ethanol bath for 3 h, transferred onto glass plates, dried in a 56 C oven, and autoradiographed for 16 h on Kodirex X-ray film. All autoradiograms were scanned in a Joyce-Loebl scanning densitometer, using the same sensitivity throughout. Labeling of virus-specified polypeptides. Monolayer cultures of clone G8 cells were grown almost to confluency in glass scintillation vials. During and after infection cells were maintained in protein labeling medium (Eagle minimum essential medium [Glasgow modification] lacking methionine, 5% dialyzed, heat-inactivated fetal calf serum, and 1 t&g of actinomycin D per ml, buffered with 20 mm HEPES) and incubated at 28 C in a recirculating water bath. At the specified time after infection fluids were replaced with fresh labeling medium containing 50,uCi of [us]methionine per ml. At the end of the labeling period fluids were replaced with fresh medium containing 1 mm "cold" methionine and incubated for a

3 40 LOGAN further 1 h at 280C. Labeled polypeptides were extracted as described previously (5) and analyzed on 17.5-cm slab gels of 7.5% (wt/vol) acrylamide plus 0.2% (wt/vol) N,N'-methylenebisacrylamide, using the discontinuous buffer system described by Laemmli (22). After electrophoresis, gels were dried in vacuo and autoradiographed for 24 h on Kodirex X-ray film. RESULTS Serial undiluted passage of SFV in A. albopictus (clone G8) cells. A number of clones of A. albopictus cells have been isolated in this laboratory, and the responses of these cloned cell lines to infection by SFV have been studied (Logan, manuscript in preparation). One of these clones, designated G8, produced much higher yields of virus than the parent cell line and demonstrated a marked cytopathic effect 24 h after infection by SFV. Due to the higher rate of virus synthesis in these cells it seemed possible that the rate of generation of DI particles would also be increased. To test for the generation of DI particles, cultures of clone G8 cells were infected with plaque-purified SFV at a high multiplicity (100 PFU/cell) followed by serial undiluted passage of the progeny virus. The infectious titers of SFV produced during one such experiment are shown in Table 1. Although high titers of virus were found initially, yields of virus dropped markedly during serial passage, and passage 3 yields of infectious virus were reduced almost 1,000-fold. In addition, only a slight cytopathic effect was observed in cultures of clone G8 cells infected with passage 3 virus. To determine whether the drop in infectious virus production was due to the accumulation of DI particles during serial passage, an interference analysis was perforned on passage 3 virus. In these experiments plaque-purified SFV was used as the standard virus and plaque-purified Sindbis virus was included as a heterologous control. Sindbis plaques were easily distinguished from SFV plaques as described previously (2). The results (Table 2) show that the replication of wild-type TABLE 1. Infectious virus yields during undiluted passage in clone G8 cells Passage no. Infectious titer (PFU/ml)a x X 10l 3 1.7X X x x x 107 a Twenty-hour harvests from parallel cultures, each with approximately 5 x 107 cells in 25 ml of medium per 150-cm2 flask. TABLE 2. Interference analysis on passage 3 virus Multiplicity of infection (PFU/ cell) J. VIROL. Titer of infectious progeny virus'a SFV Sindbis Passage SFV 3 (PFU/ml) X x x x x 109 a Twenty-four-hour harvests from parallel cultures with approximately 4 x 106 cells in 5 ml of medium per 5-cm petri dish. SFV was markedly inhibited by coinfection with passage 3 virus. Furthermore, this interference was specific for the homologous virus, there being no significant decrease in the yields of Sindbis virus after infection with passage 3 virus. The specificity of this interference strongly suggests the presence of DI particles. To confirm this, the particle/infectivity ratios of (i) passage 3 virus and virus stocks from (ii) chick cells or (iii) clone G8 cells infected with standard virus were determined. Virus particles were concentrated and counted, and the particle infectivity ratios were subsequently calculated to be (i) 442, (ii) 10, and (iii) 14 particles/pfu, respectively. The large increase in noninfectious particles thus confirms the presence of DI particles of SFV in passage 3 virus. Nature of the RNA species associated with DI particles. Purified DI particles of SFV generated in BHK cells contain species of RNA smaller than 42S virion RNA (2). To determine whether mosquito cell-derived DI particles had a similar structure, the RNA species associated with purified virus progeny were examined. As noted by other workers, it was not possible to separate labeled DI particles from standard virus particles by sucrose density gradient centrifugation (15, 26, 35). Therefore, RNA species were isolated from total 32P-labeled virus particles in passage 2 and 4 fluids and, after extraction, were analyzed on 2% agarose gels (Fig. 1). Passage 2 virus, in addition to virion 42S RNA, contained one major and one minor RNA species. By passage 4, the levels of 42S RNA were barely detectable, and the two smaller RNA species were the major RNA components of purified particles. Thus, a drop in virion-associated 42S RNA parallels the drop in infectiousvirus production during serial passage. Intracellular, virus-specified RNA species. To determine what effect the infection of DI particles has on normal viral RNA synthesis in SFV-infected clone G8 cells, confluent cultures were infected with either standard virus or

4 VOL. 30, 1979 DI PARTICLES OF SFV IN MOSQUITO CELLS of these RNAs with those of 42S (4.2 x lob) and 26S (1.8 x 106) RNA. The largest species had a molecular weight of 1.2 x 106, and the two smaller species had molecular weights of 0.78 x 106 and 0.61 x 106, respectively, and comigrated with the RNA species extracted from passage 4 virus. The nature of the larger RNA species is unknown, although it could be a defective RNA species that cannot be encapsidated into virus progeny. Interestingly, these species of RNA were also detected in small amounts in clone G8 cells infected wnith standard virus, the larger species appearing as a shoulder on the 26S RNA 41 I DISTANCE MIGRATED (cm) FIG. 1. Analysis ofthe RNA species extracted from purified passage 2 and 4 viruses. Confluent cultures ofclone G8 cells were infected with undilutedpassage 1 or 3 virus for I h at 28 C. Cultures were labeled with 100,ICi of 32Pi per ml in the presence of actinomycin D (0.1,ug/ml). Fluids were harvested after 24 h at 28 C, and virus was purified as described (21). Viral RNA was extracted from purified virus pellets and analyzed on a 2% agarose gel. Migration is from left to right. RNA extracted from: (A) purified passage 2 virus; (B) purified passage 4 virus. Autoradiograms were scanned by a Joyce-Loebl densitometer. passage 3 virus. Intracellular RNA was labeled with 32Pi in the presence of actinomycin D (1,Lg/ ml) and analyzed. The results (Fig. 2) show that 42S and 26S RNA were the major RNA species in cells infected with standard SFV. The minor RNA components, 38S and 33S RNA, are considered to be conformational variants of 42S and 26S RNA, respectively (21). In contrast, the amounts of 42S RNA and 26S RNA in cels infected with passage 3 virus, at the same multiplicity, were greatly reduced, and three smaller RNA species were predominant. The molecular weights of these smaller intracellular RNA species were estimated by comparing the mobilities DISTANCE MIRATED (cm) FIG. 2. Analysis on 2%1b agarose gels of RNA species synthesized in clone G8 cells infected with standard SFV or passage 3 virus. Monolayer cultures of clone G8 cells were infected with approximately 5 PFU of virus per cell and labeled with 1 mci of 32Pi from 2 to 8 h in the presence of actinomycin D (1.tg/ ml). RNA from: (A) standard SFV-infected cells; (B) passage 3 virus-infected cells; (C) mock-infected cells.

5 42 LOGAN peak. It appears, therefore, that DI particles are generated during the first passage and subsequently amplified by serial passage. No viral RNA species were detected in mock-infected cells under the conditions used. Virus polypeptides synthesized in clone G8 cells infected with SFV DI particles. To determine whether DI RNAs are translated in infected mosquito cells, the polypeptide species synthesized in clone G8 cells infected with either standard virus or serially passaged virus were analyzed. In these experiments the same multiplicity of infection (in terms of infectious particles per milliliter) of all virus stocks was used. The electrophoretic profiles of [35S]methionine-labeled polypeptides (Fig. 3) show that clone G8 cells infected with standard virus contain substantial amounts of virus-specified proteins at 4 h postinfection. The envelope proteins (El and E2) and core protein comprise the major structural proteins of the virion, whereas pol90 and pol63 are the putative virus polymerase components (5, 8). The results (Fig. 3) show that, like standard virus-specified RNA synthesis (Fig. 2), all the standard virus polypeptides are synthesized in markedly reduced amounts in cells infected with passage 3 virus. Moreover, the observation that no other virus-specified polypeptides can be detected in cells infected with serially passaged virus suggests that DI RNAs found in these cells do not act as functional mrna's. Studies concerning the synthesis of the virus-specified proteins in clone G8 mosquito cells and their modes of cleavage from larger precursors will be reported elsewhere (Logan, manuscript in preparation). Ability of DI particles of SFV generated in mosquito ceils to interfere in chick ceils. I investigated whether mosquito cell-derived DI particles of SFV were able to interfere with standard virus replication in cultures of vertebrate cells, as it had been previously reported that vertebrate cell-derived DI particles of SFV do not interfere with virus replication in mosquito cells (11, 19). Chicken embryo cells were infected with either standard virus or passage 3 virus, and the 32P-labeled viral RNAs were extracted and analyzed (Fig. 4). The results show that the same DI RNA species were found in passage 3 virusinfected cells. Furthermore, the synthesis of these DI RNAs occurs at the expense of 42S and 26S RNA synthesis. The 13-h yields of infectious virus from parallel cultures of chick cells infected with the same multiplicity of either standard virus or passage 3 virus were 1.1 x 109 and 3.3 x 107 PFU/ml, respectively. Thus, passage 3 DI par- U SFV P 1,fom. t3 *.1,11,.. P2 P3 J. VIROL.... _WlW i:-:l-.-:- -pol 90 _m. ei * -pol 63 ihb11 -Core FIG. 3. Polyacrylanide gel electrophoresis of the polypeptides synthesized in clone G8 mosquito cells either mock infected or infected with serially passaged virus. At 4 h after infection cultures were labeled for 20 min with 50,iCi of I3S]methionine per ml. After a 1-h chase period polypeptides were extracted and analyzed. Approximately the same number of counts in thepolypeptides from uninfected cells (U), standard virus-infected cells (SFV), and cells infected with passage 1, -2, and -3 viruses (P1, P2, P3), respectively were applied to the gel. See text for identity of the marked bands. Due to host-specified background envelope proteins El and E2 are not easily resolved in this gel. ticles are replicated and interfere with standard virus multiplication in both cultured vertebrate and mosquito cells. DISCUSSION Experiments described in this report have demonstrated that serial undiluted passage of SFV in a high virus-yielding clone of A. albopictus cells leads to the rapid accumulation of DI particles. The presence of these DI particles was suggested initially by a marked decrease in in-.;.f.:. k/s:',."', '14... =El/ E2

6 VOL. 30, 1979 A) 42S S DISTANCE MIGRATED (cm) FIG. 4. Analysis of the RNA species synthesized in chick cells infected with standard SFV or passage 3 virus. Cells were infected at 37 C and labeled with 32Pi from 2 to 6 h postinfection. RNA was extracted and analyzed. RNA species from: (A) standard virusinfected cells; (B) passage 3 virus-infected cells. fectious virus yields such that by passage 3 yields are depressed almost 1,000-fold. I have shown that passage 3 DI particles interfere with virus replication in both mosquito cells and vertebrate cells. These experiments have revealed a number of similarities between the DI particles of SFV generated in clone G8 mosquito cells reported here and the DI particles isolated from vertebrate cells described previously (2, 3). Of particular interest is the observation that two RNA species, of molecular weights 0.78 x 10' and 0.61 x 106, are the major RNA components of purified passage 3 virus. These compare with values of 0.81 x 106 and 0.75 x 106 obtained by other workers for DI RNA species isolated from DI particles of SFV generated in BHK cells (2, 3). Thus, regardless of the cell type in which the DI particles are generated, there appears to be a constraint on the size of DI RNA produced during serial passage. In order that a DI RNA is amplified during serial passage, it must be capable of being both replicated and packaged into virions. It is possible that these requirements impose the size constraints on DI RNA species. DI PARTICLES OF SFV IN MOSQUITO CELLS Since little wild-type virus RNA synthesis can be detected in passage 3 virus-infected clone G8 cells, the interference in standard virus replication by passage 3 DI particles occurs at the level of RNA synthesis. The marked decrease in structural protein synthesis observed in cells infected with passage 3 virus is therefore a secondary effect caused by the decrease in 26S RNA synthesis. In contrast to the results reported here, Igarashi and Stollar (19) have demonstrated that serial undiluted passage of Sindbis virus in an uncloned population of A. albopictus cells did not lead to the accumulation of DI particles. The precise reason for this discrepancy is not known. It might be explained by the difference in virus used and/or the conditions of cell growth in the two laboratories. However, as a cloned subline of mosquito cells has been used throughout this study, it cannot be ruled out that a hostcell-specific function is required for the generation and replication of DI particles in infected cells. Recent evidence suggests that the host cell type can directly influence the generation of DI particles of a number of viruses, such as vesicular stomatitis virus (20) and SFV (31). The observation that cultures of A. albopictus cells can be cloned to give subpopulations of cells which respond differently, in terms of virus yields and the extent of cytopathic effect (17, 25), is evidence that even standard virus synthesis can be influenced by the host cell. At present it is not clear what cellular functions might be involved in the control of both standard virus and DI particle replication in infected mosquito cells. Evidence suggests that the SFV RNA polymerase contains at least one host-specified component (8). Furthermore, intracellular virus RNA synthesis is known to occur on virus-modified host cell membranes (14). Thus, it is conceivable that the host cell might affect the activity of the virus RNA polymerase either directly or by altering the arrangement of the polymerase-template complexes bound to intracellular membranes. ACKNOWLEDGMENTS I thank Ian Kennedy for his support and for providing the cell clone. I also thank Derek Burke and members of the Warwick Interferon Group for support and critical appraisal during the preparation of the manuscript. K.B.L. was in receipt of a Medical Research Council Scholarship. 43 LITERATURE CITED 1. Atkins, G. J., J. Samuels, and S. I. T. Kennedy Isolation and characterisation of temperature-sensitive mutants of Sindbis virus strain AR 339. J. Gen. Virol. 25: Bruton, C. J., and S. I. T. Kennedy Defectiveinterfering particles of Semliki Forest virus: structural

7 44 LOGAN differences between standard virus and defective-interfering particles. J. Gen. Virol. 31: Bruton, C. J., A. Porter, and S. I. T. Kennedy Defective-interfering particles of Semliki Forest virus: intracellular events during interference. J. Gen. Virol. 31: Cancedda, R., and M. J. Schlesinger Formation of Sindbis virus capsid protein in mammalian cell-free extracts programmed with viral messenger RNA. Proc. Natl. Acad. Sci. U.S.A. 71: Clegg, J. C. S., H. Brzeski, and S. I. T. Kennedy RNA polymerase components in Semliki Forest virus infected celis: synthesis from large precursors. J. Gen. Virol. 32: Clegg, J. C. S., and S. I. T. Kennedy In vitro synthesis of structural proteins of Semliki Forest virus directed by 26S RNA from infected cells. FEBS Lett. 42: Clegg, J. C. S., and S. I. T. Kennedy Translation of Semliki Forest virus intracellular 26S RNA. Characterisation of the products synthesised in vitro. Eur. J. Biochem. 53: Clewley, J. P., and S. I. T. Kennedy Purification and polypeptide composition of Semliki Forest virus RNA polymerase. J. Gen. Virol. 32: Davey, M. W., D. P. Dennett, and L. Dalgarno The growth of two togaviruses in cultured mosquito and vertebrate cells. J. Gen. Virol. 20: Eagle, H Amino acid metabolism in mammalian cell cultures. Science 130: Eaton, B. T Defective-interfering particles of Semliki Forest virus generated in BHK cells do not interfere with viral RNA synthesis in Aedes albopictus cells. Virology 68: Eaton, B. T Evidence for the synthesis of defectiveinterfering particles by Aedes albopictus cells persistently infected with Sindbis virus. Virology 77: Eaton, B. T., and P. Faulkner Altered pattern of viral RNA synthesis in cells infected with standard and defective Sindbis virus. Virology 51: Grimley, P. M., L K Berezesky, and R. M. Friedman Cytoplasmic structures associated with an arbovirus infection: loci and viral ribonucleic acid synthesis. J. Virol. 2: Guild, G. M., and V. Stollar Defective-interfering particles of Sindbis virus. III. Intracellular viral RNA species in chick embryo cell cultures. Virology 67: Guild, G. M., and V. Stollar Defective-interfering particles of Sindbis virus. V. Sequence relationships between SV.s-j) 42S RNA and intracellular defective viral RNAs. Virology 77: Igarashi, A Isolation of a Singh's Aedes albopictus cell clone sensitive to Dengue and Chikungunya viruses. J. Gen. Virol. 40: Igarashi, A., R. Koo, and V. Stollar Evolution and properties ofaedes albopictus cell cultures persistently infected with Sindbis virus. Virology 82: Igarashi, A., and V. Stollar Failure of defectiveinterfering particles of Sindbis virus produced in BHK J. VIROL. or chicken cells to affect viral replication in Aedes albopictus cells. J. Virol. 19: Kang, C. Y., and R. Allen Host function-dependent induction of defective-interfering particles of vesicular stomatitis virus. J. Virol. 25: Kennedy, S. I. T Sequence relationships between the genome and intracellular RNA species of standard and defective-interfering Semliki Forest virus. J. Mol. Biol. 108: Laemmli, U. K Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: Morser, J. M., S. I. T. Kennedy, and D. C. Burke Virus-specified polypeptides in cells infected with Semliki Forest virus. J. Gen. Virol. 21: Peleg, J Inapparent persistent virus infection in continuously grown Aedes aegypti mosquito cells. J. Gen. Virol. 5: Sarver, N., and V. Stollar Sindbis virus-induced cytopathic effect in clones of Aedes albopictus (Singh) cells. Virology 80: SchleInger, S., M. Schlesinger, and B. W. Burge Defective virus particles from Sindbis virus. Virology 48: Shenk, T. E., and V. Stollar Defective-interfering particles of Sindbis virus. I. Isolation and some chemical and biological properties. Virology 53: Shenk, T. E., and V. Stollar Defective-interfering particles of Sindbis virus. II. Homologous interference. Virology 55: Simmons, D. T., and J. H. Strauss Translation of Sindbis virus 26S RNA and 49S RNA in lysates of rabbit reticulocytes. J. Mol. Biol. 86: Singh, K. R. P Cell cultures derived from larvae of Aedes albopictus (Skuse) and Aedes aegypti (L). Curr. Sci. 36: Stark, C., and S. I. T. Kennedy The generation and propagation of defective-interfering particles of Semliki Forest virus in different cell types. Virology 89: Stevens, T. M Arbovirus replication in mosquito cell lines (Singh) grown in monolayer or suspension culture. Proc. Soc. Exp. Biol. Med. 134: Stollar, V., and T. E. Shenk Homologous viral interference in Aedes albopictus cultures chronically infected with Sindbis virus. J. Virol. 11: Stollar, V., T. E. Shenk, R. Koo, A. Igarashi, and R. W. Schlesinger Observations on Aedes albopictus cell cultures persistently infected with Sindbis virus. Ann. N.Y. Acad. Sci. 266: Weiss, B., and S. Schlesinger Defective-interfering passages of Sindbis virus: chemical composition, biological activity, and mode of interference. J. Virol. 12: Wengler, G., M. Beato, and B.-A. Hackemack Translation of 26S virus specific RNA from Semliki Forest virus-infected cells in vitro. Virology 61: Wengler, G, and G. Wengler Localisation of the 26S RNA sequence on the viral genome type 428 RNA isolated from SFV-infected cells. Virology 73:

Superinfection with Vaccinia Virus

Superinfection with Vaccinia Virus JOURNAL OF VIROLOGY, Aug. 1975, p. 322-329 Copyright 1975 American Society for Microbiology Vol. 16, No. 2 Printed in U.S.A. Abortive Infection of a Rabbit Cornea Cell Line by Vesicular Stomatitis Virus:

More information

Induction of Interferon in Chick Cells by Temperaturesensitive Mutants of Sindbis Virus

Induction of Interferon in Chick Cells by Temperaturesensitive Mutants of Sindbis Virus J. gen. ViroL 0974), 25, 381-39o Printed in Great Britain 38I Induction of Interferon in Chick Cells by Temperaturesensitive Mutants of Sindbis Virus By G. J. ATKINS, M. D. JOHNSTON, LINDA M. WESTMACOTT

More information

Replication of Sindbis Virus V. Polyribosomes and mrna in Infected Cells

Replication of Sindbis Virus V. Polyribosomes and mrna in Infected Cells JOURNAL OF VIROLOGY, Sept. 1974, p. 552-559 Vol. 14, No. 3 Copyright @ 1974 American Society for Microbiology Printed in U.S.A. Replication of Sindbis Virus V. Polyribosomes and mrna in Infected Cells

More information

D. J. Dargan,* C. B. Gait and J. H. Subak-Sharpe

D. J. Dargan,* C. B. Gait and J. H. Subak-Sharpe Journal of General Virology (1992), 73, 407-411. Printed in Great Britain 407 The effect of cicloxolone sodium on the replication in cultured cells of adenovirus type 5, reovirus type 3, poliovirus type

More information

Role of Interferon in the Propagation of MM Virus in L Cells

Role of Interferon in the Propagation of MM Virus in L Cells APPLIED MICROBIOLOGY, Oct. 1969, p. 584-588 Copyright ( 1969 American Society for Microbiology Vol. 18, No. 4 Printed in U S A. Role of Interferon in the Propagation of MM Virus in L Cells DAVID J. GIRON

More information

Defective Interfering Particles of Respiratory Syncytial Virus

Defective Interfering Particles of Respiratory Syncytial Virus INFECTION AND IMMUNITY, Aug. 1982, p. 439-444 0019-9567/82/080439-06$02.00/0 Vol. 37, No. 2 Defective Interfering Particles of Respiratory Syncytial Virus MARY W. TREUHAFTl* AND MARC 0. BEEM2 Marshfield

More information

Quantitative Assay of Paravaccinia Virus Based

Quantitative Assay of Paravaccinia Virus Based APPrU MICROBIOLOGY, JUly 1972, p. 138-142 Copyright 1972 American Society for Microbiology Vol. 24, No. 1 Printed in U.S.A. Quantitative Assay of Paravaccinia Virus Based on Enumeration of Inclusion-Containing

More information

(Mosquito) Cells: Establishment of a Persistent Viral Infection

(Mosquito) Cells: Establishment of a Persistent Viral Infection JOURNAL OF VIROLOGY, June 1981, p. 1015-1024 Vol. 38, No. 3 0022-538X/81/061015-10$02.00/0 Bunyamwera Virus Replication in Cultured Aedes albopictus (Mosquito) Cells: Establishment of a Persistent Viral

More information

Materials and Methods , The two-hybrid principle.

Materials and Methods , The two-hybrid principle. The enzymatic activity of an unknown protein which cleaves the phosphodiester bond between the tyrosine residue of a viral protein and the 5 terminus of the picornavirus RNA Introduction Every day there

More information

Temperature-Sensitive Mutants Isolated from Hamster and

Temperature-Sensitive Mutants Isolated from Hamster and JOURNAL OF VIROLOGY, Nov. 1975, p. 1332-1336 Copyright i 1975 American Society for Microbiology Vol. 16, No. 5 Printed in U.S.A. Temperature-Sensitive Mutants Isolated from Hamster and Canine Cell Lines

More information

Mengovirus Virions. growth (48-h cultures) were infected with a. cell at a density of 107 cells per ml of ABM42-

Mengovirus Virions. growth (48-h cultures) were infected with a. cell at a density of 107 cells per ml of ABM42- JOURNAL OF VIROLOGY, Mar. 1977, p. 1256-1261 Copyright 1977 American Society for Microbiology Vol. 21, No. 3 Printed in U.S.A. Factors Affecting Composition and Thermostability of Mengovirus Virions CLIFFORD

More information

Unique Peptide Maps of the Three Largest Proteins Specified by the Flavivirus Kunjin

Unique Peptide Maps of the Three Largest Proteins Specified by the Flavivirus Kunjin JOURNAL OF VIROLOGY, Nov. 1977, p. 651-661 Copyright 1977 American Society for Microbiology Vol. 24, No. 2 Printed in U.S.A. Unique Peptide Maps of the Three Largest Proteins Specified by the Flavivirus

More information

NEUTRALIZATION OF REOVIRUS: THE GENE RESPONSIBLE FOR THE NEUTRALIZATION ANTIGEN* BY HOWARD L. WEINER~ AN~ BERNARD N. FIELDS

NEUTRALIZATION OF REOVIRUS: THE GENE RESPONSIBLE FOR THE NEUTRALIZATION ANTIGEN* BY HOWARD L. WEINER~ AN~ BERNARD N. FIELDS NEUTRALIZATION OF REOVIRUS: THE GENE RESPONSIBLE FOR THE NEUTRALIZATION ANTIGEN* BY HOWARD L. WEINER~ AN~ BERNARD N. FIELDS (From the Department of Microbiology and Molecular Genetics, Harvard Medical

More information

Inhibition of Sindbis Virus Replication in HeLa Cells by

Inhibition of Sindbis Virus Replication in HeLa Cells by ANTIMICROBIAL AGENTS AND CHEMOrHERAPY, Jan. 1974, p. 55-62 Copyright 0 1974 American Society for Microbiology Vol. 5, No. 1 Printed in U.S.A. Inhibition of Sindbis Virus Replication in HeLa Cells by Poliovirus

More information

Infectious Process of the Parvovirus H-1: Correlation of Protein Content, Particle Density, and Viral Infectivity

Infectious Process of the Parvovirus H-1: Correlation of Protein Content, Particle Density, and Viral Infectivity JOURNAL OF VIROLOGY, Sept. 1981, P. 800-807 Vol. 39, No. 3 0022-538X/81/090800-08$02.00/0 Infectious Process of the Parvovirus H-1: Correlation of Protein Content, Particle Density, and Viral Infectivity

More information

Effects of Chloroquine and Cytochalasin B on the Infection of

Effects of Chloroquine and Cytochalasin B on the Infection of JOURNAL OF VIROLOGY, Mar. 1981, p. 1060-1065 Vol. 37, No. 3 0022-538X/81/031060-06$02.00/0 Effects of Chloroquine and Cytochalasin B on the Infection of Cells by Sindbis Virus and Vesicular Stomatitis

More information

Effects of Cell Culture and Laboratory Conditions on Type 2 Dengue Virus Infectivity

Effects of Cell Culture and Laboratory Conditions on Type 2 Dengue Virus Infectivity JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1979, p. 235-239 0095-1137/79/08-0235/05$02.00/0 Vol. 10, No. 2 Effects of Cell Culture and Laboratory Conditions on Type 2 Dengue Virus Infectivity JARUE S. MANNING*

More information

Received 3 September 2002/Accepted 15 January 2003

Received 3 September 2002/Accepted 15 January 2003 JOURNAL OF VIROLOGY, Apr. 2003, p. 4646 4657 Vol. 77, No. 8 0022-538X/03/$08.00 0 DOI: 10.1128/JVI.77.8.4646 4657.2003 Copyright 2003, American Society for Microbiology. All Rights Reserved. Ability of

More information

Glycoprotein Synthesis by D-Glucosamine Hydrochloride

Glycoprotein Synthesis by D-Glucosamine Hydrochloride JOURNAL OF VIROLOGY, Apr. 1974, p. 775-779 Copyright 0 1974 American Society for Microbiology Vol. 13, No. 4 Printed in U.S.A. Selective Inhibition of Newcastle Disease Virus-Induced Glycoprotein Synthesis

More information

Tryptic Peptide Analysis of Nonstructural and Structural

Tryptic Peptide Analysis of Nonstructural and Structural JOURNAL OF VIROLOGY, Dec. 1975, P. 1615-1629 Copyright X3 1975 American Society for Microbiology Vol. 16, No. 6 Printed in U.SA. Tryptic Peptide Analysis of Nonstructural and Structural Precursor Proteins

More information

Ethylenediaminetetraacetate

Ethylenediaminetetraacetate APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1980, p. 1148-1153 0099-2240/80/06-1148/06$02.00/0 Vol. 39, No. 6 Comparative Study on the Mechanisms of Rotavirus Inactivation by Sodium Dodecyl Sulfate and

More information

Persistent Infection of MDCK Cells by Influenza C Virus: Initiation and Characterization

Persistent Infection of MDCK Cells by Influenza C Virus: Initiation and Characterization J. gen. Virol. (199), 70, 341-345. Printed in Great Britain 341 Key words: influenza C virus/interferon/persistent infection Persistent Infection of MDCK Cells by Influenza C Virus: Initiation and Characterization

More information

(;[rowth Charaeteristies of Influenza Virus Type C in Avian Hosts

(;[rowth Charaeteristies of Influenza Virus Type C in Avian Hosts Archives of Virology 58, 349--353 (1978) Archives of Virology by Springer-Verlag 1978 (;[rowth Charaeteristies of Influena Virus Type C in Avian Hosts Brief Report By M ~R A~N D. AUSTIn, A. S. MONTO, and

More information

Formation of an Infectious Virus-Antibody Complex with Rous

Formation of an Infectious Virus-Antibody Complex with Rous JOURNAL OF VIROLOGY, Mar. 1976, p. 163-167 Copyright 1976 American Society for Microbiology Vol. 17, No. 3 Printed in U.S.A. Formation of an Infectious Virus-Antibody Complex with Rous Sarcoma Virus and

More information

EVALUATION OF THE EFFECTIVENESS OF A 7% ACCELERATED HYDROGEN PEROXIDE-BASED FORMULATION AGAINST CANINE PARVOVIRUS

EVALUATION OF THE EFFECTIVENESS OF A 7% ACCELERATED HYDROGEN PEROXIDE-BASED FORMULATION AGAINST CANINE PARVOVIRUS Final report submitted to Virox Technologies, Inc. EVALUATION OF THE EFFECTIVENESS OF A 7% ACCELERATED HYDROGEN PEROXIDE-BASED FORMULATION AGAINST CANINE PARVOVIRUS Syed A. Sattar, M.Sc., Dip. Bact., M.S.,

More information

Transcriptional Mapping of Rabies Virus In Vivo

Transcriptional Mapping of Rabies Virus In Vivo JOURNAL OF VIROLOGY, Nov. 1978, P. 518-523 0022-538X/78/0028-0518$02.00/0 Copyright 1978 American Society for Microbiology Transcriptional Mapping of Rabies Virus In Vivo Vol. 28, No. 2 Printed in U.S.A.

More information

ISOLATION OF A SARCOMA VIRUS FROM A SPONTANEOUS CHICKEN TUMOR

ISOLATION OF A SARCOMA VIRUS FROM A SPONTANEOUS CHICKEN TUMOR ISOLATION OF A SARCOMA VIRUS FROM A SPONTANEOUS CHICKEN TUMOR Shigeyoshi ITOHARA, Kouichi HIRATA, Makoto INOUE, Masanori Veterinary Pathology, Faculty of Agriculture, Yamaguchi University* HATSUOKA, and

More information

hemagglutinin and the neuraminidase genes (RNA/recombinant viruses/polyacrylamide gel electrophoresis/genetics)

hemagglutinin and the neuraminidase genes (RNA/recombinant viruses/polyacrylamide gel electrophoresis/genetics) Proc. Natl. Acad. Sci. USA Vol. 73, No. 6, pp. 242-246, June 976 Microbiology Mapping of the influenza virus genome: Identification of the hemagglutinin and the neuraminidase genes (RNA/recombinant viruses/polyacrylamide

More information

Effect of Exogenous Interferon on Rubella Virus Production in Carrier Cultures of Cells Defective in Interferon Production

Effect of Exogenous Interferon on Rubella Virus Production in Carrier Cultures of Cells Defective in Interferon Production INFECTION AND IMMUNITY, Aug. 1970, p. 132-138 Copyright 1970 American Society for Microbiology Vol. 2, No. 2 Printed in U.S.A. Effect of Exogenous Interferon on Rubella Virus Production in Carrier Cultures

More information

Amantadine in Tissue Culture'

Amantadine in Tissue Culture' JOURNAL OF BACTERIOLOGY, Sept., 1965 Copyright 1965 American Society for Microbiology Vol. 90, No. 3 Printed in U.S.A. Mode of Action of the Antiviral Activity of Amantadine in Tissue Culture' C. E. HOFFMANN,

More information

Encapsidation of Sendai Virus Genome RNAs by Purified

Encapsidation of Sendai Virus Genome RNAs by Purified JOURNAL OF VIROLOGY, Mar. 1988, p. 834-838 22-538X/88/3834-5$2./ Copyright C) 1988, American Society for Microbiology Vol. 62, No. 3 Encapsidation of Sendai Virus Genome RNAs by Purified NP Protein during

More information

Host Restriction of Friend Leukemia Virus. Role of the Viral Outer Coat (mice/fv-1 locus/vesicular stomatitis virus)

Host Restriction of Friend Leukemia Virus. Role of the Viral Outer Coat (mice/fv-1 locus/vesicular stomatitis virus) Proc. Nat. Acad. Sci. USA Vol. 70, No. 9, pp. 2549-2553, September 1973 Host Restriction of Friend Leukemia Virus. Role of the Viral Outer Coat (mice/fv-1 locus/vesicular stomatitis virus) THEODORE G.

More information

Identification of Polysomal RNA in BHK Cells Infected by Sindbis Virus

Identification of Polysomal RNA in BHK Cells Infected by Sindbis Virus JOURNAL OF VIROLOGY, Apr. 1973, p. 53-543 Copyright 1973 American Society for Microbiology Vol. 11, No. 4 Plrintd in U.S.A. Identification of Polysomal RNA in BHK Cells Infected by Sindbis Virus DEBORAH

More information

THE ROLE OF INTERFERON IN VACCINIA VIRUS INFECTION OF MOUSE EMBRYO TISSUE CULTURE

THE ROLE OF INTERFERON IN VACCINIA VIRUS INFECTION OF MOUSE EMBRYO TISSUE CULTURE THE ROLE OF INTERFERON IN VACCINIA VIRUS INFECTION OF MOUSE EMBRYO TISSUE CULTURE BY LOWELL A. GLASGOW, M.D., A~rD KARL HABEL, M.D. (From the Laboratory of Biology of Viruses, National Institute of Allergy

More information

Replication in Tissue Culture

Replication in Tissue Culture JOURNAL OF VIROLOGY, Jan 1977, p. 277-283 Copyright C 1977 American Society for Microbiology Vol. 21, No. 1 Printed in U.S.A. Effect of Cyclophosphamide In Vitro and on Vaccinia Virus Replication in Tissue

More information

Synthesis of Plus- and Minus-Strand RNA in Rotavirus-Infected Cells

Synthesis of Plus- and Minus-Strand RNA in Rotavirus-Infected Cells JOURNAL OF VIROLOGY, Nov. 1987, p. 3479-3484 0022-538X/87/113479-06$02.00/0 Copyright 1987, American Society for Microbiology Vol. 61, No. 11 Synthesis of Plus- and Minus-Strand RNA in Rotavirus-Infected

More information

PERSISTENT INFECTIONS WITH HUMAN PARAINFLUENZAVIRUS TYPE 3 IN TWO CELL LINES

PERSISTENT INFECTIONS WITH HUMAN PARAINFLUENZAVIRUS TYPE 3 IN TWO CELL LINES 71 PERSISTENT INFECTIONS WITH HUMAN PARAINFLUENZAVIRUS TYPE 3 IN TWO CELL LINES Harold G. Jensen, Alan J. Parkinson, and L. Vernon Scott* Department of Microbiology & Immunology, University of Oklahoma

More information

BY F. BROWN, B. CARTWRIGHT AND DOREEN L. STEWART Research Institute (Animal Virus Diseases), Pirbright, Surrey. (Received 22 August 1962) SUMMARY

BY F. BROWN, B. CARTWRIGHT AND DOREEN L. STEWART Research Institute (Animal Virus Diseases), Pirbright, Surrey. (Received 22 August 1962) SUMMARY J. gen. Microbial. (1963), 31, 179186 Prinied in Great Britain 179 The Effect of Various Inactivating Agents on the Viral and Ribonucleic Acid Infectivities of FootandMouth Disease Virus and on its Attachment

More information

Mechanism of Pock Formation by Shope Fibroma

Mechanism of Pock Formation by Shope Fibroma JOURNAL OF BACTERIOLOGY, Sept., 1966 Copyright ( 1966 American Society for Microbiology Vol. 92, No. 3 Printed in U.S.A. Mechanism of Pock Formation by Shope Fibroma Virus on Monolayers of Rabbit Cells

More information

Effect of Low-NaCl Medium on the Envelope Glycoproteins of Sindbis Virus

Effect of Low-NaCl Medium on the Envelope Glycoproteins of Sindbis Virus JOURNAL OF VIROLOGY, Mar. 1978, p. 764-769 0022-538X/78/0025-0764$02.00/0 Copyright i 1978 American Society for Microbiology Vol. 25, No. 3 Printed in U.S.A. Effect of Low-NaCl Medium on the Envelope Glycoproteins

More information

Transfection of Sf9 cells with recombinant Bacmid DNA

Transfection of Sf9 cells with recombinant Bacmid DNA Transposition Bacmid DNA Mini Culturing baculo cells Transfection of Sf9 cells with recombinant Bacmid DNA Amplification of the virus Titration of baculo stocks Testing the expression Transposition 1.

More information

Overview of virus life cycle

Overview of virus life cycle Overview of virus life cycle cell recognition and internalization release from cells progeny virus assembly membrane breaching nucleus capsid disassembly and genome release replication and translation

More information

Genome RNAs and Polypeptides of Reovirus Serotypes

Genome RNAs and Polypeptides of Reovirus Serotypes JOURNAL OF VIROLOGY, June 1977, p. 726-733 Copyright 1977 American Society for Microbiology Vol. 22, No. 3 Printed in U.S.A. Genome RNAs and Polypeptides of Reovirus Serotypes 1, 2, and 3 ROBERT F. RAMIG,*

More information

Effect of Puromycin and Actinomycin D on a Persistent Mumps Virus Infection In Vitro

Effect of Puromycin and Actinomycin D on a Persistent Mumps Virus Infection In Vitro JOURNAL OF VIROLOGY, Aug. 1969, p. 133-143 Copyright 1969 American Society for Microbiology Vol. 4, No. 2 Printed in U.S.A. Effect of Puromycin and Actinomycin D on a Persistent Mumps Virus Infection In

More information

Polypeptides of Respiratory Syncytial Virus

Polypeptides of Respiratory Syncytial Virus JOURNAL OF VIROLOGY, Jan. 1977, p. 427-431 Vol. 21, No. 1 Copyright C 1977 American Society for Microbiology Printed in U.S.A. Polypeptides of Respiratory Syncytial Virus SEYMOUR LEVINE Department ofimmunology

More information

Inhibition of the Multiplication of Vesicular Stomatitis and

Inhibition of the Multiplication of Vesicular Stomatitis and JOURNAL OF VIROLOGY, June 1974, p. 1186-1193 Copyright 1974 American Society for Microbiology Vol. 13, No. 6 Printed in U.S.A. Inhibition of the Multiplication of Vesicular Stomatitis and Newcastle Disease

More information

Annexure III SOLUTIONS AND REAGENTS

Annexure III SOLUTIONS AND REAGENTS Annexure III SOLUTIONS AND REAGENTS A. STOCK SOLUTIONS FOR DNA ISOLATION 0.5M Ethylene-diamine tetra acetic acid (EDTA) (ph=8.0) 1M Tris-Cl (ph=8.0) 5M NaCl solution Red cell lysis buffer (10X) White cell

More information

In Vitro Cultivation of Human Rotavirus in MA 104 Cells

In Vitro Cultivation of Human Rotavirus in MA 104 Cells Acute Diarrhea: Its Nutritional Consequences in Children, edited by J. A. Bellanti. Nestle, Vevey/Raven Press, New York 1983. ETIOLOGIC AGENTS OF ACUTE DIARRHEA In Vitro Cultivation of Human Rotavirus

More information

Chapter 6- An Introduction to Viruses*

Chapter 6- An Introduction to Viruses* Chapter 6- An Introduction to Viruses* *Lecture notes are to be used as a study guide only and do not represent the comprehensive information you will need to know for the exams. 6.1 Overview of Viruses

More information

Translation of Sindbis virus mrna: analysis of sequences downstream of the initiating AUG codon that enhance translation.

Translation of Sindbis virus mrna: analysis of sequences downstream of the initiating AUG codon that enhance translation. Translation of Sindbis virus mrna: analysis of sequences downstream of the initiating AUG codon that enhance translation. I Frolov and S Schlesinger J. Virol. 1996, 70(2):1182. CONTENT ALERTS Updated information

More information

The reovirus genome comprises 10 segments of doublestranded

The reovirus genome comprises 10 segments of doublestranded Reovirus reverse genetics: Incorporation of the CAT gene into the reovirus genome Michael R. Roner* and Wolfgang K. Joklik *Department of Biological Sciences, Center for Molecular Biology and Biotechnology,

More information

Recombinant Virus Vaccine for Bluetongue Disease in Sheep

Recombinant Virus Vaccine for Bluetongue Disease in Sheep JOURNAL OF VIROLOGY, May 1990, p. 1998-2003 Vol. 64, No. 5 0022-538X/90/051998-06$02.00/0 Copyright 1990, American Society for Microbiology Recombinant Virus Vaccine for Bluetongue Disease in Sheep P.

More information

ELECTRON MICROSCOPIC STUDIES ON EQUINE ENCEPHALOSIS VIRUS

ELECTRON MICROSCOPIC STUDIES ON EQUINE ENCEPHALOSIS VIRUS Onderstepoort]. vet. Res. 40 (2), 53-58 (1973) ELECTRON MICROSCOPIC STUDIES ON EQUINE ENCEPHALOSIS VIRUS G. LECATSAS, B. J. ERASMUS and H. J. ELS, Veterinary Research Institute, Onderstepoort ABSTRACT

More information

Leukocytes and Interferon in the Host Response to Viral Infections

Leukocytes and Interferon in the Host Response to Viral Infections JOURNAL OF BACTERIOLOGY, June, 1966 Copyright 1966 American Society for Microbiology Vol. 91, No. 6 Printed in U.S.A. Leukocytes and Interferon in the Host Response to Viral Infections IL. Enhanced Interferon

More information

Berne Virus Is Not 'Coronavirus-fike'

Berne Virus Is Not 'Coronavirus-fike' J. gen. Virol. (1984), 65, 645~i49. Printed in Great Britain 645 Key words: Berne virus/polypeptides/inhibitors Berne Virus Is Not 'Coronavirus-fike' By M. C. HORZINEK,* M. WEISS 1 AND J. EDERVEEN Institute

More information

Sindbis virus-induced inhibition of protein synthesis is partially reversed by medium containing an elevated potassium concentration

Sindbis virus-induced inhibition of protein synthesis is partially reversed by medium containing an elevated potassium concentration Journal of General Virology (1994), 75, 411~415. Printed in Great Britain 411 Sindbis virus-induced inhibition of protein synthesis is partially reversed by medium containing an elevated potassium concentration

More information

A New Role for ns Polyprotein Cleavage in Sindbis Virus Replication

A New Role for ns Polyprotein Cleavage in Sindbis Virus Replication JOURNAL OF VIROLOGY, July 2008, p. 6218 6231 Vol. 82, No. 13 0022-538X/08/$08.00 0 doi:10.1128/jvi.02624-07 Copyright 2008, American Society for Microbiology. All Rights Reserved. A New Role for ns Polyprotein

More information

Lecture 2: Virology. I. Background

Lecture 2: Virology. I. Background Lecture 2: Virology I. Background A. Properties 1. Simple biological systems a. Aggregates of nucleic acids and protein 2. Non-living a. Cannot reproduce or carry out metabolic activities outside of a

More information

Genetic Complementation among Poliovirus Mutants Derived

Genetic Complementation among Poliovirus Mutants Derived JOURNAL OF VIROLOGY, Dec. 1986, p. 1040-1049 0022-538X/86/121040-10$02.00/0 Copyright C) 1986, American Society for Microbiology Vol. 60, No. 3 Genetic Complementation among Poliovirus Mutants Derived

More information

Stomatitis Virus. in continuous suspension culture maintained at 105 to 4 X 105 cells/ml in Eagle medium modified for

Stomatitis Virus. in continuous suspension culture maintained at 105 to 4 X 105 cells/ml in Eagle medium modified for JOURNAL OF VIROLOGY, Aug. 1969, p. 15-161 Vol., No. Copyright @ 1969 American Society for Microbiology Printed in U.S.A. Ribonucleic Acid Synthesis of Vesicular Stomatitis Virus I. Species of Ribonucleic

More information

virus-i (RAV-1) or Rous associated virus-2 (RAV-2), do not transform but do produce

virus-i (RAV-1) or Rous associated virus-2 (RAV-2), do not transform but do produce ISOLATION OF NONINFECTIOUS PARTICLES CONTAINING ROUS SARCOMA VIRUS RNA FROM THE MEDIUM OF ROUS SARCOMA VIRUS-TRANSFORMED NONPRODUCER CELLS* BY HARRIET LATHAM ROBINSONt VIRUS LABORATORY, UNIVERSITY OF CALIFORNIA,

More information

Chromatin IP (Isw2) Fix soln: 11% formaldehyde, 0.1 M NaCl, 1 mm EDTA, 50 mm Hepes-KOH ph 7.6. Freshly prepared. Do not store in glass bottles.

Chromatin IP (Isw2) Fix soln: 11% formaldehyde, 0.1 M NaCl, 1 mm EDTA, 50 mm Hepes-KOH ph 7.6. Freshly prepared. Do not store in glass bottles. Chromatin IP (Isw2) 7/01 Toshi last update: 06/15 Reagents Fix soln: 11% formaldehyde, 0.1 M NaCl, 1 mm EDTA, 50 mm Hepes-KOH ph 7.6. Freshly prepared. Do not store in glass bottles. 2.5 M glycine. TBS:

More information

Analysis of Viral and Defective-Interfering Nucleocapsids in Acute and Persistent Infection by Rhabdoviruses

Analysis of Viral and Defective-Interfering Nucleocapsids in Acute and Persistent Infection by Rhabdoviruses J. gen. Virol. (1982), 60, 87-97. Printed in Great Britain 87 Key words: nucleocapsms/persistence/dlparticles/rabies Analysis of Viral and Defective-Interfering Nucleocapsids in Acute and Persistent Infection

More information

Estimations of the Molecular Weight of the Influenza Virus Genome

Estimations of the Molecular Weight of the Influenza Virus Genome o r. gem Viral. &97I), H, Io3-Io9 103 Printed in Great Britain Estimations of the Molecular Weight of the Influenza Virus Genome By J. J. SKEHEL National Institute for Medical Research, Mill Hill, London

More information

Introduction.-Cytopathogenic viruses may lose their cell-destroying capacity

Introduction.-Cytopathogenic viruses may lose their cell-destroying capacity AN INHIBITOR OF VIRAL ACTIVITY APPEARING IN INFECTED CELL CULTURES* BY MONTO Hot AND JOHN F. ENDERS RESEARCH DIVISION OF INFECTIOUS DISEASES, THE CHILDREN'S MEDICAL CENTER, AND THE DEPARTMENT OF BACTERIOLOGY

More information

THE CYTOPATHOGENIC ACTION OF BLUETONGUE VIRUS ON TISSUE CULTURES AND ITS APPLICATION TO THE DETECTION OF ANTIBODIES IN THE SERUM OF SHEEP.

THE CYTOPATHOGENIC ACTION OF BLUETONGUE VIRUS ON TISSUE CULTURES AND ITS APPLICATION TO THE DETECTION OF ANTIBODIES IN THE SERUM OF SHEEP. Onderstepoort Journal of Veterinary Research, Volume 27, Number 2, October, 1956. The Government Printer. THE CYTOPATHOGENIC ACTION OF BLUETONGUE VIRUS ON TISSUE CULTURES AND ITS APPLICATION TO THE DETECTION

More information

Identification of the Virucidal Agent in Wastewater Sludge

Identification of the Virucidal Agent in Wastewater Sludge APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1977, p. 860-864 Copyright X) 1977 American Society for Microbiology Vol. 33, No. 4 Printed in U.S.A. Identification of the Virucidal Agent in Wastewater Sludge

More information

Wilmington, Delaware cells were harvested in the cold and pelleted. The cell. pellet was suspended in 2 ml of cold buffer consisting

Wilmington, Delaware cells were harvested in the cold and pelleted. The cell. pellet was suspended in 2 ml of cold buffer consisting JOURNAL OF VIROLOGY, June 1969, p. 599-64 Vol. 3, No. 6 Copyright 1969 American Society for Microbiology Printed in U.S.A. Sindbis Virus-induced Viral Ribonucleic Acid Polymerasel T. SREEVALSAN' AND FAY

More information

Effects of 2-Deoxyglucose, Glucosamine, and Mannose on Cell Fusion and the Glycoproteins of Herpes Simplex Virus

Effects of 2-Deoxyglucose, Glucosamine, and Mannose on Cell Fusion and the Glycoproteins of Herpes Simplex Virus JOURNAL OF VIROLOGY, May 1976, p. 644-651 Copyright 1976 American Society for Microbiology Vol. 18, No. 2 Printed in U.S.A. Effects of 2-Deoxyglucose, Glucosamine, and Mannose on Cell Fusion and the Glycoproteins

More information

The Infectious Cycle. Lecture 2 Biology W3310/4310 Virology Spring You know my methods, Watson --SIR ARTHUR CONAN DOYLE

The Infectious Cycle. Lecture 2 Biology W3310/4310 Virology Spring You know my methods, Watson --SIR ARTHUR CONAN DOYLE The Infectious Cycle Lecture 2 Biology W3310/4310 Virology Spring 2016 You know my methods, Watson --SIR ARTHUR CONAN DOYLE The Infectious Cycle Virologists divide the infectious cycle into steps to facilitate

More information

Cell-Free Synthesis of Herpes Simplex Virus-Coded

Cell-Free Synthesis of Herpes Simplex Virus-Coded JOURNAL OF VIROLOGY, Sept. 1977, p. 455-460 Copyright C 1977 American Society for Microbiology Vol. 23, No. 3 Printed in U.S.A. Cell-Free Synthesis of Herpes Simplex Virus-Coded Pyrimidine Deoxyribonucleoside

More information

of an Infectious Form of Rous Sarcoma Virus*

of an Infectious Form of Rous Sarcoma Virus* Proceedings of the National Academy of Sciences Vol. 66, No. 2, pp. 314-321, June 1970 A Cell-Associated Factor Essential for Formation of an Infectious Form of Rous Sarcoma Virus* H. Hanafusa, T. Miyamoto,

More information

Recipes for Media and Solution Preparation SC-ura/Glucose Agar Dishes (20mL/dish, enough for 8 clones)

Recipes for Media and Solution Preparation SC-ura/Glucose Agar Dishes (20mL/dish, enough for 8 clones) Protocol: 300 ml Yeast culture preparation Equipment and Reagents needed: Autoclaved toothpicks Shaker Incubator set at 30 C Incubator set at 30 C 60 mm 2 sterile petri dishes Autoclaved glass test tubes

More information

Dental Research Institute, Faculty of Dentistry, University of Toronto, Toronto, Canada *For correspondence:

Dental Research Institute, Faculty of Dentistry, University of Toronto, Toronto, Canada *For correspondence: Zymogram Assay for the Detection of Peptidoglycan Hydrolases in Streptococcus mutans Delphine Dufour and Céline M. Lévesque * Dental Research Institute, Faculty of Dentistry, University of Toronto, Toronto,

More information

STUDIES OF THE HEMAGGLUTININ OF HAEMOPHILUS PERTUSSIS HIDEO FUKUMI, HISASHI SHIMAZAKI, SADAO KOBAYASHI AND TATSUJI UCHIDA

STUDIES OF THE HEMAGGLUTININ OF HAEMOPHILUS PERTUSSIS HIDEO FUKUMI, HISASHI SHIMAZAKI, SADAO KOBAYASHI AND TATSUJI UCHIDA STUDIES OF THE HEMAGGLUTININ OF HAEMOPHILUS PERTUSSIS HIDEO FUKUMI, HISASHI SHIMAZAKI, SADAO KOBAYASHI AND TATSUJI UCHIDA The National Institute of Health, Tokyo, Japan (Received: August 3rd, 1953) INTRODUCTION

More information

(From the Laboratory of Cell Biology, National Institute of Allergy and Infectious Diseases, National Instil/utes of Health, Bahesda, Maryland)

(From the Laboratory of Cell Biology, National Institute of Allergy and Infectious Diseases, National Instil/utes of Health, Bahesda, Maryland) Published Online: 1 September, 1959 Supp Info: http://doi.org/10.1084/jem.110.3.445 Downloaded from jem.rupress.org on December 1, 2018 THE EFFECT OF CELL POPULATION DENSITY ON THE AMINO ACID REQUIREMENTS

More information

HIV-1 Virus-like Particle Budding Assay Nathan H Vande Burgt, Luis J Cocka * and Paul Bates

HIV-1 Virus-like Particle Budding Assay Nathan H Vande Burgt, Luis J Cocka * and Paul Bates HIV-1 Virus-like Particle Budding Assay Nathan H Vande Burgt, Luis J Cocka * and Paul Bates Department of Microbiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, USA

More information

Adenovirus Manual 1. Table of Contents. Large Scale Prep 2. Quick MOI Test 4. Infection of MNT-1 Cells 8. Adenovirus Stocks 9

Adenovirus Manual 1. Table of Contents. Large Scale Prep 2. Quick MOI Test 4. Infection of MNT-1 Cells 8. Adenovirus Stocks 9 Adenovirus Manual 1 Table of Contents Large Scale Prep 2 Quick MOI Test 4 TCID 50 Titration 5 Infection of MNT-1 Cells 8 Adenovirus Stocks 9 CAUTION: Always use filter tips and bleach everything!!! Adenovirus

More information

Cytomegalovirus Based upon Enhanced Uptake of Neutral

Cytomegalovirus Based upon Enhanced Uptake of Neutral JOURNAL OF CUNICAL MICROBIOLOGY, JUlY 1976, p. 61-66 Copyright 1976 American Society for Microbiology Vol. 4, No. 1 Printed in U.S.A. Plaque Reduction Neutralization Test for Human Cytomegalovirus Based

More information

Purification of Theiler's Murine Encephalomyelitis Virus and

Purification of Theiler's Murine Encephalomyelitis Virus and JOURNAL OF VIROLOGY, Mar. 1980, p. 1165-1172 0022-538X/80/03-1165/08$02.00/0 Vol. 33, No. 3 Purification of Theiler's Murine Encephalomyelitis Virus and Analysis of the Structural Virion Polypeptides:

More information

Effect of Pactamycin on Synthesis of Poliovirus

Effect of Pactamycin on Synthesis of Poliovirus JOURNAL OF VIROLOGY, OCt. 1971, p. 395-41 Copyright 1971 American Society for Microbiology Vol. 8, No. 4 Printed in U.S.A. Effect of Pactamycin on Synthesis of Poliovirus Proteins: a Method for Genetic

More information

SUPPLEMENTARY MATERIAL

SUPPLEMENTARY MATERIAL SUPPLEMENTARY MATERIAL Purification and biochemical properties of SDS-stable low molecular weight alkaline serine protease from Citrullus Colocynthis Muhammad Bashir Khan, 1,3 Hidayatullah khan, 2 Muhammad

More information

INTRABULBAR INOCULATION OF JAPANESE ENCEPHALITIS VIRUS TO MICE

INTRABULBAR INOCULATION OF JAPANESE ENCEPHALITIS VIRUS TO MICE THE KURUME MEDICAL JOURNAL Vol. 15, No. 1, 1968 INTRABULBAR INOCULATION OF JAPANESE ENCEPHALITIS VIRUS TO MICE TOSHINORI TSUCHIYA Department of Microbiology, and Department of Ophthalmology, Kurume University

More information

Molecular Biology of Rotaviruses I. Characterization of Basic Growth Parameters and Pattern of

Molecular Biology of Rotaviruses I. Characterization of Basic Growth Parameters and Pattern of JOURNAL OF VIROLOGY, Aug. 1981, p. 490-496 Vol. 39, No. 2 0022-538X/81/080490-07$02.00/0 Molecular Biology of Rotaviruses I. Characterization of Basic Growth Parameters and Pattern of Macromolecular Synthesis

More information

Protocol for Gene Transfection & Western Blotting

Protocol for Gene Transfection & Western Blotting The schedule and the manual of basic techniques for cell culture Advanced Protocol for Gene Transfection & Western Blotting Schedule Day 1 26/07/2008 Transfection Day 3 28/07/2008 Cell lysis Immunoprecipitation

More information

Genomic Alterations Associated with Persistent Infections by Equine Infectious Anaemia Virus, a Retrovirus

Genomic Alterations Associated with Persistent Infections by Equine Infectious Anaemia Virus, a Retrovirus J. gen. Virol. (1984), 65, 1395-1399. Printed in Great Britain 1395 Key words: EIA V/retrovirus persistence~antigenic variation/oligonucleotide mapping Genomic Alterations Associated with Persistent Infections

More information

Ultrastructure of Mycoplasmatales Virus laidlawii x

Ultrastructure of Mycoplasmatales Virus laidlawii x J. gen. Virol. (1972), I6, 215-22I Printed in Great Britain 2I 5 Ultrastructure of Mycoplasmatales Virus laidlawii x By JUDY BRUCE, R. N. GOURLAY, AND D. J. GARWES R. HULL* Agricultural Research Council,

More information

Plaque Assay of Sendai Virus in Monolayers of a Clonal Line

Plaque Assay of Sendai Virus in Monolayers of a Clonal Line JOURNAL OF CUNICAL MICROBIOLOGY, Feb. 1976. p. 91-95 Copyright 1976 American Society for Microbiology Vol. 3, No. 2 Printed in U.SA. Plaque Assay of Sendai Virus in Monolayers of a Clonal Line of Porcine

More information

BIL 256 Cell and Molecular Biology Lab Spring, Tissue-Specific Isoenzymes

BIL 256 Cell and Molecular Biology Lab Spring, Tissue-Specific Isoenzymes BIL 256 Cell and Molecular Biology Lab Spring, 2007 Background Information Tissue-Specific Isoenzymes A. BIOCHEMISTRY The basic pattern of glucose oxidation is outlined in Figure 3-1. Glucose is split

More information

Accelerated Cytopathology in HeLa Cells Induced

Accelerated Cytopathology in HeLa Cells Induced INFECTION AND IMMUNITY, Dec. 197, p. 75-71 Copyright 197 American Society for Microbiology Vol., No. Printed in U.S.A. Accelerated Cytopathology in HeLa Cells Induced by Reovirus and Cycloheximide PHILIP

More information

Size nm m m

Size nm m m 1 Viral size and organization Size 20-250nm 0.000000002m-0.000000025m Virion structure Capsid Core Acellular obligate intracellular parasites Lack organelles, metabolic activities, and reproduction Replicated

More information

Specific Sindbis Virus-Coded Function for Minus-Strand RNA

Specific Sindbis Virus-Coded Function for Minus-Strand RNA JOURNAL OF VIROLOGY, Aug. 1981, p. 348-358 Vol. 39, No. 2 0022-538X/81/080348-11$02.00/0 Specific Sindbis Virus-Coded Function for Minus-Strand RNA Synthesis DOROTHA L. SAWICKI,`* STANLY G. SAWICKI,' SIRKKA

More information

G. W. WOOD J. C. MUSKETT and D. H. THORNTON MAFF, Central Veterinary Laboratory, New Haw, Weybridge, Surrey, U.K.

G. W. WOOD J. C. MUSKETT and D. H. THORNTON MAFF, Central Veterinary Laboratory, New Haw, Weybridge, Surrey, U.K. J. Comp. Path. 1986 vol. 96 OBSERVATIONS ON THE ABILITY OF AVIAN REOVIRUS VACCINMATION OF HENS TO PROTECT THEIR PROGENY AGAINST THE EFFECTS OF CHALLENGE WITH HOMOLOGOUS AND HETEROLOGOUS STRAINS By G. W.

More information

STRUCTURE, GENERAL CHARACTERISTICS AND REPRODUCTION OF VIRUSES

STRUCTURE, GENERAL CHARACTERISTICS AND REPRODUCTION OF VIRUSES STRUCTURE, GENERAL CHARACTERISTICS AND REPRODUCTION OF VIRUSES Introduction Viruses are noncellular genetic elements that use a living cell for their replication and have an extracellular state. Viruses

More information

The Schedule and the Manual of Basic Techniques for Cell Culture

The Schedule and the Manual of Basic Techniques for Cell Culture The Schedule and the Manual of Basic Techniques for Cell Culture 1 Materials Calcium Phosphate Transfection Kit: Invitrogen Cat.No.K2780-01 Falcon tube (Cat No.35-2054:12 x 75 mm, 5 ml tube) Cell: 293

More information

Norovirus Report. Can copper and silver ionisation kill norovirus? A Study Report

Norovirus Report. Can copper and silver ionisation kill norovirus? A Study Report Norovirus Report Can copper and silver ionisation kill norovirus? A Study Report Can copper and silver ionisation kill norovirus? A Study Report Introduction Norovirus is the leading cause of non-bacterial

More information

Unique Features of Hepatitis C Virus Capsid Formation Revealed by De Novo Cell-Free Assembly

Unique Features of Hepatitis C Virus Capsid Formation Revealed by De Novo Cell-Free Assembly JOURNAL OF VIROLOGY, Sept. 2004, p. 9257 9269 Vol. 78, No. 17 0022-538X/04/$08.00 0 DOI: 10.1128/JVI.78.17.9257 9269.2004 Copyright 2004, American Society for Microbiology. All Rights Reserved. Unique

More information

SOME PROPERTIES OF ECHO AND COXSACKIE VIRUSES IN TISSUE CULTURE AND VARIATIONS BY HEAT

SOME PROPERTIES OF ECHO AND COXSACKIE VIRUSES IN TISSUE CULTURE AND VARIATIONS BY HEAT THE KURUME MEDICAL JOURNAL Vol. 9, No. 1, 1962 SOME PROPERTIES OF ECHO AND COXSACKIE VIRUSES IN TISSUE CULTURE AND VARIATIONS BY HEAT SHIGERU YAMAMATO AND MASAHISA SHINGU Department of Microbiology, Kurume

More information

Antigenic Variation between Human Respiratory Syncytial Virus Isolates

Antigenic Variation between Human Respiratory Syncytial Virus Isolates J. gen. Virol. (1986), 67, 863-870. Printed in Great Britain 863 Key words: RS virus/antigenic variation/phosphoprotein Antigenic Variation between Human Respiratory Syncytial Virus Isolates By H. B. GIMENEZ,*

More information

Hepatitis B Antiviral Drug Development Multi-Marker Screening Assay

Hepatitis B Antiviral Drug Development Multi-Marker Screening Assay Hepatitis B Antiviral Drug Development Multi-Marker Screening Assay Background ImQuest BioSciences has developed and qualified a single-plate method to expedite the screening of antiviral agents against

More information