The replication cycle of tanapox virus in owl monkey kidney cells

1999 ◽  
Vol 45 (1) ◽  
pp. 92-96 ◽  
Author(s):  
Sangita Mediratta ◽  
Karim Essani
Keyword(s):  
Latent Period ◽  
Growth Curves ◽  
Single Step ◽  
Replication Cycle ◽  
Monkey Kidney ◽  
Kidney Cells ◽  
Extracellular Virus ◽  
Tanapox Virus ◽  
Owl Monkey ◽  
Step Growth

The growth kinetics of tanapox virus in owl monkey kidney cells was elucidated by single-step growth curves at multiplicities of 10, 1.0, and 0.1 plaque forming units (pfu) per cell at 37 and 33°C. Virus replicated equally well at both temperatures and produced a cytopathic effect that was characterized by densely packed rounded cells with retrogressed monolayer and granular vacuolated cytoplasm. Single-step growth curves revealed that the eclipse period varied from 24 h postinfection (hpi) at a multiplicity of infection of 10 pfu/cell to 48 hpi at 0.1 pfu/cell. The length of the latent period also varied from 36 hpi at 10 pfu/cell to 48 hpi at 0.1 pfu/cell. The intracellular virus, extracellular virus, and total virus titers reached their maximums relatively early at 10 pfu/cell as compared with 0.1 pfu/cell. About 78% of the mature progeny virion is retained intracellularly at 10 pfu/cell at 96 hpi. We conclude that tanapox virus replication is similar to other poxviruses, but the replication cycle is longer when compared with vaccinia virus.Key words: tanapox virus, single-step growth curve, eclipse period, latent period.

scholarly journals Autographa californica Nucleopolyhedrovirus orf69 Encodes an RNA Cap (Nucleoside-2′-O)-Methyltransferase

2003 ◽  
Vol 77 (6) ◽  
pp. 3430-3440 ◽  
Author(s):  
Xiaofeng Wu ◽  
Linda A. Guarino
Keyword(s):  
Late Phase ◽  
Growth Curves ◽  
Immunoblot Analysis ◽  
Single Step ◽  
Coding Region ◽  
Methyltransferase Activity ◽  
Autographa Californica Nucleopolyhedrovirus ◽  
High Conservation ◽  
Step Growth

ABSTRACT The AcNPV orf69 gene encodes a protein that contains an S-adenosylmethionine (AdoMet)-dependent methyltransferase signature motif. More significantly, ORF69 shows high conservation at residues diagnostic for (nucleoside 2′-O)-methyltransferase activity. To analyze the function of this protein, which was renamed MTase1, it was overexpressed in Escherichia coli and purified to homogeneity. Photo cross-linking experiments showed that MTase1 bound AdoMet, and functional assays demonstrated cap 0-dependent methyltransferase activity. In vivo expression assays in insect cells showed that MTase1 was synthesized during the late phase of infection and that its expression was dependent on viral DNA replication. Primer extension analysis identified a late promoter motif, ATAAG, at the transcription start site. A mutant virus was constructed by inserting the lacZ gene into the coding region of mtase1. Immunoblot analysis confirmed that MTase1 was not synthesized in these cells, and single-step growth curves revealed that the rate of virus replication in tissue culture was not affected by the absence of MTase1.

scholarly journals Reverse Genetics System Demonstrates that Rotavirus Nonstructural Protein NSP6 Is Not Essential for Viral Replication in Cell Culture

2017 ◽  
Vol 91 (21) ◽  
Author(s):  
Satoshi Komoto ◽  
Yuta Kanai ◽  
Saori Fukuda ◽  
Masanori Kugita ◽  
Takahiro Kawagishi ◽  
...  
Keyword(s):  
Cell Culture ◽  
Viral Replication ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Growth Curves ◽  
Single Step ◽  
Replication Cycle ◽  
Nonstructural Proteins ◽  
Virus Growth ◽  
Severe Diarrhea

ABSTRACT The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6-deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmid-only-based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches.

scholarly journals Remote Activation of Host Cell DNA Synthesis in Uninfected Cells Signaled by Infected Cells in Advance of Virus Transmission

2015 ◽  
Vol 89 (21) ◽  
pp. 11107-11115 ◽  
Author(s):  
Nora Schmidt ◽  
Thomas Hennig ◽  
Remigiusz A. Serwa ◽  
Magda Marchetti ◽  
Peter O'Hare
Keyword(s):  
Virus Infection ◽  
Infected Cell ◽  
Dna Synthesis ◽  
Spatial Organization ◽  
Virus Transmission ◽  
Growth Curves ◽  
Uninfected Cell ◽  
Single Step ◽  
Host Interaction ◽  
Step Growth

ABSTRACTViruses modulate cellular processes and metabolism in diverse ways, but these are almost universally studied in the infected cell itself. Here, we study spatial organization of DNA synthesis during multiround transmission of herpes simplex virus (HSV) using pulse-labeling with ethynyl nucleotides and cycloaddition of azide fluorophores. We report a hitherto unknown and unexpected outcome of virus-host interaction. Consistent with the current understanding of the single-step growth cycle, HSV suppresses host DNA synthesis and promotes viral DNA synthesis in spatially segregated compartments within the cell. In striking contrast, during progressive rounds of infection initiated at a single cell, we observe that infection induces a clear and pronounced stimulation of cellular DNA replication in remote uninfected cells. This induced DNA synthesis was observed in hundreds of uninfected cells at the extended border, outside the perimeter of the progressing infection. Moreover, using pulse-chase analysis, we show that this activation is maintained, resulting in a propagating wave of host DNA synthesis continually in advance of infection. As the virus reaches and infects these activated cells, host DNA synthesis is then shut off and replaced with virus DNA synthesis. Using nonpropagating viruses or conditioned medium, we demonstrate a paracrine effector of uninfected cell DNA synthesis in remote cells continually in advance of infection. These findings have significant implications, likely with broad applicability, for our understanding of the ways in which virus infection manipulates cell processes not only in the infected cell itself but also now in remote uninfected cells, as well as of mechanisms governing host DNA synthesis.IMPORTANCEWe show that during infection initiated by a single particle with progressive cell-cell virus transmission (i.e., the normal situation), HSV induces host DNA synthesis in uninfected cells, mediated by a virus-induced paracrine effector. The field has had no conception that this process occurs, and the work changes our interpretation of virus-host interaction during advancing infection and has implications for understanding controls of host DNA synthesis. Our findings demonstrate the utility of chemical biology techniques in analysis of infection processes, reveal distinct processes when infection is examined in multiround transmission versus single-step growth curves, and reveal a hitherto-unknown process in virus infection, likely relevant for other viruses (and other infectious agents) and for remote signaling of other processes, including transcription and protein synthesis.

STUDIES OF INFECTIOUS BOVINE RHINOTRACHEITIS VIRUS: 1. PLAQUE ASSAY AND SOME CHARACTERISTICS IN BOVINE KIDNEY CELLS

1963 ◽  
Vol 9 (4) ◽  
pp. 567-576 ◽  
Author(s):  
Leslie R. Sabina ◽  
Raymond C. Parker
Keyword(s):  
Infectious Virus ◽  
Plaque Assay ◽  
Growth Curves ◽  
Kidney Cells ◽  
Infectious Bovine Rhinotracheitis ◽  
High Input ◽  
Bovine Kidney ◽  
One Step ◽  
Step Growth ◽  
Infectious Bovine Rhinotracheitis Virus

A reproducible plaquing procedure for infectious bovine rhinotracheitis virus (IBRV) in an established bovine kidney cell line is reported. The validity of this system for quantitative analysis has been established by conventional methods.After infection at different multiplicities, one-step growth curves have shown that the eclipse period for IBRV lasts approximately 4 hours and that the infectious virus increases at a logarithmic rate for 12 to 14 hours. The virus yield with the low and high input is 30 PFU and 210 PFU per cell, respectively. Only 1 to 9% of the total virus is released at 24 hours postinfection. The data presented indicate the half-life of IBRV at 37 °C and 42 °C to be 16 and 3.5 hours, respectively. A comparison of hyperimmune bovine and rabbit sera has shown that 92% of the infective particles are neutralized within 30 minutes.

Lamella-Particle Complexes in Nuclei of Owl Monkey Kidney Cells Infected With Herpesvirus saimiri

1977 ◽  
Vol 58 (5) ◽  
pp. 1421-1425 ◽  
Author(s):  
William G. Banfield ◽  
Cecil W. Lee ◽  
Tommie Sue Tralka ◽  
Alan S. Rabson
Keyword(s):  
Monkey Kidney ◽  
Herpesvirus Saimiri ◽  
Kidney Cells ◽  
Owl Monkey

scholarly journals Characterization of Bacteriophage against Enterococcus faecium Resistant to Vancomycin Isolated from Chicken Skin

2019 ◽  
pp. 1-14
Author(s):  
Hazzierah Syaffieqah An Nadiah Azlan ◽  
Muhajir Hamid ◽  
Adelene Ai-Lian Song
Keyword(s):  
Latent Period ◽  
Growth Kinetics ◽  
Enterococcus Faecium ◽  
Burst Size ◽  
Single Step ◽  
Vancomycin Resistant Enterococcus ◽  
Chicken Skin ◽  
Step Growth ◽  
Vancomycin Resistant ◽  
Faecium Strain

Aims: To characterize bacteriophages with strong in vitro lytic activity against vancomycin resistant Enterococcus faecium before testing on the chicken skin for their efficacy. Study Design: An experimental was carried out to characterize two isolated bacteriophages against Enterococcus faecium and test for their efficacy on chicken skin. Study Place: The study was carried out in Laboratory of Vaccine and Immunotherapeutics, Institue of Bioscience, Universiti Putra Malaysia in Selangor, which is the most populous state in Malaysia. Methodology: Two host specific lytic phages against vancomycin resistant Enterococcus faecium strain FM8, designated as FM8-P1 and FM8-P2 were physiological characterized. This includes determination of their adsorption rate, multiplicity of infection, and single step growth kinetics. The optimum pH and temperature for both bacteriophages activity were also determined before tested on chicken skin at 4°C and 25°C, which represent chiller and room temperature in poultry production line. Results: Based on the result of single-step growth kinetics, the latent period of FM8-P1 was 35 min with a burst size of 460 particles per infected cells, while FM8-P2 has a shorter latent period (20 min) but a smaller burst size of 60 particles. The highest adsorption rate for FM8-P1 was 83% and FM8-P2 was 90% at 2 min and 4 min respectively. Both bacteriophages also exihibited a wide range of pH and temperature for their activity. Conclusion: The specificity, lytic activity and stability of FM8-P1 and FM8-P2 emphasized their potential in effectively eliminating the vancomycin resistant Enterococcus faecium strain FM8. However, further works are required to validate their in situ reliability.

scholarly journals Herpes Simplex Virus Type 1 Propagation, Titration and Single-step Growth Curves

BIO-PROTOCOL ◽  
2019 ◽  
Vol 9 (23) ◽  
Author(s):  
Linda Grosche ◽  
Katinka Döhner ◽  
Alexandra Düthorn ◽  
Ana Hickford-Martinez ◽  
Alexander Steinkasserer ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Growth Curves ◽  
Single Step ◽  
Step Growth ◽  
Simplex Virus

scholarly journals Baculovirus lef-12 Is Not Required for Viral Replication

2002 ◽  
Vol 76 (23) ◽  
pp. 12032-12043 ◽  
Author(s):  
Linda A. Guarino ◽  
Toni-Ann Mistretta ◽  
Wen Dong
Keyword(s):  
Trichoplusia Ni ◽  
Late Phase ◽  
Growth Curves ◽  
Single Step ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Infected Cells ◽  
Step Growth

ABSTRACT The baculovirus lef-12 (orf41) gene is required for transient expression of baculovirus late genes. To analyze the role of LEF-12 in the context of infected cells, two mutant viruses were constructed. Both mutants were viable in Trichoplusia ni High 5 and Spodoptera frugiperda Sf9 cells. Single-step growth curves, however, indicated that virus yields were reduced approximately fivefold in the absence of LEF-12. Pulse-labeling of infected cells revealed that LEF-12 mutant viruses entered the late phase and synthesized late proteins at levels equivalent to or only twofold lower than those of wild-type virus-infected cells. Western blot analyses confirmed that LEF-12 was not synthesized in cells infected with mutant virus. In wild-type virus-infected cells, LEF-12 was not detected until 18 h postinfection, and accumulation of LEF-12 peaked at 24 to 36 h postinfection. Primer extension mapping revealed that lef-12 mRNA was synthesized by 12 h postinfection and peaked between 18 and 24 h postinfection. Furthermore, synthesis of lef-12 mRNA and LEF-12 protein were inhibited by the addition of aphidicolin, indicating that lef-12 is expressed after DNA replication.

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