Bid is involved in apoptosis induced by Chinese giant salamander iridovirus and contributes to the viral replication in an amphibian cell line

2021 ◽  
Vol 116 ◽  
pp. 103935
Author(s):  
Yiqun Li ◽  
Yanan Liu ◽  
Yong Zhou ◽  
Wenzhi Liu ◽  
Yuding Fan ◽  
...  
Keyword(s):  
Viral Replication ◽  
Cell Line ◽  
Chinese Giant Salamander

A fish cell line CHSE-sp exposed to long-term cold temperature retains viability and ability to support viral replication

1994 ◽  
Vol 30 (3) ◽  
pp. 148-150 ◽  
Author(s):  
Natsuko Araki ◽  
Masahiko Yamaguchi ◽  
Hideki Nakano ◽  
Yasushi Wada ◽  
Masahide Hasobe
Keyword(s):  
Viral Replication ◽  
Cell Line ◽  
Cold Temperature ◽  
Fish Cell Line ◽  
Fish Cell

scholarly journals Contemporary circulating enterovirus D68 strains show differential viral entry and replication in human neuronal cells

2018 ◽  
Author(s):  
David M Brown ◽  
Alison M Hixon ◽  
Lauren M Oldfield ◽  
Yun Zhang ◽  
Mark Novotny ◽  
...  
Keyword(s):  
Cell Culture ◽  
Viral Replication ◽  
Cell Line ◽  
Viral Entry ◽  
Neuronal Cells ◽  
Neuronal Cell ◽  
Cell Culture Model ◽  
Acute Flaccid Myelitis ◽  
Culture Model ◽  
Enterovirus D68

AbstractHistorically, enterovirus D68 (EV-D68) has primarily been associated with respiratory illnesses. However, in the summers of 2014 and 2016 EV-D68 outbreaks coincided with a spike in polio-like acute flaccid myelitis/paralysis (AFM/AFP) cases. This raised concerns that the EV-D68 virus could be the causative agent of AFM during these recent outbreaks. To assess the neurotropic capacity of EV-D68, we explored the use of the neuroblastoma-derived neuronal cell line, SH-SY5Y, as a tissue culture model to determine if differential infection permissibility is observed for different EV-D68 strains. In contrast to HeLa and A549 cells, which support viral infection of all EV-D68 strains tested, SH-SY5Y cells only supported infection by a subset of contemporary EV-D68 strains, including members from the 2014 outbreak. Viral replication and infectivity in SH-SY5Y was assessed using four different assays – infectious virus production, cytopathic effects, cellular ATP release, and VP1 capsid protein production – with similar results. Similar differential neurotropism was also observed in differentiated SH-SY5Y cells, primary human neuron cultures, and a mouse paralysis model. Using the SH-SY5Y cell culture model, we determined that barriers to viral entry was at least partly responsible for the differential infectivity phenotype, since transfection of genomic RNA into SH-SY5Y generated virions for all EV-D68 isolates, but only a single round of replication was observed from strains which could not directly infect SH-SY5Y. In addition to supporting virus replication and other functional studies, this cell culture model may help confirm epidemiological associations between EV-D68 strains and AFM and allow for the rapid identification of emerging neurotropic strains.Author SummarySince the outbreak during the summer of 2014, EV-D68 has been linked to a type of limb paralysis referred to as acute flaccid myelitis (AFM), with evidence mounting for the causal link of EV-D68 to AFM. Among these AFM cases, concurrent EV-D68 infection was confirmed in several independent epidemiological clusters in four continents. In this report, we describe a neuronal cell culture model (SH-SY5Y cells) where only a subset of contemporary 2014 outbreak strains of EV-D68 show infectivity in neuronal cells, or neurotropism, based on four different assays of viral replication and infection. We further confirmed the observed difference in neurotropismin vitrousing primary human neuron cell cultures andin vivowith a mouse paralysis model. Using the SH-SY5Y cell model, we determined that a barrier to viral entry is at least partly responsible for neurotropism. SH-SY5Y cells may be useful in determining if specific EV-D68 genetic determinants are associated with neuropathogenesis, and replication in this cell line could be used as rapid screening tool for identifying neurotropic EV-D68 strains. This may assist with better understanding of pathogenesis and epidemiology, and with the development of potential therapies.

scholarly journals Nef enhances HIV-1 replication and infectivity independently of SERINC3 and SERINC5 in CEM T cells

2020 ◽  
Author(s):  
Peter W. Ramirez ◽  
Aaron A. Angerstein ◽  
Marissa Suarez ◽  
Thomas Vollbrecht ◽  
Jared Wallace ◽  
...  
Keyword(s):  
Growth Rate ◽  
T Cells ◽  
T Cell ◽  
Viral Replication ◽  
Cell Line ◽  
Host Protein ◽  
Target Cells ◽  
Viral Fusion ◽  
Viral Growth ◽  
Hiv 1

AbstractThe lentiviral nef gene encodes several discrete activities aimed at co-opting or antagonizing cellular proteins and pathways to defeat host defenses and maintain persistent infection. Primary functions of Nef include downregulation of CD4 and MHC class-I from the cell surface, disruption or mimicry of T-cell receptor signaling, and enhancement of viral infectivity by counteraction of the host antiretroviral proteins SERINC3 and SERINC5. In the absence of Nef, SERINC3 and SERINC5 incorporate into virions and inhibit viral fusion with target cells, decreasing infectivity. However, whether Nef’s counteraction of SERINC3 and SERINC5 is the cause of its positive influence on viral growth-rate in CD4-positive T cells is unclear. Here, we utilized CRISPR/Cas9 to knockout SERINC3 and SERINC5 in a leukemic CD4-positive T cell line (CEM) that displays robust nef-related infectivity and growth-rate phenotypes. As previously reported, viral replication was severely attenuated in CEM cells infected with HIV-1 lacking Nef (HIV-1ΔNef). This attenuated growth-rate phenotype was observed regardless of whether or not the coding regions of the serinc3 and serinc5 genes were intact. Moreover, knockout of serinc3 and serinc5 failed to restore the infectivity of HIV-1ΔNef virions produced from infected CEM cells in single-cycle replication experiments using CD4-positive HeLa cells as targets. Taken together, our results corroborate a recent study using another T-lymphoid cell line (MOLT-3) and suggest that Nef modulates a still unidentified host protein(s) to enhance viral growth rate and infectivity in CD4-positive T cells.ImportanceHIV-1 Nef is a major pathogenicity factor in vivo. A well-described activity of Nef is the enhancement of virion-infectivity and viral propagation in vitro. The infectivity-effect has been attributed to Nef’s ability to prevent the cellular, antiretroviral proteins SERINC3 and SERINC5 from incorporating into viral particles. While the activity of the SERINCs as inhibitors of retroviral infectivity has been well-documented, the role these proteins play in controlling HIV-1 replication is less clear. We report here that genetic disruption of SERINC3 and SERINC5 rescues neither viral replication-rate nor the infectivity of cell-free virions produced from CD4-positive T cells of the CEM lymphoblastoid line infected with viruses lacking Nef. This indicates that failure to modulate SERINC3 and SERINC5 is not the cause of the virologic attenuation of nef-negative HIV-1 observed using this system.

scholarly journals Establishment of a stable SARS-CoV-2 replicon system for application in high-throughput screening.

2021 ◽  
Author(s):  
Tomohisa Tanaka ◽  
Akatsuki Saito ◽  
Tatsuya Suzuki ◽  
Yoichi Miyamoto ◽  
Kazuo Takayama ◽  
...  
Keyword(s):  
Viral Replication ◽  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Luciferase Activity ◽  
Fusion Gene ◽  
Viral Proteins ◽  
Renilla Luciferase ◽  
Interferon Β ◽  
Subgenomic Replicon

Experiments with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are limited by the need for biosafety level 3 (BSL3) conditions. A SARS-CoV-2 replicon system rather than an in vitro infection system is suitable for antiviral screening since it can be handled under BSL2 conditions and does not produce infectious particles. However, the reported replicon systems are cumbersome because of the need for transient transfection in each assay. In this study, we constructed a bacterial artificial chromosome vector (the replicon-BAC vector) including the SARS-CoV-2 replicon and a fusion gene encoding Renilla luciferase and neomycin phosphotransferase II, examined the antiviral effects of several known compounds, and then established a cell line stably harboring the replicon-BAC vector. Several cell lines transiently transfected with the replicon-BAC vector produced subgenomic replicon RNAs (sgRNAs) and viral proteins, and exhibited luciferase activity. In the transient replicon system, treatment with remdesivir or interferon-β but not with camostat or favipiravir suppressed the production of viral agents and luciferase, indicating that luciferase activity corresponds to viral replication. VeroE6/Rep3, a stable replicon cell line based on VeroE6 cells, was successfully established and continuously produced viral proteins, sgRNAs and luciferase, and their production was suppressed by treatment with remdesivir or interferon-β. Molnupiravir, a novel coronavirus RdRp inhibitor, inhibited viral replication more potently in VeroE6/Rep3 cells than in VeroE6-based transient replicon cells. In summary, our stable replicon system will be a powerful tool for the identification of SARS-CoV-2 antivirals through high-throughput screening.

scholarly journals Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication.

1997 ◽  
Vol 41 (8) ◽  
pp. 1715-1720 ◽  
Author(s):  
S K Ladner ◽  
M J Otto ◽  
C S Barker ◽  
K Zaifert ◽  
G H Wang ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Viral Replication ◽  
Cell Line ◽  
Large Scale ◽  
Hbv Replication ◽  
Compound Libraries ◽  
Human Hepatitis ◽  
B Virus ◽  
Potential Inhibitors

We report the development and isolation of a cell line, termed HepAD38, that replicates human hepatitis B virus (HBV) under conditions that can be regulated with tetracycline. In the presence of the antibiotic, this cell line is free of virus due to the repression of pregenomic (pg) RNA synthesis. Upon removal of tetracycline from the culture medium, the cells express viral pg RNA, accumulate subviral particles in the cytoplasm that contain DNA intermediates characteristic of viral replication, and secrete virus-like particles into the supernatant. Since the HepAD38 cell line can produce high levels of HBV DNA, it should be useful for analyses of the viral replication cycle that depend upon viral DNA synthesis in a synchronized fashion. In addition, this cell line has been formatted into a high-throughput, cell-based assay that permits the large-scale screening of diverse compound libraries for new classes of inhibitors of HBV replication.

scholarly journals An Aedes Aegypti-Derived Ago2 Knockout Cell Line to Investigate Arbovirus Infections

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1066
Author(s):  
Christina Scherer ◽  
Jack Knowles ◽  
Vattipally B. Sreenu ◽  
Anthony C. Fredericks ◽  
Janina Fuss ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Viral Replication ◽  
Cell Line ◽  
Small Rna Sequencing ◽  
Sequencing Data ◽  
Rna Sequences ◽  
Argonaute 2 ◽  
Si Rna ◽  
Sirna Pathway ◽  
The Impact

Mosquitoes are known as important vectors of many arthropod-borne (arbo)viruses causing disease in humans. These include dengue (DENV) and Zika (ZIKV) viruses. The exogenous small interfering (si)RNA (exo-siRNA) pathway is believed to be the main antiviral defense in arthropods, including mosquitoes. During infection, double-stranded RNAs that form during viral replication and infection are cleaved by the enzyme Dicer 2 (Dcr2) into virus-specific 21 nt vsiRNAs, which are subsequently loaded into Argonaute 2 (Ago2). Ago2 then targets and subsequently cleaves complementary RNA sequences, resulting in degradation of the target viral RNA. Although various studies using silencing approaches have supported the antiviral activity of the exo-siRNA pathway in mosquitoes, and despite strong similarities between the siRNA pathway in the Drosophila melanogaster model and mosquitoes, important questions remain unanswered. The antiviral activity of Ago2 against different arboviruses has been previously demonstrated. However, silencing of Ago2 had no effect on ZIKV replication, whereas Dcr2 knockout enhanced its replication. These findings raise the question as to the role of Ago2 and Dcr2 in the control of arboviruses from different viral families in mosquitoes. Using a newly established Ago2 knockout cell line, alongside the previously reported Dcr2 knockout cell line, we investigated the impact these proteins have on the modulation of different arboviral infections. Infection of Ago2 knockout cell line with alpha- and bunyaviruses resulted in an increase of viral replication, but not in the case of ZIKV. Analysis of small RNA sequencing data in the Ago2 knockout cells revealed a lack of methylated siRNAs from different sources, such as acute and persistently infecting viruses-, TE- and transcriptome-derived RNAs. The results confirmed the importance of the exo-siRNA pathway in the defense against arboviruses, but highlights variability in its response to different viruses and the impact the siRNA pathway proteins have in controlling viral replication. Moreover, this established Ago2 knockout cell line can be used for functional Ago2 studies, as well as research on the interplay between the RNAi pathways.

scholarly journals Isolation and Characterization of vΔI3 Confirm that Vaccinia Virus SSB Plays an Essential Role in Viral Replication

2017 ◽  
Vol 92 (2) ◽  
Author(s):  
Matthew D. Greseth ◽  
Maciej W. Czarnecki ◽  
Matthew S. Bluma ◽  
Paula Traktman
Keyword(s):  
Viral Replication ◽  
Vaccinia Virus ◽  
Cell Line ◽  
Infectious Virus ◽  
Binding Activity ◽  
Dna Viruses ◽  
Ssdna Binding ◽  
Fold Reduction ◽  
Ssdna Binding Protein ◽  
A Cell

ABSTRACTVaccinia virus is unusual among DNA viruses in replicating exclusively in the cytoplasm of infected cells. The single-stranded DNA (ssDNA) binding protein (SSB) I3 is among the replication machinery encoded by the 195-kb genome, although direct genetic analysis of I3 has been lacking. Herein, we describe a complementing cell line (CV1-I3) that fully supports the replication of a null virus (vΔI3) lacking the I3 open reading frame (ORF). In noncomplementing CV1-CAT cells, vΔI3 shows a severe defect in the production of infectious virus (≥200-fold reduction). Early protein synthesis and core disassembly occur normally. However, DNA replication is profoundly impaired (≤0.2% of wild-type [WT] levels), and late proteins do not accumulate. When several other noncomplementing cell lines are infected with vΔI3, the yield of infectious virus is also dramatically reduced (168- to 1,776-fold reduction). Surprisingly, the residual levels of DNA accumulation vary from 1 to 12% in the different cell lines (CV1-CAT < A549 < BSC40 < HeLa); however, any nascent DNA that can be detected is subgenomic in size. Although this subgenomic DNA supports late protein expression, it does not support the production of infectious virions. Electron microscopy (EM) analysis of vΔI3-infected BSC40 cells reveals that immature virions are abundant but no mature virions are observed. Aberrant virions characteristic of a block to genome encapsidation are seen instead. Finally, we demonstrate that a CV1 cell line encoding a previously described I3 variant with impaired ssDNA binding activity is unable to complement vΔI3. This report provides definitive evidence that the vaccinia virus I3 protein is the replicative SSB and is essential for productive viral replication.IMPORTANCEPoxviruses are of historical and contemporary importance as infectious agents, vaccines, and oncolytic therapeutics. The cytoplasmic replication of poxviruses is unique among DNA viruses of mammalian cells and necessitates that the double-stranded DNA (dsDNA) genome encode the viral replication machinery. This study focuses on the I3 protein. As a ssDNA binding protein (SSB), I3 has been presumed to play essential roles in genome replication, recombination, and repair, although genetic analysis has been lacking. Herein, we report the characterization of an I3 deletion virus. In the absence of I3 expression, DNA replication is severely compromised and viral yield profoundly decreased. The production of infectious virus can be restored in a cell line expressing WT I3 but not in a cell line expressing an I3 mutant that is defective in ssDNA binding activity. These data show conclusively that I3 is an essential viral protein and functions as the viral replicative SSB.

Evaluation of hepatitis B viral replication and proteomic analysis of HepG2.2.15 cell line after knockdown of HBx

2011 ◽  
Vol 10 (3) ◽  
pp. 295-302 ◽  
Author(s):  
Hai-Yang Xie ◽  
Jun Cheng ◽  
Chun-Yang Xing ◽  
Jin-Jin Wang ◽  
Rong Su ◽  
...  
Keyword(s):  
Hepatitis B ◽  
Viral Replication ◽  
Cell Line ◽  
Proteomic Analysis

scholarly journals The Major Immediate-Early Gene ie3 of Mouse Cytomegalovirus Is Essential for Viral Growth

2000 ◽  
Vol 74 (23) ◽  
pp. 11129-11136 ◽  
Author(s):  
Ana Angulo ◽  
Peter Ghazal ◽  
Martin Messerle
Keyword(s):  
Viral Replication ◽  
Cell Line ◽  
Normal Mouse ◽  
Regulatory Protein ◽  
Immediate Early Gene ◽  
Early Gene ◽  
Immediate Early ◽  
Deletion Mutants ◽  
Mouse Cytomegalovirus ◽  
Mouse Fibroblasts

ABSTRACT The significance of the major immediate-early gene ie3of mouse cytomegalovirus (MCMV) and that of the correspondingie2 gene of human cytomegalovirus to viral replication are not known. To investigate the function of the MCMV IE3 regulatory protein, we generated two different MCMV recombinants that contained a large deletion in the IE3 open reading frame (ORF). The mutant genomes were constructed by the bacterial artificial chromosome mutagenesis technique, and MCMV ie3 deletion mutants were reconstituted on a mouse fibroblast cell line that expresses the MCMV major immediate-early genes. The ie3 deletion mutants failed to replicate on normal mouse fibroblasts even when a high multiplicity of infection was used. The replication defect was rescued when the IE3 protein was provided in trans by a complementing cell line. A revertant virus in which the IE3 ORF was restored was able to replicate with wild-type kinetics in normal mouse fibroblasts, providing evidence that the defective growth phenotype of theie3 mutants was due to disruption of the ie3gene. To characterize the point of restriction in viral replication that is controlled by ie3, we analyzed the pattern of expression of selective early (β) and late (γ) genes. While we could detect transcripts for the immediate-early gene ie1in cells infected with the ie3 mutants, we failed to detect transcripts for representative β and γ genes. These data demonstrate that the MCMV transactivator IE3 plays an indispensable role during viral replication in tissue culture, implicating a similar role for the human CMV ie2 gene product. To our knowledge, the ie3 deletion mutants represent the first MCMV recombinants isolated that contain a disruption of an essential gene.

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