Establishment of a cell line persistently infected with bovine herpesvirus-4 by use of a recombinant virus

Bovine herpesvirus-4 (BHV-4), a gammaherpesvirus lacking a clear disease association, productively infects multiple cell lines of various species and causes cell death. A human rhabdomyosarcoma cell line, RD-4, infected with BHV-4 produced low levels of early and late viral RNAs and infectious virus, but exhibited no cytopathic effect. Using a recombinant BHV-4 containing a neomycin-resistance gene, we established RD-4-derived cell lines persistently infected with BHV-4. The viral genome in these cells was predominantly circular. Because of drug selection, every cell contained a viral genome. In addition, all cells stained with a BHV-4-specific antiserum. Therefore, these cell lines are not carrier cultures. These cells produced infectious virus at all passages tested. Even though cells were selected and maintained at a concentration of geneticin at least 2·5 times that necessary to kill uninfected RD-4 cells, selected cells contained only approximately one viral genome per diploid host cell genome. Persistently infected cells grew more slowly than uninfected cells, even in the absence of drug. The slower growth of these cells suggests that any growth advantage conferred by multiple copies of the neomycin-gene-carrying viral genome might be offset by the detrimental effects of viral gene expression. This situation contrasts with other gammaherpesviruses, which are able to growth-transform cells.

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A bovine macrophage cell line supports bovine herpesvirus-4 persistent infection

Journal of General Virology ◽

10.1099/0022-1317-82-5-1181 ◽

2001 ◽

Vol 82 (5) ◽

pp. 1181-1185 ◽

Cited By ~ 34

Author(s):

Gaetano Donofrio ◽

Vicky L. van Santen

Keyword(s):

Viral Genome ◽

Bovine Herpesvirus ◽

Macrophage Cell Line ◽

Macrophage Cell ◽

Persistently Infected ◽

Bovine Herpesvirus 4 ◽

Bovine Macrophage ◽

Herpesvirus 4

Although bovine herpesvirus-4 (BHV-4), a gammaherpesvirus lacking a clear disease association, has been demonstrated in many tissues during persistent BHV-4 infection, a likely site of virus persistence is in cells of the monocyte/macrophage lineage. To establish an in vitro model of persistent infection potentially useful for examining the molecular mechanisms of BHV-4 persistence/latency, we infected the bovine macrophage cell line BOMAC. Following extensive cell death, surviving cells were found to be persistently infected, maintaining the viral genome over many passages and producing low levels of infectious virus. Although selection was unnecessary for the maintenance of the viral genome, cells persistently infected with recombinant BHV-4 containing a neomycin-resistance gene could be selected with geneticin, thus confirming that persistent BHV-4 infection was compatible with cell survival and replication. Furthermore, persistent BHV-4 infection caused no decrease in the growth rate of BOMAC cells. Sodium butyrate, which reactivates latent gammaherpesviruses in vitro, or dexamethasone, which reactivates latent BHV-4 in vivo, increased viral DNA by 10- to 15-fold in persistently infected BOMAC cells. This suggests that reactivation of latent BHV-4 by dexamethasone in vivo might involve direct action of dexamethasone on latently infected cells.

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Bovine herpesvirus 4 is tropic for bovine endometrial cells and modulates endocrine function

Reproduction ◽

10.1530/rep-07-0065 ◽

2007 ◽

Vol 134 (1) ◽

pp. 183-197 ◽

Cited By ~ 57

Author(s):

Gaetano Donofrio ◽

Shan Herath ◽

Chiara Sartori ◽

Sandro Cavirani ◽

Cesidio Filippo Flammini ◽

...

Keyword(s):

Epithelial Cells ◽

Stromal Cells ◽

Bovine Herpesvirus ◽

Endocrine Function ◽

Uterine Disease ◽

Endometrial Stromal Cells ◽

Persistently Infected ◽

Endometrial Cells ◽

Bovine Herpesvirus 4 ◽

Herpesvirus 4

Bovinepostpartumuterine disease, metritis, affects about 40% of animals and is widely considered to have a bacterial aetiology. Although the γ-herpesvirus bovine herpesvirus 4 (BoHV-4) has been isolated from several outbreaks of metritis or abortion, the role of viruses in endometrial pathology and the mechanisms of viral infection of uterine cells are often ignored. The objectives of the present study were to explore the interaction, tropism and outcomes of BoHV-4 challenge of endometrial stromal and epithelial cells. Endometrial stromal and epithelial cells were purified and infected with a recombinant BoHV-4 carrying an enhanced green fluorescent protein (EGFP) expression cassette to monitor the establishment of infection. BoHV-4 efficiently infected both stromal and epithelial cells, causing a strong non-apoptotic cytopathic effect, associated with robust viral replication. The crucial step for the BoHV-4 endometriotropism appeared to be after viral entry as there was enhanced transactivation of the BoHV-4 immediate early 2 gene promoter following transient transfection into the endometrial cells. Infection with BoHV-4 increased cyclooxygenase 2 protein expression and prostaglandin estradiol secretion in endometrial stromal cells, but not epithelial cells. Bovine macrophages are persistently infected with BoHV-4, and co-culture with endometrial stromal cells reactivated BoHV-4 replication in the persistently infected macrophages, suggesting a symbiotic relationship between the cells and virus. In conclusion, the present study provides evidence of cellular and molecular mechanisms, supporting the concept that BoHV-4 is a pathogen associated with uterine disease.

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Herpes Simplex Virus Latency Is Noisier the Closer We Look

Journal of Virology ◽

10.1128/jvi.01701-19 ◽

2019 ◽

Vol 94 (4) ◽

Cited By ~ 4

Author(s):

Navneet Singh ◽

David C. Tscharke

Keyword(s):

Gene Expression ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Viral Genome ◽

Infectious Virus ◽

Viral Gene Expression ◽

Viral Gene ◽

Peripheral Neurons ◽

Virus Latency ◽

Simplex Virus

ABSTRACT During herpes simplex virus (HSV) latency, the viral genome is harbored in peripheral neurons in the absence of infectious virus but with the potential to restart infection. Advances in epigenetics have helped explain how viral gene expression is largely inhibited during latency. Paradoxically, at the same time, the view that latency is entirely silent has been eroding. This low-level noise has implications for our understanding of HSV latency and should not be ignored.

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Activation of bovine herpesvirus 4 lytic replication in a non-permissive cell line by overexpression of BoHV-4 immediate early (IE) 2 gene

Journal of Virological Methods ◽

10.1016/j.jviromet.2003.11.019 ◽

2004 ◽

Vol 116 (2) ◽

pp. 203-207 ◽

Cited By ~ 13

Author(s):

Gaetano Donofrio ◽

Sandro Cavirani ◽

Simone Taddei ◽

Cesidio Filippo Flammini

Keyword(s):

Cell Line ◽

Bovine Herpesvirus ◽

Lytic Replication ◽

Immediate Early ◽

Permissive Cell ◽

Bovine Herpesvirus 4 ◽

Herpesvirus 4

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Multiple RSV strains infecting HEp-2 and A549 cells reveal cell line-dependent differences in resistance to RSV infection.

10.1101/2021.06.15.448622 ◽

2021 ◽

Author(s):

Anubama Rajan ◽

Felipe-Andres Piedra ◽

Letisha Aideyan ◽

Trevor McBride ◽

Matthew J Robertson ◽

...

Keyword(s):

Gene Expression ◽

Cell Line ◽

Cell Lines ◽

A549 Cells ◽

Viral Gene Expression ◽

Model Systems ◽

Viral Gene ◽

Transformed Cell ◽

Syncytial Virus ◽

Transformed Cell Lines

Respiratory syncytial virus (RSV) is a leading cause of pediatric acute respiratory infection worldwide. There are currently no approved vaccines or antivirals to combat RSV disease. A few transformed cell lines and two historic strains have been extensively used to study RSV. Here we report a thorough molecular and cell biological characterization of HEp-2 and A549 cells infected with four strains of RSV representing both major subgroups as well as historic and more contemporaneous genotypes -- [RSV/A/Tracy (GA1), RSV/A/Ontario (ON), RSV/B/18537 (GB1), RSV/B/Buenos Aires (BA)] -- via measurements of viral replication kinetics and viral gene expression, immunofluorescence-based imaging of gross cellular morphology and cell-associated RSV, and measurements of host response including transcriptional changes and levels of secreted cytokines and growth factors. Our findings strongly suggest 1) the existence of a conserved difference in gene expression between RSV subgroups A and B; 2) the A549 cell line is a more stringent and natural host of replicating RSV than the HEp-2 cell line; and 3) consistent with previous studies, determining the full effects of viral genetic variation in RSV pathogenesis requires model systems as tractable as transformed cell lines but better representative of the human host.

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NAD-linked mechanisms of gene de-repression and a novel role for CtBP in persistent adenovirus infection of lymphocytes

Virology Journal ◽

10.1186/s12985-019-1265-y ◽

2019 ◽

Vol 16 (1) ◽

Cited By ~ 1

Author(s):

Megan L. Dickherber ◽

Charlie Garnett-Benson

Keyword(s):

Gene Expression ◽

Epithelial Cells ◽

Cell Lines ◽

Lymphocyte Activation ◽

Viral Gene Expression ◽

Productive Infection ◽

Viral Gene ◽

Global Changes ◽

Persistently Infected ◽

Infected Cells

Abstract Background Adenovirus (AdV) infection is ubiquitous in the human population and causes acute infection in the respiratory and gastrointestinal tracts. In addition to lytic infections in epithelial cells, AdV can persist in a latent form in mucosal lymphocytes, and nearly 80% of children contain viral DNA in the lymphocytes of their tonsils and adenoids. Reactivation of latent AdV is thought to be the source of deadly viremia in pediatric transplant patients. Adenovirus latency and reactivation in lymphocytes is not well studied, though immune cell activation has been reported to promote productive infection from latency. Lymphocyte activation induces global changes in cellular gene expression along with robust changes in metabolic state. The ratio of free cytosolic NAD+/NADH can impact gene expression via modulation of transcriptional repressor complexes. The NAD-dependent transcriptional co-repressor C-terminal Binding Protein (CtBP) was discovered 25 years ago due to its high affinity binding to AdV E1A proteins, however, the role of this interaction in the viral life cycle remains unclear. Methods The dynamics of persistently- and lytically-infected cells are evaluated. RT-qPCR is used to evaluate AdV gene expression following lymphocyte activation, treatment with nicotinamide, or disruption of CtBP-E1A binding. Results PMA and ionomycin stimulation shifts the NAD+/NADH ratio in lymphocytic cell lines and upregulates viral gene expression. Direct modulation of NAD+/NADH by nicotinamide treatment also upregulates early and late viral transcripts in persistently-infected cells. We found differential expression of the NAD-dependent CtBP protein homologs between lymphocytes and epithelial cells, and inhibition of CtBP complexes upregulates AdV E1A expression in T lymphocyte cell lines but not in lytically-infected epithelial cells. Conclusions Our data provide novel insight into factors that can regulate AdV infections in activated human lymphocytes and reveal that modulation of cellular NAD+/NADH can de-repress adenovirus gene expression in persistently-infected lymphocytes. In contrast, disrupting the NAD-dependent CtBP repressor complex interaction with PxDLS-containing binding partners paradoxically alters AdV gene expression. Our findings also indicate that CtBP activities on viral gene expression may be distinct from those occurring upon metabolic alterations in cellular NAD+/NADH ratios or those occurring after lymphocyte activation.

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The Core 2 β-1,6-N-Acetylglucosaminyltransferase-Mucin Encoded by Bovine Herpesvirus 4 Was Acquired from an Ancestor of the African Buffalo

Journal of Virology ◽

10.1128/jvi.77.3.1784-1792.2003 ◽

2003 ◽

Vol 77 (3) ◽

pp. 1784-1792 ◽

Cited By ~ 23

Author(s):

N. Markine-Goriaynoff ◽

J.-P. Georgin ◽

M. Goltz ◽

W. Zimmermann ◽

H. Broll ◽

...

Keyword(s):

Phylogenetic Analyses ◽

Bovine Herpesvirus ◽

Viral Gene ◽

African Buffalo ◽

M Gene ◽

Nucleotide Substitutions ◽

Strong Constraint ◽

Bovine Herpesvirus 4 ◽

Gene Acquisition ◽

Herpesvirus 4

ABSTRACT The Bo17 gene of bovine herpesvirus 4 (BoHV-4) is the only viral gene known to date that encodes a homologue of the cellular core 2 β-1,6-N-acetylglucosaminyltransferase-mucin type (C2GnT-M). To investigate the origin and evolution of the Bo17 gene, we analyzed its distribution among BoHV-4 strains and determined the sequences of Bo17 from nine representative strains and of the C2GnT-M gene from six species of ruminants expected to encompass the group within which the gene acquisition occurred. Of 34 strains of BoHV-4, isolated from four different continents, all were found to contain the Bo17 gene. Phylogenetic analyses indicated that Bo17 was acquired from a recent ancestor of the African buffalo, implying that cattle subsequently acquired BoHV-4 by cross-species transmission. The rate of synonymous nucleotide substitution in Bo17 was estimated at 5 × 10−8 to 6 × 10−8 substitutions/site/year, consistent with previous estimates made under the assumption that herpesviruses have cospeciated with their hosts. The Bo17 gene acquisition was dated to around 1.5 million years ago. Bo17 sequences from BoHV-4 strains from African buffalo and from cattle formed two separate clades, estimated to have split about 700,000 years ago. Analysis of the ratio of nonsynonymous to synonymous nucleotide substitutions revealed a burst of amino acid replacements subsequent to the transfer of the cellular gene to the viral genome, followed by a return to a strong constraint on nonsynonymous changes during the divergence of contemporary BoHV-4 strains. The Bo17 gene represents the most recent of the known herpesvirus gene acquisitions and provides the best opportunity for learning more about this important process of viral evolution.

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