A Model for Persistent Murine Coronavirus Infection Involving Maintenance via Cytopathically Infected Cell Centres

Macintyre, Georgina; Wong, Foong Ying; Anderson, Robert

doi:10.1099/0022-1317-70-3-763

Cited by 10 publications

(9 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Likewise, phosphorothioate antisense oligomers, while somewhat effective in cell culture (1,26), are subject to degradation in cells and are most effective against shorter sequences with inherently lower target specificity due to RNase H promiscuity (74). Proteinase inhibitors, including EST (E-64d), have been used to treat coronavirus infection in cell culture (36), but EST was found to achieve a tenfold reduction in titer at a concentration of approximately 1,200 M. Treatment with HYG reduced viral titers in murine (46)(47)(48)(49), feline (8), and bovine (33,34) coronavirus experimental systems and appears to have the broadest anticoronaviral activity of the small molecule antivirals tested. However, there exist reports of incomplete inhibition of virus growth and strains of bovine coronavirus apparently not susceptible to HYG-mediated inhibition (33,34).…”

Section: Discussionmentioning

confidence: 99%

Antisense Morpholino-Oligomers Directed against the 5′ End of the Genome Inhibit Coronavirus Proliferation and Growth†

Neuman

Stein

Kroeker

et al. 2004

J Virol

View full text Add to dashboard Cite

Conjugation of a peptide related to the human immunodeficiency virus type 1 Tat represents a novel method for delivery of antisense morpholino-oligomers. Conjugated and unconjugated oligomers were tested to determine sequence-specific antiviral efficacy against a member of the Coronaviridae, Mouse hepatitis virus (MHV). Specific antisense activity designed to block translation of the viral replicase polyprotein was first confirmed by reduction of luciferase expression from a target sequence-containing reporter construct in both cell-free and transfected cell culture assays. Peptide-conjugated morpholino-oligomers exhibited low toxicity in DBT astrocytoma cells used for culturing MHV. Oligomer administered at micromolar concentrations was delivered to >80% of cells and inhibited virus titers 10-to 100-fold in a sequence-specific and dose-responsive manner. In addition, targeted viral protein synthesis, plaque diameter, and cytopathic effect were significantly reduced. Inhibition of virus infectivity by peptide-conjugated morpholino was comparable to the antiviral activity of the aminoglycoside hygromycin B used at a concentration fivefold higher than the oligomer. These results suggest that this composition of antisense compound has therapeutic potential for control of coronavirus infection.Coronaviruses are medically and economically important pathogens of humans, livestock, and birds. Coronavirus infections cause a wide range of symptoms, including upper respiratory illness, gastroenteritis, myocarditis, nephritis, central nervous system demyelination, encephalitis, hepatitis, and peritonitis, and can have a debilitating effect on the host immune system, leading to secondary microbial infection. In humans a coronavirus infection marked by aerosol transmissibility and a high degree of immunopathology-related mortality has been linked with the outbreak of severe acute respiratory syndrome (52, 64). Other human coronaviruses cause approximately one-third of all cases of common colds (20,29,56

show abstract

Section: Discussionmentioning

confidence: 99%

Antisense Morpholino-Oligomers Directed against the 5′ End of the Genome Inhibit Coronavirus Proliferation and Growth†

Neuman

Stein

Kroeker

et al. 2004

J Virol

View full text Add to dashboard Cite

show abstract

“…Persistently infected cultures are resistant to superinfection with homologous viruses but not with heterologous viruses such as vesicular stomatitis virus (VSV) (13). Numerous mechanisms have been suggested for the establishment and/or maintenance of persistence in vitro that include roles for mutant or interfering viruses (15,26), for latently infected cell populations (25), and for a host cell-regulated subpopulation of cytopathically infected cells in a carrier culture (13,16,17). In this report, we show that the establishment of a persistent infection in cultured cells results from the epigenetic expression of MHVR.…”

mentioning

confidence: 99%

Persistent infection of cultured cells with mouse hepatitis virus (MHV) results from the epigenetic expression of the MHV receptor

Sawicki

Holmes

1995

J Virol

View full text Add to dashboard Cite

The A59 strain of murine coronavirus mouse hepatitis virus (MHV) can cause persistent infection of 17C1-1 cells and other murine cell lines. Persistently infected cultures released large amounts of virus (10 7 to 10 8 PFU/ml) and were resistant to superinfection with MHV but not to infection with unrelated Semliki Forest and vesicular stomatitis viruses. The culture medium from persistently infected cultures did not contain a soluble inhibitor such as interferon that protected uninfected cells from infection by MHV or vesicular stomatitis virus. The persistent infection was cured if fewer than 100 cells were transferred during subculturing, and such cured cultures were susceptible to reinfection and the reestablishment of persistent infection. Cultures of 17C1-1 cells that had been newly cloned from single cells consisted of a mixture of MHV-resistant and -susceptible cells. 17C1-1/#97 cells, which were cured by subcloning after 97 passages of a persistently infected culture over a 1-year period, contained 5 to 10% of their population as susceptible cells, while 17C1-1/#402 cells, which were cured by subcloning after 402 passages over a 3-year period, had less than 1% susceptible cells. Susceptibility to infection correlated with the expression of MHV receptor glycoprotein (MHVR [Bgp1 a ]). Fluorescenceactivated cell sorter analysis with antibody to MHVR showed that 17C1-1/#97 cells contained a small fraction of MHVR-expressing cells. These MHVR-expressing cells were selectively eliminated within 24 h after challenge with MHV-A59, and pretreatment of 17C1-1/#97 cells with monoclonal antibody CC1, which binds to the N-terminal domain of MHVR, blocked infection. We conclude that the subpopulation of MHVR-expressing cells were infected and killed in acutely or persistently infected cultures, while the subpopulation of MHVRnonexpressing cells survived and proliferated. The subpopulation of MHVR-negative cells produced a small proportion of progeny cells that expressed MHVR and became infected, thereby maintaining the persistent infection as a steady-state carrier culture. Thus, in 17C1-1 cell cultures, the unstable or epigenetic expression of MHVR permitted the establishment of a persistent, chronic infection. MATERIALS AND METHODSCells and viruses. The spontaneously transformed mouse BALB/c 3T3 cells, 17C1-1 cells (28), and the A59 strain of MHV were kindly provided by L. Sturman. The 17C1-1 cells used in this study were cloned from a single cell and grown at 37ЊC and 7.5% CO 2 in Dulbecco's modified Eagle medium containing 6% fetal bovine serum (FBS), 5% tryptose phosphate broth (TPB), 500 U of penicillin per ml, and 100 U of streptomycin per ml (neutral pH growth medium). The cultures were routinely passaged every 3 days at a dilution of 1:60. The cells were detached from the monolayer by incubation in Hanks' balanced salt * Corresponding author.

show abstract

“…Along these lines, we have previously presented evidence (9) in support of the antiviral properties of hygromycin B against acute and persistent in vitro infections of mouse hepatitis virus (MHV-A59). The studies described in this report attempt to examine the effect of hygromycin B in systems more relevant to natural MHV infection.…”

mentioning

confidence: 97%

“…This difference may be due to selective accumulation of hygromycin B in certain organs, as has been described with other aminoglycosides (3,13,15,16 The mechanisms underlying the antiviral effects of hygromycin B remain incompletely understood. Hygromycin B does not inactivate the infectivity of MHV to which it is exposed (9). As a translational inhibiting drug to which normal cells are impermeable, hygromycin B has been suggested to selectively penetrate cells which have been rendered permeable by virus infection (2,5).…”

mentioning

confidence: 99%

Hygromycin B therapy of a murine coronaviral hepatitis

Macintyre

Curry

Wong

et al. 1991

Antimicrob Agents Chemother

View full text Add to dashboard Cite

Hepatitis caused by mouse hepatitis virus (MHV-A59), a murine coronavirus, is accompanied by direct infection and replication of virus within the liver. We demonstrate here that the aminoglycoside hygromycin B is able to eliminate MHV-A59 infection from mouse peritoneal macrophages and cultured liver cells in vitro and is also able to reduce levels of virus replication and necrotic liver foci in vivo.The proposed selective entry of hygromycin B into cytopathically infected cells (reviewed in reference 5) and the resultant death of these cells by the inhibition of cell protein synthesis suggests that this compound may be useful as an antiviral drug. Along these lines, we have previously presented evidence (9) in support of the antiviral properties of hygromycin B against acute and persistent in vitro infections of mouse hepatitis virus (MHV-A59). The studies described in this report attempt to examine the effect of hygromycin B in systems more relevant to natural MHV infection. Evidence is presented from both in vitro studies, using mouse peritoneal macrophages and liver cells, and in vivo studies which suggest that hygromycin B has a therapeutic effect in limiting MHV replication and in reducing the numbers of hepatic lesions produced during an in vivo infection.In vitro studies with peritoneal macrophages and cultured liver cells. Macrophages and liver cells are important cell targets for MHV. Since the infectability of mouse macrophages often correlates with in vivo susceptibility to MHV (1, 17), we examined MHV-A59 infection in cultures of peritoneal macrophages. Two strains of mice were used, BALB/c and A/J, both of which have been shown to be susceptible to MHV-A59 infection (7,14). Starch-activated peritoneal macrophages (6) were harvested from 4-monthold BALB/c and A/J mice by repeated washing of the peritoneal cavity with minimal essential medium (MEM) containing 20% fetal calf serum (FCS). The total peritoneal cavity wash was centrifuged at 1,000 x g for 1 min, and the pellet was washed with MEM supplemented with 20% FCS. The final pellet was resuspended in MEM (plus 10% FCS), plated out in eight-well microtiter slides, and incubated overnight to allow the macrophages to adhere to the wells. The monolayers were then infected with MHV-A59 (multiplicity of infection, 0.1) and were treated with various concentrations of hygromycin B. Immunofluorescence was carried out on 95% ethanol-5% acetic acid-treated cultures by using a mouse monoclonal antibody directed against the MHV-A59 large envelope glycoprotein (designated S) that was probed with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G. Virus titers in the range of i05 to 106 were obtained between 12 and 36 h postinfection (p.i.) from MHV-infected peritoneal macrophage cultures obtained from both BALB/c and A/J mice ( Fig. 1A and B and A/J peritoneal macrophage cultures was extensive, with 100% fusion of the A/J and BALB/c monolayers occurring by 24 h p.i. (Fig. 1D) and 36 h p.i. (Fig. 1C), respectively. This study indicates that ...

show abstract

A Model for Persistent Murine Coronavirus Infection Involving Maintenance via Cytopathically Infected Cell Centres

Cited by 10 publications

References 21 publications

Antisense Morpholino-Oligomers Directed against the 5′ End of the Genome Inhibit Coronavirus Proliferation and Growth†

Antisense Morpholino-Oligomers Directed against the 5′ End of the Genome Inhibit Coronavirus Proliferation and Growth†

Persistent infection of cultured cells with mouse hepatitis virus (MHV) results from the epigenetic expression of the MHV receptor

Hygromycin B therapy of a murine coronaviral hepatitis

Contact Info

Product

Resources

About