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Recombinant Marburg virus expressing EGFP allows rapid screening of virus growth and real-time visualization of virus spread.

ABSTRACT: The generation of recombinant enhanced green fluorescent protein (EGFP)-expressing viruses has significantly improved the study of their life cycle and opened up the possibility for the rapid screening of antiviral drugs. Here we report rescue of a recombinant Marburg virus (MARV) expressing EGFP from an additional transcription unit (ATU). The ATU was inserted between the second and third genes, encoding VP35 and VP40, respectively. Live-cell imaging was used to follow virus spread in real time. EGFP expression was detected at 32 hours postinfection (hpi), and infection of neighboring cells was monitored at 55 hpi. Compared to the parental virus, production of progeny rMARV-EGFP was reduced 4-fold and lower protein levels of VP40, but not nucleoprotein, were observed, indicating a decrease in downstream protein expression due to the insertion of an ATU. Interestingly, EGFP concentrated in viral inclusions in infected cells. This was reproduced by transient expression of both EGFP and other fluorescent proteins along with filovirus nucleocapsid proteins, and may suggest that a general increase in protein synthesis occurs at viral inclusion sites. In conclusion, the EGFP-expressing MARV will be a useful tool not only to monitor virus spread and screen for antiviral compounds, but also to investigate the biology of inclusion body formation.

SUBMITTER: Schmidt KM

PROVIDER: S-EPMC3218667 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Recombinant Marburg virus expressing EGFP allows rapid screening of virus growth and real-time visualization of virus spread.

Schmidt Kristina Maria KM Schümann Michael M Olejnik Judith J Krähling Verena V Mühlberger Elke E

The Journal of infectious diseases 20111101

The generation of recombinant enhanced green fluorescent protein (EGFP)-expressing viruses has significantly improved the study of their life cycle and opened up the possibility for the rapid screening of antiviral drugs. Here we report rescue of a recombinant Marburg virus (MARV) expressing EGFP from an additional transcription unit (ATU). The ATU was inserted between the second and third genes, encoding VP35 and VP40, respectively. Live-cell imaging was used to follow virus spread in real time ...[more]

PMID: 21987762

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Project description:Hand, foot, and mouth disease (HFMD) is a reemerging illness caused by a variety of enteroviruses. The main causative agents are enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and, most recently, coxsackievirus A6 (CVA6). Enterovirus infections can vary from asymptomatic infections to those with a mild fever and blisters on infected individuals' hands, feet, and throats to infections with severe neurological complications. Viral persistence for weeks postinfection (wpi) has also been documented by the demonstration of virus in children's stools. However, little is known about disease progression, viral spread, and tissue tropism of these viruses. These types of studies are limited because many recently developed mouse models mimic the severe neurological complications that occur in a small percentage of enterovirus infections. In the present study, we documented real-time EV71 infection in two different mouse strains by the use of in vivo imaging. Infection of BALB/c mice with a bioluminescent mouse-adapted EV71 construct (mEV71-NLuc) resulted in a lack of clinical signs of disease but in relatively high viral replication, as visualized by luminescence, for 2 wpi. In contrast, mEV71-NLuc infection of AG129 mice (alpha/beta and gamma interferon receptor deficient) showed rapid spread and long-term persistence of the virus in the brain. Interestingly, AG129 mice that survived infection maintained luminescence in the brain for up to 8 wpi. The results we present here will allow future studies on EV71 antiviral drug susceptibility, vaccine efficacy, transmissibility, and pathogenesis. IMPORTANCE We report here that a stable full-length enterovirus 71 (EV71) reporter construct was used to visualize real-time viral spread in AG129 and BALB/c mice. To our knowledge, this is the first report of in vivo imaging of infection with any member of the Picornaviridae family. The nanoluciferase (NLuc) gene, one of the smallest luciferase genes currently available, was shown to be stable in the EV71 genome for eight passages on rhabdomyosarcoma cells. Real-time visualization of EV71 infection in mice identified areas of tropism that would have been missed by traditional methods, including full characterization of EV71 replication in BALB/c mice. Additionally, the bioluminescent construct allowed for increased speed and sensitivity of cell culture assays and will allow future studies involving various degrees of enterovirus infection in mice, not just severe infections. Our data suggest that interferon plays an important role in controlling EV71 infection in the central nervous system of mice.

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Recombinant Marburg virus expressing EGFP allows rapid screening of virus growth and real-time visualization of virus spread.

Publications

Recombinant Marburg virus expressing EGFP allows rapid screening of virus growth and real-time visualization of virus spread.

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