This research was funded by a Wellcome Trust Senior Fellowship awarded to Ian Goodfellow, and a Marie Curie Intra European Fellowship (FP7 European Research Council) awarded to Armando Arias. We would like to thank Io Hong Cheong, Dr Rebecca Robey and Dr Mike Skinner for giving us permission to use their Agilent bioanalyser and helping with running RNA samples.

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We have nothing to disclose.

Here we have illustrated two different reverse genetic approaches that allow the recovery of infectious MNV in cell culture. Both approaches effectively bypass the absolute requirement for the covalently linkage of VPg to the 5' end of the viral RNA genome via the generation of capped MNV transcripts that are then recognised by the cellular ribosomes. In vitro transcription followed by enzymatically capping is more efficient in the recovery of infectious MNV than transcription of infectious plasmids in cells expressing T7 RNA polymerase, in which the transcripts may be capped by the FPV capping enzymes. Virus titres recovered with these reverse genetics systems are similar to these obtained by transfection of viral VPg-linked RNA purified from infected cell cultures 17 (Figure 5). The transfection of capped MNV RNA into permissive Raw264.7 cells renders a virus titre only 1-log lower than experiments involving the transfection of total RNA from infected cells containing viral VPg-linked RNA (Figure 5). This fact encourages further investigations to determine whether the addition of a VPg molecule to the 5' end of transcripts generated by these systems could result in increased virus yields which may reveal functional aspects underlying MNV infectivity in cells associated to VPg. Nevertheless, we regard this reverse genetics system as a highly efficient one comparable to other RNA viruses reverse genetics systems currently used in which the in vitro transcribed RNA allows the recovery of titres only 10-100 lower than in real infections with virions 19, 20.
Overall, the current methodologies constitute a significant step forward in the field of norovirus molecular biology and provide us with the tools to investigate the functional roles of proteins and conserved RNA motifs in norovirus genomes. These approaches have already been combined with current mouse model available and have shown that MNV recovered from infectious cDNA is able to cause lethal infection of &gt;80% STAT1-/- mice in less than 10 days 4, 21. Making use of this system we have recovered viable murine norovirus mutants of the capsid protein and of a polypyrimidine tract involved in the binding of different host factors (PTB and PCBP) that display a somewhat attenuated phenotypes in vivo 21, 22. In addition, we have recently demonstrated that viruses lacking the ability to express the VF1 protein from ORF4 efficiently replicate in cell culture but again have reduced virulence in mice with respect to WT MNV 8. These studies encourage us to design attenuated versions of human noroviruses based on MNV studies which could be investigated as potential vaccine candidates.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Lithium chloride precipitation solution</td>
 <td>Ambion</td>
 <td>AM9480</td>
 </tr>
 <tr>
 <td>RNA storage solution</td>
 <td>Ambion</td>
 <td>AM7000</td>
 </tr>
 <tr>
 <td>MEGAscript T7 script</td>
 <td>Ambion</td>
 <td>AM1333</td>
 </tr>
 <tr>
 <td>ScriptCap m7G Capping System</td>
 <td>Epicentre Biotechnologies</td>
 <td>SCCE0610</td>
 </tr>
 <tr>
 <td>Neon transfection system</td>
 <td>Invitrogen</td>
 <td>MPK5000</td>
 </tr>
 <tr>
 <td>Neon transfection system kit</td>
 <td>Invitrogen</td>
 <td>MPK1025</td>
 </tr>
 <tr>
 <td>Opti-MEM I</td>
 <td>Invitrogen</td>
 <td>31985070</td>
 </tr>
 <tr>
 <td>Lipofectamine 2000 Transfection Reagent</td>
 <td>Invitrogen</td>
 <td>11668-027</td>
 </tr>
 <tr>
 <td>Agilent RNA 600 Nano kit</td>
 <td>Agilent</td>
 <td>5067-1511</td>
 </tr>
 <tr>
 <td>Agilent 2100 bioanalyzer</td>
 <td>Agilent</td>
 <td>G2939AA</td>
 </tr>
 <tr>
 <td>Illustra GFX PCR DNA and Gel Band Purification Kit</td>
 <td>GE Healthcare</td>
 <td>28-9034-70</td>
 </tr>
 <tr>
 <td>RiboMAX Large Scale RNA Production System-T7</td>
 <td>Promega</td>
 <td>P1300</td>
 </tr>
 </tbody>
</table>

reverse genetics mediated recovery of infectious murine norovirus

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1, 2. During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3. The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4 but which can be propagated in cells 5 has opened new avenues for the investigation of these pathogens 6, 7. 
MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) (Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8.
Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery 9-11. Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs 12-14. This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches. 
Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis 15-17.

Watch this Scientific Journal Video about Reverse Genetics Mediated Recovery of Infectious Murine Norovirus at JoVE.com

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close ...

reverse-genetics-mediated-recovery-of-infectious-murine-norovirus

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JoVE Journal

Immunology and Infection

16.4K visualizações.  Imperial College London. Os norovírus são a principal causa de gastroenterite ainda técnicas moleculares para sua caracterização são ainda relativamente novo. Aqui relatamos duas diferentes abordagens genética reversa para a recuperação eficiente de norovírus murino (MNV), o único membro do gênero que podem ser propagadas em cultura de células.