Recovery of genetically defined murine norovirus in tissue culture by using a fowlpox virus expressing T7 RNA polymerase

Abstract

Despite the significant disease burden caused by human norovirus infection, an efficient tissue-culture system for these viruses remains elusive. Murine norovirus (MNV) is an ideal surrogate for the study of norovirus biology, as the virus replicates efficiently in tissue culture and a low-cost animal model is readily available. In this report, a reverse-genetics system for MNV is described, using a fowlpox virus (FWPV) recombinant expressing T7 RNA polymerase to recover genetically defined MNV in tissue culture for the first time. These studies demonstrated that approaches that have proved successful for other members of the family Caliciviridae failed to lead to recovery of MNV. This was due to our observation that vaccinia virus infection had a negative effect on MNV replication. In contrast, FWPV infection had no deleterious effect and allowed the recovery of infectious MNV from cells previously transfected with MNV cDNA constructs. These studies also indicated that the nature of the 3'-terminal nucleotide is critical for efficient virus recovery and that inclusion of a hepatitis delta virus ribozyme at the 3' end can increase the efficiency with which virus is recovered. This system now allows the recovery of genetically defined noroviruses and will facilitate the analysis of the effects of genetic variation on norovirus pathogenesis.

Fig. 1.

Diagrammatic representation of the MNV genome and the constructs used during this study. (a) Proteolytic-cleavage map of the MNV 1 genome as demonstrated by Sosnovtsev et al. (2006). Text in italics highlights the nomenclature as proposed by Sosnovtsev et al. (2006). The caspase 3 cleavage sites in NS1/2 and the position of the subgenomic RNA are also indicated. (b) Schematic representation of the constructs used during this study. The positions of the restriction sites used during this study, the active site in the viral RNA-dependent RNA polymerase NS7 and the 3′-terminal nucleotide upstream of the poly-A tail are indicated. See Methods for specific details of each construct.

Fig. 2.

Analysis of the effect of poxvirus infection on MNV replication. (a) BHK cells were mock-infected (M) or infected with either VACV (MVA-T7) or FWPV (FPV-T7) expressing T7 RNA polymerase and subsequently transfected with MNV VPg-linked RNA. Levels of the viral RNA-dependent RNA polymerase (NS7) and minor capsid protein (VP2) were analysed by Western blotting with rabbit polyclonal antisera. In parallel, the virus yield was determined and expressed as TCID₅₀ per 35 mm dish. Transfections were carried out in triplicate; error bars represent sd. (b) BHK cells were either mock-infected (M) or infected with MVA-T7 or FPV-T7 and subsequently transfected with a cDNA construct containing the entire MNV genome under the control of a T7 RNA polymerase promoter (pT7 : MNV-G). NS7 expression levels were subsequently analysed by Western blotting.

Fig. 3.

Recovery of genetically defined noroviruses in tissue culture. (a) Western blot analysis of BHK cells transfected with either purified VPg-linked MNV RNA (vRNA) or plasmids expressing the cDNA encompassing the MNV subgenomic RNA [pT7 : MNV-SG (SG)], genomic RNA [pT7 : MNV-G (G)], genomic RNA containing a frame-shift mutation in the region coding for NS7 [pT7 : MNV-G^FS (G^FS)] or the repaired derivative [pT7 : MNV-G^FS/R (G^FS/R)]. Samples where infectious virus was recovered are indicated by +. (b) RT-PCR analysis demonstrating the presence of nuclease-resistant MNV RNA in nuclease-treated supernatants. PCRs were carried out with and without the prior addition of reverse transcriptase (RT). Positive and negative controls for PCR amplification (PCR− and PCR+) contained nuclease-free water or a plasmid encoding the MNV genomic RNA, respectively. Size of molecular mass markers is indicated.

Fig. 4.

Recovery of genetically marked MNV. (a) Schematic representation of the mutated region of the MNV genome. The additional BglII restriction site introduced in the recombinant virus CW1-Bgl is indicated by an asterisk. (b) RT-PCR amplification and subsequent digestion of the amplified region, indicating the presence of an additional BglII site in the mutated virus CW1-Bgl that is absent in the parental recombinant CW1 virus derived from cDNA (CW1-R) or tissue culture-adapted MNV (CW1). Size of molecular mass markers is indicated (in bp). (c) Sequencing chromatogram of the RT-PCR products encompassing the mutated region of CW1 and CW1-Bgl. The nucleotide change introduced in CW1-Bgl is underlined; the introduced BglII site is indicated by a black line.

Fig. 5.

Growth characteristics of noroviruses recovered from cDNA. Plaque phenotype (a) and one-step growth-curve analysis (b) of wild-type MNV (CW1, ○), MNV recovered entirely from cDNA (CW1-R, □) and the recombinant MNV containing the additional BglII site (CW1-Bgl, ▵).