Journal of Virological Methods 190 (2013) 17–19 Contents lists available at SciVerse ScienceDirect Journal of Virological Methods jou rn al hom ep age: www.elsevier.com/locate/jviromet Short communication Development of a strand specific SYBRGreen RT-PCR for a GIII.2 bovine norovirus Mette Myrmel ∗ Norwegian School of Veterinary Science, P.B. 8146 Dep., 0033 Oslo, Norway Article history: Received 18 December 2012 Received in revised form 8 March 2013 Accepted 13 March 2013 Available online 26 March 2013 Keywords: Bovine norovirus RT-PCR Strand specific a b s t r a c t A strand specific SYBRGreen RT-PCR was developed for a bovine norovirus (GIII.2). HEK293 cells were transfected with a plasmid containing the complete virus genome and copy DNA was produced with viral RNA strand-specific primers that introduced nucleotide changes. Amplicons from the negative and positive viral RNA strands, and from potential transcripts made by sequence independent transcription, were separated by melting curve analysis. The RT-PCR showed high strand specificity and could be a useful tool to study virus replication in replicon and reverse genetic systems and in screening for low levels of virus replication in norovirus permissive cell lines. © 2013 Elsevier B.V. All rights reserved. Noroviruses (NVs) are common enteric pathogens that have been found in several species. Bovine NVs (BoNVs) are very com- mon among calves and cause mild, self-limiting diarrhea (Jor et al., 2010; Oliver et al., 2007), while human NVs (HuNVs) are among the most common viral enteric pathogens in people and may cause sig- nificant disease with vomiting and diarrhea (Matthews et al., 2012). These viruses are not efficiently propagated in cell culture and this has hampered functional studies on their replication. Noroviruses belong to the Caliciviridae and have a linear sin- gle stranded positive-sense RNA genome of 7.5 kb. A virus protein (VPg) is covalently linked to the 5 ′ UTR (untranslated region), whereas the 3 ′ UTR is polyadenylated (Green et al., 2001). The NV genome generally encodes three open reading frames (ORFs). The non-structural polyprotein is encoded by ORF1 (Liu et al., 1996), while the major capsid protein, VP1, and the minor capsid protein, VP2, are encoded by ORF 2 and ORF 3, respectively. Replication of viral RNA is by transcription of the positive strand, resulting in the production of a negative strand from which positive genomic and sub genomic RNA are made. The NV genus is divided into five dif- ferent genogroups (G). Human NVs are found in GI, GII, and GIV, porcine NVs belong to GII, BoNVs belong to GIII and murine NVs (MuNVs) belong to GV. The identification of MuNV, which replicates efficiently in tissue culture and has a small animal model available, has increased our knowledge of NV biology (Karst et al., 2003). However, MuNVs dif- fer from the HuNVs in several aspects. They have four ORFs, infect macrophage-like cells in vivo, and replicate in cultured primary dendritic cells and macrophages (Wobus et al., 2004). Murine NVs ∗ Tel.: +47 22964771. E-mail address: mette.myrmel@nvh.no cause severe and lethal disease in STAT1 -/- deficient mice, but only modest clinical signs in immune competent mice (fecal inconsis- tency) (Kahan et al., 2011). Bovine NVs could be a better model for HuNVs than MuNVs and calves might also be used as an animal model of infection. Serology indicates that BoNVs infect humans (Vildevall et al., 2010;Widdowson et al., 2005), and gnotobiotic calves developed gastrointestinal symptoms after inoculation with a human GII.4 NV strain (Souza et al., 2008). In order to establish an additional system for the study of NV replication, a BoNV replicon (plasmid containing a complete BoNV GIII.2 genome; pHMBoNV) was developed (manuscript in preparation). The replicon was based on transcription (production of positive stranded viral RNA) by cel- lular RNA polymerase 1, translation of viral mRNA and production of negative stranded viral RNA by viral RNA dependent RNA poly- merase. The present communication describes the development of a strand-specific RT-PCR to enable the study of this replicon. A general approach with strand-specific RT-primers in sepa- rate tubes does not provide the necessary results due to copy DNA (cDNA) production of the incorrect strand by sequence inde- pendent priming (Haddad et al., 2007). Different strategies have been used to avoid this problem, including running RT reactions at high temperatures and using tagged RT-primers and tag-specific primers in the PCR (Vashist et al., 2012). Also, carryover of RT- primers into the PCR must be avoided. The specificity of the present assay is ensured by (1) strand-specific RT primers that introduce nucleotide substitutions into the amplicons (Feng et al., 2012); (2) RT at a relatively high temperature; (3) low concentrations of RT primers, and (4) diluted cDNA to prevent carryover of RT primers (Vashist et al., 2012). Copy DNA from correctly primed positive and negative strand viral RNA can thereby be identified and separated from cDNA resulting from sequence-independent priming during a SYBRGreen PCR with final melting curve analysis. 0166-0934/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2013.03.012