Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA

BACKGROUND: Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. RESULTS: A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that modifications in the E2 protein coding sequence were stably maintained. A single amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence. CONCLUSIONS: These results show that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses

Introduction: building the history of language learning and teaching (HoLLT)

The papers presented in this issue are the result of a workshop held at the University of Nottingham in December 2012 as part of an Arts and Humanities Research Council research network Towards a History of Modern Foreign Language Teaching and Learning (2012–14) intended to stimulate historical research into language teaching and learning. This, the first workshop in the programme, focused on exchanging information on the history of language learning and teaching (HoLLT) across the different language traditions, for it had become clear to us that scholars working within their own language disciplines were often relatively unaware of work outside these. We hope that this special issue — with overview articles on the history of English, French, German, and Spanish as second/foreign languages — will help overcome that lack of awareness and facilitate further research collaboration. Charting the history of language teaching and learning will, in turn, make us all better informed in facing challenges and changes to policy and practice now and in the future. It is instructive in the current climate, for example, to realize that grave doubts were held about whether second foreign languages could survive alongside French in British schools in the early twentieth century (McLelland, forthcoming), or to look back at earlier attempts to establish foreign languages in primary schools (Bayley, 1989; Burstall et al., 1974; Hoy, 1977). As we write, language learning in England is undergoing yet more radical change. Language teaching for all children from the age of seven is being made compulsory in primary schools from 2014, while at Key Stage 3 (up to age 16), where a foreign language has not been compulsory since 2002, the most recent programme of study for England has virtually abandoned the recent focus on intercultural competence and now requires learners to ‘read great literature in the original language’,1 a radical change in emphasis compared to the previous half-century, which seems to reflect a very different view of what language learning is for. We seem to be little closer in 2014 than we were at the dawn of the twentieth century to answering with any certainty the questions that lie at the very foundations of language teaching: who should learn a foreign language, why learners learn, what they need to learn, and what we want to teach them — answers that we need before we can consider how we want to teach. The research programme begun under our research network is intended to help us to take ‘the long view’ on such questions

Analysis of classical swine fever virus RNA replication determinants using replicons

Self-replicating RNAs (replicons), with or without reporter gene sequences, derived from the genome of the Paderborn strain of classical swine fever virus (CSFV) have been produced. The full-length viral cDNA, propagated within a bacterial artificial chromosome (BAC), was modified by targeted recombination within E. coli. RNA transcripts were produced in vitro and introduced into cells by electroporation. The translation and replication of the replicon RNAs could be followed by the accumulation of luciferase (from Renilla reniformis or Gaussia princeps) protein expression (where appropriate), as well as by detection of the CSFV NS3 protein production within the cells. Inclusion of the viral E2 coding region within the replicon was advantageous for the replication efficiency. Production of chimeric RNAs, substituting the NS2 and NS3 coding regions (as a unit) from the Paderborn strain with the equivalent sequences from the highly virulent Koslov strain or the vaccine strain Riems, blocked replication. However, replacing the Paderborn NS5B coding sequence with the RNA polymerase coding sequence from the Koslov strain greatly enhanced expression of the reporter protein from the replicon. In contrast, replacement with the Riems NS5B sequence significantly impaired replication efficiency. Thus these replicons provide a system for determining specific regions of the CSFV genome required for genome replication without the constraints of maintaining infectivity