5.6. Full-length infectious virus clones

A supremely powerful tool in RNA virus research is cloning full-length genomic sequences of the virus into bacterial vectors. The naked RNA transcribed from such clones is usually infectious when introduced into a suitable host (Yunus et al., 2010), especially for positive-stranded RNA viruses (i.e. the majority of known bee viruses). Such clones can be manipulated by site-directed mutagenesis and recombination for functional analysis of different open reading frames or control regions. Reporter genes such as green fluorescent protein can be inserted to make fusion proteins with viral genes or to study promoter function in real-time (Ongus et al., 2006) and of course they can function as a genetically pure source of infectious virus, rather than having to rely on biological propagation, with the danger of contamination with other viruses and the changing genetic constitution of the virus, through evolution.

Full-length viral cDNAs are also an important tool in studying the genetic complexity of virus populations, since they make it possible to identify complete sequences of individual viruses within the population, including natural recombinants between major variants (Palacios et al., 2007; Moore et al., 2011).

5.6.2. Protocol