10.1. Introduction

The detection of viral replication is crucial for differentiating between an active infection and just the presence of virus particles in a host, or between a mechanical (virus non-replicating) and a biological (virus replicating) vector of a virus. Evidence that a virus is actively infecting a host includes the presence of viral particles and structures within host cells, revealed by electron microscopy, preferably including a specific nucleic acid or serological probe to positively identify the virus. Another approach is to detect the non-structural proteins involved in virus replication, which for most positive-stranded RNA viruses are only produced after invasion and mark the start of a replication cycle. Negative-stranded RNA viruses often carry their replicative proteins within the particle. A related philosophy, which is more sensitive and accessible, is to specifically detect the replicative strand RNA of a virus. Most of the described bee viruses are single- and plus-strand RNA viruses which replicate through a negative-strand RNA intermediate serving as template for the generation of new viral plus-strand RNA genomes. The specific detection of viral negative strand RNAs can therefore serve as a marker of active replication of these RNA viruses in a certain host, tissue or cell type. Below are outlined two methods for strand-specific detection of RNA virus sequences.