Viruses cause more than 50% of acute gastroenteritis, with norovirus (NoV) the most prevalent in all ages. More than 36 genotypes of NoV have been identified, however viruses from the Genogroup II, genotype 4 (GII.4) lineage cause over 70% of all NoV infections with pandemics every 2-3 years. The most recent GII.4 variant, Sydney 2012, emerged in 2012 and continues to cause the majority of NoV outbreaks to the present day.
The aim of this study was to use real time, reverse transcriptase – polymerase chain reaction (RT-PCR) and sequencing to determine the molecular epidemiology of NoV genotypes in Australia over the last year. We also developed a frozen sphere of known MS2 RNA and plaque forming units (pfu) levels, called a BioSphere. BioSpheres are easily handled and can validate each step in viral detection assays with no interference to RNA extraction, RT and PCR. In addition, MS2 bacteriophage is a good candidate for a process control as it is prone to the same degradation as RNA viruses and is similar in size, shape and properties.
The GII.4 variant Sydney 2012 was found to be the dominant cause of NoV-associated acute gastroenteritis cases and outbreaks, accounting for 74.5% (73/98) of all NoV infections. The majority of non-GII.4 variants detected were inter-genotypic recombinants (15/25, 60%), with GII.Pb/GII.3 viruses the most common (24%). MS2 RNA levels within the BioSpheres were approximately 250,000 genomes by real time RT-PCR and viral titres of 10,000 pfu/BioSphere by plaque assay. Validation of the NoV detection assay was achieved when the threshold cycle (Ct) values matched BioSphere control measurements.
In summary, this study shows GII.4 Sydney 2012 variant continues to be the predominant cause acute gastroenteritis in NSW, Australia. The present study also demonstrates that the MS2 BioSphere is an ideal process control for viral RNA detection assays.