Using RNA-coat protein crosslinking we have shown that the principal RNA recognition surface on the interior of infectious MS2 virions overlaps with the known peptides that bind the high affinity translational operator, TR, within the phage genome. The data also reveal the sequences of genomic fragments in contact with the coat protein shell. These show remarkable overlap with previous predictions based on the hypothesis that virion assembly is mediated by multiple sequences-specific contacts at RNA sites termed Packaging Signals (PSs). These PSs are variations on the TR stem-loop sequence and secondary structure. They act co-operatively to regulate the dominant assembly pathway and ensure cognate RNA encapsidation. In MS2, they also trigger conformational change in the dimeric capsomere creating the A/B quasi-conformer, 60 of which are needed to complete the T=3 capsid. This is the most compelling demonstration to date that this ssRNA virus, and by implications potentially very many of them, assemble via a PS-mediated assembly mechanism.

Picornaviruses are important viral pathogens, but despite extensive study, the assembly process of their infectious virions is still incompletely understood, preventing the development of anti-viral strategies targeting this essential part of the life cycle. We report the identification, via RNA SELEX and bioinformatics, of multiple RNA sites across the genome of a typical enterovirus, enterovirus-E (EV-E), that each have affinity for the cognate viral capsid protein (CP) capsomer. Many of these sites are evolutionarily conserved across known EV-E variants, suggesting they play essential functional roles. Cryo-electron microscopy was used to reconstruct the EV-E particle at ~2.2 Å resolution, revealing extensive density for the genomic RNA. Relaxing the imposed symmetry within the reconstructed particles reveals multiple RNA-CP contacts, a first for any picornavirus. Conservative mutagenesis of the individual RNA-contacting amino acid side chains in EV-E, many of which are conserved across the enterovirus family including poliovirus, is lethal but does not interfere with replication or translation. Anti-EV-E and anti-poliovirus aptamers share sequence similarities with sites distributed across the poliovirus genome. These data are consistent with the hypothesis that these RNA-CP contacts are RNA Packaging Signals (PSs) that play vital roles in assembly and suggest that the RNA PSs are evolutionarily conserved between pathogens within the family, augmenting the current protein-only assembly paradigm for this family of viruses.

HighlightsThis is the first study of rotavirus genotypes circulating in UK pigs.Rotavirus transmission between pigs and humans is not thought to be common in the UK.Human rotavirus genotype P[8] found in a UK pig.The uncommon rotavirus genotype P[32] is widespread in UK pig herds.

Significance and impact of study:Infection of rotavirus has a negative impact on the health and growth of pigs in production. Given that the virus is transmitted faecal orally, use of an effective disinfectant on farm, which works even in high organic matter, has the potential to save costs in terms of outbreaks of disease and viral contamination. Here we test a number of commercial disinfectants of which one a phenolic compound, Bio-OO-cyst, shows effectivity even in high organic matter, implying its use could have a huge impact in reducing viral contamination and preventing losses in production. Abstract:Rotavirus is an enteric pathogen that causes morbidity and mortality in young mammals, including pigs. Outbreaks of rotavirus on commercial farms have a significant economic impact in terms of losses in production. Effective cleaning and disinfection along with good farm management can reduce rotavirus contamination in the environment, and decrease the chance of outbreaks of disease. This study investigated the efficacy of six commercial disinfectants against MS2 bacteriophage and Group A porcine rotavirus, in the presence of high and low levels of organic matter to simulate the farm environment. A phenolic-based disinfectant (Bi-OO-cyst) was effective at all levels of organic matter concentrations. Iodophore based disinfectants did not have a significant virucidal effect against rotavirus under any conditions. For peroxygen compound-based disinfectants and glutaraldehydebased disinfectants, organic matter load made a significant difference in reducing efficacy. This highlights the importance of thorough cleaning with detergent before disinfection to reduce viral contamination on the farm and decrease rotavirus disease incidence in pigs.

Graphical abstract