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.

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.

Rotavirus is endemic in pig farms where it causes a loss in production. This study is the first to characterise porcine rotavirus circulating in UK pigs. Samples from diarrheic pigs with rotavirus enteritis obtained between 2010 and 2012 were genotyped in order to determine the diversity of group A rotavirus (GARV) in UK pigs. A wide range of rotavirus genotypes were identified in UK pigs: six G types (VP7); G2, G3, G4, G5, G9 and G11 and six P types (VP4); P[6], P[7], P[8], P[13], P[23], and P[32]. With the exception of a single P[8] isolate, there was less than 95% nucleotide identity between sequences from this study and any available rotavirus sequences. The G9 and P[6] genotypes are capable of infecting both humans and pigs, but showed no species cross-over within the UK as they were shown to be genetically distinct, which suggested zoonotic transmission is rare within the UK. We identified the P[8] genotype in one isolate, this genotype is almost exclusively found in humans. The P[8] was linked to a human Irish rotavirus isolate in the same year. The discovery of human genotype P[8] rotavirus in a UK pig confirms this common human genotype can infect pigs and also highlights the necessity of surveillance of porcine rotavirus genotypes to safeguard human as well as porcine health.

Group A rotaviruses (GARV) are a significant cause of enteritis in young pigs. The aim of this study was to extend our understanding of the molecular epidemiology of porcine GARV in the UK by investigating the genetic diversity of GARV on a conventional farrow-to-finish farm. Faecal samples were obtained from six batches of pigs in 2009 and 8 batches in 2010, when the pigs were 2, 3 (time point omitted in 2009), 4, 5, 6 and 8 weeks of age. Presence of rotavirus was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in 89% and 80% of samples from 2009 and 2010, respectively. A combination of multiplex PCRs and sequencing identified four VP7 genotypes (G2, G3, G4 and G5) and three VP4 genotypes (P[6], P[7] and P[32]) present in almost every combination over the 2 years. The predominant genotype combination was G5P[32] in 2009 and G4P[32] in 2010. Conservation among the P[32] sequences between 2009 and 2010 suggests that reassortment may have led to the different genotype combinations. There were significant changes in the predominant VP7 genotype prior to weaning at 4 weeks, and post weaning when pigs were moved to a different building. Phylogenetic analysis indicated that introduction of new viruses onto the farm was limited. Taken together, these findings suggest that genetically diverse GARV strains persist within the farm environment.

Single-stranded RNA viral genomes (gRNA) are dynamic molecules that permit packaging into virions and their subsequent extrusion during infection. For viruses with such genomes, we discovered a previously unsuspected mechanism that regulates their assembly. This regulation is the result of multiple cognate coat protein (CP)–gRNA contacts distributed across the RNA. Collectively, these interactions make the assembly highly efficient and specific. The regions of the gRNA packaging signals (PSs) driving this assembly are potential drug targets, whilst the manipulation of PS–CP contacts with nonviral RNA cargos is a route towards bespoke virus-like particles. Infectivity depends on the virions being able to transfer their gRNAs into host cells. The starting point for this transfer appears to be an encapsidated RNA with a defined three-dimensional structure, especially around the PSs. A combination of asymmetric cryo-electron microscopy structure determination and X-ray synchrotron footprinting were used to define these contacts and structures in a number of viral examples, including hepatitis B virus and enteroviruses. These tools allow us to look beyond the outer CP layer of the virion shell and to see the functional, asymmetric components that regulate viral infectivity. This revealed yet more unexpected aspects of critical infection mechanisms, such as the RNA conformational changes required for encapsidation, the details of PS–CP contacts regulating the assembly, and the conformational “memory” imposed by encapsidation.