The recent discovery of novel alphacoronaviruses (alpha-CoVs) in European and Asian rodents revealed that rodent coronaviruses (CoVs) sampled worldwide formed a discrete phylogenetic group within this genus. To determine the evolutionary history of rodent CoVs in more detail, particularly the relative frequencies of virus-host co-divergence and cross-species transmission, we recovered longer fragments of CoV genomes from previously discovered European rodent alpha-CoVs using a combination of PCR and high-throughput sequencing. Accordingly, the full genome sequence was retrieved from the UK rat coronavirus, along with partial genome sequences from the UK field vole and Poland-resident bank vole CoVs, and a short conserved ORF1b fragment from the French rabbit CoV. Genome and phylogenetic analysis showed that despite their diverse geographic origins, all rodent alpha-CoVs formed a single monophyletic group and shared similar features, such as the same gene constellations, a recombinant beta-CoV spike gene, and similar core transcriptional regulatory sequences (TRS). These data suggest that all rodent alpha CoVs sampled so far originate from a single common ancestor, and that there has likely been a long-term association between alpha CoVs and rodents. Despite this likely antiquity, the phylogenetic pattern of the alpha-CoVs was also suggestive of relatively frequent host-jumping among the different rodent species.

Orthohantaviruses are globally distributed viruses, associated with rodents and other small mammals. However, data on the circulation of orthohantaviruses within the UK, particularly the UK-endemic Tatenale virus, is sparse. In this study, 531 animals from five rodent species were collected from two locations in northern and central England and screened using a degenerate, pan- orthohantavirus RT-PCR assay. Tatenale virus was detected in a single field vole (Microtus agrestis) from central England and twelve field voles from northern England. Unbiased high-throughput sequencing of the central English strain resulted in the recovery of the complete coding sequence of a novel strain of Tatenale virus, whilst PCR-primer walking of the northern English strain recovered almost complete coding sequence of a previously identified strain. These findings represented the detection of a third lineage of Tatenale virus in the United Kingdom and extended the known geographic distribution of these viruses from northern to central England. Furthermore, the recovery of the complete coding sequence revealed that Tatenale virus was sufficiently related to the recently identified Traemersee virus, to meet the accepted criteria for classification as a single species of orthohantavirus.

Hantaviruses are a diverse group of single-stranded, negative-sensed RNA viruses, known to cause sporadic outbreaks of potentially fatal human disease. To date, only two Orthohantavirus species have been detected in the UK - Seoul virus and Tatenale. Whilst Seoul is known to be pathogenic in humans, only partial fragments of Tatenale have been recovered, precluding any accurate analysis of its phylogeny or potential pathogenicity. To overcome this shortfall we used a degenerate primer PCR method to identify Tatenale-infection in rodents living in two separate locations in the UK. PCR positive samples were then subjected to either unbiased high-throughput sequencing or overlapping PCR product sequencing to recover the complete coding sequence of the Tatenale virus. This analysis provided in-depth insight into the evolutionary origins of this recently identified UK Orthohantavirus and unequivocally showed that Tatenale virus meets the established criteria for classification as a novel species. Crucially, our data will facilitate in vitro investigation into the zoonotic potential of Tatenale virus.

Validated inactivation protocols are required for the safe handling and disposal of virus samples. This is particularly important for hazard group 3 agents and above. Various methods are employed for virus inactivation, some of which include the use of heat or chemical chaotropic agents such as guanidine hydrochloride. It is generally accepted that these processes are sufficient to denature all viruses. While inactivation of certain viruses with such methods have been reported, validation of their activity against a wide of range of viruses is required. Here, we examined the inactivation of a panel of hazard group 2 viruses (Rhabdoviridae, Togaviridae, Peribunyaviridae, and Flaviviridae) using a combination of heat, guanidine hydrochloride-containing buffer, and ethanol. Viruses were treated with proteinase K and guanidine hydrochloride-containing buffer, and heated at 56 °C for 30 min. This was followed by additional treatment with absolute ethanol. Resulting virus–buffer–ethanol mixtures were column-purified to remove residual ethanol and other toxic reagents, before being introduced to cells. Column purification was confirmed to be insufficient for virus removal. Cultures were incubated at 37 °C for 1 h, after which media supplemented with 2% foetal bovine serum was added. Cultures were then observed daily for cytopathic effects. Samples that showed no evidence of cytopathic effects were passaged thrice to confirm absence of cytopathic effects and complete inactivation of viruses. Cultures infected with control viruses that had not been treated with buffer, heat, and ethanol but column-purified developed cytopathic effects, while cultures infected with treated viruses showed no cytopathic effects even after three passages, thus confirming complete virus inactivation. Results from this study provide evidence of the use of a combination of heat, guanidine hydrochloride-containing buffer, and ethanol for the complete inactivation of all members of the four families investigated.