Subclinical hepatitis E virus infection in laboratory ferrets in the UK

Ferrets are widely used for experimental modelling of viral infections. However, background disease in ferrets could potentially confound intended experimental interpretation. Here we report the detection of a subclinical infection of ferret hepatitis E virus (FRHEV) within a colony sub-group of female laboratory ferrets that had been enrolled on an experimental viral infection study (non-hepatitis). Lymphoplasmacytic cuffing of periportal spaces was identified on histopathology but was negative for the RNA and antigens of the administered virus. Follow-up viral metagenomic analysis conducted on liver specimens revealed sequences attributed to FRHEV and these were confirmed by reverse-transcriptase polymerase chain reaction. Further genomic analysis revealed contiguous sequences spanning 79-95 % of the FRHEV genome and that the sequences were closely related to those reported previously in Europe. Using in situ hybridization by RNAScope, we confirmed the presence of HEV-specific RNA in hepatocytes. The HEV open reading frame 2 (ORF2) protein was also detected by immunohistochemistry in the hepatocytes and the biliary canaliculi. In conclusion, the results of our study provide evidence of background infection with FRHEV in laboratory ferrets. As this infection can be subclinical, we recommend routine monitoring of ferret populations using virological and liver function tests to avoid incorrect causal attribution of any liver disease detected in in vivo studies

Assessing rabies vaccine protection against a novel lyssavirus, kotalahti bat lyssavirus

Rabies is a fatal encephalitis caused by an important group of viruses within the Lyssavirus genus. The prototype virus, rabies virus, is still the most commonly reported lyssavirus and causes approximately 59,000 human fatalities annually. The human and animal burden of the other lyssavirus species is undefined. The original reports for the novel lyssavirus, Kotalahti bat lyssavirus (KBLV), were based on the detection of viral RNA alone. In this report we describe the successful generation of a live recombinant virus, cSN-KBLV; where the full-length genome clone of RABV vaccine strain, SAD-B19, was constructed with the glycoprotein of KBLV. Subsequent in vitro characterisation of cSN-KBLV is described here. In addition, the ability of a human rabies vaccine to confer protective immunity in vivo following challenge with this recombinant virus was assessed. Naïve or vaccinated mice were infected intracerebrally with a dose of 100 focus-forming units/30 µL of cSN-KBLV; all naïve mice and 8% (n = 1/12) of the vaccinated mice succumbed to the challenge, whilst 92% (n = 11/12) of the vaccinated mice survived to the end of the experiment. This report provides strong evidence for cross-neutralisation and cross-protection of cSN-KBLV using purified Vero cell rabies vaccine

Evidence for overwintering and autochthonous transmission of Usutu virus to wild birds following its redetection in the United Kingdom

Usutu virus (USUV) is an emerging zoonotic arbovirus in Europe, where it primarily impacts Eurasian blackbirds (Turdus merula). For mosquito-borne viruses to persist in temperate areas, transovarial transmission in vectors or overwintering in either hosts or diapausing vectors must occur to facilitate autochthonous transmission. We undertook surveillance of hosts and vectors in 2021 to elucidate whether USUV had overwintered in the United Kingdom (UK) following its initial detection there in 2020. From 175 dead bird submissions, we detected 1 case of USUV infection, in a blackbird, from which a full USUV genome was derived. Using a molecular clock analysis, we demonstrate that the 2021 detection shared a most recent common ancestor with the 2020 Greater London, UK, USUV sequence. In addition, we identified USUV-specific neutralizing antibodies in 10 out of 86 serum samples taken from captive birds at the index site, demonstrating in situ cryptic infection and potential sustained transmission. However, from 4966 mosquitoes, we detected no USUV RNA suggesting that prevalence in the vector community was absent or low during sampling. Combined, these results suggest that USUV overwintered in the UK, thus providing empirical evidence for the continued northward expansion of this vector-borne viral disease. Currently, our detection indicates geographically restricted virus persistence. Further detections over time will be required to demonstrate long-term establishment. It remains unclear whether the UK, and by extension other high-latitude regions, can support endemic USUV infection

Tissue distribution of angiotensin-converting enzyme 2 (ACE2) receptor in wild animals with a focus on artiodactyls, mustelids and phocids

Natural cases of zooanthroponotic transmission of SARS-CoV-2 to animals have been reported during the COVID-19 pandemic, including to free-ranging white-tailed deer (Odocoileus virginianus) in North America and farmed American mink (Neovison vison) on multiple continents. To understand the potential for angiotensin-converting enzyme 2 (ACE2)-mediated viral tropism we characterised the distribution of ACE2 receptors in the respiratory and intestinal tissues of a selection of wild and semi-domesticated mammals including artiodactyls (cervids, bovids, camelids, suids and hippopotamus), mustelid and phocid species using immunohistochemistry. Expression of the ACE2 receptor was detected in the bronchial or bronchiolar epithelium of several European and Asiatic deer species, Bactrian camel (Camelus bactrianus), European badger (Meles meles), stoat (Mustela erminea), hippopotamus (Hippopotamus amphibious), harbor seal (Phoca vitulina), and hooded seal (Cystophora cristata). Further receptor mapping in the nasal turbinates and trachea revealed sparse ACE2 receptor expression in the mucosal epithelial cells and occasional occurrence in the submucosal glandular epithelium of Western roe deer (Capreolus capreolus), moose (Alces alces alces), and alpaca (Vicunga pacos). Only the European badger and stoat expressed high levels of ACE2 receptor in the nasal mucosal epithelium, which could suggest high susceptibility to ACE2-mediated respiratory infection. Expression of ACE2 receptor in the intestinal cells was ubiquitous across multiple taxa examined. Our results demonstrate the potential for ACE2-mediated viral infection in a selection of wild mammals and highlight the intra-taxon variability of ACE2 receptor expression, which might influence host susceptibility and infection

Detection of Highly Pathogenic Avian Influenza Virus H5N1 Clade 2.3.4.4b in Great Skuas:A Species of Conservation Concern in Great Britain

The UK and Europe have seen successive outbreaks of highly pathogenic avian influenza across the 2020/21 and 2021/22 autumn/winter seasons. Understanding both the epidemiology and transmission of these viruses in different species is critical to aid mitigating measures where outbreaks cause extensive mortalities in both land- and waterfowl. Infection of different species can result in mild or asymptomatic outcomes, or acute infections that result in high morbidity and mortality levels. Definition of disease outcome in different species is of great importance to understanding the role different species play in the maintenance and transmission of these pathogens. Further, the infection of species that have conservation value is also important to recognise and characterise to understand the impact on what might be limited wild populations. Highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b has been detected in great skuas (Stercorarius skua) across different colonies on islands off the shore of Scotland, Great Britain during summer 2021. A large number of great skuas were observed as developing severe clinical disease and dying during the epizootic and mortalities were estimated to be high where monitored. Of eight skuas submitted for post-mortem examination, seven were confirmed as being infected with this virus using a range of diagnostic assays. Here we overview the outbreak event that occurred in this species, listed as species of conservation concern in Great Britain and outline the importance of this finding with respect to virus transmission and maintenance

Observational evidence for gravitationally trapped massive axion(-like) particles

Unexpected astrophysical observations can be explained by gravitationally captured massive particles, which are produced inside the Sun or other Stars and are accumulated over cosmic times. Their radiative decay in solar outer space would give rise to a `self-irradiation' of the whole star, providing the time-independent component of the corona heating source. In analogy with the Sun-irradiated Earth atmosphere, the temperature and density gradient in the corona - chromosphere transition region is suggestive for an omnipresent irradiation of the Sun. The same scenario fits other astrophysical X-ray observations. The radiative decay of a population of such elusive particles mimics a hot gas. X-ray observatories, with an unrivalled sensitivity below ~10 keV, can search for such particles. The elongation angle relative to the Sun is the relevant new parameter.Comment: 35 pages, LaTeX, 9 figures. Accepted by Astroparticle Physic

Combining host and vector data informs emergence and potential impact of an Usutu virus outbreak in UK wild birds

Following the first detection in the United Kingdom of Usutu virus (USUV) in wild birds in 2020, we undertook a multidisciplinary investigation that combined screening host and vector populations with interrogation of national citizen science monitoring datasets to assess the potential for population impacts on avian hosts. Pathological findings from six USUV-positive wild passerines were non-specific, highlighting the need for molecular and immunohistochemical examinations to confirm infection. Mosquito surveillance at the index site identified USUV RNA in Culex pipiens s.l. following the outbreak. Although the Eurasian blackbird (Turdus merula) is most frequently impacted by USUV in Europe, national syndromic surveillance failed to detect any increase in occurrence of clinical signs consistent with USUV infection in this species. Furthermore, there was no increase in recoveries of dead blackbirds marked by the national ringing scheme. However, there was regional clustering of blackbird disease incident reports centred near the index site in 2020 and a contemporaneous marked reduction in the frequency with which blackbirds were recorded in gardens in this area, consistent with a hypothesis of disease-mediated population decline. Combining results from multidisciplinary schemes, as we have done, in real-time offers a model for the detection and impact assessment of future disease emergence events

Incursion of European Bat Lyssavirus 1 (EBLV-1) in Serotine Bats in the United Kingdom.

Lyssaviruses are an important genus of zoonotic viruses which cause the disease rabies. The United Kingdom is free of classical rabies (RABV). However, bat rabies due to European bat lyssavirus 2 (EBLV-2), has been detected in Daubenton's bats (Myotis daubentonii) in Great Britain since 1996, including a fatal human case in Scotland in 2002. Across Europe, European bat lyssavirus 1 (EBLV-1) is commonly associated with serotine bats (Eptesicus serotinus). Despite the presence of serotine bats across large parts of southern England, EBLV-1 had not previously been detected in this population. However, in 2018, EBLV-1 was detected through passive surveillance in a serotine bat from Dorset, England, using a combination of fluorescent antibody test, reverse transcription-PCR, Sanger sequencing and immunohistochemical analysis. Subsequent EBLV-1 positive serotine bats have been identified in South West England, again through passive surveillance, during 2018, 2019 and 2020. Here, we confirm details of seven cases of EBLV-1 and present similarities in genetic sequence indicating that emergence of EBLV-1 is likely to be recent, potentially associated with the natural movement of bats from the near continent

Development of immunohistochemistry and in situ hybridisation for the detection of SARS-CoV and SARS-CoV-2 in formalin-fixed paraffin-embedded specimens

The rapid emergence of SARS-CoV-2, the causative agent of COVID-19, and its dissemination globally has caused an unprecedented strain on public health. Animal models are urgently being developed for SARS-CoV-2 to aid rational design of vaccines and therapeutics. Immunohistochemistry and in situ hybridisation techniques that facilitate reliable and reproducible detection of SARS-CoV and SARS-CoV-2 viral products in formalin-fixed paraffin-embedded (FFPE) specimens would be of great utility. A selection of commercial antibodies generated against SARS-CoV spike protein and nucleoprotein, double stranded RNA, and RNA probe for spike genes were evaluated for the ability to detect FFPE infected cells. We also tested both heat- and enzymatic-mediated virus antigen retrieval methods to determine the optimal virus antigen recovery as well as identifying alternative retrieval methods to enable flexibility of IHC methods. In addition to using native virus infected cells as positive control material, the evaluation of non-infected cells expressing coronavirus (SARS, MERS) spike as a biosecure alternative to assays involving live virus was undertaken. Optimized protocols were successfully applied to experimental animal-derived tissues. The diverse techniques for virus detection and control material generation demonstrated in this study can be applied to investigations of coronavirus pathogenesis and therapeutic research in animal models