SARS-CoV-2 infection and transmission via the skin to oro-nasal route with the production of bioaerosols in the ferret model.

Direct and indirect transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been attributed to virus survival in droplets, bioaerosols and on fomites including skin and surfaces. Survival of SARS-CoV-2 variants of concern (Alpha, Beta, Gamma, and Delta) on the skin and virus transference following rounds of skin-to-skin contact were assessed on porcine skin as a surrogate for human skin. SARS-CoV-2 variants were detectable on skin by RT-qPCR after 72 h at biologically relevant temperatures (35.2 °C) with viral RNA (vRNA) detected after ten successive skin-to-skin contacts. Skin-to-skin virus transmission to establish infection in ferrets as a model for mild/asymptomatic SARS-CoV-2 infection in mustelids and humans was also investigated and compared to intranasal ferret inoculation. Naïve ferrets exposed to Delta variant SARS-CoV-2 in a 'wet' or 'dry' form on porcine skin resulted in robust infection with shedding detectable for up to 14 days post-exposure, at comparable viral loads to ferrets inoculated intranasally. Transmission of SARS-CoV-2 to naïve ferrets in direct contact with infected ferrets was achieved, with environmental contamination detected from ferret fur swabs and air samples. Genetic substitutions were identified in bioaerosol samples acquired following single contact passage in ferrets, including Spike, ORF1ab, and ORF3a protein sequences, suggesting a utility for monitoring host adaptation and virus evolution via air sampling. The longevity of SARS-CoV-2 variants survival directly on the skin and skin-to-skin transference, enabling subsequent infection via the skin to oro-nasal contact route, could represent a pathway for SARS-CoV-2 infection with implications to public and veterinary health.

Detection of Usutu virus infection in wild birds in the United Kingdom, 2020.

In August 2020, as part of a long-term disease surveillance programme, Usutu virus was detected in five Eurasian blackbirds (Turdus merula) and one house sparrow (Passer domesticus) from Greater London, England. This was initially detected by reverse transcription-PCR and was confirmed by virus isolation and by immunohistochemical detection of flavivirus in tissues. Phylogenetic analysis identified Usutu virus African 3.2 lineage, which is prevalent in the Netherlands and Belgium, suggesting a potential incursion from mainland Europe.

Clinical Characteristics of Ratborne Seoul Hantavirus Disease.

Although Seoul orthohantavirus is the only globally spread hantavirus pathogen, few confirmed human infections with this virus have been reported in Western countries, suggesting lower medical awareness of the milder, transient, and often chameleon-like symptoms of this zoonosis. We describe lesser known clinical and laboratory characteristics to help improve underreporting of this virus.

Molecular Epidemiology and Evolution of European Bat Lyssavirus 2.

Bat rabies cases in Europe are mainly attributed to two lyssaviruses, namely European Bat Lyssavirus 1 (EBLV-1) and European Bat Lyssavirus 2 (EBLV-2). Prior to the death of a bat worker in Finland in 1985, very few bat rabies cases were reported. Enhanced surveillance in the two subsequent years (1986-1987) identified 263 cases (more than a fifth of all reported cases to date). Between 1977 and 2016, 1183 cases of bat rabies were reported, with the vast majority (>97%) being attributed to EBLV-1. In contrast, there have been only 39 suspected cases of EBLV-2, of which 34 have been confirmed by virus typing and presently restricted to just two bat species; Myotis daubentonii and Myotis dasycneme. The limited number of EBLV-2 cases in Europe prompted the establishment of a network of European reference laboratories to collate all available viruses and data. Despite the relatively low number of EBLV-2 cases, a large amount of anomalous data has been published in the scientific literature, which we have here reviewed and clarified. In this review, 29 EBLV-2 full genome sequences have been analysed to further our understanding of the diversity and molecular evolution of EBLV-2 in Europe. Analysis of the 29 complete EBLV-2 genome sequences clearly corroborated geographical relationships with all EBLV-2 sequences clustering at the country level irrespective of the gene studied. Further geographical clustering was also observed at a local level. There are high levels of homogeneity within the EBLV-2 species with nucleotide identities ranging from 95.5-100% and amino acid identities between 98.7% and 100%, despite the widespread distribution of the isolates both geographically and chronologically. The mean substitution rate for EBLV-2 across the five concatenated genes was 1.65 × 10-5, and evolutionary clock analysis confirms the slow evolution of EBLV-2 both between and within countries in Europe. This is further supported by the first detailed EBLV-2 intra-roost genomic analysis whereby a relatively high sequence homogeneity was found across the genomes of three EBLV-2 isolates obtained several years apart (2007, 2008, and 2014) from M. daubentonii at the same site (Stokesay Castle, Shropshire, UK).

Lyssavirus in Indian Flying Foxes, Sri Lanka.

A novel lyssavirus was isolated from brains of Indian flying foxes (Pteropus medius) in Sri Lanka. Phylogenetic analysis of complete virus genome sequences, and geographic location and host species, provides strong evidence that this virus is a putative new lyssavirus species, designated as Gannoruwa bat lyssavirus.

Discovery of hantavirus circulating among Rattus rattus in French Mayotte island, Indian Ocean.

Hantaviruses are emerging zoonotic viruses that cause human diseases. In this study, sera from 642 mammals from La Réunion and Mayotte islands (Indian Ocean) were screened for the presence of hantaviruses by molecular analysis. None of the mammals from La Réunion island was positive, but hantavirus genomic RNA was discovered in 29/160 (18 %) Rattus rattus from Mayotte island. The nucleoprotein coding region was sequenced from the liver and spleen of all positive individuals allowing epidemiological and intra-strain variability analyses. Phylogenetic analysis based on complete coding genomic sequences showed that this Murinae-associated hantavirus is a new variant of Thailand virus. Further studies are needed to investigate hantaviruses in rodent hosts and in Haemorrhagic Fever with Renal Syndrome (HFRS) human cases.

Global population divergence and admixture of the brown rat (Rattus norvegicus).

Native to China and Mongolia, the brown rat (Rattus norvegicus) now enjoys a worldwide distribution. While black rats and the house mouse tracked the regional development of human agricultural settlements, brown rats did not appear in Europe until the 1500s, suggesting their range expansion was a response to relatively recent increases in global trade. We inferred the global phylogeography of brown rats using 32 k SNPs, and detected 13 evolutionary clusters within five expansion routes. One cluster arose following a southward expansion into Southeast Asia. Three additional clusters arose from two independent eastward expansions: one expansion from Russia to the Aleutian Archipelago, and a second to western North America. Westward expansion resulted in the colonization of Europe from which subsequent rapid colonization of Africa, the Americas and Australasia occurred, and multiple evolutionary clusters were detected. An astonishing degree of fine-grained clustering between and within sampling sites underscored the extent to which urban heterogeneity shaped genetic structure of commensal rodents. Surprisingly, few individuals were recent migrants, suggesting that recruitment into established populations is limited. Understanding the global population structure of R. norvegicus offers novel perspectives on the forces driving the spread of zoonotic disease, and aids in development of rat eradication programmes.

Current status of rabies and prospects for elimination.

Rabies is one of the most deadly infectious diseases, with a case-fatality rate approaching 100%. The disease is established on all continents apart from Antarctica; most cases are reported in Africa and Asia, with thousands of deaths recorded annually. However, the estimated annual figure of almost 60,000 human rabies fatalities is probably an underestimate. Almost all cases of human rabies result from bites from infected dogs. Therefore, the most cost-effective approach to elimination of the global burden of human rabies is to control canine rabies rather than expansion of the availability of human prophylaxis. Mass vaccination campaigns with parenteral vaccines, and advances in oral vaccines for wildlife, have allowed the elimination of rabies in terrestrial carnivores in several countries worldwide. The subsequent reduction in cases of human rabies in such regions advocates the multidisciplinary One Health approach to rabies control through the mass vaccination of dogs and control of canine populations.

Detection and genetic characterization of Seoul virus from commensal brown rats in France.

BACKGROUND: Hantaviruses are single-stranded RNA viruses, which are transmitted to humans primarily via inhalation of aerosolised virus in contaminated rodent urine and faeces. Whilst infected reservoir hosts are asymptomatic, human infections can lead to two clinical manifestations, haemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), with varying degrees of clinical severity. The incidence of rodent and human cases of Seoul virus (SEOV) in Europe has been considered to be low, and speculated to be driven by the sporadic introduction of infected brown rats (Rattus norvegicus) via ports. METHODS: Between October 2010 and March 2012, 128 brown rats were caught at sites across the Lyon region in France. RESULTS: SEOV RNA was detected in the lungs of 14% (95% CI 8.01-20.11) of brown rats tested using a nested pan-hantavirus RT-PCR (polymerase gene). Phylogenetic analysis supports the inclusion of the Lyon SEOV within Lineage 7 with SEOV strains originating from SE Asia and the previously reported French & Belgian SEOV strains. Sequence data obtained from the recent human SEOV case (Replonges) was most similar to that obtained from one brown rat trapped in a public park in Lyon city centre. We obtained significantly improved recovery of virus genome sequence directly from SEOV infected lung material using a simple viral enrichment approach and NGS technology. CONCLUSIONS: The detection of SEOV in two wild caught brown rats in the UK and the multiple detection of SEOV infected brown rats in the Lyon region of France, suggests that SEOV is circulating in European brown rats. Under-reporting and difficulties in identifying the hantaviruses associated with HFRS may mask the public health impact of SEOV in Europe.

Investigating the emergence of a zoonotic virus: phylogenetic analysis of European bat lyssavirus 1 in the UK.

European bat lyssavirus 1 (EBLV-1, Lyssavirus hamburg) is predominantly detected in serotine bats (Eptesicus serotinus) and is responsible for the majority of bat rabies cases in mainland Europe. A passive bat rabies surveillance scheme detected the virus in a serotine bat in the UK for the first time in October 2018. As of May 2024, 34 cases have been reported, 20 of which involved contact with an animal and 5 reported human contact. We investigated the emergence of EBLV-1 by undertaking comprehensive sequence analysis and Bayesian phylogenetics, based on complete virus genomes of 33 UK sequences and 108 sequences covering six countries in mainland Europe (1968-2023), including 21 French EBLV-1-positive RNA samples sequenced for this study. Sequence analysis revealed extreme similarity among UK EBLV-1 sequences (99.9%-100%), implying a single source of introduction rather than multiple independent introductions. Bayesian analysis revealed that the UK EBLV-1 sequences shared their most recent common ancestor with an EBLV-1 sequence from a serotine bat detected in Brittany, France, in 2001, with an estimated date of divergence of 1997. Within the UK sequences, the earliest divergence was estimated to occur in 2007. This study provides valuable insights into the molecular epidemiology of an emerging zoonotic pathogen and improved understanding of the risks posed to public and animal health.

Detection of SARS-CoV-2 Delta Variant (B.1.617.2) in Domestic Dogs and Zoo Tigers in England and Jersey during 2021.

Reverse zoonotic transmission events of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been described since the start of the pandemic, and the World Organisation for Animal Health (WOAH) designated the detection of SARS-CoV-2 in animals a reportable disease. Eighteen domestic and zoo animals in Great Britain and Jersey were tested by APHA for SARS-CoV-2 during 2020-2023. One domestic cat (Felis catus), three domestic dogs (Canis lupus familiaris), and three Amur tigers (Panthera tigris altaica) from a zoo were confirmed positive during 2020-2021 and reported to the WOAH. All seven positive animals were linked with known SARS-CoV-2 positive human contacts. Characterisation of the SARS-CoV-2 variants by genome sequencing indicated that the cat was infected with an early SARS-CoV-2 lineage. The three dogs and three tigers were infected with the SARS-CoV-2 Delta variant of concern (B.1.617.2). The role of non-human species in the onward transmission and emergence of new variants of SARS-CoV-2 remain poorly defined. Continued surveillance of SARS-CoV-2 in relevant domestic and captive animal species with high levels of human contact is important to monitor transmission at the human-animal interface and to assess their role as potential animal reservoirs.

Next generation sequencing of viral RNA genomes.

BACKGROUND: With the advent of Next Generation Sequencing (NGS) technologies, the ability to generate large amounts of sequence data has revolutionized the genomics field. Most RNA viruses have relatively small genomes in comparison to other organisms and as such, would appear to be an obvious success story for the use of NGS technologies. However, due to the relatively low abundance of viral RNA in relation to host RNA, RNA viruses have proved relatively difficult to sequence using NGS technologies. Here we detail a simple, robust methodology, without the use of ultra-centrifugation, filtration or viral enrichment protocols, to prepare RNA from diagnostic clinical tissue samples, cell monolayers and tissue culture supernatant, for subsequent sequencing on the Roche 454 platform. RESULTS: As representative RNA viruses, full genome sequence was successfully obtained from known lyssaviruses belonging to recognized species and a novel lyssavirus species using these protocols and assembling the reads using de novo algorithms. Furthermore, genome sequences were generated from considerably less than 200 ng RNA, indicating that manufacturers' minimum template guidance is conservative. In addition to obtaining genome consensus sequence, a high proportion of SNPs (Single Nucleotide Polymorphisms) were identified in the majority of samples analyzed. CONCLUSIONS: The approaches reported clearly facilitate successful full genome lyssavirus sequencing and can be universally applied to discovering and obtaining consensus genome sequences of RNA viruses from a variety of sources.

Interspecies protein substitution to investigate the role of the lyssavirus glycoprotein.

European bat lyssaviruses type 1 (EBLV-1) and type 2 (EBLV-2) circulate within bat populations throughout Europe and are capable of causing disease indistinguishable from that caused by classical rabies virus (RABV). However, the determinants of viral fitness and pathogenicity are poorly understood. Full-length genome clones based on the highly attenuated, non-neuroinvasive, RABV vaccine strain (SAD-B19) were constructed with the glycoprotein (G) of either SAD-B19 (SN), of EBLV-1 (SN-1) or EBLV-2 (SN-2). In vitro characterization of SN-1 and SN-2 in comparison to wild-type EBLVs demonstrated that the substitution of G affected the final virus titre and antigenicity. In vivo, following peripheral infection with a high viral dose (10(4) f.f.u.), animals infected with SN-1 had reduced survivorship relative to infection with SN, resulting in survivorship similar to animals infected with EBLV-1. The histopathological changes and antigen distribution observed for SN-1 were more representative of those observed with SN than with EBLV-1. EBLV-2 was unable to achieve a titre equivalent to that of the other viruses. Therefore, a reduced-dose experiment (10(3) f.f.u.) was undertaken in vivo to compare EBLV-2 and SN-2, which resulted in 100 % survivorship for all recombinant viruses (SN, SN-1 and SN-2) while clinical disease developed in mice infected with the EBLVs. These data indicate that interspecies replacement of G has an effect on virus titre in vitro, probably as a result of suboptimal G-matrix protein interactions, and influences the survival outcome following a peripheral challenge with a high virus titre in mice.

Complete genome sequence of Ikoma lyssavirus.

Lyssaviruses (family Rhabdoviridae) constitute one of the most important groups of viral zoonoses globally. All lyssaviruses cause the disease rabies, an acute progressive encephalitis for which, once symptoms occur, there is no effective cure. Currently available vaccines are highly protective against the predominantly circulating lyssavirus species. Using next-generation sequencing technologies, we have obtained the whole-genome sequence for a novel lyssavirus, Ikoma lyssavirus (IKOV), isolated from an African civet in Tanzania displaying clinical signs of rabies. Genetically, this virus is the most divergent within the genus Lyssavirus. Characterization of the genome will help to improve our understanding of lyssavirus diversity and enable investigation into vaccine-induced immunity and protection.

Evaluation of an iELISA for detection and quantification of rabies antibodies in domestic dog sera.

Many rabies endemic-countries have recognized rabies as a public health problem that can be eliminated. As a result, some countries have started implementing small-scale vaccination programs with the aim of scaling them up. Post-vaccination serological monitoring is crucial to assess the efficacy of these programs. The recommended serological tests, the rapid fluorescent focus inhibition test, and the fluorescent antibody virus neutralization (FAVN) are accurate; however, the procedures require considerable expertise and must be carried out in high containment facilities, which are often not available in rabies endemic countries. Given these constraints, enzyme linked immunosorbent assays (ELISAs) have been considered as alternative methods to neutralization tests. This is the first study to evaluate, under field conditions, the performance of the commercial rabies indirect-ELISA (iELISA), the PlateliaTM Rabies II kit ad usum Veterinarium kit, using sera from domestic dogs. Serum samples were collected from two groups of community dogs in northern Tanzania: i) dogs with no history of vaccination against rabies (n = 100) and ii) dogs vaccinated with the Nobivac Canine Rabies® vaccine (n = 101) four weeks previously. When compared to the gold standard FAVN test, the iELISA was found to be 99% specific and 98% sensitive and there was a significant correlation between the two tests (p < 0.001, r = 0.92). Given these findings, we conclude that the PlateliaTM Rabies II kit ad usum Veterinarium can be considered a valuable tool for the rapid assessment of vaccination status of animals in vaccination programs.

A simplified method for measuring neutralising antibodies against rabies virus.

Rabies virus (RABV) causes a fatal encephalitis that can be prevented through timely vaccination. The levels of virus neutralising antibodies against rabies virus induced by vaccination can be measured using the fluorescent antibody virus neutralisation (FAVN) test. Following incubation of live virus with sera, this method involves the fixation of cell monolayers and staining of rabies virus-specific antigen using fluorescein isothiocyanate (FITC) -conjugated antibody to enable visualisation of rabies virus antigen using a fluorescence microscope. To simplify this procedure, a fluorescent recombinant rabies virus was constructed using reverse genetics by inserting the gene for the mCherry fluorescent protein in front of the ribonucleoprotein gene of the SAD B-19 genome and replacing its glycoprotein with that of the Challenge Virus Standard (CVS)-11 RABV strain to ensure antigenic authenticity with the FAVN. This new recombinant virus (termed mCCCG) expressed the mCherry protein to high levels enabling direct observation of infected cells. In vitro growth kinetics of mCCCG were indistinguishable from that of CVS-11. The stability of the recombinant virus was assessed by sequencing several passages of the rescued virus and only minor changes were detected. Comparative assessment of the virus neutralisation test using mCherry producing virus (NTmCV) against the FAVN demonstrated that test results were equivalent to each other; therefore, mCCCG can be used as an alternative to CVS-11 for measuring antibody titres against the rabies virus. The use of NTmCV removes the need for expensive antibody conjugates and significantly reduces assay time. This would be particularly beneficial for RABV serological assessment in resource limited settings. Moreover, the reading of the plates can be automatically using a cell imaging reader.

Ikoma lyssavirus, highly divergent novel lyssavirus in an African civet.

Evidence in support of a novel lyssavirus was obtained from brain samples of an African civet in Tanzania. Results of phylogenetic analysis of nucleoprotein gene sequences from representative Lyssavirus species and this novel lyssavirus provided strong empirical evidence that this is a new lyssavirus species, designated Ikoma lyssavirus.

JMM Profile: Louping ill virus.

Louping ill virus (LIV) is a single-stranded, positive-sense RNA virus within the genus Flavivirus that is transmitted to vertebrate hosts by bites from infected ticks, the arthropod vector. The virus affects livestock in upland areas of Great Britain and Ireland, resulting in a febrile illness that can progress to fatal encephalitis. Prevention of the disease is facilitated by combining acaricide treatment, land management and vaccination strategies. However, vaccines have been discontinued in recent years. Although rare, LIV can be transmitted to and cause disease in humans. Consequently, LIV infection is a threat to human and veterinary health and can impact on the rural economy.

Generalized Modules for Membrane Antigens (GMMA), an outer membrane vesicle-based vaccine platform, for efficient viral antigen delivery.

Vaccine platforms enable fast development, testing, and manufacture of more affordable vaccines. Here, we evaluated Generalized Modules for Membrane Antigens (GMMA), outer membrane vesicles (OMVs) generated by genetically modified Gram-negative bacteria, as a vaccine platform for viral pathogens. Influenza A virus hemagglutinin (HA), either physically mixed with GMMA (HA+STmGMMA mix), or covalently linked to GMMA surface (HA-STmGMMA conjugate), significantly increased antigen-specific humoral and cellular responses, with HA-STmGMMA conjugate inducing further enhancement than HA+STmGMMA mix. HA-STmGMMA conjugate protected mice from lethal challenge. The versatility for this platform was confirmed by conjugation of rabies glycoprotein (RABVG) onto GMMA through the same method. RABVG+STmGMMA mix and RABVG-STmGMMA conjugate exhibited similar humoral and cellular response patterns and protection efficacy as the HA formulations, indicating relatively consistent responses for different vaccines based on the GMMA platform. Comparing to soluble protein, GMMA was more efficiently taken up in vivo and exhibited a B-cell preferential uptake in the draining lymph nodes (LNs). Together, GMMA enhances immunity against viral antigens, and the platform works well with different antigens while retaining similar immunomodulatory patterns. The findings of our study imply the great potential of GMMA-based vaccine platform also against viral infectious diseases.

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.