Highly pathogenic avian influenza virus H5N6 (clade 2.3.4.4b) has a preferable host tropism for waterfowl reflected in its inefficient transmission to terrestrial poultry.

Highly-pathogenic avian influenza virus (HPAIV) H5N6 (clade 2.3.4.4b) incurred into Europe in late 2017 and was predominantly detected in wild birds, with very few terrestrial poultry cases. Pekin ducks directly-infected with a UK virus (H5N6-2017) were donors of infection to investigate contact transmission to three recipient species: Ducks, chickens and turkeys. H5N6-2017 transmission to ducks was 100% efficient, but transmission to in-contact galliforme species was infrequent and unpredictable, thereby reflecting the European 2017-2018 H5N6 epidemiology. Although only two of 28 (7%) infected ducks died, the six turkeys and one chicken which became infected all died and displayed systemic H5N6-2017 dissemination, while pathogenesis in ducks was generally milder. Analysis of H5N6-2017 progeny in the contacts revealed no emergent polymorphisms in an infected duck, but the galliforme species included changes in the polymerase (PB2 A199T, PA D347A), matrix (M1 T218A) and neuraminidase genes (T88I). H5N6-2017 environmental contamination was associated with duck shedding.

Assessing the risks of SARS-CoV-2 in wildlife.

The novel coronavirus SARS-CoV-2 likely emerged from a wildlife source with transmission to humans followed by rapid geographic spread throughout the globe and severe impacts on both human health and the global economy. Since the onset of the pandemic, there have been many instances of human-to-animal transmission involving companion, farmed and zoo animals, and limited evidence for spread into free-living wildlife. The establishment of reservoirs of infection in wild animals would create significant challenges to infection control in humans and could pose a threat to the welfare and conservation status of wildlife. We discuss the potential for exposure, onward transmission and persistence of SARS-CoV-2 in an initial selection of wild mammals (bats, canids, felids, mustelids, great apes, rodents and cervids). Dynamic risk assessment and targeted surveillance are important tools for the early detection of infection in wildlife, and here we describe a framework for collating and synthesising emerging information to inform targeted surveillance for SARS-CoV-2 in wildlife. Surveillance efforts should be integrated with information from public and veterinary health initiatives to provide insights into the potential role of wild mammals in the epidemiology of SARS-CoV-2.

Cytokine Expression at Different Stages of Influenza A(H1N1)pdm09 Virus Infection in the Porcine Lung, Using Laser Capture Microdissection.

Pandemic influenza A(H1N1)pdm09 virus has retained its ability to infect swine whilst developing the ability to transmit effectively between humans, thus making the pig a valuable model for studying disease pathogenesis in both species. Lung lesions in pigs caused by infection with influenza A viruses vary in both their severity and distribution with individual lung lobes exhibiting lesions at different stages of infection pathogenic development and disease resolution. Consequently, investigating interactions between the virus and host and their implications for disease pathogenesis can be complicated. Studies were undertaken to investigate the discrete expression of pro- and anti-inflammatory mediators during lung lesion formation in pigs during infection with influenza A(H1N1)pdm09 (A/Hamburg/05/09) virus. Laser capture microdissection was used to identify and select lung lobules containing lesions at different stages of development. Dissected samples were analysed using quantitative RT-PCR to assess pro- and anti-inflammatory cytokine mRNA transcripts. Differential expression of the immune mediators IL-8, IL-10 and IFN-γ was observed depending upon the lesion stage assessed. Upregulation of IFN-γ, IL-8 and IL-10 mRNA was observed in stage 2 lesions, whereas decreased mRNA expression was observed in stage 3 lesions, with IL-8 actively downregulated when compared with controls in both stage 3 and stage 4 lesions. This study highlighted the value of using laser capture microdissection to isolate specific tissue regions and investigate subtle differences in cytokine mRNA expression during lesion development in pigs infected with influenza A(H1N1)pdm09.

Highly Pathogenic Avian Influenza H5N8 Clade 2.3.4.4 Virus: Equivocal Pathogenicity and Implications for Surveillance Following Natural Infection in Breeder Ducks in the United Kingdom.

Since early 2014, several outbreaks involving novel reassortant highly pathogenic avian influenza (HPAI) A(H5N8) viruses have been detected in poultry and wild bird species in Asia, Europe and North America. These viruses have been detected in apparently healthy and dead wild migratory birds, as well as in domestic chickens, turkeys, geese and ducks. In this study, we describe the pathology of an outbreak of H5N8 HPAIV in breeder ducks in the UK. A holding with approximately 6000 breeder ducks, aged approximately 60 weeks, showed a gradual reduction in egg production and increased mortality over a 7-day period. Post-mortem examination revealed frequent fibrinous peritonitis, with severely haemorrhagic ovarian follicles and occasional splenic and pancreatic necrosis and high incidence of mycotic granulomas in the air sacs and lung. Low-to-moderate levels of HPAI H5N8 virus were detected mainly in respiratory and digestive tract, with minor involvement of other organs. Although histopathological examination confirmed the gross pathology findings, intralesional viral antigen detection by immunohistochemistry was not observed. Immunolabelled cells were rarely only present in inflamed air sacs and serosa, usually superficial to granulomatous inflammation. Abundant bacterial microcolonies were observed in haemorrhagic ovaries and oviduct. The limited viral tissue distribution and presence of inter-current fungal and bacterial infections suggest a minor role for HPAIV H5N8 in clinical disease in layer ducks.

Pathobiology of rabies virus and the European bat lyssaviruses in experimentally infected mice.

A comparison of the clinicopathology of European bat lyssavirus (EBLV) types-1 and -2 and of rabies virus was undertaken. Following inoculation of mice at a peripheral site with these viruses, clinical signs of rabies and distribution of virus antigen in the mouse brain were examined. The appearance of clinical signs of disease varied both within and across the different virus species, with variation in incubation periods and weight loss throughout disease progression. The distribution of viral antigen throughout the regions of the brain examined was similar for each of the isolates during the different stages of disease progression, suggesting that antigen distribution was not associated with clinical presentation. However, specific regions of the brain including the cerebellum, caudal medulla, hypothalamus and thalamus, showed notable differences in the proportion of virus antigen positive cells present in comparison to other brain regions suggesting that these areas are important in disease development irrespective of virus species.

Pathology associated with a human case of rabies in the United Kingdom caused by European bat lyssavirus type-2.

Human infection with the rabies-related virus European bat lyssavirus type-2 (EBLV-2) has only been reported on two occasions. Here we report the pathology observed within spinal cord and visceral tissues associated with EBLV-2 infection for the first time. Neuronal labelling with an anti-rabies nucleocapsid monoclonal antibody was observed and appeared indistinguishable from the labelling reported from human infection with rabies virus.

Targeted surveillance for European bat lyssaviruses in English bats (2003-06).

In 2003-06, targeted (active) surveillance for European bat lyssaviruses (EBLVs) was undertaken throughout England, focusing on two species most likely to host these viruses, Myotis daubentonii and Eptesicus serotinus. Blood was sampled for the detection of EBLV-specific neutralizing antibodies and oropharyngeal swabs were taken for the detection of viral RNA or infectious virus in saliva. Between 2003 and 2006, 273 E. serotinus and 363 M. daubentonii blood samples were tested by the EBLV-1 or EBLV-2 specific modified fluorescent antibody neutralization test. The EBLV-2 antibody prevalence estimate was 1.0-4.1% (95% confidence interval [CI]; mean=2.2%) for M. daubentonii. European bat lyssavirus type 1-specific antibodies were detected only in a single E. serotinus. Other nontarget species (n=5) were sampled in small numbers (n=24), with no EBLV-specific antibody detected. No viral RNA or live virus was detected in any of the oropharyngeal swabs analyzed. Host RNA was detected from 83% of the oropharyngeal swabs analyzed (total swabs 2003-06: n=766). These data show that EBLV-2 is present in M. daubentonii in England. In contrast, there is insufficient evidence to suggest that EBLV-1 is present in E. serotinus in England, although further research is warranted.

Comparative pathological study of the murine brain after experimental infection with classical rabies virus and European bat lyssaviruses.

European bat lyssaviruses (EBLVs) types 1 (EBLV-1) and 2 (EBLV-2) cause rabies in terrestrial species, but the pathological changes associated with neuroinvasion have yet to be fully elucidated. Swiss OF-1 mice were inoculated peripherally with strain RV61 (classical rabies virus), RV1423 (EBLV-1) or RV1332 (EBLV-2) to compare the nature and extent of histopathological changes produced. Inoculated animals developed varying degrees of non-suppurative encephalitis, and lyssavirus infection was confirmed by the detection of viral antigen. The lesions produced, which included perivascular cuffs and gliosis, were more severe after RV1423 or RV1332 infection than after RV61 infection. Perivascular cuffs were mainly localized to caudal brain regions, irrespective of the infecting strain; after RV1332 infection, however, they were particularly abundant, being composed of large numbers of inflammatory cells. T cells were the predominant lymphocytic component of the inflammatory infiltrate in both the Virchow-Robin space and the brain parenchyma. Viral antigen, which was widespread throughout the brain, was apparently unrelated to the degree of cuffing. The study suggested that there was increased immune activation after inoculation with strain RV1423 or RV1332, particularly the latter, but that this did not affect the final outcome.

Inflammatory responses in the nervous system of mice infected with a street isolate of rabies virus.

Rabies virus causes severe encephalitis that is invariably fatal for the victim. However, the contribution of the virus and the host to damage of the CNS is unclear. In order to investigate this we studied the neuropathology and CNS gene expression patterns in a murine model of rabies using a 'street' isolate RV61. This virus was derived from a human case of disease. In this model, infection of the CNS progresses rapidly following inoculation in the periphery, leading to extensive virus replication in the brain and the development of disease. However, previous studies have found little evidence of inflammation and lymphocyte infiltration in many regions of the CNS of infected mice. During the current study virus replication was detected in the dorsal root ganglia (DRG), spinal cord, brain and salivary gland at 11 days postinfection (dpi). Mononuclear cell infiltration was observed in the DRG and to a lesser extent, the spinal cord. Immunolabelling demonstrated that T-lymphocytes were the dominant population of infiltrating cells. Murine innate immune response gene transcripts were detected in the brain as early as 5 dpi. At 11 dpi, coincidentwith the onset of disease, elevated levels of mRNA transcripts were recorded for type-1 (alpha and beta) and type-2 interferon (gamma) and certain chemokines (CCL5 and CXCL10) with chemotactic properties for T-cells. We suggest that damage to the DRG and spinal cord could be due to a combination of both virus infection and the infiltration of T-cells.

Experimental infection of foxes with European bat lyssaviruses type-1 and -2.

Experimental studies have been undertaken to assess the susceptibility of silver foxes to bat variants of rabies virus, namely European Bat Lyssaviruses (EBLVs). Both EBLV-1 and EBLV-2 have been isolated in European bats since 1954, in Eptesicus serotinus and Myotis species, respectively. Since 2000, the number of reported cases has increased largely due to the improvement of the surveillance of bat rabies virus throughout Europe. Although over >800 EBLVs cases have been reported in bats in Europe, EBLV-1 and -2 viruses are rarely reported to infect humans and terrestrial animals. The study presented here shows that the sensitivity of silver foxes is low when infected with EBLVs via the intramuscular route; in contrast all animals infected via intracranial inoculation succumbed to the experimental challenge. The mortality rate was 100% for both EBLV-1 (approximately 4.5 log) and EBLV-2 (approximately 3.0 log). This data suggests that the susceptibility of foxes to EBLV-1 and EBLV-2 is low and that the transmission (spillover) and adaptation of EBLVs from a bat to a fox may be theoretically possible but unlikely.

The application of genetic markers for EBLV surveillance in European bat species.

The United Kingdom has performed passive surveillance for European bat lyssaviruses (EBLVs) since 1987, and species-targeted surveillance since 2003. One critical component of these studies is the accurate identification of bats either submitted for testing or sampled in the field. Identification is dependent on numerous morphological characteristics. Whilst this is an effective means of bat identification, a number of problems remain with this approach. It relies on the experience of bat specialists and can lead to problems in differentiating members of the Myotis genus, particularly between Myotis mystacinus (whiskered bat) and Myotis brandtii (Brandt's bat), and between the most common species of the genus Pipistrellus. Furthermore, degradation of bats submitted for testing can also lead to problems in making an accurate species identification. Comparison of genetic sequence data could offer an alternative approach to differentiating bat species when morphological characterisation is not possible. Using tissue samples from UK resident bat species, sequence analysis of the mitochondrial DNA cytochrome b gene, and the beta-actin gene allowed for identification of many of the most common bat species in the UK, and genetic separation of two morphologically cryptic species. Application of this approach identified the species of a bat infected with EBLV-2 in Surrey as Myotis daubentoni (Daubenton's bat).

Passive surveillance (1987 to 2004) of United Kingdom bats for European bat lyssaviruses.

Passive surveillance for European bat lyssaviruses (eblvs) in the uk began in 1987, and between 1987 and 2004, 4,883 bats of European origin (4,871 belonging to 17 UK resident species and 12 belonging to seven non-uk resident species) were tested. The proportions and numbers of each species submitted from different regions varied considerably, partly owing to inherent biases in the passive surveillance, and there were seasonal variations in the numbers, sex and age of the bats. Contact with cats was reported in approximately 30 per cent of the bats submitted. Daubenton's bat (Myotis daubentonii) was the only species found to be positive for lyssavirus infection, with four cases of eblv type 2 identified, in 1996, 2002, 2003 and 2004. No active infection with eblv type 1 was recorded.

Occupational lyssavirus risks and post-vaccination monitoring.

In the UK, rabies pre-exposure vaccination involves a 3-dose course (DO, 7 & 28) and reinforcing doses at a 2-3 year intervals. This booster schedule had been implemented following scientific evidence indicating that a reduction in the previous regime interval of 3-5 years was warranted. The regime changes were particularly relevant to high risk groups that may encounter rabies virus. Those at known high risk of exposure to rabies and other Lyssaviruses include laboratory staff and a diverse group of animal handlers. Since the detection of EBLV-2 in the UK in four Daubenton's bats between 1996-2004 and a human case in 2002, those who now come into contact with bats are also considered at risk. Prior to 2002, data indicated that less than 50 % (n = 193) of all bat handlers had been vaccinated against rabies and 25 % of this group had exceeded the recommended booster interval. Following the human fatality, the vaccine became free to all UK bat handlers and all licensed and the majority of unlicensed bat handlers were vaccinated. Vaccinated laboratory workers are serologically monitored and boosted following a blood test of < 1.0 IU/ml of neutralising antibodies (FAVN-CVS). An analysis of laboratory staff (n = 25) indicated that 28 % (n = 7) maintained a suitable antibody level for greater than 5 years after their final boost, but 16 % (n = 4) had a titre of 1.0 IU/ml for < 12 months. Of the 25 individuals, 70 % required a booster in 2.5 years. In 2002, internal VLA policy was amended to increase antibody monitoring from 2 to 4 times per annum and to insist that all laboratory workers maintained a RABV titre of > 5.0 IU/ml for those in contact with non-RABV lyssaviruses in order to ensure that all individuals had antibody levels capable of neutralising the phylogroup I lyssaviruses.

Ability of rabies vaccine strains to elicit cross-neutralising antibodies.

Two European Bat Lyssaviruses (EBLV-1 and EBLV-2) have been identified (n > 750 cases) in European bat species. In addition, EBLVs have been detected as "spillover" cases in three humans, one stone marten and four sheep. A further concern is the interaction of companion animals with infected bats and the possibility of subsequent lyssavirus infection. Cat-bat interactions represent approximately 32 % (n = 398) of passive lyssavirus surveillance submissions at the Veterinary Laboratories Agency (VLA) (1987-2004). Cats therefore represent a potentially significant spillover host. This study evaluated the ability of rabies vaccine antibodies to neutralise EBLVs using modified fluorescent antibody virus neutralisation (FAVN) assays (EBLV-1, EBLV-2). We examined vaccinated human, dog and cat sera in two classes; (i) FAVN-CVS titres > or = 0.5-5.0 IU/ml (n = 34, 79 and 54, respectively), and (ii) > 5.0 IU/ml (n = 22, 21 and 32 respectively). Most sera (approximately 80 %) with higher titres were able to neutralise both EBLV-1 and -2 regardless of the vaccine received. Only a proportion of those with low titres were capable of neutralising either EBLV-1 (27-92 %) or EBLV-2 (38-92 %) or both EBLVs (27-79 %). The cat sera constitute the lower end of each range. More animals that had received LEP based vaccines were able to neutralise EBLVs than those that had received PV based vaccines. The continuing occurrence of non-classical lyssaviruses in Europe emphasizes the need for continual surveillance of bats and other species.

EBLV-2 prevalence in the United Kingdom as determined by surveillance testing.

Five cases of EBLV-2 have been detected in the UK since 1996, with all wildlife cases in the Daubenton's bat: one on the south coast in Sussex in 1996, one in Lancashire in 2002, another in 2003, one in Surrey in 2004 and a human fatality in Angus, Scotland, in 2002. As a result of the human case, a seroprevalence study, aimed primarily at the Daubenton's bat was conducted in 2003 in Scotland and at some sites in England. In Scotland, 198 Daubenton's, 20 Natterer's and 6 pipistrelles were caught at 19 sites and analysed, while in England 67 Daubenton, 2 Brandts/ Whiskered and 4 pipistrelle bats were analysed from four sites in Lancashire. Analysis of blood was performed by a modified fluorescent antibody virus neutralisation test (mFAVN) to determine antibody titre to EBLV-2. Ignoring those sites where we had a priori reason to expect infected bats, the overall seroprevalence was between 0.7-5.1 % (95 % confidence interval), with a maximum likelihood estimate of 2.2 %. Mouth swabs were taken and tested for virus genome by RT-PCR and live virus by tissue culture isolation. All of the PCR and isolation results were negative suggesting that none of the bats sampled were actively excreting virus. This suggests a low level of active infection in Britain and raises the possibility that bats may recover following exposure to EBLV-2.

European bat lyssaviruses: Distribution, prevalence and implications for conservation.

Worldwide, there are more than 1100 species of the Order Chiroptera, 45 of which are present in Europe, and 16 in the UK. Bats are reservoirs of, or can be infected by, several viral diseases, including rabies virus strains (in the Lyssavirus genus). Within this genus are bat variants that have been recorded in Europe; European bat lyssavirus 1 (EBLV-1), European bat lyssavirus 2 (EBLV-2) and, four currently unclassified isolates. Since 1977, 783 cases of EBLVs (by isolation of viral RNA) have been recorded in Europe. EBLV-1 or EBLV-2 has been identified in 12 bat species, with over 95% of EBLV-1 infections identified in Eptesicus serotinus. EBLV-2 is associated with Myotis species (Myotis daubentonii and Myotis dasycneme). A programme of passive surveillance in the United Kingdom between 1987 and 2004 tested 4871 bats for lyssaviruses. Of these, four M. daubentonii (3.57% of submitted M. daubentonii) were positive for EBLV-2. Potential bias in the passive surveillance includes possible over-representation of synanthropic species and regional biases caused by varying bat submission numbers from different parts of the UK. In 2003, active surveillance in the UK began, and has detected an antibody prevalence level of 1-5% of EBLV-2 in M. daubentonii (n = 350), and one bat with antibodies to EBLV-1 in E. serotinus (n = 52). No cases of live lyssavirus infection or lyssavirus viral RNA have been detected through active surveillance. Further research and monitoring regarding prevalence, transmission, pathogenesis and immunity is required to ensure that integrated bat conservation continues throughout Europe, whilst enabling informed policy decision regarding both human and wildlife health issues.

Rabies human diploid cell vaccine elicits cross-neutralising and cross-protecting immune responses against European and Australian bat lyssaviruses.

The ability of antibodies elicited against the rabies human diploid cell vaccine (HDCV) to neutralise European bat Lyssaviruses (EBLV types-1 and -2), Australian bat Lyssavirus and classical rabies virus (RABV) has been evaluated using modified fluorescent antibody virus neutralisation (mFAVN) assays. Ninety-six percent (48 of 50) of the human post-vaccinated sera tested cross-neutralised these viruses (>or=0.5 IU/ml). Cross-protection experiments using inbred mice (RIII, k/k haplotype) were also assessed. Mice were given HDCV (twice by the intra-peritoneal route) and challenged (intra-cranially or peripherally) with a lethal dose (25 MLD(50)) of the individual viruses. The vaccine conferred statistically significant protection in 80--100% of animals challenged via the peripheral route. Levels of protection were lower following intra-cranial (i.c.) challenge. Our data provides strong evidence for broad spectrum cross-neutralisation and cross-protection of phylogroup I lyssaviruses using rabies HDCV.

Detection and strain differentiation of European bat lyssaviruses using in situ hybridisation.

A protocol suitable for the detection of rabies virus and the related European bat lyssaviruses type 1 and 2 is described. In situ hybridisation, employing digoxigenin labelled riboprobes was used for the detection of lyssavirus RNA in mouse-infected brain tissue. The principal advantage of this technique, compared to routine methods used for histopathology, is that this method is robust, highly sensitive, and specific for assessing the presence of RNA in different tissues. An additional advantage is that there is no longer any requirement for high laboratory bio-containment, once the tissue under investigation has been safely fixed. Using this method, both genomic and messenger RNA were detected. The ability to detect messenger RNA is indicative of the presence of replicating virus and therefore, this technique is a powerful diagnostic tool for the routine detection of strains of rabies virus including the European bat lyssaviruses.

Isolation of a European bat lyssavirus type 2 from a Daubenton's bat in the United Kingdom.

European bat lyssavirus type 2 (EBLV-2) has been isolated once previously from a bat in the UK in June 1996. In September 2002, a Daubenton's bat (Myotis daubentonii) found in Lancashire developed abnormal behaviour, including unprovoked aggression, while it was in captivity. Brain samples from the bat were tested for virus of the Lyssavirus genus, which includes EBLV-2 (genotype 6), and classical rabies virus (genotype 1). A positive fluorescent antibody test confirmed that it was infected with a lyssavirus, and PCR and genomic sequencing identified the virus as an EBLV-2a. Phylogenetic comparisons with all the published sequences from genotype 6 showed that it was closely related to the previous isolate of EBLV-2 in the UK and suggested links to isolates from bats in The Netherlands. The isolation of EBLV-2 from a bat found on the west coast of England provides evidence that this virus may be present within the UK Daubenton's bat population at a low prevalence level.