Extension of the known distribution of a novel clade C betacoronavirus in a wildlife host.

Disease surveillance in wildlife populations presents a logistical challenge, yet is critical in gaining a deeper understanding of the presence and impact of wildlife pathogens. Erinaceus coronavirus (EriCoV), a clade C Betacoronavirus, was first described in Western European hedgehogs (Erinaceus europaeus) in Germany. Here, our objective was to determine whether EriCoV is present, and if it is associated with disease, in Great Britain (GB). An EriCoV-specific BRYT-Green® real-time reverse transcription PCR assay was used to test 351 samples of faeces or distal large intestinal tract contents collected from casualty or dead hedgehogs from a wide area across GB. Viral RNA was detected in 10.8% (38) samples; however, the virus was not detected in any of the 61 samples tested from Scotland. The full genome sequence of the British EriCoV strain was determined using next generation sequencing; it shared 94% identity with a German EriCoV sequence. Multivariate statistical models using hedgehog case history data, faecal specimen descriptions and post-mortem examination findings found no significant associations indicative of disease associated with EriCoV in hedgehogs. These findings indicate that the Western European hedgehog is a reservoir host of EriCoV in the absence of apparent disease.

Antigenic site changes in the rabies virus glycoprotein dictates functionality and neutralizing capability against divergent lyssaviruses.

Lyssavirus infection has a near 100 % case fatality rate following the onset of clinical disease, and current rabies vaccines confer protection against all reported phylogroup I lyssaviruses. However, there is little or no protection against more divergent lyssaviruses and so investigation into epitopes within the glycoprotein (G) that dictate a neutralizing response against divergent lyssaviruses is warranted. Importantly, the facilities required to work with these pathogens, including wild-type and mutated forms of different lyssaviruses, are scarcely available and, as such, this type of study is inherently difficult to perform. The relevance of proposed immunogenic antigenic sites within the lyssavirus glycoprotein was assessed by swapping sites between phylogroup-I and -II glycoproteins. Demonstrable intra- but limited inter-phylogroup cross-neutralization was observed. Pseudotype viruses (PTVs) presenting a phylogroup-I glycoprotein containing phylogroup-II antigenic sites (I, II III or IV) were neutralized by antibodies raised against phylogroup-II PTV with the site II (IIb, aa 34-42 and IIa, aa 198-200)-swapped PTVs being efficiently neutralized, whilst site IV-swapped PTV was poorly neutralized. Specific antibodies raised against PTV-containing antigenic site swaps between phylogroup-I and -II glycoproteins neutralized phylogroup-I PTVs efficiently, indicating an immunodominance of antigenic site II. Live lyssaviruses containing antigenic site-swapped glycoproteins were generated and indicated that specific residues within the lyssavirus glycoprotein dictate functionality and enable differential neutralizing antibody responses to lyssaviruses.

Assessing the impact of public education on a preventable zoonotic disease: rabies.

Effective methods to increase awareness of preventable infectious diseases are key components of successful control programmes. Rabies is an example of a disease with significant impact, where public awareness is variable. A recent awareness campaign in a rabies endemic region of Azerbaijan provided a unique opportunity to assess the efficacy of such campaigns. A cluster cross-sectional survey concerning rabies was undertaken following the awareness campaign in 600 households in 38 randomly selected towns, in districts covered by the campaign and matched control regions. This survey demonstrated that the relatively simple awareness campaign was effective at improving knowledge of rabies symptoms and vaccination schedules. Crucially, those in the awareness campaign group were also 1·4 times more likely to report that they had vaccinated their pets, an essential component of human rabies prevention. In addition, low knowledge of appropriate post-exposure treatment and animal sources of rabies provide information useful for future public awareness campaigns in the region and other similar areas.

Passive surveillance of United Kingdom bats for lyssaviruses (2005-2015).

Passive surveillance for lyssaviruses in UK bats has been ongoing since 1987 and has identified 13 cases of EBLV-2 from a single species; Myotis daubentonii. No other lyssavirus species has been detected. Between 2005 and 2015, 10 656 bats were submitted, representing 18 species, creating a spatially and temporally uneven sample of British bat fauna. Uniquely, three UK cases originate from a roost at Stokesay Castle in Shropshire, England, where daily checks for grounded and dead bats are undertaken and bat carcasses have been submitted for testing since 2007. Twenty per cent of Daubenton's bats submitted from Stokesay Castle since surveillance began, have tested positive for EBLV-2. Phylogenetic analysis reveals geographical clustering of UK viruses. Isolates from Stokesay Castle are more closely related to one another than to viruses from other regions. Daubenton's bats from Stokesay Castle represent a unique opportunity to study a natural population that appears to maintain EBLV-2 infection and may represent endemic infection at this site. Although the risk to public health from EBLV-2 is low, consequences of infection are severe and effective communication on the need for prompt post-exposure prophylaxis for anyone that has been bitten by a bat is essential.

First Complete Genomic Sequence of a Rabies Virus from the Republic of Tajikistan Obtained Directly from a Flinders Technology Associates Card.

A brain homogenate derived from a rabid dog in the district of Tojikobod, Republic of Tajikistan, was applied to a Flinders Technology Associates (FTA) card. A full-genome sequence of rabies virus (RABV) was generated from the FTA card directly without extraction, demonstrating the utility of these cards for readily obtaining genetic data.

Pathobiological investigation of naturally infected canine rabies cases from Sri Lanka.

BACKGROUND: The recommended screening of rabies in 'suspect' animal cases involves testing fresh brain tissue. The preservation of fresh tissue however can be difficult under field conditions and formalin fixation provides a simple alternative that may allow a confirmatory diagnosis. The occurrence and location of histopathological changes and immunohistochemical (IHC) labelling for rabies in formalin fixed paraffin embedded (FFPE) canine brain is described in samples from 57 rabies suspect cases from Sri-Lanka. The presence of Negri bodies and immunohistochemical detection of rabies virus antigen were evaluated in the cortex, hippocampus, cerebellum and brainstem. The effect of autolysis and artefactual degeneration of the tissue was also assessed. RESULTS: Rabies was confirmed in 53 of 57 (93%) cases by IHC. IHC labelling was statistically more abundant in the brainstem. Negri bodies were observed in 32 of 53 (60.4%) of the positive cases. Although tissue degradation had no effect on IHC diagnosis, it was associated with an inability to detect Negri bodies. In 13 cases, a confirmatory Polymerase chain reaction (PCR) testing for rabies virus RNA was undertaken by extracting RNA from fresh frozen tissue, and also attempted using FFPE samples. PCR detection using fresh frozen samples was in agreement with the IHC results. The PCR method from FFPE tissues was suitable for control material but unsuccessful in our field cases. CONCLUSIONS: Histopathological examination of the brain is essential to define the differential diagnoses of behaviour modifying conditions in rabies virus negative cases, but it is unreliable as the sole method for rabies diagnosis, particularly where artefactual change has occurred. Formalin fixation and paraffin embedding does not prevent detection of rabies virus via IHC labelling even where artefactual degeneration has occurred. This could represent a pragmatic secondary assay for rabies diagnosis in the field because formalin fixation can prevent sample degeneration. The brain stem was shown to be the site with most viral immunoreactivity; supporting recommended sampling protocols in favour of improved necropsy safety in the field. PCR testing of formalin fixed tissue may be successful in certain circumstances as an alternative test.

Free-Roaming Dogs in Nepal: Demographics, Health and Public Knowledge, Attitudes and Practices.

In Nepal, most dogs are free to roam and may transmit diseases to humans and animals. These dogs often suffer from malnutrition and lack basic health care. Minimal information is available about their demographics and about public attitudes concerning dogs and diseases. We carried out a study in Chitwan District (central Nepal), to collect baseline data on free-roaming owned dog demographics, assess knowledge, attitudes and practices of dog owners concerning dogs and rabies, evaluate rabies vaccination coverage and anthelmintic treatment of dogs, measure dogs' response to rabies vaccination and assess dog health through body condition scores and parasites. We conducted household interviews with owners of free-roaming female dogs (n = 60) and administered dogs with rabies vaccination and anthelmintics. Dog owners regularly fed free-roaming dogs but provided minimal health care; 42% of respondents did not claim ownership of the dog for which they provided care. We collected skin, faecal and blood samples for parasite identification and for measuring rabies virus-specific antibodies. Ninety-two per cent of dog owners were aware of the routes of rabies virus transmission, but only 35% described the correct post-exposure prophylaxis (PEP) following a dog bite. Twenty-seven per cent of the dogs had measurable rabies virus-specific antibody titres and 14% had received anthelmintics in the previous year. Following rabies vaccination, 97% of dogs maintained an adequate antibody titre for ≥6 months. Most dogs appeared healthy, although haemoprotozoans, endoparasites and ectoparasites were identified in 12%, 73% and 40% of the dogs, respectively. Poor skin condition and parasite load were associated. Seventy-four per cent of the females had litters in 1 year (mean litter size = 4.5). Births occurred between September and February; we estimated 60% mortality in puppies. We concluded that vaccination coverage, PEP awareness and anthelmintic treatment should be emphasized in educational programmes focussed on animal welfare, veterinary and public health.

Effective vaccination against rabies in puppies in rabies endemic regions.

In rabies endemic regions, a proportionally higher incidence of rabies is often reported in dogs younger than 12 months of age, which includes puppies less than 3 months of age; this presents a serious risk to public health. The higher incidence of rabies in young dogs may be the effect of low vaccination coverage in this age class, partly as a result of the perception that immature immune systems and maternal antibodies inhibit seroconversion to rabies vaccine in puppies less than three months of age. Therefore, to test this perception, the authors report the virus neutralising antibody titres from 27 dogs that were vaccinated with high quality, inactivated rabies vaccine aged three months of age and under as part of larger serological studies undertaken in Gauteng Province, South Africa, and the Serengeti District, Tanzania. All of these dogs seroconverted to a single dose of vaccine with no adverse reactions reported and with postvaccinal peak titres ranging from 2.0 IU/ml to 90.5 IU/ml. In light of these results, and the risk of human beings contracting rabies from close contact with puppies, the authors recommend that all dogs in rabies endemic regions, including those less than three months of age, are vaccinated with high quality, inactivated vaccine.

Epidemiological characteristics of human and animal rabies in Azerbaijan.

The Caucasus is a region of geopolitical importance, in the gateway between Europe and Asia. This geographical location makes the region equally important in the epidemiology and control of transboundary infectious diseases such as rabies. Azerbaijan is the largest country in the Caucasus, and although rabies is notifiable and considered endemic, there is little information on the burden of human and animal rabies. Here, we describe a cross-disciplinary international collaboration aimed at improving rabies control in Azerbaijan. Partial nucleoprotein gene sequences were obtained from animal rabies cases for comparison with those from surrounding areas. Reported human and animal rabies cases between 2000 and 2010 were also reviewed and analysed by region and year. Comparison of rabies virus strains circulating in Azerbaijan demonstrates more than one lineage of rabies virus circulating concurrently in Azerbaijan and illustrates the need for further sample collection and characterization. Officially reported rabies data showed an increase in human and animal rabies cases, and an increase in animal bites requiring provision of post-exposure prophylaxis, since 2006. This is despite apparently consistent levels of dog vaccination and culling of stray dogs.

Antigenic variation of foot-and-mouth disease virus serotype A.

The current measures to control foot-and-mouth disease (FMD) include vaccination, movement control and slaughter of infected or susceptible animals. One of the difficulties in controlling FMD by vaccination arises due to the substantial diversity found among the seven serotypes of FMD virus (FMDV) and the strains within these serotypes. Therefore, vaccination using a single vaccine strain may not fully cross-protect against all strains within that serotype, and therefore selection of appropriate vaccines requires serological comparison of the field virus and potential vaccine viruses using relationship coefficients (r1 values). Limitations of this approach are that antigenic relationships among field viruses are not addressed, as comparisons are only with potential vaccine virus. Furthermore, inherent variation among vaccine sera may impair reproducibility of one-way relationship scores. Here, we used antigenic cartography to quantify and visualize the antigenic relationships among FMD serotype A viruses, aiming to improve the understanding of FMDV antigenic evolution and the scope and reliability of vaccine matching. Our results suggest that predicting antigenic difference using genetic sequence alone or by geographical location is not currently reliable. We found co-circulating lineages in one region that were genetically similar but antigenically distinct. Nevertheless, by comparing antigenic distances measured from the antigenic maps with the full capsid (P1) sequence, we identified a specific amino acid substitution associated with an antigenic mismatch among field viruses and a commonly used prototype vaccine strain, A22/IRQ/24/64.

Deciphering serology to understand the ecology of infectious diseases in wildlife.

The ecology of infectious disease in wildlife has become a pivotal theme in animal and public health. Studies of infectious disease ecology rely on robust surveillance of pathogens in reservoir hosts, often based on serology, which is the detection of specific antibodies in the blood and is used to infer infection history. However, serological data can be inaccurate for inference to infection history for a variety of reasons. Two major aspects in any serological test can substantially impact results and interpretation of antibody prevalence data: cross-reactivity and cut-off thresholds used to discriminate positive and negative reactions. Given the ubiquitous use of serology as a tool for surveillance and epidemiological modeling of wildlife diseases, it is imperative to consider the strengths and limitations of serological test methodologies and interpretation of results, particularly when using data that may affect management and policy for the prevention and control of infectious diseases in wildlife. Greater consideration of population age structure and cohort representation, serological test suitability and standardized sample collection protocols can ensure that reliable data are obtained for downstream modeling applications to characterize, and evaluate interventions for, wildlife disease systems.

Comparative assay of fluorescent antibody test results among twelve European National Reference Laboratories using various anti-rabies conjugates.

Twelve National Reference Laboratories (NRLs) for rabies have undertaken a comparative assay to assess the comparison of fluorescent antibody test (FAT) results using five coded commercial anti-rabies conjugates (Biorad, Bioveta, Fujirebio, Millipore, and SIFIN conjugates). Homogenized positive brain tissues infected with various lyssavirus species as well as negative samples were analyzed blindly using a standardized FAT procedure. Conjugates B, C, D, and E were found to be significantly more effective than conjugate A for GS7 (French RABV) diluted samples (1/8 and 1/100) while the frequency of concordant results of conjugates C and D differ significantly from conjugates A, B and E for CVS 27. For detection of EBLV-1 strains, conjugates C and D also presented a significantly lower frequency of discordant results compared to conjugates A, B and E. Conjugates B, C and D were found to be significantly more effective than conjugates E and A for EBLV-2 and ABLV samples. In view of these results, conjugates C and D set themselves apart from the others and appeared as the most effective of this 5-panel conjugates. This study clearly demonstrates that the variability of conjugates used by National Reference Laboratories can potentially lead to discordant results and influence assay sensitivity. In case of false negative results this could have a dramatic impact if the animal under investigation is responsible for human exposure. To avoid such situations, confirmatory tests should be implemented.

Molecular epidemiology of bat lyssaviruses in Europe.

Bat rabies cases in Europe are principally attributed to two lyssaviruses, namely European bat lyssavirus type 1 (EBLV-1) and European bat lyssavirus type 2 (EBLV-2). Between 1977 and 2011, 961 cases of bat rabies were reported to Rabies Bulletin Europe, with the vast majority (>97%) being attributed to EBLV-1. There have been 25 suspected cases of EBLV-2, of which 22 have been confirmed. In addition, two single isolations of unique lyssaviruses from European insectivorous bats were reported in south-west Russia in 2002 (West Caucasian bat virus) and in Germany in 2010 (Bokeloh bat lyssavirus). In this review, we present phylogenetic analyses of the EBLV-1 and EBLV-2 using partial nucleoprotein (N) gene sequences. In particular, we have analysed all EBLV-2 cases for which viral sequences (N gene, 400 nucleotides) are available (n = 21). Oropharyngeal swabs collected from two healthy Myotis daubentonii during active surveillance programmes in Scotland and Switzerland also yielded viral RNA (EBLV-2). Despite the relatively low number of EBLV-2 cases, a surprisingly large amount of anomalous data has been published in the scientific literature and Genbank, which we have collated and clarified. For both viruses, geographical relationships are clearly defined on the phylogenetic analysis. Whilst there is no clear chronological clustering for either virus, there is some evidence for host specific relationships, particularly for EBLV-1 where more host variation has been observed. Further genomic regions must be studied, in particular for EBLV-1 isolates from Spain and the EBLV-2 isolates to provide support for the existence of sublineages.

Epidemiological perspectives on West Nile virus surveillance in wild birds in Great Britain.

West Nile virus (WNV) is a zoonotic arthropod-borne pathogen with continued geographical expansion in Europe. We present and evaluate data on the temporal, spatial and bird species focus of the WNV surveillance programme in dead wild birds in Great Britain (2002-2009). During this period all bird samples tested negative for WNV. Eighty-two per cent of the 2072 submissions occurred during the peak period of vector activity with 53% tested during April-July before human and equine infection would be expected. Samples were received from every county, but there was significant geographical clustering (nearest neighbour index=0·23, P<0·001). Over 240 species were represented, with surveillance more likely to detect WNV in resident bird species (92% of submissions) than migrants (8%). Evidence indicates that widespread avian mortality is not generally a reported feature of WNV in Europe and hence additional activities other than dead bird surveillance may maximize the ability to detect WNV circulation before the onset of human and equine infections.

Integrating the landscape epidemiology and genetics of RNA viruses: rabies in domestic dogs as a model.

Landscape epidemiology and landscape genetics combine advances in molecular techniques, spatial analyses and epidemiological models to generate a more real-world understanding of infectious disease dynamics and provide powerful new tools for the study of RNA viruses. Using dog rabies as a model we have identified how key questions regarding viral spread and persistence can be addressed using a combination of these techniques. In contrast to wildlife rabies, investigations into the landscape epidemiology of domestic dog rabies requires more detailed assessment of the role of humans in disease spread, including the incorporation of anthropogenic landscape features, human movements and socio-cultural factors into spatial models. In particular, identifying and quantifying the influence of anthropogenic features on pathogen spread and measuring the permeability of dispersal barriers are important considerations for planning control strategies, and may differ according to cultural, social and geographical variation across countries or continents. Challenges for dog rabies research include the development of metapopulation models and transmission networks using genetic information to uncover potential source/sink dynamics and identify the main routes of viral dissemination. Information generated from a landscape genetics approach will facilitate spatially strategic control programmes that accommodate for heterogeneities in the landscape and therefore utilise resources in the most cost-effective way. This can include the efficient placement of vaccine barriers, surveillance points and adaptive management for large-scale control programmes.

A quantitative release assessment for the noncommercial movement of companion animals: risk of rabies reintroduction to the United kingdom.

In 2004, the European Union (EU) implemented a pet movement policy (referred to here as the EUPMP) under EU regulation 998/2003. The United Kingdom (UK) was granted a temporary derogation from the policy until December 2011 and instead has in place its own Pet Movement Policy (Pet Travel Scheme (PETS)). A quantitative risk assessment (QRA) was developed to estimate the risk of rabies introduction to the UK under both schemes to quantify any change in the risk of rabies introduction should the UK harmonize with the EU policy. Assuming 100 % compliance with the regulations, moving to the EUPMP was predicted to increase the annual risk of rabies introduction to the UK by approximately 60-fold, from 7.79 × 10(-5) (5.90 × 10(-5), 1.06 × 10(-4)) under the current scheme to 4.79 × 10(-3) (4.05 × 10(-3), 5.65 × 10(-3)) under the EUPMP. This corresponds to a decrease from 13,272 (9,408, 16,940) to 211 (177, 247) years between rabies introductions. The risks associated with both the schemes were predicted to increase when less than 100 % compliance was assumed, with the current scheme of PETS and quarantine being shown to be particularly sensitive to noncompliance. The results of this risk assessment, along with other evidence, formed a scientific evidence base to inform policy decision with respect to companion animal movement.

Endemic Lagos bat virus infection in Eidolon helvum.

Phylogenetic analyses suggest lyssaviruses, including Rabies virus, originated from bats. However, the role of bats in the maintenance, transmission and evolution of lyssaviruses is poorly understood. A number of genetically diverse lyssaviruses are present in Africa, including Lagos bat virus (LBV). A high seroprevalence of antibodies against LBV was detected in Eidolon helvum bats. Longitudinal seroprevalence and age-specific seroprevalence data were analysed and capture-mark-recapture (CMR) analysis used to follow 98 bats over 18 months. These data demonstrate endemic infection, with evidence of horizontal transmission, and force of infection was estimated for differing age categories. The CMR analysis found survival probabilities of seronegative and seropositive bats were not significantly different. The lack of increased mortality in seropositive animals suggests infection is not causing disease after extended incubation. These key findings point towards acute transmission of bat lyssaviruses in adapted bat hosts that occurs at a far higher rate than the occurrence of disease.

Antigenic and genetic evolution of equine influenza A (H3N8) virus from 1968 to 2007.

Equine influenza virus is a major respiratory pathogen in horses, and outbreaks of disease often lead to substantial disruption to and economic losses for equestrian industries. The hemagglutinin (HA) protein is of key importance in the control of equine influenza because HA is the primary target of the protective immune response and the main component of currently licensed influenza vaccines. However, the influenza virus HA protein changes over time, a process called antigenic drift, and vaccine strains must be updated to remain effective. Antigenic drift is assessed primarily by the hemagglutination inhibition (HI) assay. We have generated HI assay data for equine influenza A (H3N8) viruses isolated between 1968 and 2007 and have used antigenic cartography to quantify antigenic differences among the isolates. The antigenic evolution of equine influenza viruses during this period was clustered: from 1968 to 1988, all isolates formed a single antigenic cluster, which then split into two cocirculating clusters in 1989, and then a third cocirculating cluster appeared in 2003. Viruses from all three clusters were isolated in 2007. In one of the three clusters, we show evidence of antigenic drift away from the vaccine strain over time. We determined that a single amino acid substitution was likely responsible for the antigenic differences among clusters.

Laboratory virulence and orchard efficacy of entomopathogenic nematodes against the lesser peachtree borer (Lepidoptera: Sesiidae).

The lesser peachtree borer, Synanthedon pictipes (Grote & Robinson) (Lepidoptera: Sesiidae), is indigenous to eastern North America. It is a pest of commercially grown Prunus spp., especially to southeastern peach orchards where earlier regulatory changes affected pesticide use on peach leading to increased S. pictipes damage. Pest management practices are now having a positive effect toward control of this pest, but cost-competitive biological control solutions that promote environmental stewardship are needed. Here, we tested four Steinernema species and five Heterorhabditis species of entomopathogenic nematodes against larval S. pictipes. Included were four strains of S. carpocapsae (All, DD136, Sal, and Hybrid2) and three strains of S. riobrave (3-8b, 7-12, and 355). Larvae treated with any strain of S. carpocapsae always resulted in <20% survival, whereas larval survival was always >50% when treated with any other Steinernema or Heterorhabditis spp. These differences were always significant for the Hybrid2 strain of S. carpocapsae and similarly for other tested S. carpocapsae strains except for when larvae were treated with the 3-8b strain of S. riobrave. In addition, we determined the susceptibility of different size S. pictipes larvae, because they occur simultaneously in orchards, and we found that larvae rated as "medium" and "large" were significantly more susceptible than "small" larvae. Last, we demonstrated that moisture-retaining covers (placed over S. pictipes-infested wounds on peach limbs) increased efficacy of nematode treatments against larval S. pictipes. Even when using highly virulent nematodes against S. pictipes, it is likely that an aboveground application will require an environmental modification to remain efficacious.