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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.

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.

The U.K. has not yet experienced a confirmed outbreak of mosquito-borne virus transmission to people or livestock despite numerous autochthonous epizootic and human outbreaks of mosquito-borne diseases on the European mainland. Indeed, whether or not British mosquitoes are competent to transmit arboviruses has not been established. Therefore, the competence of a local (temperate) British mosquito species, Ochlerotatus detritus (=Aedes detritus) (Diptera: Culicidae) for transmission of a member of the genus Flavivirus, Japanese encephalitis virus (JEV) as a model for mosquito-borne virus transmission was assessed. The JEV competence in a laboratory strain of Culex quinquefasciatus (Diptera: Culicidae), a previously incriminated JEV vector, was also evaluated as a positive control. Ochlerotatus detritus adults were reared from field-collected juvenile stages. In oral infection bioassays, adult females developed disseminated infections and were able to transmit virus as determined by the isolation of virus in saliva secretions. When pooled at 7-21days post-infection, 13% and 25% of O.detritus were able to transmit JEV when held at 23 degrees C and 28 degrees C, respectively. Similar results were obtained for C.quinquefasciatus. To our knowledge, this study is the first to demonstrate that a British mosquito species, O.detritus, is a potential vector of an exotic flavivirus.

Summary 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.

Rabies kills many people throughout the developing world every year. The murine monoclonal antibody (mAb) 62-71-3 was recently identified for its potential application in rabies postexposure prophylaxis (PEP). The purpose here was to establish a plant-based production system for a chimeric mouse-human version of mAb 62-71-3, to characterize the recombinant antibody and investigate at a molecular level its interaction with rabies virus glycoprotein. Chimeric 62-71-3 was successfully expressed in Nicotiana benthamiana. Glycosylation was analyzed by mass spectroscopy; functionality was confirmed by antigen ELISA, as well as rabies and pseudotype virus neutralization. Epitope characterization was performed using pseudotype virus expressing mutagenized rabies glycoproteins. Purified mAb demonstrated potent viral neutralization at 500 IU/mg. A critical role for antigenic site I of the glycoprotein, as well as for two specific amino acid residues (K226 and G229) within site I, was identified with regard to mAb 62-71-3 neutralization. Pseudotype viruses expressing glycoprotein from lyssaviruses known not to be neutralized by this antibody were the controls. The results provide the molecular rationale for developing 62-71-3 mAb for rabies PEP; they also establish the basis for developing an inexpensive plant-based antibody product to benefit low-income families in developing countries.—Both, L., van Dolleweerd, C., Wright, E., Banyard, A. C., Bulmer-Thomas, B., Selden, D., Altmann, F., Fooks, A. R., Ma, J. K.-C. Production, characterization, and antigen specificity of recombinant 62-71-3, a candidate monoclonal antibody for rabies prophylaxis in humans.

Rabies is the oldest known zoonotic disease and was also the first recognized bat associated infection in humans. To date, four different lyssavirus species are the causative agents of rabies in European bats: the European Bat Lyssaviruses type 1 and 2 (EBLV-1, EBLV-2), the recently discovered putative new lyssavirus species Bokeloh Bat Lyssavirus (BBLV) and the West Caucasian Bat Virus (WCBV). Unlike in the new world, bat rabies cases in Europe are comparatively less frequent, possibly as a result of varying intensity of surveillance. Thus, the objective was to provide an assessment of the bat rabies surveillance data in Europe, taking both reported data to the WHO Rabies Bulletin Europe and published results into account. In Europe, 959 bat rabies cases were reported to the RBE in the time period 1977-2010 with the vast majority characterized as EBLV-1, frequently isolated in the Netherlands, North Germany, Denmark, Poland and also in parts of France and Spain. Most EBLV-2 isolates originated from the United Kingdom (UK) and the Netherlands, and EBLV-2 was also detected in Germany, Finland and Switzerland. Thus far, only one isolate of BBLV was found in Germany. Published passive bat rabies surveillance comprised testing of 28 of the 52 different European bat species for rabies. EBLV-1 was isolated exclusively from Serotine bats (Eptesicus serotinus and Eptesicus isabellinus), while EBLV-2 was detected in 14 Daubenton's bats (Myotis daubentonii) and 5 Pond bats (Myotis dasycneme). A virus from a single Natterer's bat (Myotis nattereri) was characterized as BBLV. During active surveillance, only oral swabs from 2 Daubenton's bats (EBLV-2) and from several Eptesicus bats (EBLV-1) yielded virus positive RNA. Virus neutralizing antibodies against lyssaviruses were detected in various European bat species from different countries, and its value and implications are discussed.

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.

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.

Two common bat lyssavirus species have been identified in many European countries: European bat lyssavirus type-1 and -2 (EBLV-1 and EBLV-2). Only limited knowledge on the susceptibility of the natural EBLV-hosts, insectivorous bats, to lyssavirus infection is available. Our study was undertaken to evaluate the susceptibility and pathology associated with an EBLV-1 infection in Eptesicus fuscus following different routes of virus inoculation including intracranial (n = 6), intramuscular (n = 14), oral (n = 7) and intranasal (n = 7). Blood and saliva samples were collected from all bats on a monthly basis. Four bats inoculated intracranially developed rabies with a mean of 11 days to death, whilst seven bats inoculated intramuscularly developed rabies, with an extended incubation period prior to death. We did not observe any mortality in the oral (p.o.) or intranasal (i.n.) groups and both groups had detectable levels of virus neutralizing antibodies (data not shown). Virus shedding was demonstrated in the saliva by virus isolation and the detection of viral RNA in ill bats, particularly immediately prior to the development of disease. In addition, the presence of virus and viral RNA was detected in the thyroid gland in bats challenged experimentally with EBLV-1, which exceeded that detected in all other extra-neural tissue. The significance of detecting EBLV-1 in the thyroid gland of rabid bats is not well understood. We speculate that the infection of the thyroid gland may cause subacute thyroiditis, a transient form of thyroiditis causing hyperthyroidism, resulting in changes in adrenocortical activity that could lead to hormonal dysfunction, thereby distinguishing the clinical presentation of rabies in the rabid host.

Summary 1. As outbreaks of infectious diseases have emerged as a threat to small populations, conservation managers are increasingly making decisions regarding whether and how to intervene in such situ ations. Past controversies and lack of knowledge and firm guidelines may inhibit this process. We present data on a vaccination campaign against a rabies outbreak in endangered Ethiopian wolves as a case study of a disease-control intervention in a threatened population. 2. Ethiopian wolves on the periphery of the outbreak area were trapped to administer a dose of injectable rabies vaccine and to assess the magnitude and duration of the immune response. The expansion of an established population monitoring programme allowed us to assess the factors influencing the probability of capturing particular animals and to evaluate the overall success of the intervention. 3. All wolves sampled 1 month after vaccination had protective levels of serum antibody titres. A booster dose administered within 1-6 months appeared to be necessary to maintain these levels. Females were less likely to be trapped than expected, if dispersing females were included in the population. Animals captured in the first trapping session were more likely to be recaptured if the

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.

European bat lyssavirus type 1 (EBLV-1, genotype 5) is known to endemically circulate in insectivorous bat populations in Germany. In August 2001, a rabies suspect stone marten (Martes foina) was found in the city of Burg (Saxony-Anhalt, Germany) and was sent to the regional veterinary laboratory for routine rabies diagnosis. Whereas brain samples repeatedly tested negative in the fluorescent antibody test for classical rabies virus (genotype 1), the mouse inoculation test and the rabies tissue culture inoculation test yielded positive results. Rabies viral RNA was also detected in the stone marten brain sample both by nested and heminested RT-PCR specific for the nucleoprotein gene and for the nucleoprotein phosphoprotein junction of rabies virus. The amplification products were sequenced to genotype the isolate. Sequence data obtained from the first-round RT-PCR products were analysed and the suspect stone marten isolate was confirmed as a rabies related virus (EBLV-1a). Phylogenetic comparison with sequences from recent genotype five isolates from Germany and Denmark showed that it was closely related to a previous isolate of EBLV-1 from a serotine bat in Saxony-Anhalt obtained in the same year in an area adjacent to the place where the EBLV-1 infected stone marten was found. Both EBLV-1 isolates share a 99.5% identity. This is the first report of an EBLV-1a spill-over from an insectivorous bat into wildlife in Europe.

A 55-year-old bat conservationist was admitted to Ninewells Hospital, Dundee, Scotland, on November 11, 2002, with an acute haematemesis. He gave a 5-day history of pain and paraesthesia in the left arm, followed by increasing weakness of his limbs with evidence of an evolving encephalitis with cerebellar involvement. The patient had never been vaccinated against rabies and did not receive postexposure treatment. Using a hemi-nested reverse transcriptase-polymerase chain reaction (RT-PCR), saliva samples taken intravitam from different dates proved positive for rabies. A 400-bp region of the nucleoprotein gene was sequenced for confirmation and identified a strain of European bat lyssavirus (EBLV) type 2a. The diagnosis was confirmed using the fluorescent antibody test (FAT) and by RT-PCR on three brain samples (cerebellum, medulla, and hippocampus) taken at autopsy. In addition, a mouse inoculation test (MIT) was performed. Between 13 and 17 days postinfection, clinical signs of a rabies-like illness had developed in all five inoculated mice. Brain smears from each infected animal were positive by the FAT and viable virus was isolated. This fatal incident is only the second confirmed case of an EBLV type-2 infection in a human after exposure to bats.

The authors have detected West Nile virus-specific antibodies in a horse in the UK with signs of neurological disease. The horse had recently been imported from Europe and does not represent a threat for onward transmission itself, but highlights the need for vigilance for this zoonotic vector-borne disease in the UK.

TO further investigate four locally acquired cases of canine babesiosis (Fig 1) in Essex ( VR , February 13, 2016, vol 178, p 172; March 5, 2016, vol 178, pp 243–244), a tick survey was completed on March 11, 2016, in the area where all four dogs had been regularly exercised. The survey aimed to detect an active population of Dermacentor reticulatus within this area and to determine if questing ticks collected at the site were positive for Babesia canis . Locations along a cycle path in central Harlow were surveyed by dragging cloth over the vegetation to attract questing ticks. FIG 1: Giemsa-stained blood smear from a dog from Essex with babesiosis Eight female and nine male ticks were collected from one area close …

CRIMEAN Congo haemorrhagic fever (CCHF) is a tickborne zoonotic disease that is non-pathogenic to many animals, but is highly pathogenic to humans and a serious threat to public health (Ergonul 2006). The causative agent is the CCHF virus (CCHFV), and the disease has the widest geographical distribution of any tickborne viral disease, with outbreaks having been recorded in Greece, Turkey, the Balkans, former Soviet Union countries, through the Middle East, Africa and Asia. Human infection may progress from early febrile symptoms to a severe haemorrhagic condition associated with high mortality. Although CCHFV is transmitted to animals by the bite of infected ticks, most human infections are via direct contact with blood or tissues from infected livestock, nosocomial transmission or through crushing infected ticks. CCHFV is transmitted in enzootic cycles to vertebrates by hard ticks of the genus Hyalomma , in particular Hyalomma marginatum marginatum . The main potential source of introduction to the UK of this virus has been through the attachment of Hyalomma nymphs to migratory birds (Foley-Fisher and others 2012, Jameson and others 2012 …

AS announced by Defra on June 30, the UK is to harmonise its pet movement rules with those of other member states of the European Union (EU). As a result, dogs, cats and ferrets entering the country from EU member states and listed non-EU countries will no longer require a blood test to confirm seroconversion following rabies vaccination (Gibbens and Locke 2011). Pets being imported from unlisted ‘third countries’ will no longer have to enter six months' quarantine in the UK, but once vaccinated, blood tested a month later, and following a further three-month wait in the country of origin, they may enter the UK with the appropriate certification. The whole procedure therefore takes four months from the time of vaccination. Quarantine in the UK may still be an option for those pet animals that cannot comply with these requirements but will be reduced in line with the new measures, to up to four months after vaccination. As with any major policy change where there may be an impact on animal or public health, Defra uses all available scientific evidence to advise policy changes. In the case of rabies, …

We have recently isolated European bat lyssavirus type 2 (EBLV-2) from a Daubenton's bat ( Myotis daubentonii ) in West Lothian, Scotland. EBLV-2 has been isolated multiple times throughout England since 1996, but this is the first isolation of live virus from a bat in Scotland, seven years after

FROM a veterinary and medical standpoint we have witnessed the successful elimination of rabies in many countries, along with growing confidence that the disease will no longer present a major threat to public health in those countries in which the disease has been eradicated. The disease, however, while diminishing in Europe, remains a major threat to human health in Asia and Africa. Rabies should therefore be considered a disease of poverty, especially in countries with poor infrastructures and limited resources to devote to combating infectious disease. It is notable that two-thirds of the world’s population lives in a rabies-endemic area. Estimates for the number of human deaths worldwide suggest that in excess of 50,000 cases occur each year, principally in Africa and Asia, with 99·9 per cent of human cases being the result of rabies transmission caused by a dog bite. These figures are likely to be underestimates due to under-reporting of

SIR, — Since 1987, we have undertaken passive surveillance in Great Britain for bat variants of rabies virus ([Harris and others 2006a][1]). In Europe, bat variants of rabies virus are classified as genotypes 5 and 6 (European bat lyssavirus [eblv] type 1 [eblv-1] and type 2 [eblv-2], respectively

AS a consequence of climate change, international transport and other global environmental changes, new diseases are likely to threaten UK livestock. Arthropod-borne viruses (arboviruses) in particular are highly responsive to environmental change, as illustrated by outbreaks of bluetongue virus in

SIR, — We report a case of classical rabies virus (genotype 1) in a 10-week-old dog imported from Sri Lanka to the uk. This case of rabies in an imported dog is the first in the uk since 1990. The dog was part of a single consignment of 13 animals that was imported to the uk by an animal charity