Louping ill virus: an endemic tick-borne disease of Great Britain.

In Europe and Asia, Ixodid ticks transmit tick-borne encephalitis virus (TBEV), a flavivirus that causes severe encephalitis in humans but appears to show no virulence for livestock and wildlife. In the British Isles, where TBEV is absent, a closely related tick-borne flavivirus, named louping ill virus (LIV), is present. However, unlike TBEV, LIV causes a febrile illness in sheep, cattle, grouse and some other species, that can progress to fatal encephalitis. The disease is detected predominantly in animals from upland areas of the UK and Ireland. This distribution is closely associated with the presence of its arthropod vector, the hard tick Ixodes ricinus. The virus is a positive-strand RNA virus belonging to the genus Flavivirus, exhibiting a high degree of genetic homology to TBEV and other mammalian tick-borne viruses. In addition to causing acute encephalomyelitis in sheep, other mammals and some avian species, the virus is recognized as a zoonotic agent with occasional reports of seropositive individuals, particularly those whose occupation involves contact with sheep. Preventative vaccination in sheep is effective although there is no treatment for disease. Surveillance for LIV in Great Britain is limited despite an increased awareness of emerging arthropod-borne diseases and potential changes in distribution and epidemiology. This review provides an overview of LIV and highlights areas where further effort is needed to control this disease.

Antibody response in horses following experimental infection with West Nile Virus lineages 1 and 2.

West Nile virus (WNV) has re-emerged as an important pathogen for humans and horses, which are considered to be incidental 'dead-end' hosts. We have demonstrated that horses are susceptible to experimental infection with WNV and that horses infected with either WNV lineage 1 or lineage 2 elicit a similar antibody profile in serum samples. These data suggest that virus-neutralizing antibody responses persist for longer than WNV-specific IgM levels in serum and that there are not any notable differences in the antibody profile following experimental infection of horses with either WNV lineage 1 and lineage 2 viruses. Furthermore, the duration of IgM appears to be short-lived in horses and may be useful for identifying and differentiating recent infections from previously exposed animals.

A new outbreak of rabies in rare Ethiopian wolves (Canis simensis).

Between October 2008 and May 2009, five brain samples from the carcasses of the rare Ethiopian wolf (Canis simenensis) were submitted for rabies virus testing. Rabies virus was detected in all five samples, and this confirmed that a further outbreak of rabies had occurred within the wolf population in the Bale Mountains of Ethiopia. Sequence comparison of a partial fragment of the nucleoprotein-coding gene demonstrated that all viruses showed 100% sequence identity, suggesting a single introduction of rabies virus.

Tick-borne encephalitis virus - a review of an emerging zoonosis.

During the last 30 years, there has been a continued increase in human cases of tick-borne encephalitis (TBE) in Europe, a disease caused by tick-borne encephalitis virus (TBEV). TBEV is endemic in an area ranging from northern China and Japan, through far-eastern Russia to Europe, and is maintained in cycles involving Ixodid ticks (Ixodes ricinus and Ixodes persulcatus) and wild vertebrate hosts. The virus causes a potentially fatal neurological infection, with thousands of cases reported annually throughout Europe. TBE has a significant mortality rate depending upon the strain of virus or may cause long-term neurological/neuropsychiatric sequelae in people affected. In this review, we comprehensively reviewed TBEV, its epidemiology and pathogenesis, the clinical manifestations of TBE, along with vaccination and prevention. We also discuss the factors which may have influenced an apparent increase in the number of reported human cases each year, despite the availability of effective vaccines.

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.

Up-regulation of chemokine gene transcripts and T-cell infiltration into the central nervous system and dorsal root ganglia are characteristics of experimental European bat lyssavirus type 2 infection of mice.

European bat lyssaviruses (EBLV) types 1 and 2 are closely related to classical rabies virus (RABV), and are capable of causing rabies in terrestrial mammals, including humans. The authors have investigated the murine host innate immune response in the brain, salivary gland, spinal cord, and blood, following peripheral inoculation with EBLV-2. In the brain, increases in Toll-like receptor-3 (TLR-3) transcript preceded overt disease, with a range of inflammatory gene transcripts increasing during the clinical stage of infection. This included transcripts for interleukin-6 (IL-6), interferon-gamma (IFN-gamma), and CXC chemokine ligand 10 (CXCL10). In the salivary gland, there was a small but significant increase of CXCL10 gene transcript and a limited increase in 2'-5' oligoadenylate synthetase (2'-5' OAS1) transcript. In the blood, there was an increase in levels of IFN-gamma and virus-neutralizing antibodies (VNAs) were detected prior to the appearance of clinical signs. These changes were associated with severe lymphocyte infiltration observed within the spinal cord and dorsal root ganglia (DRG), which was dominated by T lymphocytes and associated with widespread inflammatory changes. The authors speculate that the increase of inflammatory cytokines and chemokines in response to EBLV-2 infection leads to a dramatic increase in T-cell infiltration and provides evidence for a robust immune response to lyssavirus infection that may not commonly occur in RABV infection.

Molecular epidemiology of rabies in bat-eared foxes (Otocyon megalotis) in South Africa.

A panel of 124 rabies viruses from wildlife host species (principally the bat-eared fox, Otocyon megalotis) and domestic carnivore species were collected between 1980 and 2005 from a region of South Africa associated with endemic bat-eared fox rabies. We have studied the molecular epidemiology of bat-eared fox rabies by virtue of nucleotide sequence analyses of PCR amplicons specific to the variable G-L intergenic region as well as the conserved nucleoprotein gene of each of the rabies viruses in this South African panel. Although it was demonstrated that all of these viruses were very closely related, they could be segregated into two major phylogenetic groups. The data presented in this paper complement antigenic and surveillance data on rabies in this host species in South Africa. Most importantly our data support a hypothesis that the bat-eared fox independently maintains rabies cycles in specific geographical loci. This is the first molecular epidemiological investigation describing rabies transmission dynamics in this wildlife carnivore host species in South Africa.

Molecular epidemiological study of Arctic rabies virus isolates from Greenland and comparison with isolates from throughout the Arctic and Baltic regions.

We report a molecular epidemiological study of rabies in Arctic countries by comparing a panel of novel Greenland isolates to a larger cohort of viral sequences from both Arctic and Baltic regions. Rabies virus isolates originating from wildlife (Arctic/red foxes, raccoon-dogs and reindeer), from domestic animals (dogs/cats) and from two human cases were investigated. The resulting 400 bp N-gene sequences were compared with isolates representing neighbouring Arctic or Baltic countries from North America, the former Soviet Union and Europe. Phylogenetic analysis demonstrated similarities between sequences from the Arctic and Arctic-like viruses, which were distinct from rabies isolates originating in the Baltic region of Europe, the Steppes in Russia and from North America. The Arctic-like group consist of isolates from India, Pakistan, southeast Siberia and Japan. The Arctic group was differentiated into two lineages, Arctic 1 and Arctic 2, with good bootstrap support. Arctic 1 is mainly comprised of Canadian isolates with a single fox isolate from Maine in the USA. Arctic 2 was further divided into sub-lineages: 2a/2b. Arctic 2a comprises isolates from the Arctic regions of Yakutia in northeast Siberia and Alaska. Arctic 2b isolates represent a biotype, which is dispersed throughout the Arctic region. The broad distribution of rabies in the Arctic regions including Greenland, Canada and Alaska provides evidence for the movement of rabies across borders.

Identification of a conserved linear epitope at the N terminus of the rabies virus glycoprotein.

A novel, linear B-cell epitope has been identified at the N terminus of the rabies virus (RABV) glycoprotein. Screening of a phage-display library demonstrated that two glycoprotein-specific mAbs recognized a conserved sequence, WxxxDI, which aligned between aa 14 and 19 of the mature glycoprotein. Screening of truncated glycoprotein fragments with both mAbs confirmed the location of the epitope in the N-terminal region. Alignment of amino acid sequences from a range of RABV isolates indicated that the site was conserved in most viruses. Alignment with representatives of other lyssaviruses suggested that it is conserved within phylogroup I, which includes the European bat lyssaviruses, but not phylogroup II. A 12 aa synthetic peptide of this epitope was recognized by both mAbs and sera from a subset of rabies-vaccinated dogs. In a multimeric form, the peptide could induce an epitope-specific response following immunization in rabbits and mice.

Factors affecting the serological response of dogs and cats to rabies vaccination.

After being vaccinated against rabies some cats and dogs fail to show an antibody titre adequate to meet the requirements of the UK Pet Travel Scheme. To investigate this problem, the data derived from 16,073 serum samples submitted to the Veterinary Laboratories Agency for serological testing between 1999 and 2002, 1002 samples submitted to BioBest during March and April 2001, and 1264 samples associated with one make of vaccine submitted to BioBest between June 2001 and January 2003, were analysed. The probability of antibody titre failing to reach at least 0.5 iu/ml was analysed by logistic regression as a function of the choice of vaccine, the interval between vaccination and sampling, the sex and age of the animal, and its country of origin. In dogs, all these factors, except sex, had highly significant (P < 0.001) effects on the test failure rate, and in cats all the factors had a significant effect (P < 0.05).

Development of a qualitative indirect ELISA for the measurement of rabies virus-specific antibodies from vaccinated dogs and cats.

A protocol suitable for the detection of rabies virus-specific antibodies in serum samples from companion animals using an enzyme linked immunosorbent assay (ELISA) is described. This method has been used successfully for the qualitative assessment of rabies virus-specific antibodies in serum samples from a cohort of vaccinated dogs and cats. In two initial field studies, a variable population of field samples from the Veterinary Laboratories Agency (VLA), United Kingdom were tested. In the first study (n = 1000), the number of false-positive and false-negative results was 11 samples (1.1%) and 67 samples (6.7%), respectively. In the second study (n = 920), the number of false-positive and false-negative results was 7 samples (0.8%) and 52 samples (5.7%). In a third study, undertaken at l'Agence Française de Sécurité Sanitaire des Aliments (AFSSA), Nancy, France (n = 440), 1 false-positive sample (0.23%) and 91 (20.7%) false-negative samples were identified. Data generated using this prototype ELISA indicate a strong correlation for specificity when compared to the gold standard fluorescent antibody virus neutralisation (FAVN) test. Although the ELISA has a lower sensitivity than the FAVN test, it is a useful tool for rapidly screening serum samples from vaccinated companion animals. Using a cut-off value of 0.6 EU/ml, the sensitivity (R = % from VLA and 79% from AFSSA) and specificity (R = 97.3%) indices between the ELISA compared favourably with data generated using the FAVN test. The major advantages of the ELISA test are that it is a qualitative tool that can be completed in four hours, does not require the use of live virus and can be performed without the need for specialised laboratory containment. This contrasts with 4 days using conventional rabies antibody virus neutralisation assays. Using the current format, the ELISA assay described would be a valuable screening tool for the detection of rabies antibodies from vaccinated domestic animals in combination with other Office International des Epizooties (OIE) accepted serological tests.

Canine vaccine recipients recognize an immunodominant region of the rabies virus glycoprotein.

To investigate the immune response to anti-rabies vaccination in the principal recipient (the domestic dog), four truncated fragments of the rabies virus glycoprotein were expressed as glutathione S-transferase fusion proteins. Immune sera from vaccinated rabbits and dogs were then used to probe for reactivity with these expressed proteins. In two rabbits and four dogs tested, the dominant antibody response to non-conformational antigenic sites appeared to be directed to a region of the glycoprotein between amino acids 222 and 332. The N-terminal fragment of the glycoprotein was also significantly antigenic. Further studies to assess whether the antibody response to the internal domain could neutralize the rabies Challenge Virus Standard (CVS) strain, using antibody depletion, suggested that this fraction did contribute to the ability of post-vaccination sera to neutralize and therefore protect against infection.