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The spread of lineage 2 West Nile virus (WNV) from sub-Saharan regions to Europe and the unpredictable change in pathogenicity indicate a potential public and veterinary health threat and requires scientific awareness. To describe the results of clinical and virological investigations of the 1st outbreak of a genetic lineage 2 WNV encephalomyelitis in horses. Seventeen horses with neurologic signs. Information regarding signalment, clinical signs, and outcome was obtained for each animal. Serology was performed in 15 cases, clinicopathological examination in 7 cases, and cerebrospinal fluid was collected from 2 horses. Histopathology was carried out in 4 horses, 2 of which were assessed for the presence of WNV in their nervous system. WNV neutralizing antibody titers were between 10 and 270 (median, 90) and the results of other serological assays were in agreement with those of the plaque reduction neutralization test. Common signs included ataxia, weakness, asymmetric gait, muscle tremors, hypersensitivity, cranial nerve deficits, and recumbency. Twelve animals survived. Amplicons derived from the infection-positive specimens allowed molecular characterization of the viral strain. From our results, we conclude that this outbreak was caused by a lineage 2 WNV strain, even though such strains often are considered nonpathogenic. Neurological signs and survival rates were similar to those reported for lineage 1 virus infections. The disease occurrence was not geographically limited as had been the typical case during European outbreaks; this report describes a substantial northwestern spread of the pathogen. [ABSTRACT FROM AUTHOR]

Abstract: Usutu virus (USUV) infection was diagnosed in two free-living blackbirds and in three captive owls belonging to two different species in northern Italy in the summers of 2006–2008. Diagnosis was established by immunohistochemistry and RT-PCR. RT-PCR was performed on frozen and on paraffin-embedded tissues (PET), respectively. From the frozen samples a partial sequence of the putative USUV E and NS1 proteins (1229bp) was determined, whereas partial sequences of the putative NS3 (278bp) and NS5 (159bp) proteins were obtained from PET. Additionally, one partial sequence (163bp) of the putative 3′UTR region was determined from all samples. Sequencing of the amplification products revealed 99.8–100% nucleotide identity of the Italian USUV strains to those from other central European countries. [Copyright &y& Elsevier]

To determine whether avian bornaviruses (ABVs) were a factor in proventricular dilatation disease (PDD), we used immunohistochemistry, reverse transcription-PCR, and nucleotide sequence analysis to examine paraffin wax-embedded or frozen tissue samples of 31 psittacine birds with this disease. PDD is a fatal disease of psittacine birds associated with nonsuppurative encephalitis and ganglioneuritis of the upper intestinal tract. Tissue samples had been collected from 1999 through 2008 in Austria, Switzerland, Hungary, and Australia. Immunohistochemical demonstration of viral antigen within the brain and vegetative nerve system of the gastrointestinal tract provides strong evidence for a causative role of ABVs in this condition. Partial sequences of nucleoprotein (p40) and matrix protein (gp18) genes showed that virus in most of our cases belonged to the ABV-2 and ABV-4 groups among the 5 genogroups described so far. Viral sequences of 2 birds did not match any of the described sequences and clustered together in a new branch termed ABV-6. [ABSTRACT FROM AUTHOR]

Abstract: Usutu virus has been causing avian mortality in Austria since its emergence in 2001. Between 2003 and 2005 a total of 504 dead birds were examined by reverse-transcriptase polymerase chain reaction and immunohistochemistry for the presence of Usutu virus nucleic acid and antigen, respectively. In 2003, 92 birds (out of 177 birds) belonging to five different species were positive, while in 2004, only 11 (of 224) birds, and in 2005, 4 (of 103) birds proved positive, all of which were blackbirds (Turdus merula). Within the surveillance period the virus had spread from its initial area of emergence and circulation, the surroundings of Vienna, to large areas of the federal states of Lower Austria, Burgenland and Styria. However, the absolute numbers of Usutu virus associated avian deaths declined significantly during the course of the years. In addition, the proportion of birds with low amounts of virus in their tissues increased continuously, which may indicate developing herd immunity. [Copyright &y& Elsevier]

Usutu virus (USUV) is a mosquito-borne flavivirus of the Japanese encephalitis virus group, which has been associated with avian mortality in Austria since 2001. The affected birds are predominantly Eurasian blackbirds ( Turdus merula). In the present study, the pathogenicity of USUV for domestic geese ( Anser anser f. domestica) was investigated. Eleven 2-week-old geese were inoculated intramuscularly with 5 × 104 50% tissue culture infectious dose of USUV strain Vienna-2001 blackbird. No clinical signs were seen during the observation period. Four inoculated and one in-contact geese died without preceding clinical signs. Two of the deaths could be attributed to bacterial septicaemia and strangulation, respectively. The cause of death of two experimental and one in-contact animals remained unclear, but lack of evidence for viral lesions and viral antigen in their tissues argued against association with the USUV infection. Although in organs of the majority of inoculated geese (9/11) USUV was detected by reverse transcriptase-polymerase chain reaction, immunohistochemistry for USUV antigen was negative in all tissues of all geese. Evidence of plasma viraemia or viral excretion was found in one goose only. Seroconversion was detected in three inoculated geese 10 days post-inoculation. Geese placed in contact with inoculated geese and control animals did not exhibit USUV in their internal organs or plasma and lacked USUV-specific antibodies. This experiment shows that USUV is able to replicate in geese, but does not induce clinical disease, is unlikely to induce mortality, and only infrequently leads to viraemia or virus shedding. [ABSTRACT FROM AUTHOR]

In summer 2001, Usutu virus (USUV), a mosquito-borne flavivirus, was isolated for the first time in Europe during a mortality incident among Eurasian blackbirds ( Turdus merula ) in Austria. Chickens are frequently used as sentinel animals for arbovirus surveillance systems. In the present study, the pathogenicity of USUV for specific pathogen free chickens was investigated. Ten 2-week-old chickens were inoculated intravenously with 0.1 ml inoculum containing 10 3 median (50%) tissue culture infectious dose of USUV strain Vienna 2001-blackbird (939/01). Clinical signs, viraemia, gross and microscopic lesions, contact transmission and immunological response were evaluated. No clinical signs were observed in the USUV-inoculated animals during the experimental period. Pathological examination showed moderate splenomegaly and follicular infiltrates in the liver of several inoculated animals. Mild non-suppurative encephalitis was observed in the brain tissue of one virus-inoculated chicken examined 7 days post inoculation (d.p.i.). USUV nucleic acid was detected by reverse-transcriptase polymerase chain reaction in the organs of six inoculated chickens, although immunohistochemistry for flavivirus antigen was negative in all tissues from all chickens. Virus shedding was shown in three inoculated birds by detecting USUV RNA in cloacal swabs of two chickens at 5 d.p.i., and in the cloacal and pharyngeal swabs of one chicken at 7 d.p.i. Based on detection of viral RNA in peripheral blood mononuclear cells, viraemia was detected only in two chickens (at 7 d.p.i.). Only one of the inoculated chickens developed an antibody response. There was no evidence of virus transmission to chickens kept in contact with inoculated birds. No USUV was isolated from in-contact birds and all in-contact and control animals lacked USUV-specific antibodies. The present data suggest that domestic chickens are not at risk of developing clinical disease following USUV infection and that chickens are unlikely to be useful for sentinel purposes in USUV surveillance programmes. [ABSTRACT FROM AUTHOR]

The authors determined partial nucleic sequences of the variable regions of open-reading frame (ORF5) from 151 nucleotide to 668 nucleotide and deduced amino acid sequences of 518 nucleotide respectively of 20 equine arteritis virus (EAV) isolates. About 19 Hungarian and one Austrian EAV strains were subjected to sequence analysis, the further data of 20 EAV strains: six North American and 14 European were obtained from the GenBank. Comparative sequence analysis of the Hungarian EAV strains indicated that among the three variable regions the first has been affected mostly by point mutations. Genetic comparison of the Hungarian strains with other EAV isolates from western Europe and North America (including the Bucyrus reference strain) has been performed on the aforementioned genome region. Besides the already known genetic subgroups of EAV; phylogenetic analysis revealed a novel subgroup comprising mainly Hungarian strains. Compared with the Bucyrus virus, the overall sequence divergencies of the examined Hungarian strains ranged from 81.47 to 90.73% at nucleotide and from 84.88 to 91.86% at amino acid level. Epizootiological studies have shown that the significant part of the EAV strains having been existed in Hungary before and in 2000 belong to this unique cluster (II.D) which was not indicated in former phylogenetic studies. After 2000 new EAV strains emerged in Hungary, one of them causing abortions or neonatal death. The previously dominant‘Hungarian’ EAV genotypes were replaced by these new strains belonging to North American and European subgroups (I.A, I.B, II.A, II.B). The anamnesis of these cases revealed connections with persistent virus shedder stallions, those were imported to the country after 2000 or have been infected abroad. One of these Hungarian stallions became the source of abortion storms in Hungarian studs. [ABSTRACT FROM AUTHOR]

We conducted a complete genome analysis of a West Nile virus detected in Culex pipiens mosquitoes during a severe outbreak of human West Nile disease in Greece 2010. The virus showed closest genetic relationship to the lineage 2 strain that emerged in Hungary in 2004; increased virulence may be associated with amino acid substitution H249P. [ABSTRACT FROM AUTHOR]

Abstract: This paper offers an overview of important veterinary viral diseases of mammals stemming from aberrant immune response. Diseases reviewed comprise those due to lentiviruses of equine infectious anaemia, visna/maedi and caprine arthritis encephalitis and feline immunodeficiency. Diseases caused by viruses of feline infectious peritonitis, feline leukaemia, canine distemper and aquatic counterparts, Aleutian disease and malignant catarrhal fever. We also consider prospects of immunoprophylaxis for the diseases and briefly other control measures. It should be realised that the outlook for effective vaccines for many of the diseases is remote. This paper describes the current status of vaccine research and the difficulties encountered during their development. [Copyright &y& Elsevier]

Abstract: An update on the mosquito-borne flavivirus species including certain subtypes, as listed in the Eighth Report of the International Committee on Taxonomy of Viruses, is given. Special emphasis is placed on viruses which have been shown to cause diseases in animals, and viruses for which no pathogenicity has been proven yet. Several recent examples (Usutu virus and lineage-2 West Nile virus in central Europe, Zika virus in Micronesia) have shown that sources providing information on such scientifically largely neglected viruses are valuable tools for scientists and public health officials having to deal with such disease emergences. Furthermore the effects of global warming will lead to introduction of competent mosquito vectors into temperate climate zones and will increase efficiency of viral replication in less competent vector species. This, facilitated by rising global travel and trade activities, will facilitate introduction and permanent establishment of mosquito-borne viruses, some of which may become of public health or veterinary concern, into novel environments, e.g. industrialized countries worldwide. [Copyright &y& Elsevier]

The central European lineage of Usutu virus was isolated from a blackbird ( Turdus merula), which was found dead in the city of Brno, Czech Republic, in 2011. The virus RNA was detected in two other dead blackbirds in Brno during 2012. [ABSTRACT FROM AUTHOR]

Abstract: The emergence of lineage 2 strains of WNV in Europe as a cause of clinical disease and mortality in horses raised the question whether the existing WNV vaccines, all based on lineage 1 strains, protect against circulating lineage 2 strains of WNV. In the present paper we have determined the level of cross protection provided by the recombinant ALVAC®-WNV vaccine in a severe challenge model that produces clinical signs of WNV type 2 disease. Ten horses were vaccinated twice at 4 weeks interval with one dose of the ALVAC-WNV vaccine formulated at the minimum protective dose. A further 10 horses served as controls. Two weeks after the second vaccination, all horses were challenged intrathecally with a recent neurovirulent lineage 2 strain of WNV. The challenge produced viraemia in 10 out of 10 and encephalitis in 9 out of 10 control horses. Three horses had to be euthanized for humane reasons. In contrast, none of the vaccinated horses developed WNV disease and only 1 vaccinated horse became viraemic at a single time point at low titre. The prevalence of WNV disease and viraemia were significantly lower in the vaccinated horses than in the control horses (P <0.0001 for both). Based on these results, the ALVAC-WNV vaccine will provide veterinarians with an effective tool to control infections caused by lineage 1 and 2 strains of WNV. [Copyright &y& Elsevier]

Abstract: Proventricular dilatation disease (PDD) of psittacine birds is caused by a number of different genotypes of a novel viral species, avian bornavirus (ABV). Here we present an in situ hybridization (ISH) procedure using digoxigenin-labeled RNA probes for localizing viral genomic and mRNA of ABV-2 and ABV-4 in tissues of affected birds. Out of eleven immunohistochemically positive birds ISH signals were only found in seven. Partial sequencing of the viral genome had shown that four of them were infected with ABV-2, two with ABV-4 and one had a mixed infection with ABV-2 and ABV-4. ISH signals were present in the brain, in the vegetative nerve system, glandular epithelia and smooth muscle cells of the intestinal tract and in cardiomyocytes. Hybridization signals for viral genome were more abundant than signals for mRNA. As the probes were not strictly genotype-specific, four of the birds had hybridization signals with both, the ABV-2 and ABV-4 probes. The signals achieved with the homologous probes were more intense and more abundant than those resulting from heterologous probes. Taken together, the results of this study show that ISH can be used as a tool for localizing ABV sequences in tissues of birds with PDD and confirm the causative role of ABVs by showing viral replication in affected tissues. [ABSTRACT FROM AUTHOR]

Background noise in metagenomic studies is often of high importance and its removal requires extensive post-analytic, bioinformatics filtering. This is relevant as significant signals may be lost due to a low signal-to-noise ratio. The presence of plasmid residues, that are frequently present in reagents as contaminants, has not been investigated so far, but may pose a substantial bias. Here we show that plasmid sequences from different sources are omnipresent in molecular biology reagents. Using a metagenomic approach, we identified the presence of the (pol) of equine infectious anemia virus in human samples and traced it back to the expression plasmid used for generation of a commercial reverse transcriptase. We found fragments of multiple other expression plasmids in human samples as well as commercial polymerase preparations. Plasmid contamination sources included production chain of molecular biology reagents as well as contamination of reagents from environment or human handling of samples and reagents. Retrospective analyses of published metagenomic studies revealed an inaccurate signal-to-noise differentiation. Hence, the plasmid sequences that seem to be omnipresent in molecular biology reagents may misguide conclusions derived from genomic/metagenomics datasets and thus also clinical interpretations. Critical appraisal of metagenomic data sets for the possibility of plasmid background noise is required to identify reliable and significant signals. [ABSTRACT FROM AUTHOR]

The article describes the case of an ewe with West Nile Virus (WNV). An overview of the conducted study and the methods used are offered. Result shows that a non-suppurative encephalitis which was characterized by the brain's lymphocytic perivascular inflammatory infiltration that was accompanied by diffuse and focal gliosis.