Plasmid DNA contaminant in molecular reagents.

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.

West Nile virus lineage 2 isolated from Culex modestus mosquitoes in the Czech Republic, 2013: expansion of the European WNV endemic area to the North?

We report the detection and isolation of four almost identical strains of West Nile virus (WNV) lineage 2from Culex modestus mosquitoes collected at three fish ponds in South Moravia, Czech Republic, during August 2013. Phylogenetic analysis demonstrated that the Czech WNV strains isolated are closely related to Austrian, Italian and Serbian strains reported in 2008,2011 and 2012, respectively. Our findings show the current northernmost range of lineage 2 WNV in Europe.

Usutu virus in blackbirds (Turdus merula), Czech Republic, 2011-2012.

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.

Important mammalian veterinary viral immunodiseases and their control.

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.

Protection provided by a recombinant ALVAC(®)-WNV vaccine expressing the prM/E genes of a lineage 1 strain of WNV against a virulent challenge with a lineage 2 strain.

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.

Equine encephalomyelitis outbreak caused by a genetic lineage 2 West Nile virus in Hungary.

BACKGROUND: 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. OBJECTIVES: To describe the results of clinical and virological investigations of the 1st outbreak of a genetic lineage 2 WNV encephalomyelitis in horses. ANIMALS: Seventeen horses with neurologic signs. METHODS: 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. RESULTS: 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. CONCLUSIONS AND CLINICAL IMPORTANCE: 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.

Mosquito (Diptera: Culicidae) surveillance for arboviruses in an area endemic for West Nile (Lineage Rabensburg) and Tahyna viruses in Central Europe.

Six viral isolates were obtained from 23,243 female mosquitoes (examined in 513 pools) belonging to 16 species and collected along the lower reaches of the Dyje River in South Moravia (Czech Republic, central Europe) during 2006-2008: five isolates of Orthobunyavirus Tahyna (TAHV, California group, family Bunyaviridae: three isolations from Aedes vexans (Meigen), one from Ae. sticticus (Meigen), one from Culex modestus Ficalbi); and one isolation of Flavivirus West Nile (WNV, Japanese encephalitis group, family Flaviviridae)-strain Rabensburg (proposed lineage 3 of WNV) from Ae. rossicus (Dolbeshkin et al). All viral isolates were recovered from mosquitoes collected in 2006 (15,882 mosquitoes examined), while no virus was isolated from mosquitoes trapped in 2007 and 2008, when 1,555 and 5,806 mosquitoes were examined, respectively. The population density of local mosquitoes was very low in 2007 and 2008 because of warm and dry summer including a considerably low water table, compared with environmental conditions favorable for mosquito development in 2006. The virus isolation procedure was based on intracerebral inoculation of newborn mice. In parallel, more than one-third of the samples (183 pools consisting of 8,470 individual mosquitoes) were also examined by inoculating Vero cell cultures in Leighton tubes. However, the latter method detected only three of the six virus isolates (including WNV-Rabensburg). Ae. rossicus is a new potential vector for WNV-Rabensburg. This species feeds mostly on mammals including man; this raises the question whether this virus lineage is not adapted to an alternative mosquito-mammal cycle in the South-Moravian natural focus.

Localization of avian bornavirus RNA by in situ hybridization in tissues of psittacine birds with proventricular dilatation disease.

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.

Usutu virus in wild birds in northern Italy.

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 (1229 bp) was determined, whereas partial sequences of the putative NS3 (278 bp) and NS5 (159 bp) proteins were obtained from PET. Additionally, one partial sequence (163 bp) 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.

Zoonotic mosquito-borne flaviviruses: worldwide presence of agents with proven pathogenicity and potential candidates of future emerging diseases.

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.

Monitoring of Usutu virus activity and spread by using dead bird surveillance in Austria, 2003-2005.

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.

Application of polymerase chain reaction and virus isolation techniques for the detection of viruses in aborted and newborn foals.

The occurrence of two important pathogens, equine herpesvirus 1 (EHV1) and equine arteritis virus (EAV) causing abortions, perinatal foal mortality and respiratory disease, was investigated by polymerase chain reaction (PCR) and virus isolation to demonstrate the presence of abortigenic viruses in samples from 248 horse fetuses in Hungary. We found 26 EHV1- and 4 EAV-positive aborted or prematurely born foals from 16 and 4 outbreaks, respectively, proving that despite the widely applied vaccination, EHV1 is a far more important cause of abortions in the studs than EAV. We compared the virus content of different organs of the fetuses by PCR and isolation to identify the organ most suitable for virus demonstration. Our investigations indicate that the quantity of both viruses is highest in the lungs; therefore, according to our observations, in positive cases the probability of detection is highest from lung samples of aborted or newborn foals. Both the PCR and the virus isolation results revealed that the liver, though widely used, is not the best organ to sample either for EHV1 or for EAV detection. From the analysis of the epidemiological data, we tried to estimate the importance of the two viruses in the Hungarian horse population.

Molecular diagnosis of avian nephritis: preliminary report.

Kidney samples from chickens diagnosed with acute nephritis and gout were subjected to histological and electron microscopic examination. The investigations revealed cytoplasmic inclusion bodies in the tubular epithelial cells containing round virions of about 30 nm in diameter. Since avian nephritis virus (ANV) is known as a potential causative agent of the so-called baby chick nephropathy, an RT-PCR assay was developed for the molecular detection of ANV-specific nucleic acid in the specimen. The specificity of the assay was confirmed by direct sequencing of the amplicon obtained in the reaction. The nucleotide sequence of the PCR product showed 92% identity with the reference ANV sequence deposited in the GenBank database. After having been validated on some other suspicious cases of avian nephritis, the PCR method described in this study can be a potential tool for routine diagnostic examination of samples submitted from cases of gout and nephropathy in chickens.

Limited pathogenicity of usutu virus for the domestic goose (Anser anser f. domestica) following experimental inoculation.

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 x 10(4) 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.

Limited pathogenicity of Usutu virus for the domestic chicken (Gallus domesticus).

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.

A novel subgroup among genotypes of equine arteritis virus: genetic comparison of 40 strains.

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.

Occurrence of acute paralysis virus of the honey bee (Apis mellifera) in a Hungarian apiary infested with the parasitic mite Varroa jacobsoni.

Viruses of the honey bee have been known for a long time; however, recently the attention of scientists and apiculturalists has turned towards the relationship between these viruses and the parasitic mite Varroa jacobsoni. Although clinical symptoms indicated the presence of some of the viruses of bees in Hungary, none have previously been isolated or identified. During July unusual adult bee and brood mortality was observed in some colonies of an apiary in Budapest known to be infested with Varroa jacobsoni. Large amounts of acute paralysis virus (APV) were detected serologically in healthy honey bee pupae killed by the injection of a bacteria-free extract of diseased adult bees. Crystalline arrays of 30 nm particles were seen in ultrathin sections of the tissues of injected pupae and naturally infected adult bees. In spite of the application of acaricide treatments the bee population in several colonies had collapsed by the end of summer and the apiary suffered severe wintering losses.