Serological surveillance for Tahyna virus (California encephalitis orthobunyavirus, Peribunyaviridae) neutralizing antibodies in wild ungulates in Austria, Hungary and Romania.

A serosurvey for Tahyna virus (TAHV), a mosquito-borne California encephalitis orthobunyavirus (Peribunyaviridae) endemic to Europe, was performed to estimate the activity of TAHV on a broad geographic scale. Sera from wild boar (Sus scrofa), roe deer (Capreolus capreolus) and red deer (Cervus elaphus) were collected from Austria, Hungary and Romania. Samples were tested for neutralizing antibodies against TAHV using a virus microneutralization assay. The results demonstrate that TAHV transmission to mammals is widespread in Europe, particularly in the wild boar population where the mean rate of seroconversion is 15.2%.

A Serological Protein Microarray for Detection of Multiple Cross-Reactive Flavivirus Infections in Horses for Veterinary and Public Health Surveillance.

The genus Flavivirus in the family Flaviviridae includes some of the most important examples of emerging zoonotic arboviruses that are rapidly spreading across the globe. Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV) and Usutu virus (USUV) are mosquito-borne members of the JEV serological group. Although most infections in humans are asymptomatic or present with mild flu-like symptoms, clinical manifestations of JEV, WNV, SLEV, USUV and tick-borne encephalitis virus (TBEV) can include severe neurological disease and death. In horses, infection with WNV and JEV can lead to severe neurological disease and death, while USUV, SLEV and TBEV infections are mainly asymptomatic, however, and induce antibody responses. Horses often serve as sentinels to monitor active virus circulation in serological surveillance programmes specifically for WNV, USUV and JEV. Here, we developed and validated a NS1-antigen protein microarray for the serological differential diagnosis of flavivirus infections in horses using sera of experimentally and naturally infected symptomatic as well as asymptomatic horses. Using samples from experimentally infected horses, an IgG and IgM specificity of 100% and a sensitivity of 95% for WNV and 100% for JEV was achieved with a cut-off titre of 1 : 20 based on ROC calculation. In field settings, the microarray identified 93-100% of IgG-positive horses with recent WNV infections and 87% of TBEV IgG-positive horses. WNV IgM sensitivity was 80%. Differentiation between closely related flaviviruses by the NS1-antigen protein microarray is possible, even though we identified some instances of cross-reactivity among antibodies. However, the assay is not able to differentiate between naturally infected horses and animals vaccinated with an inactivated WNV whole-virus vaccine. We showed that the NS1-microarray can potentially be used for diagnosing and distinguishing flavivirus infections in horses and for public health purposes within a surveillance setting. This allows for fast, cheap, syndrome-based laboratory testing for multiple viruses simultaneously for veterinary and public health purposes.

Keratoconjunctivitis in a group of Icelandic horses with suspected γ-herpesvirus involvement.

REASONS FOR PERFORMING STUDY: The role of equid γ-herpesviruses on ocular surface diseases has been disputed, because the diagnosis is usually based on clinical symptoms and detection of viral DNA from samples obtained from live animals. OBJECTIVES: To describe the clinical course, results of polymerase chain reaction (PCR) analysis, in situ hybridisation, cell culture and pathohistological findings of select cases in a presumed outbreak of herpesvirus infection in a group of 15 Icelandic horses. STUDY DESIGN: Case series. METHODS: Pooled ocular and nasal swabs and peripheral blood mononuclear cells of horses diagnosed clinically with herpesvirus-associated keratoconjunctivitis were analysed for presence of equine herpesviruses (EHV)-2 and EHV-5 nucleic acid using real-time PCR. Necropsy specimens from one horse, subjected to euthanasia due to deterioration of clinical symptoms were examined histopathologically, and analysed for presence of EHV-2 and EHV-5 nucleic acid using real-time PCR. In situ hybridisation and cell culture of select samples were performed. RESULTS: All horses with symptoms of severe keratoconjunctivitis were positive for presence of either EHV-2 and/or EHV-5 nucleic acid using real-time PCR. Assessment of necropsy specimens of the most severely affected case, revealed presence of EHV-2 and/or EHV-5 nucleic acid in several ocular and extraocular anatomical locations. The remaining horses responded favourably to symptomatic treatment. CONCLUSIONS: This case series illustrates a severe outbreak of keratoconjunctivitis in a group of Icelandic horses, with suspected γ-herpesvirus involvement. For the first time equid γ-herpesviruses were detected in intraocular anatomical locations.

Phylogenetic characterization of Central/Southern European lineage 2 West Nile virus: analysis of human outbreaks in Italy and Greece, 2013-2014.

In recent years, West Nile virus (WNV) lineage 2 has been spreading and causing disease outbreaks in humans and animals in Europe. In order to characterize viral diversity, we performed full-length genome sequencing of WNV lineage 2 from human samples collected during outbreaks in Italy and Greece in 2013 and 2014. Phylogenetic analysis showed that these WNV lineage 2 genomes belonged to a monophyletic clade derived from a single introduction into Europe of the prototype Hungarian strain. Correlation of phylogenetic data with geospatial information showed geographical clustering of WNV genome sequences both in Italy and in Greece, indicating that the virus had evolved and diverged during its dispersal in Europe, leading to the emergence of novel genotypes, as it adapted to local ecological niches. These genotypes carried divergent conserved amino acid substitutions, which might have been relevant for viral adaptation, as suggested by selection pressure analysis and in silico and experimental modelling of sequence changes. In conclusion, the results of this study provide further information on WNV lineage 2 transmission dynamics in Europe, and emphasize the need for WNV surveillance activities to monitor viral evolution and diversity.

The challenge of West Nile virus in Europe: knowledge gaps and research priorities.

West Nile virus (WNV) is continuously spreading across Europe, and other continents, i.e. North and South America and many other regions of the world. Despite the overall sporadic nature of outbreaks with cases of West Nile neuroinvasive disease (WNND) in Europe, the spillover events have increased and the virus has been introduced into new areas. The high genetic diversity of the virus, with remarkable phenotypic variation, and its endemic circulation in several countries, require an intensification of the integrated and multidisciplinary research efforts built under the 7th Framework Programme of the European Union (FP7). It is important to better clarify several aspects of WNV circulation in Europe, including its ecology, genomic diversity, pathogenicity, transmissibility, diagnosis and control options, under different environmental and socio-economic scenarios. Identifying WNV endemic as well as infection-free areas is becoming a need for the development of human vaccines and therapeutics and the application of blood and organs safety regulations. This review, produced as a joint initiative among European experts and based on analysis of 118 scientific papers published between 2004 and 2014, provides the state of knowledge on WNV and highlights the existing knowledge and research gaps that need to be addressed with high priority in Europe and neighbouring countries.

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.

Middle East respiratory syndrome coronavirus (MERS-CoV) in dromedary camels, Oman, 2013.

A countrywide survey in Oman revealed Middle Eastrespiratory syndrome coronavirus (MERS-CoV) nucleicacid in five of 76 dromedary camels. Camel-derivedMERS-CoV sequences (3,754 nucleotides assembled from partial sequences of the open reading frame (ORF)1a, spike, and ORF4b genes) from Oman and Qatar were slightly different from each other, but closely related to human MERS-CoV sequences from the same geographical areas, suggesting local zoonotic transmission. High viral loads in nasal and conjunctival swabs suggest possible transmission by the respiratory route.

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.

Prevalence of linear keratopathy in a herd of Lipizzaners over an 18-month period.

The prevalence of linear keratopathy with progressing age in a closed population of a single horse breed is reported. All Lipizzaners in three federal states in Austria underwent complete ophthalmic examination four times over a period of 18 months, with six-month intervals. Findings consistent with linear keratopathy were recorded, and associated with factors such as sex, location, boarding system and level of performance throughout the study period. Logistic regression was applied to determine the influence of age on ophthalmic findings. On the first, second, third and fourth examinations, 0.8 per cent, 3.1 per cent, 4.4 per cent and 4.8 per cent (of 266, 261, 249 and 230 horses, respectively) of the study population, were diagnosed with linear keratopathy. This finding was consistently identified in the same horses, and once identified, no further progression was noted. Horses with this finding had no history of previous ocular problems or concurrent ocular abnormalities. Statistical analysis did not reveal any influence of sex, location, boarding, or level of performance; however the prevalence of linear keratopathy was found to increase with progressive age (P<0.5). The results of this study indicate that linear keratopathy was not congenital and was non-progressive in the Lipizzaner over a period of 18 months.

Progestin treatment does not affect expression of cytokines, steroid receptors, oxytocin receptor, and cyclooxygenase 2 in fetal membranes and endometrium from pony mares at parturition.

In most mammalian species, progestins have a major function in maintaining pregnancy. In humans, the physiologic initiation of parturition bears similarities with inflammatory processes and anti-inflammatory effects of progestins have been suggested to postpone birth until term. To examine if comparable effects exist in the horse, mares were treated with the synthetic progestin altrenogest from day 280 of gestation until parturition (N = 5) or were left untreated as controls (N = 7). Tissue from the amnion (AMN), allantochorion (AC), and endometrium (EM) was collected at foaling and mRNA expression of interleukin (IL)-6 and -8, cyclooxygenase 2 (COX2), estrogen receptor (ER) α, progesterone receptor, and oxytocin receptor (OTR) was analyzed. Leukocytes, steroid receptors, COX2, and OTR were also investigated by histology and immunohistochemistry. Expression of mRNA for IL-6 was higher in AMN and EM versus AC (P < 0.01). Expression of IL-8 was higher in AMN than AC and EM (P < 0.001). Steroid receptors and OTR were highly expressed in EM but not in AMN and AC (P < 0.001). Expression of COX2 was most pronounced in AC whereas IL expression was not upregulated in AC. No differences in mRNA expression existed between altrenogest-treated and control animals. Endometrial polymorphonuclear leukocytes were increased in altrenogest-treated mares. Epithelial cells of all tissues, except AC chorionic villi stained progesterone receptor-positive. Staining for ER was more pronounced in the amnion facing epithelium of the AC in altrenogest-treated versus control animals (P < 0.01). In conclusion, COX2 is highly expressed in the AC. The fetal membranes thus might play a role in the onset of labor in the horse. Altrenogest did not affect gene expression in the AMN, AC, and EM but had localized effects on inflammatory cells and ER expression. No anti-inflammatory effects of altrenogest in healthy, late pregnant pony mares could be detected.

Monitoring of West Nile virus infections in Germany.

West Nile virus (WNV) is a flavivirus that is maintained in an enzootic cycle between ornithophilic mosquitoes, mainly of the Culex genus, and certain wild bird species. Other bird species like ravens, jays and raptors are highly susceptible to the infection and may develop deadly encephalitis, while further species of birds are only going through subclinical infection. The objective of this study was to continue in years 2009-2011 the serological and molecular surveillance in wild birds in Germany (see Vector Borne Zoonotic Dis. 10, 639) and to expand these investigations for the first time also to sera from domestic poultry and horses collected between 2005 and 2009. All three cohorts function as indicators for the endemic circulation of WNV. The presence of WNV-specific antibodies was detected in all samples by virus neutralization test (VNT), indirect immunofluorescence test (IFT) and/or enzyme-linked immunosorbent assay (ELISA). The presence of WNV genomes was monitored in relevant sera using two qRT-PCRs that amplify lineage 1 and 2 strains. A total of 364 migratory and resident wild bird serum samples (with emphasis on Passeriformes and Falconiformes) as well as 1119 serum samples from domestic poultry and 1282 sera from horses were analysed. With the exception of one hooded crow, antibody carriers were exclusively found in migratory birds, but not in resident birds/domestic poultry or in local horses. Crows are facultative, short-distance winter migrants in Germany. WNV-specific nucleic acids could not be demonstrated in any of the samples. According to these data, there is no convincing evidence for indigenous WNV infections in equines and in wild/domestic birds in Germany. However, since a few years, WNV infections are endemic in other European countries such as Austria, Hungary, Greece and Italy, a state-of-the-art surveillance system for the detection of incursions of WNV into Germany deems mandatory.

Identification of mixed infections with different genotypes of avian bornaviruses in psittacine birds with proventricular dilatation disease.

Proventricular dilatation disease (PDD) is a fatal, progressive neurological disorder of psittacine birds, which is caused by a single-stranded RNA virus, the avian bornavirus (ABV). The disease pattern includes lymphoplasmacytic inflammation of the central, peripheral and autonomic nervous system. Seven avian bornavirus genotypes have been identified during the last years. So far only monoinfections with a single genotype of ABV have been attributed to PDD cases. However, after a recent survey discovered a case of a double infection with two different ABV genotypes, this seemed to indicate the need for a more systematic search for mixed infections. Brain specimens from 21 psittacine birds affected with PDD were examined. Aim of the investigation was to generate partial ABV sequences of a part of the matrix protein (M) gene and to evaluate whether sequences of more than one ABV genotype were present. RNA was extracted, and subjected to reverse transcriptase PCR with primer pairs generating a partial sequence of the matrix protein (M) gene, followed by a cloning procedure. Ten clones per case were sequenced in order to elucidate whether sequences characteristic for one or more than one genotype were present. In 19 of 21 cases clear M gene sequences could be generated; in two cases nucleic acid amplification failed. Seven birds were infected with ABV 2 and nine with ABV 4, representing the predominant genotypes in Europe. Two cases showed a mixed infection with ABV 2 and ABV 4, and one case a mixed infection with ABV 2 and ABV 6. These results suggest that the molecular cloning method is a useful tool for distinguishing between single and multiple infection events by different ABV genotypes.

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.

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.

Natural zoonotic infections of two marmosets and one domestic rabbit with herpes simplex virus type 1 did not reveal a correlation with a certain gG-, gI- or gE genotype.

Infections with herpes simplex virus type 1 (HSV-1) are not restricted to humans but infrequently may be transmitted to certain animal species, in some cases resulting in severe disease, including encephalitis and death. Recent studies demonstrate that humanderived HSV-1 field isolates can be typed according to their gG- gIand gE gene sequences. We investigated whether HSV-1 infections of animals were predominantly caused by a certain genotype. Isolates derived from two marmosets and one domestic rabbit, however, revealed different genotypes. Despite the very limited number of investigated animal-derived HSV-1 strains, this result does not point towards the existence of certain HSV-1 genotypes with a higher potential of being transmitted to animals.

Distribution of Borna disease virus antigen and RNA in tissues of naturally infected bicolored white-toothed shrews, Crocidura leucodon, supporting their role as reservoir host species.

Borna disease is a severe viral-induced disorder of the central nervous system of horses, sheep, and a few other animal species, occurring in certain areas of central Europe. Pathogenesis and epidemiology of natural Borna disease virus (BDV) infections are still not fully understood; several unique epidemiologic features, however, point toward the existence of BDV reservoir populations other than the final hosts. In this study, 69 mice and 12 shrews were trapped and examined. The virus distribution was investigated in detail in 2 BDV-positive bicolored white-toothed shrews, Crocidura leucodon, by immunohistochemistry and TaqMan real-time reverse transcription polymerase chain reaction (RT-PCR). RT-PCR amplification products were sequenced, and the sequences were compared. These shrews had been collected in a BDV-endemic geographical region using live traps and did not show obvious clinical or pathological disease signs. BDV antigen and nucleic acid were identified in several organs, including the brain, mainly in nerve tissue and neurons, respectively, but also in parenchymal cells (eg, hepatocytes, Leydig cells) and epithelial cells, particularly of the respiratory and urogenital tract.

Partial sequencing of recent Portuguese myxoma virus field isolates exhibits a high degree of genetic stability.

To study genetic changes underlying myxoma virus evolution in its new host, the European rabbit (Oryctolagus cuniculus), we sequenced selected genomic regions of nine recent virulent field strains and a live attenuated vaccine strain ("MAV", Germany). DNA was extracted from cell culture passaged myxoma virus. A total of 4863 bp (approximately 3% of the genome) of 10 regions spanning 12 genes of the myxoma viruses was sequenced and compared to the original virulent strain "Lausanne" and its attenuated field derivative strain "6918". The field strains displayed a maximum of three (strains C43, C95) and a minimum of one (strains CD01, CD05) nucleotide substitutions. These were distributed through all analysed coding regions, except gene M022L (major envelope protein), where all strains were identical to "Lausanne" and "6918". Two new single nucleotide insertions were observed in some of the field strains: within the intergenic region M014L/M015L and within gene M009L, where it leads to a frameshift. These insertions were located after homopolymeric regions. The vaccine strain displayed 37 nucleotide substitutions, predominantly (95%) located in genes M022L and M036L. Interestingly, regions M009L and M014L/M015L of the vaccine were not amplified successfully, suggesting major genomic changes that could account for its attenuated phenotype. Our results support a high degree of genetic stability of myxoma virus over the past five decades. None of the analysed genome regions by its own seems sufficient for the genetic characterisation of field strains.

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.