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.

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.

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.

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.

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.

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.

Altered gene expression may underlie prolonged duration of the QT interval and ventricular action potential in streptozotocin-induced diabetic rat heart.

Ventricular electrical conduction has been investigated in the streptozotocin (STZ)-induced diabetic rat. Diabetes was induced with a single injection of STZ (60 mg/kg bodyweight, ip). The ECG was measured continuously, in vivo, using a biotelemetry system. Left ventricular action potentials were recorded with an extracellular suction electrode. Expression of mRNA transcripts for selected ion transport proteins was measured in left ventricle with real-time RT-PCR. At 10 weeks after STZ treatment, in vivo heart rate (HR) was reduced (267 +/- 3 vs. 329 +/- 5 BPM), QRS complex duration and QT interval were prolonged in diabetic rats compared to controls. In vitro spontaneous HR was reduced and paced heart action potential repolarization was prolonged in diabetic rats compared to controls. The mRNA expression for Kcnd2 (I (to) channel) and Kcne2 (I (kr) channel) was significantly reduced in diabetic rats compared to controls. Altered gene expression and, in particular, genes that encode K(+) channel proteins may underlie delayed propagation of electrical activity in the ventricular myocardium of STZ-induced diabetic rat.

Evolution of rabbit haemorrhagic disease virus (RHDV) in the European rabbit (Oryctolagus cuniculus) from the Iberian Peninsula.

To date information on rabbit haemorrhagic disease virus (RHDV) in Spain and Portugal has been scarce, although the disease is endemic and continues to have a considerable impact on species conservation and hunting industry. We analysed RHDVs obtained between 1994 and 2007 at different geographic locations in Portugal (40 samples), Spain (3 samples) and France (4 samples) from wild European rabbits (Oryctolagus cuniculus) that succumbed to the disease. Phylogenetic analyses based on partial VP60 gene sequences allowed a grouping of these RHDVs into three groups, termed "Iberian" Groups IB1, IB2 and IB3. Interestingly, these three Iberian groups clustered separately, though not far from earlier RHDVs of Genogroup 1 (containing e.g., strain "AST89"), but clearly distinct from globally described RHDV strains of Genogroups 2-6. This result, supported by a bootstrap value of 76%, gives rise to the hypothesis that the virus evolved independently since its introduction to wild rabbit populations on the Iberian Peninsula, with the Pyrenees acting as a natural barrier to rabbit and hence to virus dispersal. No differences were observed in RHDV sequences obtained from geographic regions where the rabbit subspecies O. c. algirus prevails compared with those obtained from O. c. cuniculus.

Influence of different semen extenders and seminal plasma on PMN migration and on expression of IL-1beta, IL-6, TNF-alpha and COX-2 mRNA in the equine endometrium.

After artificial insemination or mating an inflammatory response is induced by spermatozoa and components of the inseminate or ejaculate. In order to investigate the inflammatory reaction of the endometrium to different semen extenders, phosphate buffered saline (PBS), seminal plasma (SP), skim milk-based extender (SM) or egg yolk semen extender (EY) was inoculated into the uterus of oestrous mares (n=8) during four consecutive cycles in alternating order. Twelve hours after treatment, a uterine lavage was performed and an endometrial biopsy was taken. An additional biopsy was taken in the oestrous cycle before experiments were started. No differences in volume, pH, specific density or cell count of lavage fluid were found between the treatments. A significantly (p<0.01) lower number of leukocytes in the endometrium was identified in pre-experiment biopsies (68+/-5 leukocytes per field) compared to PBS (154+/-32), SP (175+/-22), SM (193+/-29) and EY treatments (113+/-17). PMN numbers were lower (p<0.01) after infusion of EY (23+/-10) compared to PBS (59+/-21) and SM extender (69+/-21). The number of eosinophils increased after inoculation of SP (p<0.05 vs. PBS, SM and EY). All treatments increased expression of interleukins (IL)-1beta and 6, tumor necrosis factor-alpha (TNF-alpha) and cyclooxgygenase-2 (COX-2) in the endometrium compared to pre-experiment values. Expression of COX-2 mRNA was significantly higher after infusion of SM than after PBS treatment (p<0.04). In conclusion, extender alone as well as seminal plasma and PBS causes an inflammatory endometrial response with the least pronounced response induced by EY-based semen extender.

Embryo transfer induces a subclinical endometritis in recipient mares which can be prevented by treatment with non-steroid anti-inflammatory drugs.

We tested the hypothesis that subclinical endometritis occurs after embryo transfer (ET) in the horse. Recipient mares were treated with meclofenamic acid (M) or flunixin meglumin (F) after ET or were left untreated (n=9 per group). Embryos were re-collected 4 days after transfer. Endometrial biopsies were taken for histology and analysis of cyclooxygenase-2 (COX-2) by immunohistochemistry and for PCR. Bacteriological swabs were collected from the uterus and lavage fluid of donor and recipient mares. Progesterone and prostaglandin F(2alpha) release was analysed in recipient mares after ET. Four days after ET, four embryos were recovered from group M and three from group F and untreated mares, each. The number of polymorph nuclear neutrophils was reduced in treated mares (p<0.05). Expression of mRNA for inflammatory cytokines did not differ between groups. In group M, expression of endometrial prostaglandin-E-synthase was higher than in group F (p<0.05). Three out of nine control mares underwent preterm luteolysis (p<0.05 vs. treatment groups), prostaglandin release (p<0.05) and the number of COX-2 positive cells (p<0.01) were significantly higher than in treated mares. Only few bacteriological swabs were positive. In conclusion, treatment of embryo recipient mares with non-steroid anti-inflammatory drugs inhibits the inflammatory response of the endometrium after ET. Meclofenamic acid may have advantages in comparison to flunixin meglumin due to a different influence on prostaglandin synthesis that may not result in inhibition of embryonic mobility.

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%.

Expression of the oxytocin gene, but not the vasopressin gene, in the rat uterus during pregnancy: influence of oestradiol and progesterone.

Oxytocin (OT) and vasopressin (VP) are neurohypophyseal hormones with potent stimulatory actions on the uterus. In order to determine whether these hormones may have a paracrine action on the uterus, OT and VP gene expression was studied in myometrium from pregnant rats at gestational ages of 14 and 20 days, and from ovariectomized animals treated with oestradiol and progesterone. OT and VP mRNA concentrations were measured using real-time quantitative reverse transcription-PCR, and OT- and VP-like immunoreactivities were determined using RIA. OT mRNA was detected in the uterus from pregnant rats, but did not differ between the groups of different gestational ages. Oestradiol significantly (P<0.05) stimulated OT gene expression in ovariectomized rats. Progesterone alone was without effect on OT mRNA concentrations, but significantly (P<0.05) reduced the oestradiol-induced OT mRNA accumulation. The OT-like immunoreactivity in an extract of myometrium from pregnant rats was eluted from a reverse-phase HPLC column with a retention time identical to that of synthetic OT. Neither VP mRNA nor VP-like immunoreactivity was detected in the myometrium from pregnant or ovariectomized rats. The study demonstrates steroid-dependent expression of the OT gene in the rat uterus and processing of uterine preprooxytocin to the mature nonapeptide. The data support the theory that this peptide may act in a paracrine pathway. No evidence was found for the presence of VP in the uterus so that, if the hormone is involved in a stimulatory action on this tissue, it probably acts via an endocrine mechanism.

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.

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.

CTX-M-15-producing multidrug-resistant enteroaggregative Escherichia coli in the United Arab Emirates.

Extended-spectrum beta-lactamase (ESBL) production was demonstrated in five independent, multidrug-resistant isolates of enteroaggregative Escherichia coli (EAEC) from the United Arab Emirates, representing 11.3% of the EAEC isolates recovered during 1 year. All five isolates carried the bla(CTX-M-15) and the bla(TEM-1) genes, the former positioned 48 bp downstream of an ISecp1 element. In two isolates, the bla(CTX-M-15 )and bla(TEM-1) genes were located on a 95-kb plasmid. This is the first detailed description and characterisation of ESBL production in enteroaggregative E. coli and also the first report of CTX-M-producing organisms encountered on the Arabian Peninsula.

Genetic variability of encephalomyocarditis virus (EMCV) isolates.

In order to evaluate the variability of encephalomyocarditis virus (EMCV), field isolates originating from different European regions and inducing different clinical pictures in pigs have been molecularly characterised. The regions targeted were the poly(C) tract, a part of the 5'-UTR (360 nucleotides), the Leader gene (201 nucleotides), the complete capsid coding region (2502 nucleotides), the 2A gene (403 nucleotides), the end of the 3D polymerase gene (305 nucleotides) and the 3'-UTR (123 nucleotides). Analyses have also been performed on a virulent field isolate, which had been subjected to serial passages in vivo and in vitro resulting, in the case of the in vitro passaged virus, in attenuation, as demonstrated by animal experiments. The present study shows that different clinical pictures, such as acute fatal myocarditis or reproductive failure, may not only be caused by EMCV isolates which are genetically diverse but also by the same isolate. Thus no correlation could be demonstrated between genotype and clinical disease. However, the European isolate which showed the highest genetic divergence also gave rise to a more complex clinical picture. Despite EMCV having been isolated from cases of acute fatal myocarditis in pigs in certain areas of the world for many years, clinical disease, including a variety of clinical pictures and pathogenicity, has only been recognised in Europe since 1986 and thus it can be considered an emerging disease in this region. These findings, associated with the reported phenotype changes of the virus under environmental changes (passages), along with its wide distribution among vertebrate species (including higher primates), shows the validity of considering EMCV as a potential pathogen for recipients in xenotransplantation.

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.

Prevalence of neutralizing antibodies to Equine rhinitis A and B virus in horses and man.

Equine rhinitis viruses (ERVs) are the causative agents of mild to severe upper respiratory infections in horses worldwide. Immunologically, four serotypes of ERVs have been identified. Equine rhinitis A virus (ERAV) and Equine rhinitis B virus 1 (ERBV1) are the most frequent serotypes in Europe. Both viruses have a broad host range in cultured cells with ERAV being able to infect humans. Since there is neither information on the seroprevalence of ERAV and ERBV1 in Austria nor on the zoonotic potential of ERBV1, we investigated 200 horse and 137 veterinary sera for the presence of neutralizing antibodies relating to ERAV and ERBV1. One hundred and eighty (90%) and 173 (86%) horse sera neutralized ERAV and ERBV1, respectively. In contrast, only four (2.7%) and five (3.6%) human sera showed weak neutralizing activity to ERAV and ERBV1, respectively. These results indicate that ERAV and ERBV1 are widespread in the Austrian horse population; however, the risk of acquiring zoonotic infection among veterinarians appears low.

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.