Country-wide distribution of bluetongue virus with expanding host spectrum and evidence of vector competence in Hungary.

Following the introduction of bluetongue virus type 4 (BTV-4) in 2014, country-wide monitoring of bluetongue (BT) disease was performed to see whether the virus has become enzootic in Hungary. To analyse the epizootiology of BT, over 110,000 samples collected from domestic and wild ruminants were screened for the presence of BTV RNA and virus-specific antibodies using real-time RT-PCR assay and commercial ELISA kit, respectively. During laboratory analysis, specimens collected from 333 (0.8%) cattle, 79 (2.2%) sheep, 4 (0.9%) goats, and 1 (2.3%) mouflon were found to be positive by viral RNA-detection assay. In addition, antibody to BTV was detected in 5.5% (3158/57,250) of cattle, 10.1% (517/5120) of sheep, 40% (116/290) of goat, and 5.6% (16/284) of buffalo origin samples. The majority of positive samples originated from south-western counties; however, 18 out of 19 counties reported cases or antibody prevalence in the examined animals. Genome sequencing of a representative BTV-4 strain from 2015 was also performed. When comparing this strain with the isolate BTV4-HUN2014 detected only a year earlier in Hungary, mutations at 14 sites were identified within the amplified and sequenced genome. Our findings reinforce the need for continued surveillance of BT disease in Hungary. Keywords: reoviridae; orbivirus; cattle; sheep; goat; biting midge.

Resurgence of rabies in Hungary during 2013-2014: An attempt to track the origin of identified strains.

In 2013-2014, accumulation of rabies episodes raised concerns regarding ongoing elimination programme in Hungary. Nearly four dozen cases were identified over a 13-month period in the central region of the country far behind the immunization zones. Although the outbreak was successfully controlled, the origin of disease remained unknown. In this study, we sequenced the partial N and G genes from 47 Hungarian rabies virus (RV) strains isolated from the 2013-2014 outbreak. Sequencing and phylogenetic analysis of the N and G genes showed that the Hungarian RV isolates share high nucleotide similarity among each other (up to 100%). When analysing the N gene, comparable sequence similarity was seen between the outbreak strains and some historic Romanian RV strains. Unfortunately, in the lack of available sequence data from the Romanian RV strains, the genetic relationship within the G gene could not be determined. Phylogenetic analysis of Hungarian RV isolates detected in the past revealed that multiple independent RV lineages circulated in our country over the past 25 years. The parental strain of the 2013-2014 outbreak may have been imported independently perhaps from east through transborder movement of a reservoir animal. Next to the introduction, this imported RV strain seems to have spread clonally in the affected area. Our findings indicate that despite effective control measures that, overall, minimized the incidence of rabies over the past decade, field and laboratory monitoring needs to be continued to make rabies elimination programme in Hungary successful.

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.

Central autonomic control of the bone marrow: multisynaptic tract tracing by recombinant pseudorabies virus.

Bone marrow is the primary place of hematopoiesis, where the development, survival and release of multipotent stem cells, progenitors, precursors and mature cells are under continuous humoral and neural control. Dense network of nerve fibers, containing various neurotransmitters is found in the bone marrow, however, the central neuronal circuit that regulates the activities of the bone marrow through these fibers remained unexplored. Transsynaptically connected neurons were mapped by virus-based transneuronal tracing technique using two isogenic, genetically engineered pseudorabies viruses, Bartha-DupGreen and Ba-DupLac expressing green fluorescent protein and beta-galactosidase, respectively. Bartha-DupGreen was injected into the femoral bone marrow of male rats and the progression of infection was followed 4-7 days post-inoculation. Virus-labeled cells were revealed in ganglia of the paravertebral chain and in the intermediolateral cell column of the lower thoracic spinal cord. Neurons were retrogradely labeled in the C1, A5, A7 catecholaminergic cell groups and several other nuclei of the ventrolateral and ventromedial medulla, the periaqueductal gray matter, the paraventricular and other hypothalamic nuclei, and in the insular and piriform cortex. Nerve transections and double-virus tracing from the bone marrow and the surrounding muscles were used to confirm the specific spreading of the virus. These results provide anatomical evidence for the CNS control of the bone marrow and identify putative brain areas, which are involved in autonomic regulation of the hematopoiesis, the release of progenitor cells, the blood supply and the immune cell function in the bone marrow.

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.

Equine viral arteritis in a newborn foal: parallel detection of the virus by immunohistochemistry, polymerase chain reaction and virus isolation.

A 4-days-old foal died after a short course of respiratory syndrome and fever. Large areas of the alveoli, bronchioles and bronchi were partly or completely filled by hyaline membranes. Pronounced oedema and mild interstitial pneumonia were present and, in the small muscular arteries, fibrinoid necrosis and vasculitis or perivasculitis could be seen. Vasculitis was found in several other organs, and it was most severe in the thymus. The virus was detected in the lung, kidney and spleen using virus isolation and in the lung and spleen using polymerase chain reaction. The virus was also detected in several organs and cell types using both N protein-specific monoclonal antibody and horseradish peroxidase-labelled equine arteritis virus-specific equine IgG.

Restriction endonuclease analysis of Hungarian bovine herpesvirus isolates from different clinical forms of IBR, IPV and encephalitis.

The DNA of thirty-three Hungarian bovine herpesvirus isolates originating from cattle with various clinical symptoms were compared by restriction endonuclease analysis using HindIII, EcoRI and BstEII enzymes. The EcoRI and HindIII cleavage patterns were similar to those of isolates studied in other countries. Based on the cleavage patterns, the Hungarian isolates could be assorted into groups and subgroups according to the classification system proposed by Metzler et al. (1985, 1986). Based on a new BstEII cleavage pattern observed in group 1, the establishment of two new subgroups, 1a and 1b, were proposed. Three isolates belonged to subgroup 1a, sixteen to 1b, seven to 2a, five to 2b, and one to group 3, which has recently been reclassified as BHV-5. Additionally, one of the isolates showed a mixed cleavage pattern of 1a and 1b. However, no strict correlation was found between the different clinical forms and the established DNA fingerprint groups. There was no evidence of a change in the prevalence of the different genotypes when comparing isolates collected at different times of a 24-year period.

The pathology of concurrent bovine viral diarrhoea and infectious bovine rhinotracheitis virus infection in newborn calves.

Concurrent bovine viral diarrhoea (BVD) and systemic infectious bovine rhinotracheitis (IBR) are reported from two neonatal (11 and 15 days old) calves. The diseases occurred sporadically in a large-scale herd which may have been due to the calves' heterogeneous immunobiological status. Gross pathological and histopathological examinations revealed focal interstitial pneumonia with acidophilic intranuclear inclusions in the alveolar epithelial cells and necrotic foci in the liver with a few intranuclear inclusions in the hepatocytes. There were subserous haemorrhages in the forestomachs and intestine, necrotic changes in the rumen, enteritis, lymphocytic necrosis in the Peyer's patches, and fibrinoid necrosis in the wall of some of the neighbouring blood vessels. BVD virus was demonstrated by immunofluorescence (IF), whereas IBR virus by electron microscopy, immunofluorescence and virus isolation.