Bokeloh bat lyssavirus isolation in a Natterer's bat, Poland.

In recent years, Bokeloh bat lyssavirus (BBLV), a member of the novel lyssavirus genus Bokeloh bat lyssavirus in the family Rhabdoviridae, has been detected in Germany (five cases) and France (two cases). Here, we report the isolation of BBLV in a Natterer's bat (Myotis nattereri) in Poland. The bat brain tested positive for rabies using classical diagnostics tests (FAT and RTCIT) and then subsequently confirmed by molecular techniques. Viral RNA was found in all peripheral organs tested, and the highest viral loads were detected in brain, the salivary gland and bladder. Phylogenetic analysis performed on complete viral genome sequences revealed the closest homology to representatives of BBLV lineage B, isolated previously in southern Germany. This case provides further evidence that BBLV is widespread in Europe.

Genetic Analysis of the M Gene of Equine Influenza Virus Strains Isolated in Poland, in the Context of the Asian-like Group Formation.

INTRODUCTION: Traditionally, evolutionary analysis of equine influenza virus (EIV) is based on the HA gene. However, the specificity of the influenza virus enables the classification of viral strains into different phylogenetic groups, depending on the gene being analysed. The aim of the study was to analyse phylogenetic paths of EIV based on M gene with reference to the HA gene. MATERIAL AND METHODS: M gene of Polish isolates has been sequenced and analysed along with all M sequences of EIV available in GenBank database. Phylogenetic analysis was performed using BioEdit, ClustalW, and MEGA7 softwares. RESULTS: The clustering of the strains isolated not only from Asia but also from Europe into one common Asian-like group of EIV was observed. Twelve nucleotide substitutions in the M gene of strains from the Asian-like group were crucial for the evolutionary analysis. We also observed homology in the M gene of the Asian-like and H7N7 strains. CONCLUSIONS: M gene specific for the Asian-like group is present in strains recently isolated in Europe and Asia, which were classified previously in the Florida 2 clade based on HA. Therefore, Asian-like group does not seem to be assigned to a specific geographical region. Traces of H7N7 strains in more conservative genes like M of some contemporary EIV strains may indicate the link between the old phylogenetic group and recent H3N8 strains. Analysis of conservative genes may be more useful in tracking the direction of virus evolution than in the genes where the high variability rate may blur the original relationships.

Seroprevalence of bovine herpesvirus 1 related alphaherpesvirus infections in free-living and captive cervids in Poland.

To determine the occurrence of bovine herpesvirus 1 (BoHV-1) related alphaherpesvirus infections in cervids, 1194 serum samples of wild ruminants originating from 59 forest districts of Poland were tested with IBR gB ELISA and virus neutralization test (VNT) against BoHV-1 and cervid herpesvirus 1 (CvHV-1). The seroprevalence differed significantly between free-living and captive cervids (P<0.001) with a total of 89 out of 498 (17.9%) and 268 out of 696 (38.5%) seropositive animals in each type of population. In free-ranging cervids, the highest seroprevalence was found among red deer (25.6%) and in fallow deer (23.1%), while it was the lowest in roe deer (1.7%). The seroprevalence varied at the district level between 0 and 100% with the mean value of 17.4% (95% CI:10.1-24.0). Additionally, seroprevalence was associated with afforestation (χ2=7.5; P=0.006) and to some degree with the mean of cattle density in province (χ2=7.0; P=0.08). The mean antibody titre against CvHV-1 in VNT (161.8; 95%CI: 146.0-177.6) has been significantly higher (P<0.0001) than the mean titre of BoHV-1 antibodies (10.1; 95%CI: 8.9-11.4). The results showed that BoHV-1 related alphaherpesvirus infections are present in population of free-ranging and farmed cervids in Poland. Based on the VNT results and considering the low susceptibility of red deer to BoHV-1, it seems that the dominant alphaherpesvirus circulating in wild ruminants is most likely CvHV-1 and therefore it is rather unlikely that deer in Poland could play any role as a reservoir of BoHV-1 for cattle.

Diagnostic Reliability of Different RT-PCR Protocols for the Detection of Bluetongue Virus Serotype 14 (BTV-14).

INTRODUCTION: The reverse transcription polymerase chain reaction (RT-PCR) is one of the most extensively used methods for identification of animals infected with bluetongue virus (BTV). There are several RT-PCR protocols published and several real-time RT-PCR (rtRT-PCR) commercial kits available on the market. Because Poland faced BTV-14 infection in 2012, different protocols were implemented in the country to confirm the RT-PCR results positive for this virus. The article presents a comparative study of several RT-PCR protocols and discusses their diagnostic reliability and applicability. MATERIAL AND METHODS: Six rtRT-PCR/RT-PCR protocols were compared for the laboratory diagnostic of fourteen BTV-14 isolates circulating in Poland in 2012-2014. RESULTS: All 14 isolates were positive in the protocols of Shaw et al. (18), a commercial LSI NS3 kit, and Eschbaumer et al. (5). Four out of fourteen BTV-14 isolates gave positive results in Hoffmann's 2 and 6 protocols and none of the 14 isolates yielded positive results in Maan et al. (8) method. Phylogenetic study of a short fragment of 450 nt of BTV segment 2 (258-696 positions) revealed 100% identity within Polish variants and with Russian and Spanish isolates. CONCLUSION: The paper points to the possible false negative results in the diagnosis of BTV infections depending on the protocol used.

First detection of bluetongue virus serotype 14 in Poland.

Here, we present the first detected cases of bluetongue virus (BTV) in native cattle from Poland. The virus was found in animals located near the Polish-Belarusian and Polish-Lithuanian borders. The positive animals were detected through an official epidemiological surveillance program. A combination of type-specific real-time RT-PCR and phylogenetic tests revealed the presence of BTV serotype 14 (BTV-14). This serotype is highly homologous to the vaccine strain and BTV-14 present in Russia, Lithuania, and Spain (from an animal imported from Lithuania). The most probable route of virus introduction to Poland was transmission through midges. All of the cases were subclinical.

NS-gene based phylogenetic analysis of equine influenza viruses isolated in Poland.

The phylogenetic analysis of influenza virus is based mainly on the variable hemagglutinin or neuraminidase genes. However, some discrete evolutionary trends might be revealed when more conservative genes are considered. We compared all available in GenBank database full length NS sequences of equine influenza virus including Polish isolates. Four nucleotides at positions A202, A237, T672 and A714 and three amino acids at positions H59, K71 and S216 which are also present in A/eq/Pulawy/2006 and A/eq/Pulawy/2008 may be discriminating for the Florida sublineage. Threonine at position 83 seems to be characteristic for EIV strains of Florida 2 isolated after 2007. There are nine common substitutions in the NS sequences of A/eq/Pulawy/2005, A/eq/Aboyne/1/2005 and A/eq/Lincolnshire/1/2006 in relation to the reference strain A/eq/Miami/63, resulting in four amino acid changes in NS1 protein (I56, E76, K140, E179) and one in NEP (R22). We grouped these strains as "Aboyne-like". Some of the listed changes were also observed in H7N7 strains isolated between 1956 and 1966, in A/eq/Jilin/89 or in pre-divergent H3N8 strains. Two hypotheses regarding the origin of this group were postulated: three independent transfers of avian influenza viruses into the equine population or reassortation between H7N7 and H3N8 EIV. Similarities of the NS sequences of "Aboyne like" viruses to viruses isolated in the fifties or seventies can reflect a phenomenon of "frozen evolution".

Detection of the neuropathogenic variant of equine herpesvirus 1 associated with abortions in mares in Poland.

BACKGROUND: The incidence of reported cases of equine herpesvirus myeloencephalopathy (EHM) caused by infection with neuropathogenic strains of equine herpesvirus 1 (EHV-1) has markedly increased over the last decade in many Western countries. The purpose of this study was to estimate the prevalence of the neuropathogenic (G2254) and non-neuropathogenic (A2254) variants of EHV-1 among isolates associated with abortions in Polish stud farms. RESULTS: The results of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing were consistent, and showed that two out of 64 abortions (3.1%) were induced by the neuropathogenic genotype G2254. All remaining 18 EHV-1 positive abortion cases (28.1%) were caused by the non-neuropathogenic genotype A2254. CONCLUSIONS: Most of the abortions in mares in Poland from 1999 to 2012 were associated with non-neuropathogenic strains of EHV-1. However, the presented data indicate that the neuropathogenic genotype of the virus is also present in Polish stud farms. Such a presence suggests that the future emergence of EHM in Poland is probable.

First report of bovine viral diarrhoea virus-2 infection in cattle in Poland.

This report describes the first identification in Poland of bovine viral diarrhoea virus (BVDV)-2 in a dairy herd where severe clinical disease with losses of young animals was observed. The virus was readily cultivated in cell culture and a phylogenetic analysis of the nucleotide sequences and secondary structures of the viral genomic 5' untranslated region confirmed virus identity. The economic impact of the infection was significant compared to the previously prevalent BVDV-1 infections confirming that this genotype of BVDV can cause severe sickness in affected herds. The use of BVDV-1 vaccine did not prevent the infection with the BVDV-2 genotype.

First detection of Schmallenberg virus in elk (Alces alces) indicating infection of wildlife in Bialowieza National Park in Poland.

Schmallenberg virus (SBV) RNA was detected in the serum of an elk (Alces alces) calf captured on the outskirts of Bialowieza National Park (BNP) in December 2012, and shortly afterwards the calf died of acute bronchopneumonia. Serum samples from 169 animals, including bison, red and fallow deer, originating from eight locations situated in four Polish Provinces, were tested for the presence of SBV-specific antibodies between 2011 and 2013. Although no antibodies were found in samples collected up to July 2012, positive samples subsequently appeared between November 2012 and January 2013 in all of the sampled regions. The introduction of SBV infection to the European bison (Bison bonasus) population of BNP between July and November 2012 was also confirmed.

Detection of the Schmallenberg virus in nulliparous Culicoides obsoletus/scoticus complex and C. punctatus--the possibility of transovarial virus transmission in the midge population and of a new vector.

The arthropod-borne Schmallenberg virus (SBV) emerged in Europe in the late summer/autumn of 2011. SBV spread across the continent until 2012. This paper presents SBV detection in female Culicoides spp. caught in UV traps located in 23 different locations in Poland. The midges were divided into pools containing 20.5 individual insects on average according to species and parity status. The study was based on duplex real-time reverse transcription PCR (RT-PCR) for the detection of the SBV S segment and culicoid 18S gene fragments. Forty-four out of 402 midge pools tested (10.9%) were found to be positive for the presence of viral RNA. The SBV positive Culicoides came from 10 traps spread randomly across the country and were collected between August and October 2012. The timing of the SBV positive midge collections and the locations of the traps corresponded to the epizootic situation of SBV in ruminants. SBV RNA was most frequently identified in gravid midges (36.4%), while in nulliparous, blood-fed and parous midges the percentages were 10.8% 13.0% and 8.1%, respectively. The majority (82%) of SBV positive pools belonged to Culicoides obsoletus/scoticus complex; however, viral RNA was also found in 8 out of the 149 (5.4%) Culicoides punctatus pools tested. While no statistical differences in the Ct values between different parity groups were found, the bimodal distribution observed at the Ct frequency plots suggested active SBV replication, especially in parous and gravid midge females, and sub-transmissible infection in nulliparous and blood-fed insects. The most important findings included identification of C. punctatus as a new possible vector of SBV and the recovery of viral RNA from the nulliparous females which may suggest transovarial transmission in C. obsoletus/scoticus complex and C. punctatus.

Sequence analysis of ORFs 5, 6 and 7 of equine arteritis virus during persistent infection of the stallion--a 7-year study.

Nucleotide and amino acid sequences of ORFs 5, 6 and 7 of EAV during persistent infection in the stallion of the Malopolska breed were analysed in the study. A total of 11 blood and semen samples were collected between 2004 and 2011. The titre of specific EAV antibodies in this carrier stallion was maintained at a high level throughout the study and was equal approximately 1:128. The sequence analysis of ORF5 showed 16 variable sites including 12 with synonymous substitutions and 4 with non-synonymous substitutions. The degree of nucleotide sequence identity among the strains ranged from 98.92% to 100%, whereas amino acid homology ranged from 98.06% to 100%. Ten substitutions were identified including 7 with synonymous mutations and 3 with non-synonymous mutations in ORF6. The degree of similarities among the strains ranged from 94.55 to 100% and from 98.41% to 100% at the level of nucleotide and amino acid sequence, respectively. Only a single point mutation at position 255 of ORF7 (99.6% identity) was found in nucleotide sequences of these strains. Phylogenetic analysis showed that all strains present in the semen of this carrier stallion created a separate cluster of "quasi-species" within the second European subgroup of EAV.

Mass spectrometry identification of granins and other proteins secreted by neuroblastoma cells.

We used mass spectrometry-based protein identification to determine the presence of granins and other proteins in the mouse neuroblastoma secretome. We detected polypeptides derived from four members of the granin family: chromogranin A, chromogranin B, secretogranin III, and VGF. Many of them are derived from previously described biologically active regions; however, for VGF and CgB, we detected peptides not related to known bioactivities. Along with granins, we identified 115 other proteins secreted by mouse neuroblastoma cells, belonging to different functional categories. Fifty-six out of 119 detected proteins possess the signal fragments required for translocation into endoplasmic reticulum. Sequences of remaining 63 proteins were analyzed using SecretomeP algorithm to determine probability of nonclassical secretion. Identified proteins are involved in the regulation of cell cycle, proliferation, apoptosis, angiogenesis, proteolysis, and cell adhesion.

Distribution of a pathological form of prion protein in the brainstem and cerebellum in classical and atypical cases of bovine spongiform encephalopathy.

This study evaluated the distribution and signal intensity of a prion protein resistant to proteolysis (PrP(res)) in the brainstem and cerebellum of cattle affected with classical and atypical forms of bovine spongiform encephalopathy (BSE) using a Western immunoblotting technique. In both classical and atypical cases of BSE, a stronger signal was detected in the more rostral brainstem regions relative to the obex. In classical and H-type cases a significant decrease in the PrP(res) signal was found in the cerebellum when compared to that in the obex, whereas L-type BSE cases were characterised by signals of similar intensity in these regions. The uniform distribution of PrP(res) in the region rostral to the obex suggests that when autolysed samples are being tested for BSE, both classical and atypical forms are detectable, even when this target site is missing or cannot be clearly identified. The findings indicate that both the obex and rostral brainstem can be used for BSE diagnosis whereas use of the more caudal brainstem regions and cerebellum is not recommended.

Characterization of PRNP and SPRN coding regions from atypical scrapie cases diagnosed in Poland.

Scrapie, a fatal transmissible spongiform encephalopathy (TSE) occurs in two phenotypes: classical and atypical. Many authors point out that the polymorphism of three codons (136, 154, 171) of the PRNP (PrP gene) is associated with a sheep susceptibility to classical scrapie. Until now, only one PRNP gene variant coding phenylalanine at codon 141 has been found to be associated with atypical scrapie. Another recently identified and interesting candidate gene for scrapie susceptibility in sheep is an SPRN gene coding for Shadoo protein (Sho). Sho is a highly interspecies conserved protein and an insertion/deletion (indel) found in a sheep Sho gene was associated with classical scrapie occurrence. Here we determined the polymorphism of PRNP and SPRN genes in nine atypical scrapie cases (six in native born sheep and three in imported sheep) and compared these results with a control group of healthy animals comprising six corresponding Polish sheep breeds. In atypical scrapie cases five PRNP diplotypes were identified: A(136)R(154)Q(171)/ARQ, AHQ/ARQ, ARR/ARQ, ARR/AHQ and AHQ/AHQ. The ARR/AHQ diplotype was found only in imported sheep. A previously unobserved SNP in PRNP (E224K) was also found in both atypical scrapie and in a few control animals. In the ORF of the SPRN gene, six SNPs and one indel were identified. None of these variations was exclusive for scrapie animals and they were probably, naturally occurring polymorphisms. Special attention was given to the 6-bp indel SPRN polymorphism which was previously associated with classical scrapie occurrence.

Comparison of mRNA expression levels of selected genes in the brain stem of cattle naturally infected with classical and atypical BSE.

Since 2004 cases of atypical bovine spongiform encephalopathy (BSE) in older cattle are recorded on the basis of aberrant glycoprofiles of prion protein resistant to proteolysis (PrP(res)). The nature of those types of PrP(res) is still not fully understood but the epidemiological data indicate that their occurrence is rare. Hitherto, most BSE cases were studied on the basis of the features of pathological form of prion protein (PrP(Sc)) or lesions observed in the gray matter of the brain. Here we propose the gene expression profiling as a method to characterize and distinguish BSE types. Thus, the aim of the study was to compare the activity of some genes which are known to play a role in the pathogenesis of transmissible spongiform encephalopathies (TSEs). Significant differences in the expression level of the selected genes in the brain stem were observed for 7 out of 11 genes tested when the results for BSE affected and healthy control animals were compared. Significant up-regulation of caspase 3, Bax and 14-3-3 protein encoding genes was apparent in the obex of all BSE affected cattle regardless of the prion type. Significant and unique to BSE H-type up-regulation was detected in prion and SOD1 genes, while BSE C-type was characterized by higher Bcl-2 and Fyn gene expression levels in respect to other BSE types and control animals. Different gene expression profiles of bovine brains infected with classical and atypical BSE indicate possible different pathogenesis or origin of the disease.

Diagnosis of the first cases of scrapie in Poland.

This is the first report of cases of scrapie in Poland. The disease was an atypical phenotype, diagnosed in two aged sheep which were found dead. Brainstem samples from both animals were positive on the applied ELISA rapid test, while the confirmatory immunoblot indicated abnormal banding patterns of protease resistant prion protein (PrP(res)). The genotypes of these sheep were ALRQ/ALHQ and ALRQ/ALRR. The absence of premonitory clinical signs, the advanced age of the affected sheep, the higher concentration of PrP(res) in the cerebellum relative to the obex, the unusual banding profile of the prion protein and its relatively low resistance to proteolytic degradation confirmed the diagnosis of atypical scrapie (Nor98-like) in both cases.

Genetic typing of equine influenza virus isolated in Poland in 2005 and 2006.

Two equine influenza virus strains were isolated from horses during the local respiratory disease outbreaks in Poland in 2005 and 2006. The H3 equine influenza viral RNA was amplified directly from the clinical specimens with RT-PCR and HA1 fragments were sequenced. The highest homology of HA1 nucleotide sequences of A/eq/Pulawy/05 with A/eq/Aboyne/1/05 and A/eq/Pulawy/06 with A/eq/Essex/2/05 was found. The phylogenetic analysis based on amino acid sequences of HA1 fragments of 84 equine influenza virus strains isolated in Europe during the period of 1976-2007 was conducted to determine the evolutionary relationship of the two Polish and the other European isolates. The resulting phylogenetic tree clearly clustered A/eq/Pulawy/05 with the strains belonging to the European lineage of the equine influenza virus. On the other hand A/eq/Pulawy/06 was placed in the Florida sub-lineage of the American type strains. The presence of the same amino acids: methionine, asparagine and threonine at the positions 48, 159 and 163 respectively, in both Polish isolates, despite the fact that the strains are grouped in two different lineages may indicate the existence of the common ancestor. It is possible that A/eq/Pulawy/06 evolved locally rather than was introduced.

Ecology and evolution of rabies virus in Europe.

The evolution of rabies viruses of predominantly European origin was studied by comparing nucleotide sequences of the nucleoprotein and glycoprotein genes, and by typing isolates using RFLP. Phylogenetic analysis of the gene sequence data revealed a number of distinct groups, each associated with a particular geographical area. Such a pattern suggests that rabies virus has spread westwards and southwards across Europe during this century, but that physical barriers such as the Vistula river in Poland have enabled localized evolution. During this dispersal process, two species jumps took place - one into red foxes and another into raccoon dogs, although it is unclear whether virus strains are preferentially adapted to particular animal species or whether ecological forces explain the occurrence of the phylogenetic groups.