Transcriptomic Analysis of MDBK Cells Infected with Cytopathic and Non-Cytopathic Strains of Bovine Viral Diarrhea Virus (BVDV).

Bovine viral diarrhea virus (BVDV) belongs to the Flaviviridae family and the Pestivirus genus. Infection with BVDV causes a disease with a wide spectrum of clinical symptoms, most often mild, although infections with this virus constitute a serious economic problem all over the world. The virus is characterized by a high genetic variability, while the accumulation of single mutations leads to the formation of its new variants. The aim of this study was to better understand the complicated pathogenesis of this disease at the molecular level via the analysis of the transcriptome of cells infected with this virus. The bovine kidney cell line (MDBK), the cytopathic (cp) reference strain, and two non-cytopathic (ncp) BVD virus field strains were used in transcriptomic studies. The cell transcriptome was tested 24 and 72 h after infection. The results of the microarray analysis revealed changes in the expression levels of numerous genes. Genes with changed expression as a result of infection with the cp strain caused changes in the expression levels of a large number of genes and enriched a number of pathways. Genes with increased expression levels were enriched among other pathways involved in the cell cycle, while genes with reduced expression levels enriched pathways mostly related to metabolism. Genes with increased expression levels as a result of infection with ncp strains enriched a much smaller number of pathways, among them, pathways related to signaling activity 24 h post-infection and serine biosynthetic pathways both 24 and 72 h post-infection. Pathways enriched by genes with reduced expression levels were related to the innate immune response (72 h post-infection) or metabolism (24 and 72 h post-infection). The results of microarray studies can help us to better understand the host's response to BVDV infection.

Lack of Fetal Protection against Bovine Viral Diarrhea Virus in a Vaccinated Heifer.

The aim of the report was to present the circulation of BVDV (bovine viral diarrhea virus) in the cattle population and determine the cause of the failure of vaccination failure leading to the birth of the PI (persistently infected) calf. The case study was carried out at the BVDV-free animal breeding center and cattle farm, where the vaccination program against BVDV was implemented in 2012, and each newly introduced animal was serologically and virologically tested for BVDV. In this case, a blood sample was taken from a 9-month-old breeding bull. Positive RT-PCR and negative ELISA serology results were obtained. The tests were repeated at 2-week intervals, and the results confirmed the presence of the virus and the absence of specific antibodies, i.e., persistent infection. Additionally, sequencing and phylogenetic analysis were performed, and the BVDV-1d subgenotype was detected. The results of this study showed that pregnant heifers and cows that are vaccinated multiple times with the killed vaccine containing BVDV-1a may not be fully protected against infection with other subgenotypes of BVDV, including their fetuses, which can become PI calves.

Variability of E2 protein-coding sequences of bovine viral diarrhea virus in Polish cattle.

Bovine viral diarrhea virus (BVDV) belongs to the Pestivirus genus of the Flaviviridae family and has worldwide distribution, being one of the main causes of economic losses in cattle raising. The genome of pestiviruses is a single strand of positive-sense RNA with a length of 12.3 kb, which encodes one open reading frame flanked by untranslated regions. E2 glycoprotein is required for binding to cell-surface receptors and it also contains major antigenic determinants. The nucleotide sequence coding E2 is the most variable part of the viral genome. The heterogeneity that exists among circulating strains causes problems in the development of effective vaccines and reliable diagnostics. In this study, and for the first time analysis was made of the E2 glycoprotein coding sequences of 14 Polish BVDV-1 strains which belong to four subtypes: 1b (n = 7), 1f (n = 3), 1s (n = 3), and 1r (n = 1). These sequences showed evidence of strong purifying (negative) selection. However, we also identified positively selected sites. The availability of E2 sequences of Polish BVDV strains for reference, knowledge gained through epitope prediction attempts, and information on protein glycosylation sites can afford a better understanding of host-pathogen interactions.

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.

Bovine Neonatal Pancytopenia - correlation between clinical symptoms, hematological parameters and lifespan.

OBJECTIVE: To compare the hematological parameters and clinical symptoms between Bovine Neonatal Pancytopenia (BNP) diseased calves dying before and after 14 days of life. MATERIAL AND METHODS: Clinical observations included 47 calves from dams which underwent a 3-year vaccination program with the inactivated PregSure® BVD vaccine. In 25 of these 47 BNP affected calves blood examinations were performed and in 22 dead calves diagnosis was mainly based on post-mortem findings. RESULTS: Cutaneous bleeding was the predominant clinical manifestation in 32 from 47 calves (68.1%). Seven from 47 calves (14.9%) developed cutaneous bleeding as the only symptom and 17 from 47 calves (36.2%) demonstrated these alterations in combination with hemorrhagic lesions of the oral mucosa. In 66.0% (31/47) of calves petechiae of the oral mucosa were seen and petechiation without any other BNP related symptoms occurred in eight from 47 calves (17.0%). The hematological analysis revealed thrombocytopenia in all 25 cases (n = 23: PLT < 60 x 109/l, n = 2: PLT 139-164 x 109/l). Nineteen from 25 calves (76.0%) developed thrombocytopenia and leukocytopenia (WBC < 3.5 x 109/l). In nine of them a decrease of erythrocyte count (RBC < 4.5 x 109/l), hemoglobin concentration (Hb < 8 g/dl) and packed cell volume (PCV < 24%) was measured. Three BNP affected calves without clinical symptoms were identified by hematological examination. The average life time of BNP affected calves was 14.7 ± 6.2 days. Clinical findings, especially multifocal cutaneous hemorrhages were more frequently recognized in calves living longer than 14 days. CONCLUSION AND CLINICAL RELEVANCE: At the time of falling ill with BNP, older calves displayed more numerous symptoms, especially bleeding in the skin. Thrombocytopenia and erythropenia occur as well as a decreased hemoglobin concentration and a low PCV. The time between outbreak of symptoms and death of calves which fell ill later, did not differ from the survival time of BNP calves, which displayed symptoms at a younger age. A decrease of thrombocytes was the cardinal laboratory finding.

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.

Bulk tank milk somatic cell counts in dairy herds with different bovine viral diarrhoea virus status in Poland.

The aim of the study was to examine the effect of bovine viral diarrhoea virus (BVDV) infection on bulk tank milk somatic cell counts (BMSCC). Twenty nine dairy farms supplying milk to a dairy in Eastern Poland were recruited for the study. Bulk milk ELISA and RT-PCR were used to determine the BVDV infection status and the presence of PI animals in the farms. The BMSCC mean values for the BVDV seronegative (218.7 × 10(3)cells/ml; SD: 89.8) and seropositive (214.9 × 10(3)cells/ml; SD: 74.0) herds did not differ significantly. To assess the relationship between BVDV infection and BMSCC a multilevel mixed-effects linear model was used. No statistically significant effect of BVDV infection on BMSCC was found. The mean values of BMSCC for the herds with PI individuals measured before (230.1 × 10(3)cells/ml, SD: 64.9) and after (223.3 × 10(3)cells/ml, SD: 62.4) the PI removal were not statistically different. An increase in herd size was associated with a significant decrease in BMSCC. An increase in BMSCC was observed during summer (from May to September) compared to during winter (from October to April).

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.

Comparison of the performance of five different immunoassays to detect specific antibodies against emerging atypical bovine pestivirus.

Bovine pestiviruses represent a considerably variable group. In addition to the two accepted species BVDV-1 and BVDV-2, a number of atypical bovine pestiviruses have been detected both in foetal calf sera and in field samples. The sera collected during the initial six weeks of experimental infection of calves with atypical pestivirus, BVDV-1 and a combination of both viruses have been examined by routine and new diagnostic tests to validate their robustness and sensitivity. As expected, virus neutralization tests using homologous virus were able to differentiate the two groups infected by BVDV-1 or atypical pestivirus, whereas the animals inoculated with a mixture of these two viruses had a reaction pattern very similar to the homologous virus alone. It was found that immunoassays using whole virus and polyclonal antibodies are the most robust, but all tests examined were able to detect antibodies also from cattle infected with atypical pestivirus a few weeks after infection. The detection, however, was at a lower level and slightly delayed. Statistical validation of the threshold suggested by the manufacturer showed that in some cases the reduction of the cut-off values would improve the test sensitivity.

Kinetics of single and dual infection of calves with an Asian atypical bovine pestivirus and a highly virulent strain of bovine viral diarrhoea virus 1.

Atypical bovine pestiviruses related to bovine viral diarrhoea virus (BVDV) have recently been detected in cattle from South America, Asia and Europe. The purpose of this study was to compare the clinical and virological aspects of dual infection with BVDV-1 (Horton 916) and an Asian atypical bovine pestivirus (Th/04_KhonKaen) in naïve calves, in comparison to single infections. Milder clinical signs were observed in the animals infected with single Th/04_KhonKaen strain. Leukocytopenia and lymphocytopenia were observed in all infected groups at a similar level which correlated with the onset of viraemia. Co-infection with both viruses led to prolonged fever in comparison to single strain inoculated groups and simultaneous replication of concurrent viruses in blood and in the upper respiratory tract. Following the infections all the calves seroconverted against homologous strains. Atypical pestiviruses pose a serious threat to livestock health and BVDV eradication, since they may have the potential to be widely spread in cattle populations without being detected and differentiated from other BVDV infections.

Polymorphism of the prion protein gene (PRNP) in Polish cattle affected by classical bovine spongiform encephalopathy.

Recent attempts to discover genetic factors affecting cattle resistance/susceptibility to bovine spongiform encephalopathy (BSE) have led to the identification of two insertion/deletion (indel) polymorphisms, located within the promoter and intron 1 of the prion protein gene PRNP, showing a significant association with the occurrence of classical form of the disease. Because the effect of the polymorphisms was studied only in few populations, in this study we investigated whether previously described association of PRNP indel polymorphisms with BSE susceptibility in cattle is also present in Polish cattle population. We found a significant relation between the investigated PRNP indel polymorphisms (23 and 12 bp indels), and susceptibility of Polish Holstein-Friesian cattle to classical BSE (P < 0.05). The deletion variants of both polymorphisms were related to increased susceptibility, whereas insertion variants were protective against BSE.

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.

Modelling BSE trend over time in Europe, a risk assessment perspective.

BSE is a zoonotic disease that caused the emergence of variant Creuzfeldt-Jakob disease in the mid 1990s. The trend of the BSE epidemic in seven European countries was assessed and compared, using Age-Period-Cohort and Reproduction Ratio modelling applied to surveillance data 2001-2007. A strong decline in BSE risk was observed for all countries that applied control measures during the 1990s, starting at different points in time in the different countries. Results were compared with the type and date of the BSE control measures implemented between 1990 and 2001 in each country. Results show that a ban on the feeding of meat and bone meal (MBM) to cattle alone was not sufficient to eliminate BSE. The fading out of the epidemic started shortly after the complementary measures targeted at controlling the risk in MBM. Given the long incubation period, it is still too early to estimate the additional effect of the ban on the feeding of animal protein to all farm animals that started in 2001. These results provide new insights in the risk assessment of BSE for cattle and Humans, which will especially be useful in the context of possible relaxing BSE surveillance and control measures.

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.

Molecular discrimination of atypical bovine spongiform encephalopathy strains from a geographical region spanning a wide area in Europe.

Transmissible spongiform encephalopathy strains can be differentiated by their behavior in bioassays and by molecular analyses of the disease-associated prion protein (PrP) in a posttranslationally transformed conformation (PrPSc). Until recently, isolates from cases of bovine spongiform encephalopathy (BSE) appeared to be very homogeneous. However, a limited number of atypical BSE isolates have recently been identified upon analyses of the disease-associated proteinase K (PK) resistance-associated moiety of PrPSc (PrPres), suggesting the existence of at least two additional BSE PrPres variants. These are defined here as the H type and the L type, according to the higher and lower positions of the nonglycosylated PrPres band in Western blots, respectively, compared to the position of the band in classical BSE (C-type) isolates. These molecular PrPres variants, which originated from six different European countries, were investigated together. In addition to the migration properties and glycosylation profiles (glycoprofiles), the H- and L-type isolates exhibited enhanced PK sensitivities at pH 8 compared to those of the C-type isolates. Moreover, H-type BSE isolates exhibited differences in the binding of antibodies specific for N- and more C-terminal PrP regions and principally contained two aglycosylated PrPres moieties which can both be glycosylated and which is thus indicative of the existence of two PrPres populations or intermediate cleavage sites. These properties appear to be consistent within each BSE type and independent of the geographical origin, suggesting the existence of different BSE strains in cattle. The choice of three antibodies and the application of two pHs during the digestion of brain homogenates provide practical and diverse tools for the discriminative detection of these three molecular BSE types and might assist with the recognition of other variants.