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A voluntary bovine viral diarrhoea virus (BVDV) control programme, which later became a compulsory eradication programme, based on the Swedish model was introduced in Lower Austria in 1997. The persistently infected animals were detected by Ag-ELISA and all samples were re-tested by the improved single-tube RT-PCR, employing panpestivirus primers targeting the 5'-UTR of the virus genome. In 2010, the BVDV eradication programme, which became compulsory from 2004, reached the final stage with only five remaining infected herds in which BVDV was difficult to eradicate. To resolve the problem in those herds, a molecular epidemiology approach was used. No differences in the spectrum of BVDV-1 subgenotypes at the beginning and at the final stage of eradication programme were found. The genetic study revealed the importance of human risk factor when finishing an eradication programme. Molecular epidemiology was also used to analyse BVDV isolates associated with re-introductions to BVDV-free herds.

Background: Hepatitis E virus (HEV) is agent causing hepatitis worldwide. Originally considered to be limited to developing countries, this virus was also detected in developed countries. In recent years an increasing number of reports indicate that farmed domestic pigs are widely infected with HEV in several European countries. The HEV status in Slovakia is still missing., Results: In this study, the circulation of HEV among domestic swine in Slovakia and genetic diversity of the virus was studied. Overall HEV RNA was detected in 53/388 (13.7, 95% CI: 10.40-17.48%) pig rectal swabs in five production stages (age categories) with statistically significant differences among all the stages. The highest HEV prevalence was observed in weaners 24/81 (29.6, 95% CI: 19.99-40.81%) and then significantly declined in growers and fatteners. No HEV was detected in suckling piglets and sows. Twenty-eight partial sequences of ORF1 (242 bp) and seventeen of ORF2 (304 bp) were analysed. Phylogenetic analysis and p-distance comparisons confirmed in both ORFs that all Slovak HEV sequences belong to the genotype HEV-3, major clade 3abchij with higher identity to 3a and 3i subtypes. Three sequences were outside of all lastly updated HEV-3 subtypes., Conclusion: This is the first report to fill the information gap about HEV infection in pigs in Slovakia. The results suggested a lower prevalence of HEV in Slovak pig farms than observed in other European countries. While most HEV isolates were typed as HEV-3 clade 3abchij, three sequences were unclassified.

As for other European countries, IBR is a significant cause of financial losses in cattle in Slovakia. The State Veterinary and Food Administration of the Slovak Republic prepared a voluntary IBR control program for cattle farms in 1995, which was implemented in 1996. In subsequent years, 48-119 farms/year enrolled in the voluntary IBR control program. Since the end of 2006, the IBR control program became compulsory by law for all cattle farms in Slovakia. Serology was used to identify infected animals using a conventional ELISA amongst non-vaccinated cattle and a gE specific ELISA in cattle vaccinated with marker vaccine. Eradication is based on culling when the serological prevalence of IBR in a herd is below 15%. When the prevalence is higher than 15%, the culling is combined with the application of a marker vaccine. A radical method where all animals are slaughtered is used with the agreement of the farmer when appropriate, especially for very small herds. Depending upon the selected eradication method, the antibody positive cattle can be gradually replaced in the herds to eliminate financial losses due to the disease. The movement of cattle is under strict control requiring a health certificate issued by the state veterinary authority and the movement must be recorded in the central livestock registry. The next step for herds is monitoring to achieve official IBR-free status. Based on the official figures from The State Veterinary and Food Administration, 60.2% herds were free of IBR in Slovakia in 2020., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mandelik, Bires, Ozsvari, Hodnik and Vilcek.)

The aim of this study was to investigate the use of a standardized animal model subjected to antibiotic treatment, and the effects of this treatment on the course of dextran sodium sulphate (DSS)-induced colitis in mice. By decontamination with selective antibiotics and observation of pathogenesis of ulcerative colitis (UC) induced chemically by exposure of mice to various concentrations of DSS, we obtained an optimum animal PGF model of acute UC manifested by mucin depletion, epithelial degeneration and necrosis, leading to the disappearance of epithelial cells, infiltration of lamina propria and submucosa with neutrophils, cryptitis, and accompanied by decreased viability of intestinal microbiota, loss of body weight, dehydration, moderate rectal bleeding, and a decrease in the selected markers of cellular proliferation and apoptosis. The obtained PGF model did not exhibit changes that could contribute to inflammation by means of alteration of the metabolic status and the induced dysbiosis did not serve as a bearer of pathogenic microorganisms participating in development of ulcerative colitis. The inflammatory process was induced particularly by exposure to DSS and its toxic action on compactness and integrity of mucosal barrier in the large intestine. This offers new possibilities of the use of this animal model in studies with or without participation of pathogenic microbiota in IBD pathogenesis.

The aim of this work was the genetic typing of RVA isolates originating from pigs and human patients in Slovakia. Seventy-eight rectal swabs from domestic pigs and 30 stool samples from humans were collected. The whole VP7 (G genotypes), VP6 (I genotypes) and partial VP4 (P genotypes) ORFs were amplified by RT-PCR. Genetic variability was higher amongst porcine sequences, where four G genotypes (G3, G4, G5, G11), two P genotypes (P[6], P[13]) and one I5 genotype were detected. Human RVA strains were represented by two G genotypes (G1, G3), two I genotypes (I1, I2), and one P genotype (P[8]). Genetic analysis did not show a relationship between Slovakian porcine and human RVA strains, but phylogenetic grouping of some Slovakian porcine sequences with Hungarian human sequences in both G and P genotypes was observed.

A novel disease, of unknown origin, causing a deadly pneumonia of human patients was reported in December 2019 in Wuhan, Hubei province of China. Later called coronavirus disease (COVID-19), it rapidly spread across China and worldwide. Intensive research revealed that the etiological agent of the global COVID-19 pandemic was a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 genome contains typical coronavirus genes but the receptor binding domain (RBD) in the S protein is highly specific. The site for furin-like protease cleavage of the S protein into S1 and S2 subunits is also unique. Further analyses suggested that SARS-CoV-2 is of zoonotic origin. The analysis of the SARS-CoV-2 genome, especially the S gene, shows that natural evolutionary process between a bat-CoV and a pangolin-CoV or other animal coronavirus could have been important in creating SARS-CoV-2, with transmission of novel virus to the human population. On the other hand, new analyses indicate that SARS-CoV-2 is not a recombinant virus. Keywords: COVID-19; SARS-CoV-2; genome; bat coronavirus; pangolin coronavirus.

Astroviruses are widely detected in pigs but their detection in wild boars is rather sporadic. In this study, astroviruses were detected in organ homogenates of wild boars by applying nested reverse transcriptase polymerase chain reaction, and the typing was carried out by phylogenetic analysis. Overall, 30/200 (15.0%) homogenates were positive for astroviruses. Genetic typing revealed that of 13 amplicons analyzed, 8 were typed as porcine astrovirus lineage 2 (PAstV-2), 2 as lineage 4 (PAstV-4), 2 identical sequences were grouped with chicken astrovirus, and 1 sequence belonged to a bat astrovirus lineage. This first identification of chicken and bat astroviruses in wild boars indicates interspecies transmission.

Background: Surveillance and characterization of pig enteric viruses such as transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), rotavirus, astrovirus (PAstV), sapovirus (PSaV), kobuvirus and other agents is essential to evaluate the risks to animal health and determination of economic impacts on pig farming. This study reports the detection and genetic characterization of PAstV, PSaV in healthy and diarrheic domestic pigs and PEDV and TGEV in diarrheic pigs of different age groups., Results: The presence of PAstV and PSaV was studied in 411 rectal swabs collected from healthy (n = 251) and diarrheic (n = 160) pigs of different age categories: suckling (n = 143), weaned (n = 147) and fattening (n = 121) animals on farms in Slovakia. The presence of TGEV and PEDV was investigated in the diarrheic pigs (n = 160). A high presence of PAstV infections was detected in both healthy (94.4%) and diarrheic (91.3%) pigs. PSaV was detected less often, but also equally in clinically healthy (8.4%) and diarrheic (10%) pigs. Neither TGEV nor PEDV was detected in any diarrheic sample. The phylogenetic analysis of a part of the RdRp region revealed the presence of all five lineages of PAstV in Slovakia (PAstV-1 - PAstV-5), with the most frequent lineages being PAstV-2 and PAstV-4. Analysis of partial capsid genome sequences of the PSaVs indicated that virus strains belonged to genogroup GIII. Most of the PSaV sequences from Slovakia clustered with sequences originating from neighbouring countries., Conclusions: Due to no significant difference between healthy and diarrheic pigs testing of the presence of PAstV and PSaV provides no diagnostic value. Genetic diversity of PAstV was very high as all five lineages were identified in pig farms in Slovakia. PSaV strains were genetically related to the strains circulating in Central European region.

This report describes the first disease outbreak caused by chimeric swine enteric coronavirus (SeCoV) on two pig farms in Slovakia in early 2015. The infection was introduced by import of two breeding boars which were placed in provisional quarantine in a unit not strictly separated from other healthy pigs in the same building. Subsequently, loss of appetite and diarrhoea were observed in both boars during the first three days in the isolation unit. The infection gradually spread to the farrowing area and throughout the farm in two weeks and later to another nearby farm. Yellow watery diarrhoea accompanied by dehydration and death was observed in piglets with a mortality ranging from 30 to 35%. In the absence of an available vaccine, the pregnant sows were dosed by mouth with a 10% suspension prepared from the intestine and faeces of infected piglets in warm water. Three weeks after dosing, new litters of piglets were born which remained healthy with no development of diarrhoea.

The porcine kobuvirus 1 (PKV-1) is believed to be an enteric virus. To investigate the prevalence of PKV-1 in pigs, virus was detected by RT-PCR in rectal swabs originating from 414 healthy and diarrheic pigs of different age categories on farms in Slovakia. Among all ages of animals, PKV-1 was detected equally in diarrheic (63.8%) and clinically healthy (62.9%) pigs. PKV-1 was more often detected in diarrheic (74.6%) than in healthy (64.4%) suckling piglets (<28days) but data was not statistically significant. Results in weaned (28-70days) and fattening (>70days) of both healthy and diarrheic pigs were inconsistent ranging in interval 56.2% to 67.9%. This study did not confirm a clear relationship of PKV-1 infection with diarrhea in pigs. Rotavirus A infection was detected among the same animals in 39% diarrheic and 9.2% healthy suckling piglets (p<0.001) confirming rotavirus as a causative agent of diarrhea in this age group. The difference was not significant in older pigs with both diarrheic and healthy pigs being infected within a range of 0% to 12.2%. Co-infection with PKV-1 and rotavirus A was detected overall in 5.6% of healthy and in 13.5% of diarrheic pigs and was highest in suckling piglets (33.9%). The PKV-1sequences from pigs in Slovakia were analyzed at the genetic level in the partial 3D gene region for the first time. The viral sequences were grouped in phylogenetic clusters according to their farm of origin. When compared with 157 nucleotide sequences originating from pig samples of different countries around the world Slovakian PKV-1 sequences were clustered in the phylogenetic tree with Asian sequences but not with nucleotide sequences from the neighbouring countries of Czech Republic or Hungary., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Pestiviruses isolated from sheep and goats in India thus far have been bovine viral diarrhoea virus 1 (BVDV-1) or BVDV-2. During routine genetic typing of pestiviruses in the years 2009-10, border disease virus (BDV) was detected in eight Indian sheep of a flock showing clinical signs of BD by real time RT-PCR. All the samples yielded positive virus isolates in cell culture but were found negative by a BVDV antigen ELISA. A representative BDV isolate was characterized at genetic and antigenic level. Phylogenetic analysis carried out in 5'-UTR, N(pro) and E2 regions of genome typed the Indian BDV isolate as BDV-3. A more detailed analysis in N(pro) and entire region coding structural proteins showed that the N(pro) (168), C (100 aa), E(rns) (227 aa), E1 (195 aa) and E2 (373 aa) proteins were of size characteristic for BDV reference strain X818. Antigenic differences were evident between the BDV-3 isolate and previously reported BDV-1, BDV-5 and BDV-7 strains. Although origin of BDV-3 in India is not clear, the results reflect probable introduction through trade in sheep between India and other countries or BDV-3 may be more widely distributed. Additionally, this study suggests that for diagnosis of BDV infection, the commercial BVDV Ag-ELISA should be used with caution. This is the first identification of BDV in sheep in India which highlights the need for continued pestivirus surveillance and assessing its impact on sheep and goat production., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

As the number of free-living wild boars (Sus scrofa L.) continues to rise in Slovakia, the probability of pathogen transmission between susceptible species increases. We investigated the distribution and genetic characterization of porcine parvovirus type 3 (PPV3), porcine circovirus type 2 (PCV2), and their coinfection in wild boars. Among 194 animals tested, 19.1% were positive for PPV3 and 43.8% for PCV2. Similar rates of coinfection with both viruses reaching 11.0% and 11.8% were observed in juvenile and mature wild boars, respectively. Phylogenetic analysis of PPV3 sequences from VP1 and NS1 genomic regions revealed a close genetic relationship among isolates from Slovakia and those sampled worldwide. Prevalence of PCV2 in wild boars was lower than that reported in domestic pigs in Slovakia. The PCV2 variants originating from sylvatic and domestic hosts in Slovakia were grouped in the same clusters, namely PCV2b-1A/1B and PCV2a-2D.

PRRSV was confirmed by a nested RT-PCR in two out of 129 samples originating from wild boars from Eastern Slovakia. Virus isolates were genetically typed in ORF7 as PRRSV-1, EU-1 with 99.7% (sample 14WB) and 100% (sample 10WB) nucleotide sequence similarity to Lelystad reference strain and Porcilis PRRS vaccine strain, respectively. The origin of PRRSV in these wild boars is unclear but it is highly likely that virus was transmitted from vaccinated domestic pigs to wild boars.

Background: Porcine circovirus type 2 (PCV2) is an etiological agent of porcine circovirus diseases (PCVDs). Post-weaning multisystemic wasting syndrome (PMWS) as the most important PCVD is considered a multifactorial disease. It was demonstrated that not only PCV2 but several viruses are associated with PMWS. Studies of viral co-infections in PMWS pigs led often to controversial results. The aim of this work was to determine the presence of emerging (PRRSV), re-emerging (PTV) and newly-emerging (TTSuV1, TTSuV2, PBoV1) viruses in samples of dead pigs suffering from PMWS. The impact of vaccination against PCV2 and the influence of age on the occurrence of single and multiple viral infections in pigs were also investigated., Results: Viruses were detected by PCR, RT-PCR and real-time PCR in the pooled tissue samples (lymph nodes, liver and spleen) of pigs with PMWS (n = 56) which were divided into three groups: suckling piglets, post-weaning pigs and fattening pigs. In addition, lymph node samples were collected from apparently healthy fattening pigs (n = 59). The effect of vaccination against PCV2 with Ingelvac CircoFlex vaccine was also investigated. Between non-vaccinated pigs, the highest prevalence of individual viruses and multiple viral infections were found in diseased post-weaning and fattening animals with PMWS. Severe clinical disease was observed in swine co-infected with PCV2 and PRRSV. The prevalence of TTSuV1 and TTSuV2 was high in all groups of pigs and did not appear to have a significant effect on the syndrome. Simultaneous infection with TTSuV1 and PBoV1 was frequently confirmed in pigs with PMWS. No healthy pig was found to be infected with PRRSV, PTV or PBoV1. Vaccination against PCV2 did not influence the prevalence of TTSuVs, but significantly protected pigs against multiple viral infections., Conclusions: Post-weaning PMWS pigs were more often co-infected with viral pathogens than suckling or fattening pigs. Co-infection with PRRSV enforces clinical signs of PMWS, the influence of other viral co-infections is not clear. Vaccination against PCV2 significantly reduced viral co-infections in pigs.

For the pestivirus border disease virus (BDV) at least seven major genotypes have been described (BDV-1-BDV-7). So far, complete genomic sequences have been reported for four BDV genotypes (BDV-1-BDV-4). In this study we report the entire genomic sequence of the noncytopathogenic (ncp) BDV-5 reference strain Aveyron. The viral genome encompasses 12,284 nucleotides (nt) and contains one large open reading frame (11,700 nt) flanked by a 370 nt long 5'-untranslated region (UTR) and a 214 nt long 3'-UTR. The genome organization as well as the lengths of the viral polyprotein (3899 amino acids) and the 5'-UTR are very similar to the ones of other BDV strains, while the 3'-UTR of BDV Aveyron is considerably shorter when compared to other BDV strains. Comparative analysis of complete coding sequences revealed that BDV Aveyron shares nucleotide sequence identities of 76.9% to 79.0% with the other BDV strains, and less than 72% identity with other pestiviruses. In contrast to other BDV strains, a unique insertion of four amino acids (KAPD) of unknown origin is present in the C-terminal part of the viral autoprotease NS2 encoded by BDV Aveyron. Immunoblot analysis revealed that infection of cells with the ncp BDV strain Aveyron comprising this unique insertion in NS2 resulted in the expression of high amounts of NS3 and thereby showed that BDV Aveyron significantly differs from other ncp BDV strains in terms of NS2-3 processing and production of NS3., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The BVDV eradication program in Lower Austria according to the Swedish model started in 1997 as a voluntary strategy and became obligatory for all herd owners in 2004. In this paper we analyse BVDV isolates found in PI animals of the last infected cattle farms at the genetic level and describe possible risk factors for virus transmission in the final stage of the BVDV eradication program in Lower Austria, where only five infected herds were identified in 2010. Viral isolates from 23 farms on which PI animals had been detected in 2009 (n = 10), 2010 (n = 5), 2008 (n = 5) and in 2006 (n = 3) were analysed at a genetic level at the 5'-UTR. All isolates analysed by phylogenetic analysis fall into five BVDV-1 subgenotypes, i.e., b, e, f, g, and h. No new subgenotypes were identified when compared to isolates analysed at the beginning of the BVDV eradication program. The phylogenetic analysis also revealed three groups of herds with genetically identical isolates; the first group comprised two herds, the second seven herds and the third four herds. Analysis of several factors such as distance between herds, purchase of cattle, work of farmers, veterinarians, assistants of milk recording associations and animal carriers indicated the latter as the most critical factor for transmitting the same viruses. Seven herds of two groups of identical BVDV isolates were served by the same cattle carrier, six herds shared the veterinarian and five herds had the same assistant of the milk recording association who could be the risk factor for the introduction of BVDV into the herd. The analysis helped to stop virus transmission. Therefore, from early 2012 up to now, no new infection has occurred in Lower Austria. The results indicated that biosecurity is a very important factor to prevent the spreading of viruses in the final stage of the BVDV eradication program.

All twenty-nine PRRSV ORF7 nucleotide sequences obtained from clinical samples originating from the three Central European countries Austria (n = 7), Czech Republic (n = 12), and Slovakia (n = 10) belonged to type 1, subtype I (EU-1). Twenty-seven sequences encoded the typical length of the nucleocapsid protein composed of 128 amino acids. Two genetically identical ORF7s of PRRSV originating from a single farm in Slovakia showed a new length polymorphism of the nucleocapsid protein comprising 132 amino acids.

A novel, real-time PCR system for the detection of porcine circovirus type 2 (PCV2) was developed. The system employed Plexor technology and detected 10(8)-10(1) copies per reaction of PCV2 DNA within a recombinant plasmid. The examination of clinical material showed consistent diagnostic sensitivity when samples contained more than 10(3) viral copies per reaction. Specificity of Plexor real-time PCR was confirmed using the porcine viruses PCV1, PRRSV, CSFV, TTSuV1 and TTSuV2 employing the melting curve analysis of PCR products. The low values of coefficient of variation in the intra- (1.74%) and inter-assay (2.41%) analysis suggested that the assay was a highly reproducible. The Plexor real-time PCR was compared with three other real-time PCR systems (SYBR Green, TaqMan, LUX) with conclusion that it can be used as a method of choice for the detection and quantitation of PCV2., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Previous studies have shown that bovine viral diarrhoea virus type 1 (BVDV-1) subtype b is predominantly circulating in Indian cattle. During testing for exotic pestiviruses between 2007 and 2010, BVDV-2 was identified by real time RT-PCR in two of 1446 cattle blood samples originating from thirteen states of India. The genetic analysis of the isolated virus in 5' UTR, N(pro), entire structural genes (C, E(rns), E1 and E2), nonstructural genes NS2-3 besides 3' UTR demonstrated that the nucleotide and amino acid sequences showed highest similarity with BVDV-2. The entire 5' and 3' UTR consisted of 387 and 204 nucleotides, respectively, and an eight nucleotide repeat motif was found twice within the variable part of 3' UTR that may be considered as a characteristic of BVDV-2. The phylogenetic analysis revealed that the cattle isolate and earlier reported goat BVDV-2 isolate fall into separate clades within BVDV-2a subtype. Antigenic typing with monoclonal antibodies verified the cattle isolate also as BVDV-2. In addition, cross-neutralization tests using antisera raised against Indian BVDV strains circulating in ruminants (cattle, sheep, goat and yak) displayed significant antigenic differences only between BVDV-1 and BVDV-2 strains. This is the first identification of BVDV-2 in Indian cattle that may have important implications for immunization strategies and molecular epidemiology of BVD., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Of 120 clinical specimens obtained from pigs bred on 28 PMWS-affected farms in Slovakia, porcine circovirus type 2 (PCV-2) was detected by single PCR in 77 samples. A short 224 bp fragment of ORF2 was used for preliminary grouping of isolates by phylogenetic analysis. Nucleotide sequences of the entire ORF2 region provided more precise genetic typing and segregation of preselected isolates (n=10) into two known genotypes, PCV-2a (n=1) and PCV-2b (n=9). Complete genome sequencing of three selected isolates allowed their definitive grouping into genotype PCV-2b, cluster 1A or genotype PCV-2a, cluster 2D. No correlation between the mutations and the geographic origin of isolates was observed. Results confirmed that many PCV-2 isolates are genetically very stable since similar viruses circulate in Central and Western Europe., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Classical swine fever (CSF), a highly contagious viral disease of pigs, is endemic in India. As there is no information concerning the accurate genetic typing of classical swine fever virus (CSFV) isolates in India, 16 CSF viruses isolated during 2005-2007 from domestic pigs in different districts of Assam were typed in 5' UTR (150 nucleotides). To confirm the genetic typing results and to study the genetic variability, selected viruses were also analyzed in E2 (190 nt) and NS5B gene (409 nt) regions. Phylogenetic analysis revealed that all the 16 CSFV isolates analyzed belonged to group 1 and subgroup 1.1 in contrast to the situation in other Asian countries. Additionally, analysis in E2 and NS5B region placed the Indian isolates in a clearly separated clade within subgroup 1.1. The results suggest that subgroup 1.1 CSF viruses are currently circulating in India, which is important for epidemiology and control of CSF., (Copyright © 2009 Elsevier Ltd. All rights reserved.)

This report describes the full-length genome sequence of the pestivirus strain H2121 which was recently isolated from Pyrenean chamois and typed as Border disease virus (BDV) genotype 4. Comparison with full-length genomic sequences of the approved pestivirus species Bovine viral diarrhea virus-1 (BVDV-1), BVDV-2, BDV, and Classical swine fever virus, the tentative species represented by strain Giraffe-1, as well as the atypical pestivirus strain Th/04&#95;KhonKaen confirmed that the chamois pestivirus strain is most similar to BDV. The viral genome of H2121 is 12,305 nucleotides long and contains one large open reading frame. The latter encodes a polyprotein consisting of 3899 amino acids and is flanked with 376 nucleotides long 5' untranslated region (UTR) and 229 nt long 3' UTR. The genome organization of the chamois virus is reminiscent to that of other pestiviruses. Compared to other BDV strains including BDV-1 strain X818 and BDV-2 strain Reindeer-1, the 5' UTR and ORF of the chamois virus are very similar in length, while the 3' UTR of H2121 is 31-44 nucleotides shorter. In contrast to other BDV strains, the genome of the chamois virus contains a unique four amino acid insertion at the N-terminus of NS2., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

The objective of our study was to develop and evaluate a TaqMan real-time RT-PCR (RRT-PCR) assay for universal detection of influenza A (IA) viruses. The primers and LNA-modified octanucleotide probe were selected to correspond to extremely conserved regions of the membrane protein (MP) segment identified by a comprehensive bioinformatics analysis including 10,405 IA viruses MP sequences, i.e., all of the sequences of the Influenza Virus Sequence database collected as of August 20, 2009. The RRT-PCR has a detection limit of approximately five copies of target RNA/reaction and excellent reaction parameters tested in four IA viruses reference laboratories. The inclusivity of the assay was estimated at both the bioinformatic and the experimental level. Our results predicted that this RRT-PCR assay was able to detect 99.5% of known human IA virus strains, 99.84% of pandemic influenza A (H1N1) strains, 99.75% of avian strains, 98.89% of swine strains, 98.15% of equine strains, and 100% of influenza A viruses of other origin.

Light Upon eXtension real-time PCR (LUX real-time PCR) assay was developed for the detection of porcine circovirus type 2 (PCV2). The primers flanking a 114 bp fragment were selected from ORF1. The optimized assay could detect 20 viral copies of pBluescript SK+ plasmid containing inserted PCV2 DNA. The dynamic range of quantitative analysis covered a 7-order interval ranging from 20 to 2 x 10(8) genome equivalents per assay with the best results in the range from 2 x 10(2) to 2 x 10(7) viral copies. The LUX real-time PCR assay had a high specificity since it detected PCV2 but not PCV1, CSFV, PRRSV or negative samples. There was good agreement between the LUX real-time PCR and the conventional PCR when lymph nodes from PCV2 infected animals were tested. A comparison of the LUX real-time PCR with the TaqMan PCR and SYBR Green PCR indicated that the amount of viral copies determined using linear calibration curve differed from assay to assay but not more than an order. LUX real-time PCR, similar to the TaqMan PCR, was more specific for generation of fluorogenic signal than SYBR Green PCR., (Copyright 2010 Elsevier B.V. All rights reserved.)

The genetic diversity of bovine viral diarrhoea virus (BVDV) isolates in infected cattle from Tyrol and Vorarlberg (Austria) was investigated. Blood samples were collected within the compulsory Austrian BVDV control programme during 2005 and 2006. The 5'-untranslated region (5'-UTR) and partially the N-terminal autoprotease (N(pro)) were amplified by one-step reverse transcriptase-polymerase chain reaction (RT-PCR) and the PCR products were subsequently sequenced. Phylogenetic analysis based on 5'-UTR and N(pro) sequences demonstrated that almost all isolates (307/310) were of the BVDV-1 genotype. They were clustered into eight different subtypes, here listed by their frequency of occurrence: BVDV-1h (143), BVDV-1f (79), BVDV-1b (41), BVDV-1d (28), BVDV-1e (6), BVDV-1a (4), BVDV-1g (3) and BVDV1-k (3). Two pestivirus isolates were typed as BVDV-2 and one isolate as BDV closely related to Gifhorn strain (BDV-3). Correlation among isolates could only be observed at the farm level, i.e., within a herd. However, no correlation between the genetic and geographical distances could be observed above the farm level. Because of the wide distribution of certain BVDV-1 subtypes and the low prevalence of herd-specific strains, a determination of tracing routes of infection was not possible. Furthermore, recombination events were not detected.

The wide spread prevalence of bovine viral diarrhea virus type 1 (BVDV-1) in cattle and recent identification of BVDV-2 in goats in India warranted pestivirus surveillance in sheep. Nested reverse transcription-polymerase chain reaction (RT-PCR) was used to detect BVDV-2 in one of 1561 blood samples collected randomly from 78 sheep flocks in 11 states of India. Antigenic characterization of the isolated pestivirus using polyclonal and monoclonal antibodies typed the isolate as BVDV-2. When analyzed at genetic level in N(pro) (N-terminal autoprotease) and entire gene region coding structural proteins, namely capsid (C) protein and envelope proteins E(rns) (ribonuclease secreted), E1 and E2 genomic organization was the same for all pestiviruses, the nucleic acid and amino acid sequences showed highest similarity with those of BVDV-2. When compared with BVDV-2 isolate 890, the sequence homology was 83.7% for C, 84.6% for E(rns) and E1, and 81.5% for E2. The cleavage site C/E(rns) was found totally conserved while N(pro)/C was conserved only from C-terminus of N(pro), E(rns)/E1 site was conserved only from C-terminus of E(rns) and E1/E2 site was conserved only from C-terminus of E1. Phylogenetic analysis of nucleotide sequences in 5' untranslated regions (UTR), N(pro), E2, NS3 and NS5B regions placed the sheep isolate in a separate clade within BVDV-2 subtype b. This was supported by the presence of unique mutations in the structural protein coding regions beside NS3 and NS5B. To our knowledge this is the first report on the sequence analysis of the entire structural gene coding region of a BVDV-2b isolate. This is also the first occurrence of BVDV-2 subtype b in sheep, providing the evidence that this subtype can also occur in species other than cattle.

Since cattle are widely infected by bovine viral diarrhea virus (BVDV) in India, we searched for pestivirus infection in yaks. Of 71 pure and crossbred yaks from Himalayan region, pestivirus antigen was detected by Ag-ELISA in three animals. Pestivirus in leukocyte and cell culture isolated virus samples originating from positive yaks was also confirmed by RT-PCR using panpestivirus specific primers selected from 5'-untranslated region (5' UTR). The 5' UTR, N(pro) and E2 regions were sequenced and used for genetic typing. Phylogenetic analysis revealed that pestiviruses detected in three Himalayan yaks were similar genetically, belonging to BVDV-1. Antigenic characterisation of yak pestivirus also confirmed the typing as BVDV-1. This is the first report on the identification of BVDV type 1 in yaks.

The identification and genetic characterization of bovine viral diarrhea virus (BVDV) isolate 17237 detected in western Slovakia is described. The analysis of 5'-untranslated region (5'-UTR), autoprotease (Npro) gene, and structural genes (C, Erns, E1, E2) was carried out. The percentage of nucleotide and deduced amino acid identity in analyzed genes implied that the isolate was closely related to the bovine viral diarrhea virus 2 (BVDV-2). Furthermore, the phylogenetic analysis revealed that this isolate fall into BVDV-2b subtype that is sporadic in Europe. The cleavage sites between viral proteins were similar to the ones of a reference strain of BVDV-2.

Ten isolates belonging to different BVDV-1 subtypes and three BVDV-2 isolates were studied in 3'-nontranslated region (3'-NTR) by sequencing of PCR products and comparative computer-assisted nucleotide sequence analysis. The alignment of nucleotide sequences revealed that all BVDV-1 isolates except BVDV-1a isolates represented by NADL strain were characterised with a deletion of 38 nucleotides in the variable region located after the stop codon. For all BVDV-1 subtypes, a constant region at the end of viral genome was highly conserved. The variable and constant regions with no significant insertions or deletions were also identified in BVDV-2 isolates. The poly AT reach region was situated at different locations in both pestiviruses.

The purpose of this investigation was to determine the influence of communal Alpine pasturing on the spread of pestivirus infections among sheep and goats. The study included 481 sheep from 23 farms and 131 goats from 26 farms pastured on separated Alpine meadows in the western part of Austria. At the starting of pasturing on the sheep meadow, 325 (67.6%) animals were seropositive, on the goat meadows in 16 (12.2%) samples antibodies to pestiviruses were detected. At the end of pasturing, 74 seronegative sheep and two seronegative goats had seroconverted. Between the beginning and the end of pasturing the seroprevalence in sheep increased significantly from 67.6% to 83% (P<0.05). Moreover, in the peripheral blood mononuclear cells of four sheep, pestivirus-specific RNA was detected before as well as after pasturing; these animals remained serologically negative throughout the investigation. They were, therefore, identified as persistently infected. Sequence analysis in the N(pro) region revealed that the detected pestiviruses were the same at genetic level and they were grouped into the Border disease virus (BDV)-3 genotype. No pestivirus RNA was found in goat samples. The results of this survey indicate that communal Alpine pasturing does play a key role in the spread of BDV. Moreover, BDV has been identified and characterized for the first time in sheep in Austria, which until then had been regarded as being free from BD.

Pestiviruses are not strictly host-species specific and can infect not only domestic but also wild animals. The most important pestivirus, CSFV, infects domestic pigs and wild boars, which may cause a major problem for successful CSFV eradication programmes. Mainly BVDV specific antibodies have been reported in captive and free-living animals. Virus has been isolated from some of these animal species, but since BVDV can contaminate cell cultures and foetal calf serum, early reports of BVDV isolation have to be considered with caution. Genetic typing of early pestivirus isolates from wild species revealed that the majority were BVDV-1. Of the pestiviruses identified so far three species (CSFV, BVDV-1, giraffe pestivirus) and three genotypes (BDV-2, BDV-4, pronghorn) appear to circulate in wildlife animal populations. The potential for pestiviruses to spread between farm animals and free-living animals is discussed as are epidemiological and technical problems, and the future direction of research.

A one-step multiplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR) using SmartCycler technology and TaqMan probes was developed for detection and typing of bovine viral diarrhea viruses (BVDV). Common primers and type-specific (BVDV1 and BVDV2) TaqMan probes were designed in the 5'-untranslated region of the viral genome. The real-time assay was able to detect 10-100 TCID50 of virus, with correlation coefficient (r2) values of 0.998 and 0.999 for BVDV1 and BVDV2, respectively. The assay accurately typed 54 BVDV strains and field isolates and specificity of the TaqMan probes was further demonstrated by the lack of reactivity with the closely related Pestiviruses, classical swine fever virus and border disease virus. The assay was also shown to have high reproducibility. When the assay was compared with virus isolation for bovine serum samples, there was full agreement between the tests. Thus, the one-step real-time RT-PCR assay appears to be a rapid, sensitive, and specific test for detection and typing of BVDV.

The first reported outbreak of bovine viral diarrhea (BVD) in 1946 described a transmissible acute disease characterized by severe leukopenia, high fever, gastrointestinal erosions and hemorrhages. However, in the ensuing years, the most commonly observed acute form of BVD was clinically mild. There was limited viral shed and spread following these acute infections. This led to the assumptions that acute infections with BVD viruses (BVDV) were clinically unimportant, spread of the virus within a group was always due to the presence of a persistently infected animal and transmission between healthy immunocompetent cattle was insignificant. These assumptions were challenged when outbreaks of severe acute BVDV were observed in North America starting in the late 1980s. This study demonstrates that widespread outbreaks of severe acute BVD observed in 1993 in North America can be traced to a single strain of BVDV that apparently spread explosively following acute infection. These findings are notable in that they draw into question management of acute BVD infection, design of studies examining virulence and nomenclature used to identify strains for GenBank submission.

Faeces of 230 calves with and without diarrhoea collected during the winter period 2004/2005 in 100 Austrian farms (Styria and Lower Austria) were examined for viral, bacterial and parasitic enteropathogens. Torovirus-specific nucleic acid confirmed by reverse transcriptase-polymerase chain reaction was found in 12 of 230 calves (5.2%). Ten of these calves were clinically ill, several of them showing signs of dehydration and abnormal faecal consistency at the time of sampling. Computer assisted analysis of two nucleotide sequences obtained from Austrian bovine samples revealed 93% similarity to Breda strain, but only 71% or 52% similarity to Equine Berne or Porcine Markelo torovirus strains respectively. Phylogenetic analysis grouped Austrian torovirus samples into the Bovine torovirus cluster indicating the first detection of Bovine torovirus in Austria. In addition, the following agents were detected in bovine faecal samples: Bovine coronavirus, 25.7%; Escherichia coli, 17%; Cryptosporidium spp., 11.7%; Eimeria spp., 10.4%; Rotavirus, 9.1%; Clostridium perfringens, 9.1% and Giardia spp., 6.1%. Salmonella spp. was not detected.

Three Indian Bovine viral diarrhea virus 1 (BVDV-1) isolates were analyzed at genetic level in N(pro) (viral autoprotease) and entire gene region coding structural proteins, namely capsid (C) protein, E(rns), and envelope proteins E1 and E2. All these isolates were found to be of b subtype based on the entire 504 nt region of N(pro) and 1119 nt region of E2. However, in comparison with other isolates of this subtype, they were allocated inside the BVDV-1 subtype b cluster to a separate clade with a longer distance. Of six cysteine residues in N(pro) only three were totally conserved in all three isolates. The isolates showed 94.9-99.3% and 92.2-99.0% identities for the entire C-E2 gene region at nucleotide and amino acid levels, respectively. The lowest identity values (88.5-91.7%) were observed for E2 amino acid sequences. The identity of the isolates with Osloss, a reference BVDV-1 subtype b strain, was in the range of 82.1-89.9% for nucleotide and 78.6-89.2% for amino acid sequences in the C-E2 region. The N(pro)/C and E(rns)/E1 cleavage sites were highly conserved. The C/E(rns) and E1/E2 cleavage sites were more conserved from the N-end of E(rns) and the C-end of El, respectively. These findings suggest that some unique mutations have occurred in the described Indian BVDV-1 isolates, though they all belong to the BVDV-1 subtype b.

Genetic typing of bovine viral diarrhoea virus (BVDV) is important for the precise classification of viruses as well as for the development of molecular epidemiology. BVDV isolates were usually typed based on comparison of genomic sequences from the 5'-untranslated region (5'-UTR), N(pro) and E2 region. Recently we have identified 11 genetic groups (subgenotypes) of BVDV-1. Our further experiments confirmed a new subgenotype, BVDV-1k, isolated from cattle in Switzerland. BVDV isolates from India were typed as BVDV-1b whereas BVDV-1c is a predominant subgenotype in Australia. The results of genetic typing of BVDV indicate that distribution of subgenotypes has no relationship to the geographic origin of viral isolates.

Eighty-nine bovine viral diarrhoea viruses (BVDV) from Australia have been genetically typed by sequencing of the 5' untranslated region (5'-UTR) and for selected isolates the N(pro) region of the viral genome. Phylogenetic reconstructions indicated that all of the samples examined clustered within the BVDV type 1 genotype. Of the 11 previously described genetic groups of BVDV-1, 87 of the samples examined in this study clustered with the BVDV-1c, while two samples clustered with the BVDV-1a. Based on these analyses there appears to be limited genetic variation within the Australian BVDV field isolates. In addition, the phylogenetic reconstructions indicate that the clustering of Australian BVDV in the phylogenetic trees is not a result of geographic isolation.

A unique pestivirus, isolated from a pronghorn antelope (Antilocopra americana), was characterized. Serum neutralization studies suggested that this virus was antigenically related to pestiviruses. Genomic characteristics, unique to pestiviruses, indicated that this virus belongs to the Pestivirus genus. These characteristics included the organization of the 5' untranslated region (5'-UTR), the presence and length of a viral Npro coding region, conservation of cysteine residues in Npro, conservation of predicted amino acid sequences flanking the cleavage sites between viral polypeptides Npro and C and between C and Erns and conservation of predicted hydrophobicity plots of Npro protein. While this data indicated the virus belongs to the Pestivirus genus, phylogenetic analysis in 5'-UTR, Npro and E2 regions suggested that it is the most divergent of the pestiviruses identified to date. This conclusion was also supported by the amino acid identity in coding regions. The corresponding values were much lower for the comparison of pronghorn pestivirus to other pestivirus genotypes than only between previous recognized genotypes. These results suggest the virus isolated from pronghorn antelope represents a new pestivirus genotype. It also represents the only pestivirus genotype first isolated from New World wildlife.

An enteric disease characterised by diarrhoea and ill thrift affected 12 of a flock of 700 six- to 12-month-old ewe lambs in Cornwall between December 1996 and September 1997. The affected lambs were undersized, became thin and suffered an unremitting diarrhoea until they died. The illness lasted for three to 14 days, although, with hindsight, the owner considered that the lambs had been below average size before the enteric signs developed. The outbreak ceased only as a result of the dispersal sale of the flock as breeding ewes. The flock had been purchased from different sources, but 11 of the cases occurred in a group of 40 purchased from one source. Postmortem, the alimentary changes resembled mucosal disease in cattle, and immunostaining of histological sections of the affected tissues revealed pestiviral antigen. Non-cytopathic pestiviruses were isolated from the lesions of two of the affected lambs and from the blood of several clinically normal ewe lambs from the same group. All the pestivirus isolates were typed as Border disease virus.

Thirteen BVDV isolates collected in four geographic regions of India between 2000 and 2002 were typed in 5'-UTR. To confirm results of genetic typing, selected viruses were also analysed in the N(pro) region. Phylogenetic analysis revealed that all Indian BVDV isolates belong to BVDV-1b (Osloss-like group). Despite a long distance between the farms from which the viruses were isolated there was no correlation between the origin of viral isolates and their position in a phylogenetic tree. Higher genetic similarity of Indian BVDV isolates was observed most probably due to the uncontrolled movement of cattle as well as the uncontrolled use of semen from bulls for breeding of local and farm cattle in different states of India.

During investigations into recent population decreases in Pyrenean chamois (Rupicapra pyrenaica pyrenaica) 21 animals found dead or dying were necropsied. Immunohistochemistry revealed the presence of a pestivirus in organs from two of the 21 chamois. From one of these animals a pestivirus was isolated from the spleen, skin and serum. The virus had better growth in ovine than in bovine cells and was neutralized most effectively by an anti-border disease virus (BDV) reference antiserum. Using panpestivirus and genotype-specific primers selected from 5'-untranslated region (UTR) of the pestivirus genome, BDV RNA was demonstrated by RT-PCR. Comparison of the chamois sequences from 5'-UTR, entire N(pro) and E2 gene coding regions with those of other pestivirus genotypes revealed that this virus did not fall into any of the pestivirus genotypes identified so far. Results of phylogenetic analysis suggested that the chamois pestivirus was closely related to BDV and it was typed as BDV-4 genotype.

In the last decade, several studies were performed to characterise bovine viral diarrhoea virus (BVDV) isolates and define genetic groups by genotyping. Much data is now available from GenBank, predominantly sequences from the 5' untranslated region (5'-UTR). In order to find out whether genetic grouping of isolates from different countries could be harmonised, 22 new isolates from five countries were analysed in combination with published sequences. Eighteen of these isolates were typed as BVDV genotype 1 (BVDV-1), and one isolate from Argentina and three isolates from Brazil were typed as BVDV-2. BVDV-1 isolates were clustered into five previously defined genetic groups: BVDV-1a, b, d, e and f. Two isolates from Finland and one from Egypt formed a group which was tentatively labelled as BVDV-1j, since statistical support was low. By using a fragment of the Npro gene for typing, we found that these isolates fall into the same group as a deer strain, and are statistically significant. Some Swiss BVDV strains taken from GenBank were found in a new genetic group which was designated as BVDV-1k. The BVDV-2 isolates included in this study seemed to fall into two genetic groups.

When several human vaccines were tested for pestivirus contamination using a one-tube closed nested RT-PCR method employing pan-pestivirus primers selected from the 5'-untranslated region (5'-UTR) of the pestivirus genome, a 224 bp DNA product was produced from a poliovirus vaccine. Although this amplicon was of the size expected for pestiviruses, its sequence showed a 100% similarity with the corresponding reverse complement of a nucleotide sequence from the VP2 gene of the poliovirus type 1 Sabin strain. It is recommended that all positive PCR products, especially those prepared using pan-pestivirus primers, obtained from screening biological substances for pestivirus contamination should be checked by use of a specific hybridization probe and preferably by sequencing.

Eight isolates of Bovine respiratory syncytial virus (BRSV) were made from calves with severe respiratory disease on seven farms in one region of Britain. Genetic analysis of the viruses showed that seven, which were isolated between 1997 and 1999 were almost identical and were distinguishable from an earlier 1991 isolate. When compared with the available sequences of BRSVs from other countries, the recent British isolates were more closely related to US isolates than to earlier British and current mainland European isolates.

To characterise the bovine virus diarrhoea virus (BVDV) isolates circulating in the southeastern region of Austria, namely in the province of Styria, 71 blood samples collected between 1998 and 2000 from persistently infected cattle in 62 herds were subjected to genetic typing. For this, 288bp fragments from the 5' untranslated region (5'-UTR) were amplified by polymerase chain reaction after reverse transcription (RT-PCR). The products were sequenced and used for phylogenetic analysis. Seventy virus isolates were typed as BVDV species 1 (BVDV-1). Only one isolate was typed as BVDV species 2 (BVDV-2), representing the first isolate of this pestivirus genotype found in Austria. In addition, phylogenetic analysis revealed that viruses belonging to five genetic groups within BVDV-1 are circulating in Styria. Most viruses (53) were found in group BVDV-1f, nine viruses in BVDV-1h, four viruses in BVDV-1b, three viruses in BVDV-1d and one virus in BVDV-1g. No virus was found in genetic group BVDV-1a, which is dominant in the UK and widely distributed in USA. Likewise, the BVDV isolates predominating in a neighbouring country, namely Germany, belonged to different genogroups than those circulating in Styria. We conclude that in a particular region and environment certain BVDV-1 genetic groups predominate. New groups, including BVDV-2, can be introduced, e.g. by trade of animals. The low incidence of BVDV-2 in Styria is in concert with the sporadic occurrence of these viruses in other regions of Europe.

A serological survey for bovine viral diarrhoea virus (BVDV) antibodies on a collection of 1295 serum samples obtained from 6-12 months old cattle originating from 45 farms in Slovakia was carried out. On 13 farms more than 90% of the examined animals were seropositive, on 14 farms 71-90% seroprevalence was observed, on 13 farms only 50-70% animals were found to be positive for BVDV antibodies, while the remaining 5 farms showed fewer than 50% seropositive animals. The average incidence of BVDV antibodies (around 70%) was similar as determined 30 years ago. Of 84 serum samples from seronegative animals originating from 14 farms in which 70-98% seropositivity was observed, six were positive in Ag-BVDV ELISA indicating persistently infected (PI) cattle. On a farm to which animals were imported from abroad, a BVD outbreak was observed. Of 110 animals tested, four were positive in Ag-ELISA indicating the presence of PI cattle on this farm. Genetic typing of two isolates from imported animals performed by RT-PCR (324/326 primers from 5'-UTR), sequencing of PCR products and computer-assisted phylogenetic analysis revealed that they belong to BVDV-1 h group.