Genetic diversity and phenotypic associations of feline caliciviruses from cats in Switzerland.

Feline calicivirus (FCV) is a common viral pathogen in domestic cats worldwide. The variable regions of the capsid (VP1) gene of FCV have one of the highest recorded rates of molecular evolution. Understanding the genetic diversity and phylogeny of FCV is a prerequisite to exploring the epidemiology and pathogenesis of this virus and to the development of efficacious vaccine strategies. In this study, we undertook a nationwide molecular characterization of FCV using for the first time nearly complete capsid (VP1) gene sequences. Sequences from 66 FCV samples were used to investigate the correlation between viral phylogeny and several traits, including geographic origin, signalment, husbandry, FCV vaccination and co-infections. Codon-based nucleotide alignment showed that individual nucleotides and their corresponding amino acid sites were either invariant or highly variable. Using a threshold of 20 % genetic distance in variable region E, FCV samples were grouped into 52 strains, 10 of which comprised two to three samples. Significant associations between FCV phylogeny and host characteristics were found, specifically the pedigree status of the cats, and two well-supported lineages were identified in which the current FCV strain definition was confounded. No correlation between viral genetic distances and geographic distances was evident. The greater resolution of the FCV phylogeny in this study compared to previous studies can be attributed to our use of more conserved regions of the capsid (VP1) gene; nonetheless, our results were still hampered by sequence saturation. The study highlights the need for whole-genome sequences for FCV phylogeny studies.

Retrospective epidemiological evaluation of molecular and animal husbandry data within the bovine viral diarrhoea virus (BVDV) control programme in Western Austria during 2009-2014.

A retrospective epidemiological investigation of molecular and animal husbandry data collected over an observation period of five years (2009-2014) within the compulsory bovine viral diarrhoea virus (BVDV) control programme in Western Austria, covering the federal provinces of Tyrol and Vorarlberg is presented in this study. Samples collected from 232 infected calves were phylogenetically classified based on the 5' untranslated region (5'UTR). All but 13 samples, which were typed as border disease virus subtype 3 (BDV-3), belonged to the bovine viral diarrhoea virus genotype 1 (BVDV-1) and clustered within six different subtypes (1b, 1e, 1f, 1h, 1d and 1k). Movement data and survival times from infected individual animals were analysed because of their potential of passing on infection to naive herds. From the moment of submission of the laboratory results, 180 animals were culled within the first month, 13 lived longer than two but not longer than six months and seven infected animals lived longer than one year. 13 of the infected animals were born on alpine pastures and eleven infected animals were grazed on mountain pastures during summer. The movement of infected animals and the role of trade in alpine areas are a possible source for spreading the infection, thus hampering the progress of eradication.

First detection, clinical presentation and phylogenetic characterization of Porcine epidemic diarrhea virus in Austria.

BACKGROUND: Porcine epidemic diarrhea (PED) is a syndrome that is characterized by rapidly spreading watery diarrhea affecting pigs of all ages, but with major effects on suckling piglets. The disease, as well as the causative Alphacoronavirus, the Porcine epidemic diarrhea virus (PEDV), was first described in Europe in the 1970s and since then has spread over many Asian and American countries, where it recently led to devastating effects on swine health and pork industry. While the disease was seldom reported in Europe within the last few decades, a few recent reports re-emergence of PED in German pig farms. The hitherto isolated German strain seems to be closely related to a low pathogenic PEDV variant from the USA. This case report describes the first detection of PEDV in Austria. CASE PRESENTATION: Reduced feed uptake and occasional diarrhea were observed in December 2014 in a group of fattening pigs, kept on an Austrian swine farm. The concerned pigs had been recently purchased from Germany. Within a few weeks, diarrhea became apparent also in pigs of Austrian origin, which were kept in a different stable on the same farm. Gastrointestinal symptoms among fattening pigs were generally mild, quickly resolving and did not lead to death. PEDV RNA was identified by RT-qPCR in pooled feces and serum and PEDV antibodies were detectable in serum in both groups of pigs. Phylogenetic analysis of the nearly complete PEDV spike gene shows that the Austrian PEDV strain is highly similar to other strains involved in recent outbreaks in Western and Central Europe. CONCLUSION: This is the first report demonstrating the presence of PEDV in Austria. The virus was probably introduced by purchasing piglets from a German source, which underlines the significance of trans-boundary animal trade for the distribution of highly contagious diseases, such as PED.

A novel triplex quantitative PCR strategy for quantification of toxigenic and nontoxigenic Vibrio cholerae in aquatic environments.

Vibrio cholerae is a severe human pathogen and a frequent member of aquatic ecosystems. Quantification of V. cholerae in environmental water samples is therefore fundamental for ecological studies and health risk assessment. Beside time-consuming cultivation techniques, quantitative PCR (qPCR) has the potential to provide reliable quantitative data and offers the opportunity to quantify multiple targets simultaneously. A novel triplex qPCR strategy was developed in order to simultaneously quantify toxigenic and nontoxigenic V. cholerae in environmental water samples. To obtain quality-controlled PCR results, an internal amplification control was included. The qPCR assay was specific, highly sensitive, and quantitative across the tested 5-log dynamic range down to a method detection limit of 5 copies per reaction. Repeatability and reproducibility were high for all three tested target genes. For environmental application, global DNA recovery (GR) rates were assessed for drinking water, river water, and water from different lakes. GR rates ranged from 1.6% to 76.4% and were dependent on the environmental background. Uncorrected and GR-corrected V. cholerae abundances were determined in two lakes with extremely high turbidity. Uncorrected abundances ranged from 4.6×10(2) to 2.3×10(4) cell equivalents liter(-1), whereas GR-corrected abundances ranged from 4.7×10(3) to 1.6×10(6) cell equivalents liter(-1). GR-corrected qPCR results were in good agreement with an independent cell-based direct detection method but were up to 1.6 log higher than cultivation-based abundances. We recommend the newly developed triplex qPCR strategy as a powerful tool to simultaneously quantify toxigenic and nontoxigenic V. cholerae in various aquatic environments for ecological studies as well as for risk assessment programs.

Comparative evaluation of serum, FTA filter-dried blood and oral fluid as sample material for PRRSV diagnostics by RT-qPCR in a small-scale experimental study.

Recently, research into alternative sample materials, such as oral fluid or filter-dried blood has been intensified, in order to facilitate cost-effective and animal-friendly sampling of individuals or groups of pigs for diagnostic purposes. The objective of this study was to compare the sensitivity of porcine reproductive and respiratory syndrome virus (PRRSV)-RNA detection by reverse transcription quantitative real-time PCR (RT-qPCR) in serum, FTA filter-dried blood and oral fluid sampled from individual pigs. Ten PRRSV negative pigs were injected with an EU-type PRRSV live vaccine. Blood and oral fluid samples were taken from each pig before, and 4, 7, 14 and 21 days after vaccination. All samples were then analyzed by PRRSV RT-qPCR. In serum, eight often pigs tested RT-qPCR positive at different time points post infection. Absolute quantification showed low serum PRRSV-RNA loads in most samples. In comparison to serum, sensitivity of PRRSV-RNA detection was strongly reduced in matched FTA filter-dried blood and in oral fluid from the same pigs. These results indicate that with low PRRSV-RNA loads the diagnostic sensitivity of PRRSV-RNA detection by RT-qPCR achieved with serum is currently unmatched by either FTA filter-dried blood or oral fluid.

Rapid spread and association of Schmallenberg virus with ruminant abortions and foetal death in Austria in 2012/2013.

Schmallenberg virus (SBV) has emerged in summer-autumn 2011 in north-western Europe. Since then, SBV has been continuously spreading over Europe, including Austria, where antibodies to SBV, as well as SBV genome, were first detected in autumn 2012. This study was performed to demonstrate the dynamics of SBV spread within Austria, after its probable first introduction in summer 2012. True seroprevalence estimates for cattle and small ruminates were calculated to demonstrate temporal and regional differences of infection. Furthermore, the probability of SBV genome detection in foetal tissues of aborted or stillborn cattle and small ruminants as well as in allantoic fluid samples from cows with early foetal losses was retrospectively assessed. SBV first reached Austria most likely in July-August 2012, as indicated by retrospective detection of SBV antibodies and SBV genome in archived samples. From August to October 2012, a rapid increase in seroprevalence to over 98% in cattle and a contemporaneous peak in the detection of SBV genome in foetal tissues and allantoic fluid samples was noted, indicating widespread acute infections. Notably, foetal malformations were absent in RT-qPCR positive foetuses at this time of the epidemic. SBV spread within Austrian cattle reached a plateau phase as early as October 2012, without significant regional differences in SBV seroprevalence (98.4-100%). Estimated true seroprevalences among small ruminates were comparatively lower than in cattle and regionally different (58.3-95.6% in October 2012), potentially indicating an eastward spread of the infection, as well as different infection dynamics between cattle and small ruminants. Additionally, the probability of SBV genome detection over time differed significantly between small ruminant and cattle samples subjected to RT-qPCR testing.

Long-term infection of goats with bluetongue virus serotype 25.

Toggenburg Orbivirus (TOV) is the prototype of bluetongue virus serotype 25 (BTV-25). It was first detected in goats in Switzerland in 2008. The virus does not induce clinical signs in infected goats. In field samples viral RNA could be detected only in goats and never in other ruminants. BTV-25 RNA was repeatedly detected for more than one year in the blood of goats from a single flock in Principality of Liechtenstein. Since viral persistence over such a long period has never been reported for bluetongue, blood samples from 110 goats and 2 sheep of that flock were collected during a period of up to two years and analyzed for the presence of BTV-25 RNA and antibodies. Most of the animals which tested positive for BTV-25 RNA, remained positive during the whole investigation period. Moreover, five of these goats were BTV-25 RNA positive over a period of 19-25 months. A weak antibody response against BTV VP7 was commonly observed. As BTV-25 cannot be propagated in any culture system, the presence of virus could only be demonstrated in samples by viral RNA detection using RT-qPCR. To address the question of infectivity of the virus in blood from long-term positive animals, goats were experimentally infected with this blood. Viral replication was demonstrated by increasing RNA amounts. Thus, our findings provide evidence that BTV-25 can persist much longer in an infected host than known so far for other BTV serotypes. Hence, persistence of infectious BTV represents an additional important factor in BTV epidemiology.

First detection of hepatitis E virus in Austrian pigs by RT-qPCR.

Long known to cause disease outbreaks in man in countries with poor sanitary conditions, an increasing number of autochthonous HEV genotype 3 infections have been reported in industrialised countries. Genotype 3 poses an important potential zoonotic threat, with infected pigs functioning as the main reservoir. This study reports the first detected emergence of HEV in Austrian pigs. Five Austrian strains were partially sequenced and phylogenetic analysis demonstrates that they cluster within genotype 3. In addition, a reverse transcription quantitative real-time PCR (RT-qPCR) method using a MGB-hydrolysis probe was developed offering the possibility to detect the HEV genotype 3 in faeces, liquid- and tissue-samples from domestic pigs. The method was adapted to the strains found in Austria. Sensitivity of the assay was tested with different pig organs (liver, mesenteric lymph nodes and kidney) as well as with serum, bile and faeces samples. Within the dynamic range of the assay, a quantitative determination of virus loads was performed. For specificity testing several common swine pathogens were used. Results demonstrated that the proposed method allows implementation of reliable high-throughput screening of Austrian swine samples in the future.

Detection and molecular characterization of Suid herpesvirus type 1 in Austrian wild boar and hunting dogs.

Aujeszky's disease (AD), caused by Suid herpesvirus type 1 (SuHV-1), is an economically important disease in domestic swine. Thus, rigorous control programmes have been implemented and consecutively AD in domestic swine was successfully eradicated in many countries, including Austria. However, SuHV-1 continues to thrive in wild boar populations, as indicated by high seroprevalences in a number of European countries and by occasional cases of AD in hunting dogs. For the first time, SuHV-1 was detected in Austrian wild boar and a molecular characterization of SuHV-1 isolated from wild boar and hunting dogs was performed. Results of preliminary serological analyses suggest a regional SuHV-1 seroprevalence of over 30% in free-living and almost 70% in fenced wild boar from Eastern Austria. Molecular typing of Austrian SuHV-1 isolates of wild boar origin revealed the presence of two genetically distinct variants of SuHV-1, both capable of infecting dogs that have been exposed to infected wild boar during hunting.

[Evaluation of four commercial RT-qPCR test kits for detection of Bluetongue virus RNA]. / Evaluierung von vier kommerziellen RT-qPCR Testkits zum Nachweis von Bluetongue Virus RNA.

Since 2006, the occurrence of bluetongue in Northwest- and Central Europe has lead to extensive financial losses. For first-line laboratory diagnosis, reverse transcription real-time PCR (RT-qPCR) has been used increasingly, necessitating careful evaluation of all applied methods by the performing laboratory. In the course of an internal validation, four commercially available BTV RT-qPCR kits and two published reference methods were compared. Clear differences regarding reaction kinetics, analytical as well as diagnostic sensitivity and specificity were observed. Furthermore, test kits were not equally suitable for testing samples from a selection of BTV-susceptible host species. Only two commercial kits were in all examined parameters equal to, or superior than, the more demanding reference methods and thus represent a possible alternative to using published methods for BTV RT-qPCR screening.

Bluetongue virus RNA detection by RT-qPCR in blood samples of sheep vaccinated with a commercially available inactivated BTV-8 vaccine.

In 2008, a country-wide bluetongue virus 8 (BTV-8) vaccination campaign has been initiated in Austria, using a single commercial inactivated BTV-8 vaccine. Based on preliminary data, we hypothesised that vaccine-derived BTV RNA is transiently detectable by reverse transcription quantitative real-time PCR (RT-qPCR) in the blood of vaccinated animals. Thus BTV-8 vaccine was administered to five BTV-naïve adult sheep and blood samples were taken at various time-points post-vaccination. BTV RNA was detectable by several RT-qPCR methods in all five animals, mainly within the first 9 days post-vaccination. These results show that RT-qPCR based testing for BTV-infection may be influenced by vaccination with certain inactivated BTV-8 vaccines.

Phylogenetic analysis suggests independent introduction of feline immunodeficiency virus clades A and B to Central Europe and identifies diverse variants of clade B.

Phylogenetic analysis of European feline immunodeficiency virus sequences confirms a highly distinct pattern in the occurrence of FIV-subtypes within Germany and Austria, indicating little mixing and at least two independent introductions of FIV. There is also evidence for a North to South gradient of FIV subtype distribution in Europe, with subtype A being more common in the North, while subtype B is prevalent in the South of Europe. In addition, among Austrian subtype B sequences, considerable differences from classical subtype B sequences were detected by bootscanning, which either suggests recombination with so far unrecognized subtypes, or a long period of independent evolution.

Mutations in the catalytic core or the C-terminus of murine leukemia virus (MLV) integrase disrupt virion infectivity and exert diverse effects on reverse transcription.

Understanding of the structures and functions of the retroviral integrase (IN), a key enzyme in the viral replication cycle, is essential for developing antiretroviral treatments and facilitating the development of safer gene therapy vehicles. Thus, four MLV IN-mutants were constructed in the context of a retroviral vector system, harbouring either a substitution in the catalytic centre, deletions in the C-terminus, or combinations of both modifications. IN-mutants were tested for their performance in different stages of the viral replication cycle: RNA-packaging; RT-activity; transient and stable infection efficiency; dynamics of reverse transcription and nuclear entry. All mutant vectors packaged viral RNA with wild-type efficiencies and displayed only slight reductions in RT-activity. Deletion of either the IN C-terminus alone, or in addition to part of the catalytic domain exerted contrasting effects on intracellular viral DNA levels, implying that IN influences reverse transcription in more than one direction.

Phylogenetic analysis of feline immunodeficiency virus in Central Europe: a prerequisite for vaccination and molecular diagnostics.

Feline immunodeficiency virus (FIV) is a worldwide-occurring lentivirus that severely impairs the immune function of infected domestic cats. Due to structural and biological similarities, FIV represents a promising model for human immunodeficiency virus (HIV) and AIDS. A major obstacle in developing vaccines against lentiviruses is their high mutation rate. Furthermore, mutations in target sequences provide a pitfall for molecular diagnostics. It is therefore important to determine the genetic diversity of lentiviruses in any region where vaccination or implementation of new diagnostic techniques are planned. This study presents a phylogenetic analysis of 30 FIV strains derived from Central Europe. In order to improve the reliability of genotyping, DNA from two different proviral genes was amplified and comparative phylogenetic trees were inferred. The highly coincident results point to the existence of extensive virus variation with the presence of at least two highly divergent subtypes of FIV in Austria and Germany.