Nachweis und Quantifizierung von PCV-3 in österreichischen Schweinen mittels duplex Real-Time PCR

Das porzine Circovirus 3 (PCV-3) ist ein zirkuläres DNA-Virus, welches im Jahr 2015 in den USA erstmals in Schweinen nachgewiesen wurde. Es gehört gemeinsam mit den porzinen Circoviren 1, 2 und 4 zur Familie Circoviridae. PCV-3 ist global verbreitet und wurde in diversen europäischen, asiatischen und amerikanischen Ländern nachgewiesen. Die Pathogenität des Virus wird bislang kontrovers diskutiert.In der vorliegenden Arbeit wurde eine bereits publizierte PCV-3 Real-Time PCR-Methode durch Einbeziehung eines parallelen Tests zum Nachweis einer endogenen Kontrolle an die Erfordernisse der klinischen Diagnostik angepasst (PCV-3/β-Aktin duplex Real-Time PCR) und validiert. Mit dieser Methode wurden in der Folge Proben österreichischer Schweine auf das Vorkommen von PCV-3-DNA getestet.Eine sensitive (Detektionslimit: 25 Kopien/PCR) und spezifische duplex Real-Time PCR zur Detektion von PCV-3 und β-Aktin als endogene Kontrolle wurde etabliert. Anschließend wurden 27 Seren von gesunden Ebern sowie 30 Seren und Organpools von 119 kranken Schweinen verschiedenen Alters und Organpools von 41 Aborten auf PCV-3 getestet und die positiven Proben anschließend quantifiziert. Dabei wurden 15,4&nbsp;% der Seren gesunder Eber, 6,7&nbsp;% der Seren sowie 12,6&nbsp;% der Organpools kranker Schweine und 9,8&nbsp;% der Aborte positiv auf PCV-3 getestet. Die Quantifizierung der Proben gab mit 9,1&nbsp;x&nbsp;108&nbsp;Kopien/ml die höchste Viruslast in einer Abortprobe, sämtliche positiv getesteten Seren blieben unterhalb des Quantifizierungslimits von 250&nbsp;Kopien/PCR.</p

Rapid generation of replication-deficient monovalent and multivalent vaccines for bluetongue virus: protection against virulent virus challenge in cattle and sheep.

Since 1998, 9 of the 26 serotypes of bluetongue virus (BTV) have spread throughout Europe, and serotype 8 has suddenly emerged in northern Europe, causing considerable economic losses, direct (mortality and morbidity) but also indirect, due to restriction in animal movements. Therefore, many new types of vaccines, particularly subunit vaccines, with improved safety and efficacy for a broad range of BTV serotypes are currently being developed by different laboratories. Here we exploited a reverse genetics-based replication-deficient BTV serotype 1 (BTV-1) (disabled infectious single cycle [DISC]) strain to generate a series of DISC vaccine strains. Cattle and sheep were vaccinated with these viruses either singly or in cocktail form as a multivalent vaccine candidate. All vaccinated animals were seroconverted and developed neutralizing antibody responses to their respective serotypes. After challenge with the virulent strains at 21 days postvaccination, vaccinated animals showed neither any clinical reaction nor viremia. Further, there was no interference with protection with a multivalent preparation of six distinct DISC viruses. These data indicate that a very-rapid-response vaccine could be developed based on which serotypes are circulating in the population at the time of an outbreak

The 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 2 to 3 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

The 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 2 to 3 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

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