Heinz-Jürgen Thiel

Feline infectious peritonitis (FIP) is a lethal immunopathological disease caused by feline coronaviruses (FCoVs). Here, we describe a reverse genetics approach to study FIP by assessing the pathogenicity of recombinant type I and type II and chimeric type I/type II FCoVs. All recombinant FCoVs established productive infection in cats, and recombinant type II FCoV (strain 79-1146) induced FIP. Virus sequence analyses from FIP-diseased cats revealed that the 3c gene stop codon of strain 79-1146 has changed to restore a full-length open reading frame (ORF).

Vaccines play a key role in the control of viral diseases both in humans and in animals. In order to ensure the quality and consistency of vaccines they are extensively tested, including potency control of individual batches. In the case of vaccines against rabies the most widely used test for batch potency control is the National Institutes of Health (NIH) test. The NIH test is performed in mice leading to the consumption of thousands of animals every year. Protection against rabies after vaccination is associated with neutralizing antibodies directed against the viral glycoprotein (G). Therefore the amount of G-protein in vaccine preparations is an important parameter with regard to potency. Additionally the structural integrity of virus particles in vaccine preparations may be crucial for their immunogenicity. The objective of our work is the development of in vitro methods to determine the potency of vaccines against rabies. The result of this ongoing project shall be an assay panel including measurement of the antigenic content as well as parameters of antigen quality in a vaccine preparation allowing a precise prediction of the potency of rabies vaccines without using animal experiments.

A mysterious disease affecting calves, named bovine neonatal pancytopenia (BNP), emerged in 2007 in several European countries. Epidemiological studies revealed a connection between BNP and vaccination with an inactivated vaccine against bovine virus diarrhea (BVD). Alloantibodies reacting with blood leukocytes of calves were detected in serum and colostrum of dams, which have given birth to calves affected by BNP. To understand the linkage between vaccination and the development of alloantibodies, we determined the antigens reacting with these alloantibodies. Immunoprecipitation of surface proteins from bovine leukocytes and kidney cells using sera from dams with a confirmed case of BNP in their gestation history reacted with two dominant protein species of 44 and 12 kDa. These proteins were not detected by sera from dams, free of BVDV and not vaccinated against BVD, and from sera of animals vaccinated with a different inactivated BVD vaccine. The 44 kDa protein was identified by mass spectrometry analysis as MHC I, the other as β-2-microglobulin. The presence of major histocompatibility complex class I (MHC I) in the vaccine was confirmed by Western blot using a MHC I specific monoclonal antibody. A model of BNP pathogenesis is proposed.

Persistent infection of domestic cats with feline coronaviruses (FCoVs) can lead to a highly lethal, immunopathological disease termed feline infectious peritonitis (FIP). Interestingly, there are two serotypes, type I and type II FCoVs, that can cause both persistent infection and FIP, even though their main determinant of host cell tropism, the spike (S) protein, is of different phylogeny and displays limited sequence identity. In cell culture, however, there are apparent differences. Type II FCoVs can be propagated to high titers by employing feline aminopeptidase N (fAPN) as a cellular receptor, whereas the propagation of type I FCoVs is usually difficult, and the involvement of fAPN as a receptor is controversial. In this study we have analyzed the phenotypes of recombinant FCoVs that are based on the genetic background of type I FCoV strain Black but encode the type II FCoV strain 79-1146 S protein. Our data demonstrate that recombinant FCoVs expressing a type II FCoV S protein acquire the ability to efficiently use fAPN for host cell entry and corroborate the notion that type I FCoVs use another main host cell receptor. We also observed that recombinant FCoVs display a large-plaque phenotype and, unexpectedly, accelerated growth kinetics indistinguishable from that of type II FCoV strain 79-1146. Thus, the main phenotypic differences for type I and type II FCoVs in cell culture, namely, the growth kinetics and the efficient usage of fAPN as a cellular receptor, can be attributed solely to the FCoV S protein.

In this study we report the complete sequence and genome organization of the serotype I feline coronavirus (FCoV) strain Black. Furthermore, a reverse genetic system was established for this FCoV strain by cloning a full-length cDNA copy into vaccinia virus. This clone served as basis for the generation of recombinant FCoV (recFCoV) that was shown to bear the same features in vitro as the parental FCoV. Using this system, accessory 3abc genes in the FCoV genome were replaced by green fluorescent protein (recFCoV-GFP) andRenillaluciferase genes (recFCoV-RL). In addition, we showed that feline CD14⁺blood monocytes and dendritic cells can be easily detected after infection with recFCoV-GFP. Thus, our established reverse genetic system provides a suitable tool to study the molecular biology of serotype I FCoV.

Preventive vaccination against rabies virus is a highly effective method for preventing rabies in humans and animals. For travel purposes, vaccination of domestic carnivores is obligatory. In addition, some countries require testing for neutralizing antibodies against rabies. The minimal threshold level accepted by WHO/OIE is 0.5 IU/ml. Despite proper vaccination some animals do not reach the threshold. The objective of this study was to identify specific risk factors in dogs and cats for post-vaccination rabies antibody titres below 0.5 IU/ml by FAVN test. Rabies vaccination protocols and recommendations were reviewed with regard to travel regulations. Comprehensive data was collected on animals tested for rabies antibodies via a questionnaire sent to veterinarians who submitted sera for rabies titration. The questionnaire included data on species, age, sex, breed, vaccine used, date of last vaccination and blood sampling, vaccination history and further medical treatments at time of vaccination. Data on around 1,200 animals was analysed. Most animals older than one year had already received more than one rabies vaccination. The influence of breed and sex on antibody titre seems to be insignificant. Young dogs have a high risk of results below 0.5 IU/ml after their first vaccination. This risk can be minimised by the application of a second vaccination and blood sampling according to the manufacturer's recommendations. An important factor for the test outcome might be the virus strain used in the vaccine.

Gegenstand und Ziel: Die bovine Virusdiarrhö/Mucosal Disease( BVD/MD) stellt nach wievor eine der ökonomisch bedeutsamsten Virusinfektionen des Rindes dar. Aufgrund der hohen Prävalenz des Erregers und der geographischen Lage ist der Einsatzvon Impfstoffen bei der Bekämpfung der BVD/MD in Deutschland nach verbreiteter Ansicht derzeit unverzichtbar. Zentrale Bedeutung besitzt dabei der Schutz vor diapla- zentarer Übertragung des Virus. Eine Reihe von Impfstoffen gegen BVD/MD auf der Basis von vermehrungsfähigem Virus (“Lebendvakzinen”) bzw. inaktiviertem Virus (“Totvakzinen”) ist derzeit in Deutschland zugelassen, zum Teil mit dem Zusatz “fetaler Schutz”. Neben dem alleinigen Einsatz von “Lebend-” bzw. “Totvakzinen” werden beide Impfstoffarten auch kombiniert im Rahmen des so genannten zweistufigen Impfregimes verwendet. Ziel der vorliegenden Untersuchung war der Vergleich unterschiedlicher Impfstoffe und Impfregime. Material und Methoden: In einem Tierversuch wurden 60 Rinder mit unterschiedlichen Impfstoffen gegen BVD/MD immunisiert. Über einen Zeitraum von 10 Monaten wurden Serumproben der Tiere gewonnen und im Neutralisationstest gegen drei BVD-Testviren aus den Gruppen BVDV-1a,-1b und BVDV-2a getestet. Ergebnisse: Die zweimalige Immunisierung mit dem neuen Impfstoff PregSure^® BVD ergab vergleichbar hohe neutralisierende Antikörpertiter gegen BVDV-1 wie die Kombination von “Totimpfstoff” und “Lebendimpfstoff” im zweistufigen Verfahren. Hinsichtlich BVDV-2führte die zweimalige Impfung mit PregSure^® BVD sogar zu höheren Titern. Schlussfolgerungen: Es zeigte sich,dass der neue “Totimpfstoff” PregSure^® BVD auchfürden Einsatz im Rahmen zweistufiger Impfprogramme gut geeignet ist. Klinische Relevanz: Der alleinige Einsatz von “Totimpfstoffen” bietet praktische Vorteile. Wichtigstes Ziel aller Impfungen gegen BVD/MD bleibt der “fetale Schutz”.