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Forschungsstelle
BLV
Projektnummer
1.15.05
Projekttitel
Etablierung von vorsorgenden Massnahmen für PEDV Ausbrüche
Projekttitel Englisch
Establishing preparedness for PEDV outbreaks

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Erfasste Texte


KategorieText
Schlüsselwörter
(Deutsch)

       PEDV Epidemie, PEDV Diagnostik, reverse Genetik, Transkriptomanalyse, Prävention

Schlüsselwörter
(Englisch)

       PEDV epidemic, PEDV diagnostics, reverse genetics, transcriptome analysis, prevention

Kurzbeschreibung
(Deutsch)

       Porcine epidemic diarrhea (PED) ist eine verheerende enterale Krankheit in Schweinen die eine schwerwiegende ökonomische und tiergesundheitliche Bedrohung darstellt. Obwohl seit mehr als 20 Jahren keine PED-Ausbrüche in der Schweiz beobachtet wurden, zeichnet sich durch die neueren Ausbrüche in China und die Einschleppung von PEDV nach Nordamerika ab, dass PEDV auch für Europa wieder eine Gefahr darstellt. Um für diesen Fall gerüstet zu sein werden wir in diesem Projekt

(i)                die momentanen diagnostischen Nachweismethoden auf den neuesten Stand bringen um PEDV mit hoher Spezifität und Sensitivität zu detektieren und um hoch-pathogene von niedrig-pathogenen Stämmen zu unterscheiden,

(ii)              geeignete experimentelle Systeme entwickeln um die Pathogenität der aktuelle PEDV-Stämme zu beurteilen,

(iii)             zu klären ob die entwickelten diagnostische Nachweisverfahren geeignet sind die aktuellen PEDV-Stämme aus Feldproben umfassend zu detektieren,

(iv)             zu klären ob Unterschiede in biologische Signaturen zwischen hoch-pathogenen von niedrig-pathogenen PEDV Stämmen prädiktiv für die Virulenz sind.

Kurzbeschreibung
(Englisch)

       Porcine epidemic diarrhea (PED) is a devastating enteric disease of pigs that poses severe economical and animal health threats. Although PED outbreaks were not observed in Switzerland since more than 20 years, recent increase in outbreaks in China and transmission of PEDV to North-America exemplified that PEDV may become a problem in Europe. In order to raise preparedness for future PEDV outbreaks this project aims to:

(i)                to revise and update diagnostic tools in order to detect PEDV with high specificity and sensitivity and to distinguish between high-pathogenic and low-pathogenic strains,

(ii)              to establish suitable experimental systems to evaluate pathogenicity of currently circulating PEDV strains,

(iii)             to clarify if the developed diagnostic tests will be able to detect the currently circulating PEDV strains from field samples,

(iv)             to clarify if differences in biological signatures between high-pathogenic and low-pathogenic PEDV strains will be predictive for pathogenicity.

Projektziele
(Englisch)

As described above, the current outbreak of high-pathogenic PEDV in Asia and the USA raised considerable concerns in Europe. The situation became more complicated since the detection of co-circulating low-pathogenic PEDV. Therefore, this project aims to raise preparedness for possible future outbreaks of high-pathogenic PEDV with the focus of updating and further developing appropriate diagnostic tools to detect high-pathogenic PEDV and to distinguish high-pathogenic from low-pathogenic PEDV (aim 1). Importantly, we also aim to further our knowledge on critical viral and host factors that are decisive for PEDV pathogenicity (aim 2). This knowledge will allow us to predict if newly emerging PEDV strains will have a low- or high-pathogenic phenotype without the necessity of performing infection experiments in vivo. If successful, this will provide a contribution to the idea of 3R. Finally, we will perform one infection study in vivo (small scale). This study has two purposes. First, we urgently need samples from infected pigs that are comparable to field samples (such as fecal samples and serum) in order to validate the specificity and sensitivity of our diagnostic tools. It is also important to know basic parameters of PEDV transmission, for example how long virus can be shed by feces. The second purpose is to provide proof of principle that predictions of PEDV pathogenicity are valid by comparing a high-pathogenic PEDV with a low-pathogenic PEDV.

 

The specific aims are as follows:

       Aim 1: Revision and update of PCR- and serology-based diagnostic tools to detect PEDV infection.

       Based on recent publications we will evaluate the use and specificity of real-time RT-PCR protocols for the detection of currently circulating PEDV strains. This will be done in close collaboration with Prof. Mathias Ackermann and PD Dr. Monika Engels at the Institute of Virology, University of Zürich (reference laboratory for coronavirus infections), who will implement the resulting optimized real-time RT-PCR to their diagnostics division. Furthermore, Dr. Alex Räber, (Thermo Fisher Scientific, Prionics AG) has expressed interest in PEDV-specific qRT-PCR, and we will negotiate terms and conditions to participate and collaborate on this aim.

       We have already received protocols to detect PEDV by qRT-PCR form the United States Department of Agriculture (USDA) and in both laboratories, Bern and Zürich, we have already started to establish them. However, these tests are rather basic and need refinement and validation. Furthermore, these protocols do not allow discrimination between different PEDV strains. Thus, we will develop and validate appropriate qRT-PCR protocols according to these needs.

       In addition we will assess current ELISA protocols to detect PEDV-specific antibody responses in pigs. Again there will be a close collaboration with the Institute of Virology, Vetsuisse Faculty Zürich, and Thermo Fisher Scientific/Prionics AG in order to ensure efficient implementation into diagnostic routine. Thermo Fisher Scientific/Prionics AG is currently engaged in establishing a commercially available ELISA test for PEDV. We will assess this test (as soon as available) for detection of PEDV antibodies using stored serum samples. Since this ELISA test is based on the prototype PEDV-CV777 strain, we will assess its specificity for currently circulating PEDV strains once we have obtained a high-pathogenic PEDV isolate from the USDA, and later when we have established cell culture and a reverse genetics system for the recent PEDV strains (see aims 2 and 3). Based on the obtained results, we will decide if a second generation ELISA test, based on antigens derived from current PEDV strains, will be developed.

       Aim 2: Establishment of a reverse genetic system of a currently circulating (U.S.) PEDV stain and identification of predictive biological markers of PEDV pathogenicity.

       The identification of co-circulating low- and high-pathogenic PEDV strains make it necessary to discriminate between the two pathotypes. Current concepts of discrimination are solely based on sequence information, which is scarce. In particular it is unclear how many different low-pathogenic PEDV strains exist and where they are endemic. Furthermore, it is unclear if they share common sequence signatures that can be used for discrimination by using qRT-PCR. Thus, we aim to identify biological signatures that are independent from particular sequence variations, but correlate with pathogenicity. We will therefore study PEDV induced host cell responses in primary porcine cells, such as primary porcine macrophages and epithelial cells. Both cell types are known to be important target cells for PEDV. By comparing infection-induced host transcriptional changes between low- and high-pathogenic PEDV we aim to identify particular transcriptional patterns that differ between the two pathotypes.

       In addition we will generate a reverse genetic system for PEDV based on the sequence of a high-pathogenic US-PEDV strain. Recombinant PEDV generated by reverse genetics will have a defined sequence and will serve as a reference virus for the analysis of host transcriptional changes induced by infection of high-pathogenic PEDV. As reference virus for low-pathogenic PEDV will either use the cell culture-adapted PEDV strain CV777 or, if available, a recent low-pathogenic isolate form the US (we are currently in contact with the USDA to obtain samples). By comparing the genomic sequence of high- and low-pathogenic PEDV strains we will then target putative pathogenicity factors within the PEDV genome. Based on our experience, likely candidates are the PEDV non-structural protein 3, the PEDV spike protein, and the accessory ORF3 protein. By replacing the respective genomic regions in our recombinant virus by sequences derived from low-pathogenic PEDV, we aim to identify viral determinants that will turn the phenotype from high to low-pathogenic and that will induce similar host transcriptional changes as the low-pathogenic reference virus. Finally the phenotype of “engineered” low-pathogenic PEDV will be assessed in vivo (aim 3) in order to provide proof of principle that the observed host transcriptional changes are truly related to pathogenicity.

                     

       Aim 3: Experimental PEDV infection in vivo to assess clinical features, pathogenesis and shedding of low- and high-pathogenic PEDV.

In Aim 3 we will perform infection studies in piglets in order to assess the clinical features and pathogenicity of PEDV. As PEDV source for these infection studies we will use the recombinant high-pathogenic virus obtained from cloned cDNA (reverse genetics; aim 2) and will compare pathogenicity to a low-pathogenic reference strain and the recombinant PEDV containing particular genomic regions form low-pathogenic PEDV (obtained in aim 2). The in vivo experiment will have two main purposes. First, as described above, by assessing the pathogenicity of high- and low-pathogenic reference viruses in comparison with the “engineered” and presumably low-pathogenic virus we aim to obtain proof of principle that the host transcriptional changes identified in aim 2 have indeed predictive value. Second, we will obtain clinical samples that will allow us to validate the established diagnostic tools obtained in aim 1. We believe that this is absolutely essential, since cell-culture derived material has only limited value to judge on specificity and sensitivity of diagnostic test compared to field samples (e.g. feces and serum). Furthermore, it is absolute essential to assess viral loads in fecal samples and to know for how long virus is shed.

Publikationen / Ergebnisse
(Englisch)

We are currently preparing two manuscripts describing (i) the reverse genetic system and the in vivo experi-ments, and (ii) the qRT-PCR and the phylogenetic analysis.

Kristen-Burmann, C.; Ehmann, R.; Ackermann, M.; Thiel, V.; Tekes, T. (2017). Porcine epidemic diarrhea – on the hunt for its molecular pathogenesis. Poster Presentation, 27th Annual Meeting of the Society for Virol-ogy, Marburg, 22.-25.3.2017.

Rogger, P.; Stalder, H.; Kristen-Burmann, C.; Tekes, G.; Thiel, V. (2017) Phylogenetic analysis of PEDV as a requirement for the establishment of a diagnostic pan-strain PEDV real-time RT-PCR. Poster Presentation, Swiss Society for Microbiology (SGM), Basel, 30.-31.08.2017.

Kristen-Burmann, C.; Rogger, P.; Rappe, J.; Veiga, I. B.; Stalder, H.; Posthaus, H.; Ruggli, N.; Tekes, G.; Thiel, V. (2017) The role of the porcine epidemic diarrhoea virus (PEDV) spike protein in viral pathogenicity. Oral Presentation, 3rd Joint European Congress of the ESVP, ECVP and ESTP, Lyon, 1.9.2017.

Kristen-Burmann, C.; Rogger, P.; Ehmann, R.; Rappe, J.; Stalder, H.; Posthaus, H.; Veiga, I. B.; Ruggli, N.; Ackermann, M.; Thiel, V.; Tekes, G. (2017) Modification of the spike gene attenuates highly virulent por-cine epidemic diarrhea virus in piglets. Oral Presentation, Münchenwiler Students Retreat, Münchenwiler, 2.-3.11.2017.

Kristen-Burmann, C.; Rogger, P.; Stalder, H.; Rappe, J.; Veiga, I.; Posthaus, H.; Ruggli, N.; Ackermann, M.; Thiel, V.; Tekes, G. (2018) Modification of the spike gene attenuates highly virulent porcine epidemic diar-rhea virus in piglets. Poster Presentation, 7th Swissvirology Meeting, Fribourg, 30.01.2018.

Kristen-Burmann, C.; Rogger, P.; Ehmann, R.; Rappe, J.; Stalder, H.; Posthaus, H.; Veiga, I. B.; Ruggli, N.; Ackermann, M.; Thiel, V.; Tekes, G. (2018). Modification of the spike gene attenuates highly virulent por-cine epidemic diarrhea virus in piglets. 28th Annual Meeting of the Society for Virology, Würzburg, 20.-23.3.2018.

Rogger, P. (2018) Wissenstransfer, Projekt 1.15.05, BLV, Liebefeld, 7.6.2018.

Rogger, P. (2018) Dissertationsvortrag, Vetsuisse Fakultät, Bern, 27.6.2018.

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