Vergleichende Analyse von Typ 1 und Typ 2 Viren des reproduktiven und respiratorischen Syndroms des Schweins in vitro: Makrophagentropismus und genetische Determinanten der unterschiedlichen Replikationseigenschaften

• Forschungsstelle: BLV
• Projektnummer: 1.13.12
• Projekttitel Englisch: Comparative analysis of type 1 and type 2 porcine reproductive and respiratory syndrome viruses in vitro: macrophage tropism and genetic determinants of the different replication characteristics

Schlüsselwörter (Deutsch)

Arteriviridae, reproduktives und respiratorisches Syndrom Virus des Schweins, PRRS, Schwein, Makrophagen, Zelltropismus, Pathogenese, Virulenz, Diagnostik

Schlüsselwörter (Englisch)

Arteriviridae, porcine reproductive and respiratory syndrome virus, PRRS, pig, macrophages, cell tropism, pathogenesis, virulence, diagnosis

Kurzbeschreibung (Deutsch)

Die Schweiz gehört zu den wenigen Europäischen Ländern, die noch frei vom porzinen reproduktiven und respiratorischen Syndrom (PRRS) sind. Die Krankheit verursacht weltweit enorme Verluste in der Schweineproduktion. Die grösste Gefahr, das PRRS Virus (PRRSV) einzuführen kommt vom regulären Samenimport und von der illegalen Einfuhr von Schweinen. Das Virus ist nur schwer aus der Schweinepopulation auszurotten, nicht zuletzt weil die Impfstoffe einen ungenügenden Impfschutz erzeugen. Seit einiger Zeit treten vermehrt virulentere PRRSV Stämme auf, vor allem in China, USA, Ost- und Nordeuropa. Die Gründe dafür und die genetischen Determinanten der erhöhten Virulenz sowie die Pathogenitätsmechanismen sind unbekannt. Um dies zu erforschen planen wir die Etablierung der reversen Genetik und die Generierung von Prototyp Viren der weltweit zirkulierenden Europäischen Typ1 und USA und Chinesischen Typ 2 PRRSV Isolaten. Der zweite Fokus des Projekts ist die Identifizierung von Makrophagen-spezifischen Faktoren, nebst den bekannten Rezeptoren, die für die produktive Virusreplikation in Zellkultur essentiell sind. Dies ist wichtig weil PRRSV einen engen Tropismus für primäre porzine Alveolarmakrophagen (PAM) besitzt. PAM spielen eine Schlüsselrolle in der PRRS Pathogenese. Zudem möchten wir die identifizierten zellulären Faktoren in einer permanenten Zelllinie stabil integrieren, um die Replikation von PRRSV Feldisolaten unabhängig von PAM zu ermöglichen. Eine solche Zelllinie würde die Anzucht von Feldisolaten und die Impfstoffproduktion erleichtern. Die beiden Teile des Projektes sollen anschliessend verbunden werden indem die Replikationseigenschaften von PRRSV Isolaten verschiedener Genotypen und Virulenz in permissiven primären PAM sowie in den permanenten transgenen Zelllinien analysiert werden.

Kurzbeschreibung (Englisch)

Switzerland is one of the very few countries in Europe that are still free from porcine reproductive and respiratory syndrome (PRRS), the economically most important viral disease of pigs worldwide. Import of semen and illegal pig trade represent a constant threat for the introduction of the PRRS virus (PRRSV) into Switzerland. Disease eradication is very difficult, essentially due to the low efficacy of the vaccines available. The PRRS situation has further aggravated due to increasing emergence of strains of high virulence. The reasons and the genetic determinants of this emergence remain obscure. In addition, the pathomechanisms are unknown. Consequently, we will establish the reverse genetics for selected relevant isolates of the European type 1 as well as USA and Chinese type 2 isolates circulating worldwide to study the viruses at the genetic level and the molecular aspects of their interaction with the host cell. An important focus of the project is related to the restricted and peculiar macrophage tropism of PRRSV. In fact, virus isolation is hampered by the restricted tropism of many PRRSV isolates for primary porcine alveolar macrophages (PAM) that can only be obtained in small numbers, do not proliferate and require killing of pigs. In addition, the macrophage tropism of PRRSV is most likely crucial in determining the pathogenesis of PRRSV and the co-infections associated with the syndrome. The factors, besides the known receptors, determining productive replication in macrophages need to be identified. This will enable not only to establish a universal cell culture system for PRRSV isolates applicable for diagnostics and vaccine production, but also to understand disease pathogenesis and, in a more general sense, to understand the interaction of isolates of different genotype and virulence with the host. With this information we will generate a transgenic cell line supporting replication of all PRRSV isolates. The replication characteristics of PRRSV strains of different genotype and virulence will be compared on permissive ex vivo macrophage preparations and on the transgenic cell lines.

Projektziele (Englisch)

In order to be prepared in case of PRRSV emergence in Switzerland, it is important for the IVI to possess a centre of excellence in PRRS with the necessary technical and scientific expertise to control the disease. Therefore, the objectives of this study are:

1) to implement the reverse genetics of PRRSV for type 1 and type 2 prototype strains in order to study the viral and host factors that determine PRRSV tropism and virulence as well as immunopathogenesis of the disease

2) to identify macrophage-specific cellular factors apart from Sn and CD163 that support efficient replication of natural PRRSV isolates. These factors will be used to construct permanent cell lines for the isolation and propagation of PRRSV field isolates.

3) to study the interaction of type 1 and type 2 PRRSV field isolates of different virulence with primary porcine macrophages and with the permanent cell lines generated under 2), with the aim of identifying in vitro markers differentiating the various genotypes and biotypes of PRRSV.

Publikationen / Ergebnisse (Englisch)

Rappe, J.C.F.; Stalder, H.; Thiel, V.; Ruggli, N. (2015) Inhibition of Porcine Reproductive and Respiratory Syndrome Virus and Equine Torovirus Replication by Targeting the Formation of Double-Membrane Vesicles. 25^th Annual Meeting of the Society for Virology (GfV), 18-21 March 2015, Bochum, Germany, poster presentation (P84).

Rappe, J.C.F.; Thiel, V.; Ruggli, N. (2015) Inhibition of PRRSV replication by targeting the formation of double-membrane vesicles. International PRRS Congress, 3-5 June 2015, Bochum, Germany, oral presentation (O-08).

Rappe, J.C.F.; Ruggli, N. (2016). Heterogeneous antigenic properties of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid among genotype 1 strains. 74^th Annual Meeting of the Swiss Society for Microbiology, 13–15 June 2016, Bern, Switzerland, oral presentation (O-163).

Rappe, J.C.F.; Renzullo, S., Hofmann, M.A.; Ruggli,N. (2016) Porcine reproductive and respiratory syndrome virus isolate IVI-1173, complete genome. GenBank accession number KX622783.

Rappe, J.C.F.; Wernike, K.; Beer, M.; and Ruggli, N. (2016) Porcine reproductive and respiratory syndrome virus isolate RVB-581, complete genome. GenBank accession number KX650082.

García-Nicolás, O.; Auray, G.; Sautter, C.A.; Rappe, J.C.F.; McCullough, K.C.; Ruggli, N.; Summerfield, A. (2016) Sensing of porcine reproductive and respiratory syndrome virus-Infected macrophages by plasmacytoid dendritic cells. Frontiers in Microbiology 7:771.

Rappe, J.C.F.; García-Nicolás, O.; Flückiger, F.; Thür, B.; Hofmann, M.A.; Summerfield, A.; Ruggli, N. (2016) Heterogeneous antigenic properties of the porcine reproductive and respiratory syndrome virus nucleocapsid. Vet. Res. 47(1):117

Rappe, J.C.F.; de Wilde, A.H.; V’kovski, Ph.; Stalder, H.; Snijder, E.; Brinton, M.; Müller, Ch.; Ziebuhr, J.; Ruggli, N.; Thiel, V. Antiviral activity of K22 against a wide spectrum of Nidovirales

Rappe, J.C.F.; V’kovski, Ph.; Thiel, V.; Ruggli, N. Mapping of adaptive mutations conferring resistance to a chemical compound targeting the double membrane vesicles formation during PRRSV replication, in preparation.

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1.13.12 Ruggli Vergleichende Analyse von Typ 1 und Typ 2 Viren des reproduktiven und respiratorisch…