West Nile virus

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University of Veterinary Medicine Hannover

West Nile virus: Vaccination and pathogenesis studies in large falcons and mice

INAUGURAL –DISSERTATION

in partial fulfillment of the requirements of the degree of Doctor of Veterinary Medicine

-Doctor medicinae veterinariae - ( Dr. med. vet.)

submitted by Joke Henriette Angenvoort

Wesel

Hannover 2016

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Scientific supervision: Prof. Dr. Martin H. Groschup

Institute for Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut,

Federal Research Institute for Animal Health, Isle of Riems

1^st supervisor: Prof. Dr. Martin H. Groschup

2^nd supervisor: Prof. Dr. Georg Herrler

Day of the oral examination: 08.06.2016

This work was partly supported by the EU commission (projects NADIR and EDENext).

Roc Falcon S.L. provided the falcons for the animal experiment.

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To Robert

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Parts of this work have already been published in the following journals:

"Pathogenesis of West Nile virus lineage 1 and 2 in experimentally infected large falcons."

Ziegler, U., Angenvoort, J., Fischer, D., Fast, C., Eiden, M., Rodriguez, A. V., Revilla- Fernandez, S., Nowotny, N., de la Fuente, J. G., Lierz, M., Groschup, M. H.

Veterinary Microbiology (2013), volume 161, issue 3-4, pages 263-273.

Joke Angenvoort participated in study design, experimental work, data analysis and paper writing.

"Limited efficacy of West Nile virus vaccines in large falcons (Falco spp.)."

Angenvoort, J.¹, Fischer, D.¹, Fast, C., Ziegler, U., Eiden, M., de la Fuente, J. G., Lierz, M., Groschup, M. H.

Veterinary Research (2014), volume 45, page 41.

Joke Angenvoort performed the experiments at the FLI, analyzed the data and participated in study design and paper writing.

"DNA vaccines encoding the envelope protein of West Nile virus lineages 1 or 2

administered intramuscularly, via electroporation and with recombinant virus protein induce partial protection in large falcons (Falco spp.)."

Fischer, D.¹, Angenvoort, J.¹, Ziegler, U., Fast, C., Maier, K., Chabierski, S., Eiden, M., Ulbert, S., Groschup, M. H., Lierz, M.

Veterinary Research (2015), volume 46, page 87.

Joke Angenvoort performed the experiments at the FLI, analyzed the data, participated in study design and writing the manuscript.

Manuscript extracted from the doctorate project:

“Differences in pathogenicity of West Nile virus strains in experimental infected mice”

J. Angenvoort, U. Ziegler, C. Fast, M. Keller, M. Eiden, M. H. Groschup to be submitted

1 „contributed equally“

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Further publications:

"Monitoring of West Nile Virus Infections in Germany."

Ziegler, U., Seidowski, D., Angenvoort, J., Eiden, M., Müller, K., Nowotny, N., Groschup, M. H.

Zoonoses and Public Health (2012), volume 59, pages 95-101.

"Use of competition ELISA for monitoring of West Nile virus infections in horses in Germany."

Ziegler, U., Angenvoort, J., Klaus, C., Nagel-Kohl, U., Sauerwald, C., Thalheim, S., Horner, S., Braun, B., Kenklies, S., Tyczka, J., Keller, M., Groschup, M. H.

International Journal of Environmental Research and Public Health (2013), volume 10, issue 8, pages 3112-3120.

"Indigenous West Nile virus infections in horses in Albania."

Berxholi, K., Ziegler, U., Rexhepi, A., Schmidt, K., Mertens, M., Korro, K., Cuko, A., Angenvoort, J., Groschup, M. H.

Transboundary and Emerging Diseases (2013), volume 60, supplement 2, pages 45-50.

"West Nile viral infection of equids."

Angenvoort, J., Brault, A. C., Bowen, R. A., Groschup, M. H.

Veterinary Microbiology (2013), volume 167, issue 1-2, pages 168-180.

Experimental data were also published at national and international conferences:

“Infektion von Falken mit zwei verschiedenen West-Nil-Virus-Stämmen“

J. Angenvoort, U. Ziegler, C. Fast², D. Fischer, J. Garcia de la Fuente, M. Lierz, M. H.

Groschup.

54. Jahrestagung der DVG-Fachgruppe “Pathologie”, Germany, Fulda, 12./13.03.2011, oral presentation.

2 Speaker

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“Experimental infections of large falcons with West Nile virus lineage 1 and 2”

U. Ziegler, J. Angenvoort, D. Fischer, C. Fast, M. Eiden, S. Revilla-Fernández, N. Nowotny, J. G. de la Fuente, M. Lierz, M. H. Groschup.

Epizone, 5th annual meeting, Netherlands, Arnhem, 12./13.04.2011, poster presentation.

"Experimentelle Infektionsstudien an Großfalken mit dem West-Nil-Virus"

J. Angenvoort², U. Ziegler, D. Fischer, C. Fast, M. Eiden, N. Nowotny, S. Revilla-Fernández, J. G. de la Fuente, M. H. Groschup, M. Lierz.

2. DVG-Tagung über Vogel- und Reptilienkrankheiten, Germany, Hannover, 16./17./18.09.2011, oral presentation.

„Infection studies with West Nile virus lineage 1 and 2 in large falcons”

Joke Angenvoort, Ute Ziegler, Dominik Fischer, Christine Fast, Martin Eiden, Sandra

Revilla-Fernández, Norbert Nowotny, Jorge Garcia de la Fuente, Michael Lierz and Martin H.

Groschup

6th European Meeting on Viral Zoonoses, St. Raphael, France, October 1-4, 2011, poster presentation.

“Infection studies with West Nile virus lineage 1 and 2 in large falcons”

J. Angenvoort², U. Ziegler, D. Fischer, C. Fast, M. Eiden, S. Revilla-Fernández, N. Nowotny, J. Garcia de la Fuente, M. Lierz, M. H. Groschup.

National Symposium on Zoonoses Research, Germany, Berlin, 06./07.10.2011, oral presentation.

“Challenge studies with West Nile virus lineage 1 and 2 in large falcons”

U. Ziegler, J. Angenvoort, D. Fischer, C. Fast, M. Eiden, S. Revilla-Fernández, N. Nowotny, J. G. de la Fuente, M. Lierz, M. H. Groschup

122nd Annual Meeting of the Society for Virology, Germany, Essen, 14./15./16./17.03.2012, poster presentation, number 553.

“Infection studies with West Nile virus lineage 1 and 2 in large falcons”

Angenvoort, J.

First Junior Scientist Symposium Friedrich-Loeffler-Institut, Germany, Vilm, 10./11.08.2012, poster presentation.

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“Efficacy of two WNV vaccines in large falcons verified by live virus challenge”

J. H. Angenvoort³, D. Fischer³, U. Ziegler, M. Eiden, C. Fast, M. Lierz, M. H. Groschup.

National Symposium on Zoonoses Research, Germany, Berlin, 11./12.10.2012, poster presentation N 19.

„Die Effizienz einer West-Nil-Virus Vakzinierung von Großfalken aus Sicht der Pathologie“

C. Fast⁴, J. Angenvoort, D. Fischer, U. Ziegler, S. Ulbert, J. G. de la Fuente, M. H. Groschup, M. Lierz.

56. Jahrestagung der Fachgruppe Pathologie der DVG, Germany, Fulda, 9./10.03.2013, oral presentation.

3 „contributed equally“

4 speaker

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Table of contents i

List of abbreviations

arbovirus arthropod-borne virus

CNS central nervous system

DA WNV isolate Dakar

DNA deoxyribonucleic acid

dpi days post infection

ELISA enzyme linked immunosorbent assay

E protein envelope protein

EP electroporation

FCS fetal calf serum

IC2 RNA internal control RNA

IFNAR interferon alpha receptor

IFNAR -/- interferon type I receptor knock-out

IHC immunohistochemistry

ISG interferon-stimulated genes

JAK Janus kinase

JAK/STAT pathway Janus kinase / signal transducers and

activators of transcription pathway

GO WNV isolate goshawk Austria 2009

MEM minimal essential medium

M protein membrane protein

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iv List of abbreviations

ND50 Neutralization Dose 50

NS nonstructural protein

NY WNV isolate New York 1999

pfu plaque forming units

prM protein pre-membrane protein

PRNT plaque reduction neutralization test

qRT-PCR quantitative real-time reverse transcriptase

polymerase chain reaction

RNA ribonucleic acid

STAT signal transducer and activator of

transcription

TBS tris-bufferd saline solution

TCID50 Tissue Culture Infection Dose 50

UG WNV isolate Uganda

VNT micro-virus neutralization test

WNND West Nile neuroinvasive disease

WNV West Nile virus

wpv weeks post vaccination

WT wild type

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List of figures v

List of figures

Figure 1-1. Flavivirus genus. ... 2 Figure 1-2. Schematic WNV structure and genome organization. ... 3 Figure 5-1. Survival curves of IFNAR -/- and wild type (C57/Bl6) mice infected with four different WNV isolates. ... 33 Figure 5-2. Viral loads in the brains of different mouse groups determined by qRT-PCR or cell culture. ... 34 Figure 5-3. Viral loads in the hearts of different mouse groups determined by qRT-PCR or cell culture. ... 35 Figure 5-4. Viral loads in the spleens of different mouse groups determined by qRT-PCR or cell culture. ... 36 Figure 5-5. Immunohistochemistry score for brain (5-5A), spleen (5-5B) and liver (5-5C) of all mouse groups. ... 37

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vi List of tables

List of tables

Table 1-1. WNV proteins and their functions. ... 4 Table 1-2: Licensed veterinary WNV vaccines in the E.U. and U.S. ... 11 Table 5-1. Definition of immunhistochemistry (IHC) score. ... 39 Table 5-2. Mean viral loads and median IHC scores of animals which succumbed to infection (only deceased mice). ... 40

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Introduction 1

1 Introduction

West Nile virus is a Flavivirus and has been recognized since the late 1930s. After decades with only sporadic epidemics among humans and equines in Southern European countries, public health authorities were alerted by rising case numbers in these countries and by the introduction to and spread across the Americas. Now WNV is distributed worldwide except Antarctica and has impacts on public health and equine industry, but also on the natural bird population.

1.1 Taxonomy and virus structure

West Nile virus (WNV) is a member of the genus Flavivirus belonging to the family Flaviviridae. Two other genera in this family are Pestivirus and Hepacivirus. Flavivirus genus is subdivided into 14 serogroups, with WNV belonging to the Japanese encephalitis virus serocomplex encompassing seven more viruses, among them Usutu virus (Simmonds et al. 2012).

WNV isolates itself are divided into at least five genetic lineages (Bondre et al. 2007).

Lineage 1 is found in Africa, America, Asia, Australia and Europe (Lanciotti et al. 1999), and lineage 2 in Africa and Europe (Bakonyi et al. 2006; Venter u. Swanepoel 2010; Papa et al.

2011). The latter two lineages are responsible for infections with clinical importance in humans, equines and birds. Lineage three is represented by the Rabensburg virus which was isolated in the Czech Republic (Bakonyi et al. 2005), and lineage four by a tick-isolate found in the Caucasus (Bakonyi et al. 2005). In India distinct WNV lineage five isolates have been found amongst co-circulating lineage 1 isolates (Bondre et al. 2007). It remains open whether even more lineages should be discriminated (Scherret et al. 2001; J. S. Mackenzie u. Williams 2009; Vazquez et al. 2010).

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2 Introduction

Figure 1-1. Flavivirus genus. WNV is phylogenetically divided into at least five lineages (Lanciotti et al. 1999;

Bakonyi et al. 2005; Bondre et al. 2007; Papa et al. 2011), and belongs to the Japanese encephalitis virus group, which is a member of Flavivirus genus of the family Flaviviridae (Simmonds et al. 2012).

The WNV genome consists of an approximately 11 kilobase long, positive-sense single- stranded ribonucleic acid (RNA), which encodes for three structural (capsid (C), premembrane (prM) and envelope (E)) and five nonstructural (NS1, NS2a/NS2b, NS3, NS4a/NS4b and NS5) proteins (Rice et al. 1985; Lanciotti et al. 1999). The RNA is embedded in the nucleocapsid, which is composed of capsid proteins (C-protein, Figure 1-2) and surrounded by a lipid bilayer envelope in which the membrane proteins (M) and envelope proteins are anchored (Rice et al. 1985).

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Introduction 3

Figure 1-2. Schematic WNV structure and genome organization. West Nile virus has an icosahedral symmetry and is ~50 nm in diameter. The nucleocapsid is composed of multiple C-proteins, which incorporate the single stranded plus sense RNA. It is encased by a lipid bilayer in which the transmembrane anchors of M (green spheres) and E proteins (green dimers) are immersed (Mukhopadhyay et al. 2003). WNV structural protein coding RNA is preceded by a 5’ RNA cap and a 5’ noncoding region (96 nucleotides short), the RNA is terminated by a 3’ noncoding region (approximately. 600 nucleotides) (Rice et al. 1985; Lanciotti et al. 1999).

Figure adapted from (Angenvoort et al. 2013).

These WNV proteins have structural functions, but are also involved in other processes in the life cycle of WNV (Table 1-1). WNV E protein contains the major epitope for neutralizing antibodies (Beasley u. Barrett 2002). WNV nonstructural proteins have major functions in replication, which are not all fully understood, and also in immune evasion.

WNV protein Function Literature

Capsid protein (C)

Formation of capsid, translocation of premembrane protein (prM) into ER lumen (anchor C), interaction with cell proteins, modulation of cell apoptosis

(Nowak et al. 1989; Yang et al. 2002; Mukhopadhyay et al. 2003; Hunt et al. 2007;

Xu et al. 2011; Urbanowski u. Hobman 2013)

(Pre-)membrane protein (PrM)

Cleavage is prerequisite for fusion during infection of cells, interacts with cell protein (impact on late replication), virus assembly, protects E protein of premature conformational changes

(Guirakhoo et al. 1992;

Zhang et al. 2007; Tan et al.

2009; Moesker et al. 2010; J.

B. Brault et al. 2011)

Envelope protein Mediates receptor-mediated

endocytosis, (Kimura u. Ohyama 1988;

Beasley u. Barrett 2002; Chu

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4 Introduction

(E) receptor binding, membrane

fusion, target of neutralizing antibody response

et al. 2005)

Nonstructural protein 1 (NS1)

Function in early RNA replication, immune evasive potential (modulation of complement activation, inhibition of Toll-like receptor 3 signaling)

(Lindenbach u. Rice 1997;

Chung et al. 2006; Wilson et al. 2008; Avirutnan et al.

2010; Avirutnan et al. 2011)

Nonstructural protein 2A (NS2A)

Putative role in RNA replication / associated with replication complex, potential role in virus assembly, immune evasion (inhibition of interferon induction)

(J. M. Mackenzie et al. 1998;

Liu et al. 2006; Leung et al.

2008)

Nonstructural protein 2B (NS2B)

Cofactor for NS3 protease (Chambers et al. 1993; Erbel et al. 2006)

Virus protease (C|C-anchor, NS2A|NS2B, NS2B|NS3,

NS3|4A, NS4A|2K,

NS4B|NS5), RNA helicase, nucleoside triphosphatase, RNA triphosphatase

(Chambers et al. 1991;

Wengler et al. 1991; Lin et al. 1993; Yamshchikov u.

Compans 1994; Mastrangelo et al. 2007)

Nonstructural protein 4A (NS4A)

Associated with replication complex, putative cofactor of NS3 helicase

(J. M. Mackenzie et al. 1998;

Shiryaev et al. 2009)

Nonstructural protein 4B (NS4B)

Plays a role in blocking interferon signaling;

mediator of cell death, important for viral replication (by interaction with a cellular factor)

(Evans u. Seeger 2007; Puig- Basagoiti et al. 2007)

RNA-dependent RNA

polymerase; methyltrans- ferase, involved in blocking interferon signaling

(Guyatt et al. 2001; Zhou et al. 2007; Laurent-Rolle et al.

2010)

Table 1-1. WNV proteins and their functions. WNV polyprotein is cleaved into eight proteins by cellular signalases (C|prM, prM|E, E|NS1, NS4A|NS4B), cellular furin (prM->M), membrane associated cellular protease (NS1|NS2A) and viral peptidases (see above) (Nowak et al. 1989; Lin et al. 1993; Falgout u. Markoff 1995; Stadler et al. 1997).

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Introduction 5

1.2 History and epidemiology

In the 1930s WNV was first isolated from a woman from the West Nile district of Uganda suffering from a febrile illness (Smithburn et al. 1940). Subsequently, it caused sporadic epidemics in Africa, Asia, Southern Europe and the Middle East which affected humans and horses, in most cases causing subclinical to mild febrile disease (Murgue et al. 2001a;

Weinberger et al. 2001; Burt Fj Fau - Grobbelaar et al. 2002; Bondre et al. 2007). Only occasionally it caused neurological symptoms (George et al. 1984; Weinberger et al. 2001).

Therefore it was not in the focus of public health authorities as a considerable threat for human health.

Since the 1990s an increasing number of epidemics affecting humans and equines have occurred in Mediterranean and adjacent countries (Le Guenno et al. 1996; platTsai et al. 1998;

Hubalek et al. 1999; Ceianu et al. 2001; Platonov et al. 2001; Triki et al. 2001; Autorino et al.

2002; Schuffenecker et al. 2005; Barzon et al. 2009). In 1996, a major outbreak with several hundreds of people suffering from West Nile neuroinvasive disease (WNND) occurred in Romania (Tsai et al. 1998). In 1999, more than 800 humans were infected in a WNND outbreak in Volgograd, Russia (Platonov et al. 2001). In 1997, WNV lineage 1 caused neurological disease in domestic geese in Israel, which was the first time that WNV was recognized to affect birds clinically. Subsequently it also was isolated in wild birds and caused human outbreaks (Bin et al. 2001; Malkinson et al. 2002; Anis et al. 2014).

In 1999, WNV was newly introduced into New York, North America, and within only a few years spread all over the American continent (Lanciotti et al. 1999; Morales et al. 2006; Di Giallonardo et al. 2015). It is now distributed worldwide in all continents except Antarctica (Kramer et al. 2008). The outbreaks on the North American continent were accompanied by a high wild bird mortality affecting numerous species with Passerines as the most competent hosts (Lanciotti et al. 1999; Komar et al. 2003). Since its introduction several North American bird species populations have declined substantially (LaDeau et al. 2007).

WNV lineage 2 was known for long time as a cause for mild or subclinical diseases in Africa and Madagascar. It was first time discovered on the European continent in Hungary in 2004 (Bakonyi et al. 2006). This strain led to a large outbreak among birds of prey, equines and humans in 2008 and spread to larger parts of Hungary and eastern Austria. However, human

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6 Introduction

diseases were fairly mild and non-lethal. While many bird species were affected, birds of prey, especially goshawks, were particularly susceptible to WNV lineage 2 neuroinvasive infections. Apart from this intrinsic higher susceptibility, the large size and conspicuity of dead birds of prey may have lead to the impression of comparatively higher case numbers (Bakonyi et al. 2013). In retrospective studies it was shown that also in Africa WNV lineage 2 strains caused severe and fatal neuroinvasive diseases in equines (2007, 2008), which had been overlooked in the past (Venter et al. 2009). High and low virulent strains exist for lineage 1 and lineage 2, respectively (Venter u. Swanepoel 2010). In 2010, the European WNV lineage 2 strain arrived in Greece, where it caused neurological diseases with fatalities amongst elderly people. The increase in virulence was probably due to an H249P substitution in the NS3 protein (Papa et al. 2011). Over the last three decades West Nile virus has spread to new geographic regions and is considered now endemic in any parts of Southern Europe.

Interestingly both WNV lineages 1 and 2 isolates can be found in Europe sometimes in the same regions (Barzon et al. 2011; Danis et al. 2011a; European Centre for Disease Prevention and Control 2011; Savini et al. 2012). For 2015 human WNF cases have been reported in Italy, Hungary, Romania, Austria, Bulgaria, France and Portugal as well as in horses in France and Portugal (European Centre for Disease Prevention and Control 2015). In Germany wild and captive birds were monitored for WNV genome and antibodies. Essentially, WNV antibodies were found in migratory birds (migrating from Africa or Southern Europe) only.

Hence, so far there is no evidence for active WNV infections in birds in Germany (Ziegler et al. 2015).

1.3 Transmission and reservoir

WNV is transmitted in an enzootic (maintenance) cycle between ornithophilic mosquitoes and birds (A. C. Brault 2009). Important vectors are mosquitoes of the genus Culex, for example Culex pipiens in North America and Europe and Culex univittatus in Africa (Jupp u. McIntosh 1970; Tsai et al. 1998; Turell et al. 2000; Bernard et al. 2001; Calzolari et al. 2015).

Vertebrate reservoir hosts are birds with species specific differences in terms of susceptibility.

Susceptible species develop a longer and more pronounced viraemia as compared to more resistant species. The threshold viraemia titer for infectiousness of Culex mosquito species is considered to be 10⁵ plaque forming units (pfu) per ml (Turell et al. 2000; Komar et al. 2003).

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Introduction 7

Passerines (like crows and jays) were found to be most reservoir competent (Komar et al.

2003). Apart from mosquito infection, birds can also become infected per oral route or via contact to an infected cage mate (Komar et al. 2003). In addition to level and duration of viraemia also host abundance and frequency of bites of vector mosquito species influence the importance of a bird species for virus maintenance (Kilpatrick et al. 2006). Birds are the natural reservoir for WNV and spread it over long distances in the context of their seasonal migration (Malkinson et al. 2002; Dusek et al. 2009). The virus was first isolated from birds in the 1950s in Egypt (Work et al. 1953). It never was realized as the cause of clinical infection in avian hosts until 1997, when domestic geese in Israel showed neurological WNV associated disease and the virus also subsequently affected wild birds (Malkinson et al. 2002;

Banet-Noach et al. 2003). During the impact of WNV in New York and the years thereafter in the U.S. thousands of birds of over 300 species were affected and died (Lanciotti et al. 1999;

Centers for Disease Control and Prevention 2013; U.S. Department of the Interior u. 2014).

Substantial population decline has been proven for seven wild bird species in North America, most clearly in the American crow (Corvus brachyrhynchos) (LaDeau et al. 2007).

Humans and equines can become WNV infected, when bitten by infected mosquitoes (bridge vectors, also mainly Culex species) (Kilpatrick et al. 2005). Equines develop only low viraemia level lasting only few days which does not suffice to infect mosquitoes and thereby to perpetuate the infection cycle (Bunning et al. 2002). Humans develop only low viraemia levels, however infections via blood transfusion, organ transplantation and intra-uterine infections are possible (Iwamoto et al. 2003; Pealer et al. 2003). Humans and horses are therefore considered as dead-end hosts. Besides these species, also numerous other species can become infected, for example sheep, dogs, alpacas and alligators (Austgen et al. 2004;

Dunkel et al. 2004; Kutzler et al. 2004; Jacobson et al. 2005; Kecskemeti et al. 2007).

1.4 Clinical picture in humans and equines

In the majority of infections WNV leads to no or only mild disease. 80% of infected humans are only affected subclinically. The majority of diseased people (~ 20% of infected humans) develop so-called West Nile fever (Mostashari et al. 2001). It is a mild febrile disease which can include fever, headache, myalgia, arthralgia, fatigue, gastrointestinal symptoms (nausea, vomiting, diarrhea), lymphadenopathy or maculopapular rash, (Goldblum et al. 1956;

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8 Introduction

Hubalek 2001; Nash et al. 2001). Less than 1% of infected humans develop so-called WNND (Tsai et al. 1998; Mostashari et al. 2001). This is defined as meningitis, encephalitis or meningoencephalitis and can include headache, mental status changes, loss of consciousness, neck stiffness, generalized weakness to flaccid paralysis, and other symptoms (Chowers et al.

2001; Nash et al. 2001). The risk of developing WNND is higher in elderly people (Tsai et al.

1998; Chowers et al. 2001; Nash et al. 2001). WNND has often been reported in immunocompromised individuals (Chowers et al. 2001). Fatal outcome is also higher in older people (above 50 years, above 70/75 years respectively) (Tsai et al. 1998; Chowers et al.

2001; Nash et al. 2001). Fatality/case rates of recorded outbreaks varied from 4% (Romania 1996) over 14% (Israel 2000) to 17% (Greece 2010) (Tsai et al. 1998; Chowers et al. 2001;

Danis et al. 2011b).

Similarly, only about 8% of infected equines develop clinical disease (Bunning et al. 2002;

Gardner et al. 2007), the rest is infected subclinically. But in contrast to humans the main disease presentation is serious neurologic disease. The major clinical symptom is ataxia, followed by paresis, paralysis, weakness, recumbency and fever. Other reported signs were tremors and muscle fasciculation, hyperesthesia, teeth grinding and abnormal behavior and also blindness was recorded (Murgue et al. 2001b; Ostlund et al. 2001). Case fatality rate is about 22% (U.S.) to 43% (Tuscany) (Murgue et al. 2001b; Ostlund et al. 2001; Autorino et al.

2002; Schuler et al. 2004). Clinical presentation and case fatality rate of WNV lineage 2 WNND in equines are comparable to those caused by lineage 1 infections (Venter et al. 2009;

Kutasi et al. 2011).

1.5 Pathogenesis and clinic in birds

Susceptibility to WNV infection differs between bird species, with passerine species like jays, crows and sparrows being most reservoir competent and developing high WNV viraemia and also high mortality rates (Komar et al. 2003). Other species like chicken are used as sentinel species for WNV antibodies, as they develop only mild viraemia and no clinical symptoms but seroconvert reliably (Langevin et al. 2001). Natural WNV infected birds exhibit neurological symptoms such as ataxia, tremors, seizures or abnormal head posture as well as signs of general malaise such as recumbency, depression and weakness as well as a rapid loss of body condition (Steele et al. 2000; Erdelyi et al. 2007). Amongst others, common gross

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Introduction 9

pathologic changes found are calvarial and meningeal hemorrhages, myocarditis and splenomegaly, and mottled kidneys. Histological alterations and WNV antigen can be found in a wide variety of tissues and cell types, amongst others especially in brain, heart, spleen, kidney, pancreas and adrenal glands. The combination of meningoencephalitis and necrotizing myocarditis was found to be a characteristic sign of WNV infection (Steele et al.

2000; Ellis et al. 2007; Erdelyi et al. 2007; Richter et al. 2009; Wodak et al. 2011).

For raptor species numerous reports of natural WNV lineage 1 infection exist, for example in the California condor (Gymnogyps californianus), eagle species, falcon species, such as American kestrel (Falco sparverius), gyrfalcon (Falco rusticolus), merlin (Falco columbarius), peregrine falcon (Falco peregrinus) and prairie falcon (Falco mexicanus), many hawk species, osprey (Pandion haliaetus) and owl species in the U.S. and eagle species in the E.U. (Steele et al. 2000; Nusbaum et al. 2003; D'Agostino u. Isaza 2004; Wunschmann et al. 2004, 2005; N. Nemeth et al. 2006; Chang et al. 2007; Ellis et al. 2007; Harris u.

Sleeman 2007; Hofle et al. 2008; Jimenez-Clavero et al. 2008; Ip et al. 2014; Wunschmann et al. 2014). For example in the year 2001 8% of WNV affected birds in Alabama belonged to raptor species (Nusbaum et al. 2003). On the European continent WNV lineage 2 affected year after 2004 to a large extent raptor species such as goshawk (Accipiter gentilis), gyrfalcon (Falco rusticolus) and peregrine falcon (Falco peregrinus) (Bakonyi et al. 2006; Erdelyi et al.

2007; Richter et al. 2009; Wodak et al. 2011; Bakonyi et al. 2013) causing neurological infestations. Reservoir competence for American kestrels was proven after experimental infection with development of virus viraemia and shedding, although no clinical signs and no death occurred (Komar et al. 2003; N. Nemeth et al. 2006). A recent study showed that experimentally infected gyr-saker hybrid falcons (Falco rusticolus x Falco cherrug) represent no good reservoir for WNV and show no morbidity or mortality, although they develop viraemia and viral shedding (Busquets et al. 2012).

1.6 Rodent models of WNV infection

WNV infection has been studied intensively in rodent models (reviewed in (Samuel u.

Diamond 2006)). Following WNV infection, WNV replicates in mice in keratinocytes and in dendritic cells of the skin, the latter migrating to local draining lymph nodes thereafter (Johnston et al. 2000; P. Y. Lim et al. 2011). Afterwards infected mice develop peripheral

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10 Introduction

viraemia with replication in peripheral organs, sometimes followed by neuroinvasion leading to meningoencephalitis (Weiner et al. 1970). Discussed routes of neuroinvasion include infection of endothelial cells of the blood-brain-barrier, entry via WNV infected immune cells (“Trojan horse mechanism”), disruption of the blood-brain-barrier due to cytokines and matrix metalloproteinases, entry via the olfactory nerve and retrograde axonal transport (Monath et al. 1983; Samuel et al. 2007; Getts et al. 2008; Verma et al. 2009; Bai et al. 2010;

Verma et al. 2010).

Early experimental infection studies of wild type mice with WNV revealed that virulence differs with age of mice and infection route used (Weiner et al. 1970). Neuroinvasiveness of WNV in the mouse model depends on the viral genotype, whereas high virulent isolates can be found amongst both lineages 1 and 2 (Beasley et al. 2002; Beasley et al. 2005; Venter et al.

2005). Infection with WNV is detected by pathogen recognition receptors (Daffis et al. 2008;

Fredericksen et al. 2008; Town et al. 2009; Errett et al. 2013) which induces type I interferon production (Fredericksen et al. 2004; Samuel u. Diamond 2005). These bind to type I interferon receptor complexes, which causes a signal cascade of phosphorylation events of the interferon receptors, Janus kinase molecules and signal transducer and activator of transcription molecules (JAK/STAT pathway) (reviewed in (Stark u. Darnell 2012; Schneider et al. 2014)). Finally numerous interferon-stimulated genes (ISG) are expressed, creating an antiviral state (Samuel et al. 2006; Schoggins et al. ; Szretter et al. 2011; Szretter et al. 2012;

Cho et al. 2013). Using interferon type I receptor knockout mice (IFNAR -/-) it was shown that interferon type I restricts tropism and viral burden after WNV infection (Samuel u.

Diamond 2005). Additionally this approach revealed that controlling interferon response of the host is a key feature of a high virulent WNV isolate belonging to lineage 1, while a non- virulent lineage 2 isolate lacked this function (Keller et al. 2006).

1.7 Vaccines and efficacy studies

Although some vaccines for use against WNV in equines are currently licensed in Europe and the U.S. (Table 1-2), there is no licensed vaccine available for use in birds.

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Introduction 11

Trade name Antigen Adjuvant Boost Reference

Eqip® WNV, Pfizer (E.U.), WEST NILE-

INNOVATOR®, Pfizer (U.S.);

formerly Duvaxyn®

WNV Fort Dodge

WNV lineage 1 (VM-2; New York 1999), formalin inactivated

MetaStim® (SP oil, squalane, pluronic L121, polysorbate 80)

After 3–5 weeks, then annually

(Ng et al.

2003; Seino et al. 2007;

EMEA 2008;

Bowen et al.

2014) Proteq West Nile™,

Merial (E.U.), RECOMBITEK® – Equine rWNV Vaccine (U.S.)

WNV prM and E proteins of New York 1999 isolate in the recombinant live canarypox virus ALVAC

Carbomer

(polyacrylic acid) After 4–6 weeks, then annually

(Minke et al.

2004; Siger et al. 2004;

Siger et al.

2006; Seino et al. 2007; El Garch et al.

2008; Minke et al. 2011) Vetera® WNV

vaccine, Boehringer Ingelheim (U.S.)

WNV lineage 1 2005 isolate, Inactivated

Carbimmune® After 3–4 weeks, then annually Equilis West Nile,

Intervet, Equi- Nile™, MERCK (U.S.)

Inactivated flavivirus chimera with WNV prM/E proteins in YF17D backbone

Equilis West Nile:

Iscom-Matrix (saponin, cholesterol,

phosphatidylcholine) Equi-Nile™:

Havlogen®

(polyacrylic acid based)

After 3–5 weeks, then annually

(EMEA 2013)

PreveNile™, Intervet

(U.S., recalled 2010)

Live flavivirus chimera with WNV prM/E proteins in YF17D backbone

- Annually (Seino et al.

2007)

Table 1-2: Licensed veterinary WNV vaccines in the E.U. and U.S. All vaccines are approved for horses and are given at 1 ml doses intramuscular. Table based on (Angenvoort et al. 2013)

Different human vaccines (deoxyribonucleic acid (DNA) based vaccines and recombinant live vaccines) are currently under investigation (phase I and II clinical trials) (Martin et al. 2007;

Biedenbender et al. 2011; Ledgerwood et al. 2011; Dayan et al. 2012; Durbin et al. 2013).

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12 Introduction

For birds numerous vaccination approaches used in experimental studies have been published including subunit vaccines, inactivated vaccines and DNA vaccines (Turell et al. 2003;

Bunning et al. 2007; Chang et al. 2007; Okeson et al. 2007; Samina et al. 2007; M. R. Davis et al. 2008; Jarvi et al. 2008; Fassbinder-Orth et al. 2009; Kilpatrick et al. 2010; Redig et al.

2011; Wheeler et al. 2011). The equine inactivated lineage 1 WNV vaccine Duvaxyn^® was evaluated in raptor species, including peregrine falcons, American kestrels, hawks, and others, showing a moderate seroconversion rate of approximately 60% at a low titer (after triple immunization) and a good acceptance (Johnson 2005). In red-tailed hawks this vaccine failed to produce antibodies 3 weeks after the first booster vaccination (Nusbaum et al. 2003).

Efficacy after viral challenge was not evaluated in these experiments. Duvaxyn^® was also evaluated in other than raptor species (Okeson et al. 2007; Olsen et al. 2009). The recombinant live canarypox virus vector based vaccine Recombitek®, which is replication incompetent in mammals, but leads to WNV prM and E protein gene expression by the host cells, was previously tested in Western scrub-jays, and showed partial protection against WNV challenge, but also notable side effects at the vaccination sites (Wheeler et al. 2011).

An experimental DNA vaccine (pCBWN, developed at the CDC, U.S.) was shown to induce only low neutralizing titers in single birds of vaccinated red-tailed hawks (Buteo jamaicensis).

However, viraemia and shedding were significantly reduced in the non-lethal challenge model (Redig et al. 2011). In contrast, this DNA vaccine led to seroconversion of all vaccinated California condors (Gymnogyps californianus) and protection of condors during natural 2004 WNV transmission season was attributed to vaccination of the birds (Chang et al. 2007).

1.8 Goals of the studies

Large falcon species seem to be susceptible to WNV infection although detailed data on pathogenicity in these species were lacking. We wanted to evaluate susceptibility, viraemia levels, course of infection, clinical syndromes, organ viral loads and pathology of WNV infected large falcons (Falco rusticolus, Falco cherrug, Falco peregrinus and hybrids of these species). Therefore we chose the WNV lineage 1 NY99 isolate, which was associated with high wild bird mortality, and the WNV lineage 2 isolate goshawk Austria 2009, which was introduced into Europe in 2004 and thereafter was associated with numerous avian deaths amongst raptor species (Lanciotti et al. 1999; Bakonyi et al. 2013). After confirmation of high

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Introduction 13

susceptibility of large falcons to both WNV strains and after establishment of the WNV live viral challenge model, we evaluated the safety, immunogenicity and efficacy of two commercial equine WNV vaccines (inactivated vaccine Duvaxyn^® WNV and recombinant canarypoxvirus based live vaccine Recombitek^®- Equine rWNV vaccine) and two plasmid DNA vaccines at research level (against lineage 1 and 2 respectively; DNA-1, DNA-2) using different vaccination protocols in the mentioned large falcon species. Furthermore the WNV lineage 2 isolate goshawk Austria 2009 was compared to known high virulent lineage 1 isolates (NY99 and Dakar) and low virulent lineage 2 isolate Uganda in a newly established rodent model using immunocompetent wild type mice (C57/Bl6) and interferon type I receptor knock out mice (IFNAR -/-).

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14 Manuscript I

2 Manuscript I

Pathogenesis of West Nile virus lineage 1 and 2 in experimentally infected large falcons

Ute Zieglerâ, Joke Angenvoortâ, Dominik Fischer^b, Christine Fastâ, Martin Eidenâ, Ariel V.

Rodriguez^a, Sandra Revilla-Fernández^c, Norbert Nowotny^d,e, Jorge García de la Fuente^f, Michael Lierz^b, Martin H. Groschup^a,*

aFriedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Novel and Emerging Infectious Diseases, Südufer 10, 17493 Greifswald-Insel Riems, Germany,

bClinic for Birds, Reptiles, Amphibians and Fish, Justus Liebig University Giessen, Frankfurter Str. 91-93, 35392 Giessen, Germany, ^cInstitute for Veterinary Disease Control Mödling, Austrian Agency for Health and Food Safety (AGES), Robert Koch-Gasse 17, 2340

Mödling, Austria, ^dZoonoses and Emerging Infections Group, Clinical Virology, Department of Pathobiology, University of Veterinary Medicine, Vienna, Veterinärplatz 1, 1210 Vienna,

Austria, ^eDepartment of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman, ^fRoc Falcon S.L., Finca Caballera Alta,

Odèn, Lleida, Spain, ^*corresponding author

West Nile virus (WNV) is a zoonotic flavivirus that is transmitted by blood-suckling mosquitoes with birds serving as the primary vertebrate reservoir hosts (enzootic cycle).

Some bird species like ravens, raptors and jays are highly susceptible and develop deadly encephalitis while others are infected subclinically only. Birds of prey are highly susceptible and show substantial mortality rates following infection. To investigate the WNV pathogenesis in falcons we inoculated twelve large falcons, 6 birds per group, subcutaneously with viruses belonging to two different lineages (lineage 1 strain NY 99 and lineage 2 strain

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Manuscript I 15

Austria). Three different infection doses were utilized: low (approx. 500 TCID50), intermediate (approx. 4 log10 TCID50) and high (approx. 6 log10 TCID50). Clinical signs were monitored during the course of the experiments lasting 14 and 21 days. All falcons developed viraemia for two weeks and shed virus for almost the same period of time. Using quantitative real-time RT-PCR WNV was detected in blood, in cloacal and oropharyngeal swabs and following euthanasia and necropsy of the animals in a variety of neuronal and extraneuronal organs. Antibodies to WNV were first time detected by ELISA and neutralization assay after 6 days post infection (dpi). Pathological findings consistently included splenomegaly, non-suppurative myocarditis, meningoencephalitis and vasculitis. By immunohistochemistry WNV-antigens were demonstrated intralesionally. These results impressively illustrate the devastating and possibly deadly effects of WNV infection in falcons, independent of the genetic lineage and dose of the challenge virus used. Due to the relatively high virus load and long duration of viraemia falcons may also be considered competent WNV amplifying hosts, and thus may play a role in the transmission cycle of this zoonotic virus.

Published 2013 in Veterinary Microbiology

Volume 161, Issues 3 -4, pages 263 – 273 doi: 10.1016/j.vetmic.2012.07.041

link: http://www.sciencedirect.com/science/article/pii/S0378113512004439

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16 Manuscript II

3 Manuscript II

Limited efficacy of West Nile virus vaccines in large falcons (Falco spp.)

Joke Angenvoort^1†, Dominik Fischer^2†, Christine Fast¹, Ute Ziegler¹, Martin Eiden¹, Jorge Garcia de la Fuente³, Michael Lierz² and Martin H Groschup^1*

†Equal contributors, ¹Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Novel and Emerging Infectious Diseases, Südufer 10, 17493 Greifswald,

Insel Riems, Germany, ²Clinic for Birds, Reptiles, Amphibians and Fish, Justus Liebig University Giessen, Frankfurter Str. 91-93, 35392 Giessen, Germany, ³Roc Falcon S.L., Finca

Caballera Alta, 25283 Odèn, Lleida, Spain, ^*corresponding author

West Nile virus (WNV) can lead to fatal diseases in raptor species. Unfortunately, there is no vaccine which has been designed specifically for use in breeding stocks of falcons. Therefore the immunogenicity and protective capacity of two commercially available WNV vaccines, both approved for use in horses, were evaluated in large falcons. One vaccine contained adjuvanted inactivated WNV lineage 1 immunogens, while the second represented a canarypox recombinant live virus vector vaccine. The efficacy of different vaccination regimes for these two vaccines was assessed serologically and by challenging the falcons with a WNV strain of homologous lineage 1. Our studies show that the recombinant vaccine conveys a slightly better protection than the inactivated vaccine, but moderate (recombinant vaccine) or weak (inactivated vaccine) side effects were observed at the injection sites. Using the recommended 2-dose regimen, both vaccines elicited only sub-optimal antibody responses and gave only partial protection following WNV challenge. Better results were obtained for both vaccines after a third dose, i.e. alleviation of clinical signs, absence of fatalities and reduction of virus shedding and viraemia. Therefore the consequences of WNV infections in

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Manuscript II 17

falcons can be clearly alleviated by vaccination, especially if the amended triple administration scheme is used, although side effects at the vaccination site must be accepted.

Published 2014 in Veterinary Research Volume 45, Page 41 doi: 10.1186/1297-9716-45-41

link: http://www.veterinaryresearch.org/content/45/1/41

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18 Manuscript III

4 Manuscript III

DNA vaccines encoding the envelope protein of West Nile virus lineages 1 or 2 administered intramuscularly, via electroporation and with recombinant virus

protein induce partial protection in large falcons (Falco spp.)

Dominik Fischer^1*†, Joke Angenvoort^2†, Ute Ziegler², Christine Fast², Kristina Maier¹, Stefan Chabierski³, Martin Eiden², Sebastian Ulbert³, Martin H. Groschup² and Michael Lierz¹

†Equal contributors, ¹Clinic for Birds, Reptiles, Amphibians and Fish, Justus Liebig University Giessen, Frankfurter Str. 91-93, 35392 Giessen, Germany, ²Friedrich-Loeffler-

Institut, Federal Research Institute for Animal Health, Institute of Novel and Emerging Infectious Diseases, Südufer 10, 17493 Greifswald-Insel Riems, Germany, ³Fraunhofer Institute for Cell Therapy and Immunology, Perlickstraße 1, 04103 Leipzig, Germany,

*corresponding author

As West Nile virus (WNV) can cause lethal diseases in raptors, a vaccination prophylaxis of free-living and captive populations is desirable. In the absence of vaccines approved for birds, equine vaccines have been used in falcons, but full protection against WNV infection was not achieved. Therefore, two DNA vaccines encoding the ectodomain of the envelope protein of WNV lineages 1 and 2, respectively, were evaluated in 28 large falcons. Four different vaccination protocols were used, including electroporation and booster-injections of recombinant WNV domain III protein, before challenge with the live WNV lineage 1 strain NY99. Drug safety, plasmid shedding and antibody production were monitored during the vaccination period. Serological, virological, histological, immunohistochemical and molecular biological investigations were performed during the challenge trials. Antibody response following vaccination was low overall and lasted for a maximum of three weeks.

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Manuscript III 19

Plasmid shedding was not detected at any time. Viraemia, mortality and levels, but not duration, of oral virus shedding were reduced in all of the groups during the challenge trial compared to the non-vaccinated control group. Likewise, clinical scoring, levels of cloacal virus shedding and viral load in organs were significantly reduced in three vaccination groups.

Histopathological findings associated with WNV infections (meningo-encephalitis, myocarditis, and arteritis) were present in all groups, but immunohistochemical detection of the viral antigen was reduced. In conclusion, the vaccines can be used safely in falcons to reduce mortality and clinical signs and to lower the risk of virus transmission due to decreased levels of virus shedding and viraemia, but full protection was not achieved in all groups.

Published 2015 in Veterinary Research Volume 46, Page 87 doi: 10.1186/s13567-015-0220-1

link: http://www.veterinaryresearch.org/content/46/1/87

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20 Manuscript IV

5 Manuscript IV

Differences in pathogenicity of West Nile virus strains in experimental infected mice

Joke Angenvoort¹, Ute Ziegler¹, Christine Fast¹, Markus Keller¹, Martin Eiden¹, Martin H.

Groschup¹

1Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Novel and Emerging Infectious Diseases, Südufer 10, 17493 Greifswald-Insel Riems, Germany.

to be submitted

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Manuscript IV 21

5.1 Abstract

West Nile virus disease outbreaks have been observed frequently in Europe and elsewhere for many years. Clinical severity and epidemiological spread of these infections are influenced by various factors, of which the virus strain characteristics may be the most important. To characterize the European WNV lineage 2 isolate Austria 2009, we challenged immunocompetent wild type mice (C57/Bl6) and interferon type I receptor knock-out mice (IFNAR -/-) with this isolate and with two highly virulent strains (WNV lineage 1 New York 1999, WNV lineage 1 Dakar) and one non-virulent strain (WNV lineage 2 Uganda). In contrast to lineage 1 isolates, which were neuroinvasive in wild type mice causing 20% and 30% mortality, respectively, both WNV lineage 2 isolates were non-pathogenic in these mice.

In IFNAR -/- mice, all isolates were highly virulent, with lineage 2 Austria 2009 reaching viral loads in organs as high as lineage 1 isolates, while replication of lineage 2 Uganda was slightly lower. These results indicate that WNV Austria 2009 isolate is not able to overcome the interferon based immune defense in adult wild type mice, but overruns the defense in IFNAR -/- mice. Even the virulence of WNV lineage 2 Uganda strain seems to suffice to conquer these immuno-incompetent mice. However, these data are not exactly in line with the perceived higher virulence of current WNV lineage 2 strains in Europe, suggesting that there are additional interferon-independent virulence factors (i.e. environmental, hosts).

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22 Manuscript IV

5.2 Introduction

West Nile virus, a Flavivirus, is an emerging arbovirus (arthropod-borne virus), which is transmitted between mosquitoes and birds in an enzootic cycle and is distributed worldwide (May et al. 2011). It can be transmitted to dead-end hosts, e. g. humans and horses, potentially causing severe neurological diseases, especially in immuno-compromised individuals (J. S.

Mackenzie et al. 2004). In the past, virulence was assumed to be associated with the genetic lineages of West Nile virus (WNV), since all outbreaks with increased mortality rates were associated with lineage 1 WN viruses. Up to now seven lineages have been proposed (J. S.

Mackenzie u. Williams 2009). In the past, lineage 2 strains were considered as low virulent strains found in Africa and Madagascar (Lanciotti et al. 1999). However, more recent studies have revealed a much higher virulence associated with certain WNV lineage 2 isolates, and neuroinvasive diseases in humans and horses have been caused by WNV lineage 2 isolates in South Africa for example (Botha et al. 2008; Venter et al. 2009). In 2004, lineage 2 isolates first reached Europe and subsequently have led to severe outbreaks with neuroinvasive diseases in humans, other mammals and birds, mainly birds of prey (Papa et al. 2011;

Bakonyi et al. 2013). WNV lineage 2 strains are now considered as endemic in some southern parts of Europe (Papa et al. 2010; Barzon et al. 2011).

In vertebrates alpha/beta interferons (type I interferons) are major innate immune response mechanisms against viral infections (reviewed in (Samuel u. Diamond 2006)). In WNV infection they restrict tissue tropism and organ viral load and enhance survival rates and times (Samuel u. Diamond 2005). Following WNV infections, type I interferon production is induced by pathogen recognition receptors (reviewed in (Schneider et al. 2014; Lazear u.

Diamond 2015)). After binding to the interferon alpha receptor (IFNAR) complexes, type I interferons induce expression of numerous interferon-stimulated genes (ISG) that create an antiviral state (reviewed in (Schneider et al. 2014; Lazear u. Diamond 2015)). This is mediated through the JAK/STAT pathway, in which binding of interferon to the receptor complex induces a signal cascade of phosphorylation events of the receptors, Janus kinase (JAK) molecules and signal transducer and activator of transcription (STAT) molecules, which finally mediate transcription of interferon stimulated genes (reviewed in (Stark u.

Darnell 2012; Schneider et al. 2014)).

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Manuscript IV 23

Wildtype mouse challenge studies revealed virus genotype specific neuro-pathogenicity and indicated the presence of low and high virulent WNV lineage 1 and lineage 2 strains (Beasley et al. 2002; Venter et al. 2005). A Hungarian equine 2010 lineage 2 isolate was neurovirulent in adult wild type mice (Stephanie M. Lim et al. 2013). Highly virulent Texas lineage 1 and non-virulent lineage 2 Madagascar WNV strains were used to challenge wild type mice and interferon alpha/beta receptors KO mice (IFNAR -/-). Interestingly, the Madagascar lineage 2 strain was completely non-pathogenic in the wild type mice, whereas the virulent Texas lineage 1 isolate overcame interferon action by blocking JAK/STAT signaling, leading to a high lethality rate in the immunocompetent animals. In IFNAR -/- mice both strains caused high mortality rates, with virulence of Madagascar almost reaching the level of Texas lineage 1 strain (Keller et al. 2006). These results show that a major feature of a high virulent WNV strain is controlling host immune response.

In the study presented here we characterized two WNV lineage 1 (New York`99 (NY) and Dakar (DA)) and two lineage 2 strains (Uganda (UG) and goshawk Austria 2009 (GO)) using interferon type I (alpha/beta) receptor knock-out mice (IFNAR -/-) and wild type mice (WT, C57/Bl6). The aim of the study was to characterize representative high and low virulent lineage 1 and 2 isolates comprehensively with special regard to the newly emerged GO isolate.

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24 Manuscript IV

5.3 Materials and Methods

5.3.1 Animals, approval, virus strains and infection experiments

For infection studies, adult (age 10-13 weeks at day of infection) WT mice (C57Bl/6, n = 44) and IFNAR -/- mice (lacking interferon alpha/beta receptor, n = 48), bred and kept under specific pathogen free conditions, were used. The studies were approved by the competent authority of the Federal State of Mecklenburg-Western Pomerania, Germany, based on EU council directive 2010/63/EU for the protection of animals used for experiments (LALLF M- V/TSD/7221.3-1.1-038/10).

For infection each 100 Tissue Culture Infection Dose 50 (TCID50) of the respective WNV strain was diluted in 100 µl minimal essential medium (MEM) supplemented with 2% v/v fetal calf serum (FCS). Four different WNV strains were used, lineage 1 WNV New York `99 (GenBank accession no. AF196835), lineage 1 WNV Dakar (no GenBank no. available, purchased from the National Collection of Pathogenic Viruses, UK¸ the identity of the virus strains was confirmed by sequencing the viral envelope protein encoding sequence), lineage 2 WNV goshawk Austria 2009 (GenBank accession no. HM015884, kindly provided by N.

Nowotny) and lineage 2 WNV Uganda (GenBank accession no. M12294). With each strain ten WT and ten IFNAR -/- mice were infected subcutaneously in the knee fold under isoflurane anesthesia. In addition four WT and eight IFNAR -/- mice were injected with MEM and 2% FCS under the same conditions as negative controls. Mice were then housed in an ISOcage^TM (TECNIPLAST Deutschland GmbH, Hohenpeißenberg, Germany) system with negative air pressure and were checked daily for clinical symptoms. Moribund animals (those considered not to survive the next 24 hours) were decapitated under isoflurane anesthesia and subsequently necropsied, as were all remaining animals after three weeks. Brains, spleens, hearts and livers were removed, each with sterile instruments, a pinhead sized part transferred in 500 µl MEM supplemented with antibiotics (penicillin G and streptomycin), weighed and stored at -70°C until virological and molecular biological analysis. Simultaneously, parts were transferred into formaldehyde (3.7 %, neutral buffered) for immunohistochemistry (IHC).

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Manuscript IV 25

5.3.2 Virology

Organ samples were thawed, homogenized (Qiagen Tissue lyser bead mill, QIAGEN GmbH, Hilden, Germany) and centrifuged. Supernatant was subsequently used for RNA and virus isolation. For the latter, serial dilutions of supernatant in MEM supplemented with 2% FCS and antibiotics were performed and subsequently transferred onto Vero cell monolayers in 96 well cell culture plates. After 1 h of incubation at 37°C and 5% CO2, virus dilutions were removed and cells were washed once. After 7 days (d), cells were formalin fixed, stained with crystal violet and Tissue Culture Infectious Doses 50 (TCID50) were determined by reading cytopathic effects and by using the Spearman and Kaerber method for calculation (Mayr et al.

1974).

5.3.3 Molecular biology

Virus dilutions and organ sample supernatants were transferred into AVL buffer and RNA isolation was performed using the Qiagen RNA mini kit (QIAGEN) according to the manufacturer’s instructions. Simultaneously, together with each sample an external calibrator control RNA (IC2 RNA) was isolated for subsequent quantitation of RNA copy numbers (Hoffmann et al. 2006). Cycle threshold values and RNA copy numbers were determined by quantitative real-time RT-PCR (qRT-PCR) as previously described (Eiden et al. 2010). RNA copy numbers per µl RNA were converted into copy numbers per mg organ sample.

5.3.4 Immunohistochemistry

Tissue samples (brain, spleen, heart, liver) were fixed in formalin for at least two weeks.

Subsequently they were dehydrated, embedded in paraffin and 3 µm sections were cut.

Sections were rehydrated, endogenous peroxidase was blocked with 3% hydrogen peroxide in methanol (30 min) and antigen was unmasked by proteinase k treatment (4 µg/ml, 15 min).

Immunohistochemistry was performed using a polyclonal rabbit serum (rabbit vaccinated with inactivated WNV lineage 2 GO) at a dilution of 1:400 in TBS for two hours at room temperature. Goat-anti-rabbit secondary antibody (Biotinylated anti-rabbit with VECTASTAIN^® ABC KIT, Vector Laboratories, Inc., Burlingame, USA) was applied at a dilution of 1:200 (30 minutes) and diaminobenzidine was used as substrate. Depending on the proportion of IHC positive cells in a tissue sample an initial score was given and definite

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26 Manuscript IV

scores for respective organs thereafter defined as comparison to the highest score reached in a given organ (Table 5-1). Heart sections were not evaluated because of nonspecific interstitial staining reactions.

5.3.5 Statistical analysis

All statistical calculations were done using R software (R Development Core Team 2011).

For all tested variables, differences between virus strains were tested independently for IFNAR -/- mice and for C57/Bl6 mice. Each variable was assessed with multiple comparisons of always two virus strain pairs. P-values below alpha levels of 0.05 were considered statistically significant, which were adjusted for multiple tests to 0.0083 with the Bonferroni correction.

Differences in survival times were tested using the log rank test, with animals euthanized during the course of the experiment counted dead for the following day. Differences in viral loads (copies per mg brain, spleen and heart) were tested using Wilcoxon exact test. IHC scores for brain, spleen (without “positive”) and liver (without “inconclusive”) were tested using Wilcoxon exact test.

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Manuscript IV 27

5.4 Results

5.4.1 WT mouse challenge with WNV Mortality, survival times and clinical symptoms

WT mice challenged with lineage 1 NY and DA displayed 20% and 30% mortality, respectively (mean time to death 11.5 and 12.3 ± 1.2 d; no statistically significant differences for survival times). In contrast, all WT mice infected with lineage 2 WNV strains survived the whole three weeks of the experiment (Figure 5-1, supplemental Table 5-1). All diseased animals showed uniform clinical symptoms like ruffled fur, crouched body postures, slow motions, apathy which proceeded to somnolence and death usually within 24 hours after first symptoms were apparent. Sometimes whitish ocular fluids occurred.

Molecular virus detection and virus titration

All deceased NY and DA challenged WT mice showed intermediate viral loads in the central nervous system (CNS, Figure 5-2A, B and C, Table 5-2). Additionally, viral genomes were also detected in two brains of the surviving and clinically healthy NY infected WT mice. The rest of surviving NY and all surviving DA, UG and GO challenged WT mice completely cleared the infection and no genomes were detected by qRT PCR anymore.

In the heart of WNV infected WT mice low viral loads were only detected in deceased animals infected with lineage 1 strains NY and DA. However, one deceased DA infected mouse was completely negative in the heart, as were all lineage 2 infected WT mice (all p- values above 0.008; Figure 5-3 A, B and C).

Spleens of all deceased NY and DA infected WT mice and three of the survivor WT mice (two NY and one DA survivor) contained low viral loads (Figure 5-4A, B and C). However, spleens of WT mice challenged with WNV lineage 2 strains UG and GO were all virus- and viral genome free (all p-values above 0.008).

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28 Manuscript IV

Immunohistochemistry

WNV NY (n = 2/2) and DA (n = 2/3) challenged and deceased WT mice displayed moderate affections in multiple brain regions (cerebrum, endbrain, hippocampus, diencephalon, brainstem, cerebellum), and neurons, glia cells as well as dendritic structures in the neuropil showed positive WNV staining reactions in particular (Figure 5-5A).

The remaining NY and DA infected and all UG and GO infected WT mice displayed no antigen staining in the brain (no statistically significant differences between groups).

No antigen staining was found in spleen and liver of any of the WT mice (Figure 5-5B, C).

However single deceased WT mice infected with NY (n=1) and DA (n=1) revealed up to moderate randomly distributed small midzonal foci of peracute hepatocellular necrosis. Due to unspecific staining reaction clear evaluation of the heart by immunohistochemistry was not possible.

5.4.2 IFNAR -/- mouse challenge with WNV strains Mortality, survival times and clinical symptoms

All IFNAR -/- mice challenged with either WNV strain had mortality rates of 100% with a mean time to death of approximately 4 d (4 d [NY], 3.9 ± 0.3 d [DA], 4.1 ± 0.3 d [UG] and GO 4.8 ± 0.4 d; p-values were >0.008 except for IFNAR -/- mice infected with GO which survived longer). Clinical symptoms of IFNAR -/- mice correspond to those described above for WT mice.

Molecular virus detection and virus titration

All challenged IFNAR -/- mice had high (NY, DA, GO) or intermediate (UG) viral loads in the CNS (p-value for comparison of UG and NY viral loads <0.008).

High viral loads were found in spleens of all WNV challenged IFNAR -/- mice, which were always higher than those in the corresponding mouse brains (all p-values above 0.008).

Correspondingly, high viral loads were detected in the heart of all challenged IFNAR -/- mice (all p-values above 0.008).

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Manuscript IV 29

Liver samples (n=2) from deceased mice infected with both lineage 1 and 2 strains, respectively, revealed clear positive results in PCR (data not shown).

Histopathology and immunohistochemistry

Surprisingly, all IFNAR -/- mice were negative in the CNS by IHC. However, in spleen all WNV challenged IFNAR -/- mice were positive (median score NY 2, DA 2 UG 2.5 and GO 2; all p-values above 0.008) as shown by intracytoplasmatic staining reactions of dispersed mononuclear cells and in the context of randomly distributed necrotic foci.

In livers of IFNAR -/- mice infected with NY (n=2), DA (n=9), UG (n=8) and GO (n=4) up to moderate multifocal mid-zonal foci of hepatocellular necrosis were detectable. In IHC NY, DA and GO infected mice showed a clear antigen staining with a median score of 2. In contrast, the UG strain showed a median IHC liver score of 0 (statistically significant lower scores in liver than other groups with p-values < 0.008), with three clearly positive livers.

Mainly Kupffer cells stained positive for WNV antigen, rarely hepatocytes. Single animals also showed scattered positive reactions in periportal fibrocytes, inflammatory cells and/or Kupffer cells, which were not regarded for a liver IHC score > 0.

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30 Manuscript IV

5.5 Discussion and conclusions

Our findings demonstrate that the WNV lineage 2 isolates UG and GO are non-virulent in immuno-competent adult WT mice, while lineage 1 isolates NY and DA can overrun the immune system and induce a neuroinvasive disease. Interestingly, an only low viral genome load with absence of antigen detection was observed in the peripheral organs of these animals, suggesting partial virus clearance at these sites. It seems that CNS invasion led to death in these lineage 1 challenged WT mice. Additionally, our data also indicate a subclinical neuroinvasion in a few WNV lineage 1 NY challenged mice that survived the three week incubation period; viral genome was exclusively detectable in brain.

In comparison to WT mice, WNV lineage 1 isolates NY and DA were highly virulent in IFNAR -/- mice with 100% lethality, suggesting an enhanced virulence in mice lacking the interferon receptor. Interestingly, in contrast to WT mice, the viral loads in peripheral organs exceeded the viral loads in brains. Additionally, lineage 2 GO Austria isolate shows high virulence in the IFNAR-/- mice in contrast to nonvirulent phenotype in WT mice, reaching high viral loads in brain and peripheral organs comparable to the results of lineage 1 strains in IFNAR -/- mice. Curiously, survival time of GO infected IFNAR -/- mice was longer than the survival times of mice infected with the other three isolates. WNV lineage 2 UG isolate is also restored in virulence in IFNAR-/- mice, but does not reach the levels of the other three isolates completely (statistically confirmed for lower viral load in brain in comparison to NY and lower amounts of antigen in liver tissue). These results indicate that both WNV lineage 2 isolates are not capable to overwhelm the immune system of WT mice and produce clinical disease in contrast to lineage 1 isolates. However GO lineage 2 isolate seems to inhere virulence factors that allow replication similarly to lineage 1 viruses, if interferon defense is lacking.

Rapid death of IFNAR -/- mice with high viral loads after infection with WNV lineage 1 strains is in concordance with the findings of other groups which concluded that interferon type I restricts tropism and viral burden of WNV lineage 1 New York (Samuel u. Diamond 2005). In accordance to our results shown here, IFNAR -/- mice infected with a nonpathogenic lineage 2 Madagascar strain resulted in unmasked virulent phenotype, whereas in wildtype mice this isolate was nonvirulent (Keller et al. 2006).