University of Veterinary Medicine Hannover
Department of Pathology Center for Systems Neuroscience Hannover
INTRASPINAL INFECTION WITH THEILER’S MURINE ENCEPHALOMYELITIS VIRUS IN RESISTANT AND SUSCEPTIBLE MOUSE STRAINS
JIN, WEN
Hannover 2020
University of Veterinary Medicine Hannover Department of Pathology
Center for Systems Neuroscience Hannover
Intraspinal Infection with Theiler’s Murine Encephalomyelitis Virus in Resistant and Susceptible Mouse Strains
THESIS
Submitted in partial fulfilment of the requirements for the degree
DOCTOR OF PHILOSOPHY (PhD)
awarded by the University of Veterinary Medicine Hannover
by Wen Jin
Heilongjiang, P.R.China
Hannover, Germany 2020
ii
Supervisor: Prof. Dr. Wolfgang Baumgärtner, PhD
Supervision Group: Prof. Dr. Wolfgang Baumgärtner, PhD Prof. Dr. Andrea Tipold
Prof. Dr. Kirsten Haastert-Talini
1st Evaluation: Prof. Dr. Wolfgang Baumgärtner, PhD Department of Pathology,
University of Veterinary Medicine Hannover, Germany
Prof. Dr. Andrea Tipold Small Animal Clinic,
University of Veterinary Medicine Hannover, Germany
Prof. Dr. Kirsten Haastert-Talini Department of Neuroanatomy, Hannover Medical School, Germany
2nd Evaluation: Prof. Dr. Christiane Herden Justus-Liebig-Universität Gießen Fachbereich Veterinärmedizin Institut für Veterinär-Pathologie Frankfurter Str. 96
35392 Gießen Date of final exam: 09.10.2020
This thesis was in part supported by the Niedersachsen Research Network on Neuroinfectiology (N-RENNT) of the Ministry of Science and Culture of Lower Saxony, Publications were further supported by German Research Foundation and University of Veterinary Medicine Hannover, Foundation within the funding program Open Access Publishing. Wen Jin received a scholarship from the China Scholarship Council (CSC), File No.201606170127.
iii To my family
iv
“
We are all in the gutter, but some of us are looking at the stars.
”Oscar Wilde (1854 – 1900),
Lady Windermere's Fan (1893)
v
Parts of this thesis have been previously presented or published:
Oral presentations:
E. Leitzen, B. Raddatz, W. Jin, R. Ulrich, S. Goebbels, K.A. Nave, W. Baumgärtner, F. Hansmann (2017):
Characterization of inflammatory changes, viral spread and demyelination after intraspinal inoculation of Theiler’s murine encephalomyelitis virus in a resistant mouse strain
“3rd Joint European Congress of the ESVP, ESTP and ECVP”, Lyon, France, 30.08. - 02.09.2017.
W. Jin, E. Leitzen, S. Göbbels, K.A. Nave, W. Baumgärtner, F. Hansmann (2018):
Theiler´s murine encephalomyelitis virus induced lesions following intraspinal infection of susceptible mice
“11th Graduate School Day, HGNI”, Hannover, Germany, 30.11. - 01.12.2018
W. Jin, E. Leitzen, S. Göbbels, K.A. Nave, W. Baumgärtner, F. Hansmann (2020):
It’s all about location - Experimental infection with Theiler’s murine encephalomyelitis virus
“63. Jahrestagung der Fachgruppe Pathologie der Deutschen Veterinärmedizinischen Gesellschaft (DVG)”, Fulda, Germany, 07. - 08.03.2020.
Poster presentations:
W. Jin, E. Leitzen, S. Göbbels, K.A. Nave, W. Baumgärtner, F. Hansmann (2017):
Intraspinal infection of susceptible and resistant mouse strains with Theiler’s murine encephalomyelitis virus as a new model to study remyelination
“10th Graduate School Day, HGNI”, Bad Salzdetfurth, Germany, 01. - 02.12.2017.
W. Jin, E. Leitzen, S. Göbbels, K.A. Nave, W. Baumgärtner, F. Hansmann (2018):
Theiler´s murine encephalomyelitis virus induced lesions following intraspinal infection
“Fourth N-RENNT Symposium on Neuroinfectiology”, Hannover, Germany, 12. - 13.02.2018.
vi
E. Leitzen, B. Raddatz, W. Jin, R. Ulrich, S. Goebbels, K.A. Nave, W. Baumgärtner, F. Hansmann (2018):
Characterization of a new intraspinal infection model mimicking features of Guillain- Barré syndrome
“Fourth N-RENNT Symposium on Neuroinfectiology”, Hannover, Germany, 12. - 13.02.2018.
E. Leitzen, B. Raddatz, W. Jin, S. Goebbels, K.A. Nave, W. Baumgärtner, F.
Hansmann (2018):
Intraspinal TMEV infection results in transient demyelination of spinal cord and long lasting peripheral nerve damage in a resistant mouse strain
“4th International Workshop of Veterinary Neuroscience 2018”, Bern, Switzerland, 15.
- 17.02.2018.
Publications in peer-reviewed journals:
E. Leitzen, B. Raddatz, W. Jin, S. Göbbels, K.A. Nave, W. Baumgärtner, F.
Hansmann:
Virus-triggered spinal cord demyelination is followed by a peripheral neuropathy resembling features of Guilain-Barré Syndrome
Scientific Reports (2019): 9, 4588; doi.org/10.1038/s41598-019-40964-1
W. Jin, E. Leitzen, S. Göbbels, K.A. Nave, W. Baumgärtner, F. Hansmann:
Comparison of Theiler's murine encephalomyelitis virus induced spinal cord and peripheral nerve lesions following intracerebral and intraspinal Infection
International Journal of Molecular Sciences (2019): 20, 5134;
doi.org/10.3390/ijms20205134
TABLE OF CONTENTS
vii Table of Contents
Chapter 1 Summary ... 1
Chapter 2 Zusammenfassung ... 5
Chapter 3 Introduction ... 9
3.1 Theiler’s murine encephalomyelitis virus (TMEV) ... 9
3.2 TMEV structure and overview upon mouse strains susceptible/resistant to persistent TMEV infection ... 10
3.3 Different routes of experimental TMEV infection in mice ... 12
3.3.1. Intramuscular TMEV infection (gastrocnemius and tongue) ... 14
3.3.2. Intraperitoneal TMEV infection ... 15
3.3.3. Intranasal TMEV infection ... 16
3.3.4. Intranerval TMEV infection (N. hypoglossus; N. ischiadicus) ... 16
3.3.5. Intra-footpad TMEV infection ... 17
3.3.6. Intravenous TMEV infection ... 17
3.3.7. Intracerebral TMEV infection ... 17
3.3.8. Intraspinal TMEV infection ... 18
3.4. Hypothesis and Aims ... 18
Chapter 4 Comparison of Theiler's murine encephalomyelitis virus induced spinal cord and peripheral nerve lesions following intracerebral and intraspinal infection ... 21
Chapter 5 Virus-triggered spinal cord demyelination is followed by a peripheral neuropathy resembling features of Guilain-Barré Syndrome ... 23
Chapter 6 Discussion ... 25
6.1 Intracerebral versus intraspinal TMEV-infection of susceptible mice ... 25
6.2 Intraspinal TMEV-infection of susceptible mice versus resistant mice .... 27
6.3 Conclusion ... 29
Chapter 7 References ... 31
Chapter 8 Acknowledgements ... 37
LIST OF ABBREVIATIONS
ix List of abbreviations
BBB Blood-brain-barrier
BeAn BeAn 8386
BV Blood vessels
CNS Central nervous system
CP Cribriform plate
DA Daniel’s
DPI Days post infection
EC Endothelial cells
EMCV Encephalomyocarditis virus
GBS Guillain-Barré syndrome
GDVII George David VII
GL Glia limitans
GM Gray matter
IRES Internal ribosome entry site
i.c. Intracerebral
i.f. Intra-footpad
i.m. Intramuscular
i.na. Intranasal
i.ne. Intranerval
i.p. Intraperitoneal
i.s. Intraspinal
i.v. Intravenous
M Mucosa
MC Mitral cells
MN Motor neuron
MS Multiple sclerosis
OB Olfactory bulb
OE Olfactory epithelium
ORF Open reading frame
OSN Olfactory sensory neurons
OT Olfactory tract
Pol Polymerase
LIST OF ABBREVIATIONS
x
Pro Protease
PN Peripheral nerve
PNS Peripheral nervous system
SC Spinal cord
SM Skeletal muscle
TMEV Theiler’s murine encephalomyelitis virus TMEV-IDD TMEV-induced demyelinating disease
Tmevd Theiler’s murine encephalomyelitis virus demyelination Tmevp Theiler’s murine encephalomyelitis virus persistence
TO Theiler’s original
VP Viral protein
WM White matter
SUMMARY
1 Chapter 1 Summary
Intraspinal infection with Theiler’s murine encephalomyelitis virus in resistant and susceptible mouse strains
Wen Jin
Theiler’s murine encephalomyelitis virus (TMEV) infection is a murine model for a variety of human diseases including virus-induced myocarditis as well as diseases of the central nervous system (CNS) like multiple sclerosis (MS) or epilepsy. The different clinical and pathohistological outcomes after TMEV infection depend on the selected mouse and virus strain as well as the route of infection. With regard to TMEV as a model for MS, virus strains can be subdivided into two groups. The first group comprises high-neurovirulent strains including GDVII, causing a fatal encephalitis following intracerebral (i.c.) infection, independent of the genetic background of the mouse strain. The second group consists of low-neurovirulent strains including BeAn, causing a comparably mild, transient encephalitis. Moreover, mouse strains can be categorized into susceptible (e.g. SJL), intermediate (e.g. C3H) and resistant strains (e.g. C57BL/6; B6). These categories are based on the degree of susceptibility of the mouse strain developing TMEV-induced demyelinating disease (TMEV-IDD) after i.c.
infection. After i.c. infection of susceptible mice with a low-neurovirulent TMEV strain, animals develop an initial polioencephalitis followed by a demyelinating leukomyelitis with virus persistence in the spinal cord (SC), approximately 4-6 weeks after infection.
The demyelinating SC lesions show similarities with lesions of MS patients during the progressive forms of the disease.
In this thesis, a murine model for inducing demyelinating SC lesions via direct intraspinal (i.s.) TMEV infection was established and used in susceptible as well as resistant mice to answer the following questions: Do susceptible mice develop demyelinating SC lesions following i.s. TMEV-infection with similar characteristics as observed following i.c. infection and do i.s. infected mice show peripheral nerve (PN) lesions? The aims of this study were to directly compare the clinical outcome and pathohistological findings with focus on a.) the temporal development of clinical signs as well as, b.) inflammatory and degenerative changes within SC and c.) virus spread and establishment of virus persistence as well as the emergence of PN lesions after i.c. and i.s. TMEV infection of SJL mice.
SUMMARY
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The second question was whether TMEV infection of resistant B6 would result in comparable clinical signs and pathohistological lesions as seen in susceptible mice.
To answer this question resistant B6 mice were i.s. infected using a low-neurovirulent (BeAn) strain of TMEV. Investigations focused on a.) the progression of clinical signs and a deterioration of motor coordination, b.) the spatial and temporal distribution of virus protein, and c.) the emergence of inflammatory and degenerative SC and PN lesions, comparable to those seen in SJL mice.
I.s. TMEV infection of susceptible SJL mice resulted in clinical signs starting at 11 days and a deterioration of motor coordination after 14 days post infection (dpi), which was approximately 12 weeks earlier compared to i.c. infection (98 dpi). SC lesions including inflammation, demyelination and axonal damage occurred approximately 6 weeks earlier than following i.c. infection (14 dpi vs. 56 dpi). After i.s. infection, increasing numbers of TMEV-positive cells were detected in the SC at all investigated time points, indicating a virus persistence. Interestingly, TMEV was also detected within PN (starting at 7 dpi). Moreover, the presence of virus protein within PN, a phenomenon only scarcely observed after i.c. infection, was accompanied by the emergence of PN lesions.
I.s. TMEV infection of resistant B6 mice induced an early onset of clinical signs and a deterioration of motor coordination (11 dpi and 7 dpi), as seen following i.s. TMEV infection of SJL mice. Corresponding inflammatory (starting at 3 dpi), demyelinating (14 dpi) and degenerative (14 dpi) lesions within the SC white matter (WM) matched those seen in SJL mice. Low numbers of TMEV-positive cells were detected in the SC at all investigated time points. Furthermore, TMEV was occasionally detected in PN of B6 mice (7 dpi and 14 dpi). Significant PN lesions, characterized by vacuolation, inflammation, axonal damage and demyelination were detected starting at 14 dpi.
In summary, i.s. TMEV-infection of both, susceptible SJL and resistant B6 mice resulted in clinical signs and SC lesions including inflammation, demyelination and axonal damage resembling TMEV-IDD, but occurring within a comparably short time span following infection compared with i.c. infection. Moreover, the emergence of a peripheral neuropathy, characterized by inflammation, demyelination, and axonal damage was observed. These results indicate that i.s. TMEV infection offers the advantage of inducing TMEV-IDD in susceptible mice after a markedly shortened time span. This enables the investigation of virus-induced SC lesions in a time- and cost-
SUMMARY
3
efficient manner. Moreover, the i.s. infection model allows the investigation of TMEV- induced demyelinating SC lesions in resistant B6 mice. Furthermore, the emergence of PN lesions in both strains offers the opportunity to use i.s. TMEV infection as a model for human PN lesions, comparable to those seen in several subtypes of Guillain- Barré syndrome. In conclusion, i.s. TMEV infection of susceptible and resistant mice constitutes a promising model for studying demyelinating disease in both, CNS and peripheral nervous system.
ZUSAMMENFASSUNG
5 Chapter 2 Zusammenfassung
Intraspinale Infektion mit Theiler‘s murinem Enzephalomyelitis-Virus bei resistenten und empfänglichen Mausstämmen
Wen Jin
Eine Infektion von Mäusen mit Theiler’s murinem Enzephalomyelitisvirus (TMEV) dient als Modell für eine Vielzahl an humanen Erkrankungen wie beispielsweise virusinduzierten Myokarditiden oder Erkrankungen des zentralen Nervensystems (ZNS) wie z.B. Multipler Sklerose (MS) oder Epilepsie. Die unterschiedlichen klinischen und pathohistologischen Veränderungen nach einer TMEV-Infektion hängen sowohl vom verwendeten Virus- als auch vom eingesetzten Mausstamm ab.
Darüber hinaus spielt auch die gewählte Infektionsroute eine wichtige Rolle. In Bezug auf TMEV als Modell für MS können die Virusstämme in zwei Gruppen unterteilt werden. Die erste Gruppe umfasst hoch-neurovirulente Stämme wie GDVII, welche nach intrazerebraler (i.c.) Infektion, unabhängig vom genetischen Hintergrund der infizierten Mäuse, eine in der Regel tödlich verlaufende Enzephalitis verursachen. Die zweite Gruppe umfasst die schwach-neurovirulenten Stämmen z. B. BeAn, welche nach i.c. Infektion eine vergleichsweise milde, transiente Enzephalitis verursachen.
Darüber hinaus können die verwendeten Mausstämme in empfängliche (z. B. SJL), intermediäre (z. B. C3H) und resistente Stämme (z. B. C57BL/6; B6) unterteilt werden.
Diese Kategorien basieren auf der Beobachtung, ob die Tiere nach i.c. Infektion mit schwach neurovirulenten Virusstämmen die so genannte TMEV-induzierte demyelinisierende Krankheit (TMEV-induced demyelinating disease; TMEV-IDD) zu entwickeln. Nach i.c. Infektion empfänglicher Mäuse mit einem niedrig- neurovirulentem TMEV Stamm entwickeln die Tiere eine akute Polioenzephalitis auf die nach ca. 4-6 Wochen eine demyelinisierende Leukomyelitis mit Viruspersistenz im Rückenmark (RM) folgt. Die RM-Läsionen weisen morphologische Gemeinsamkeiten mit denen bei MS Patienten während der progressiven Formen auf.
In dieser Arbeit wurde eine intraspinale (i.s.) TMEV-Infektion bei empfänglichen und resistenten Mäusen durchgeführt, um folgende Fragen zu beantworten: Entwickeln empfängliche Mäuse nach i.s. TMEV-Infektion demyelinisierende RM-Läsionen ähnlich denen bei Tieren nach i.c. Infektion und zeigen i.s. infizierte Mäuse Veränderungen in den peripheren Nerven (PN)? Ziel dieser Studie war es, den
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klinischen Verlauf sowie die pathohistologischen Befunde nach i.s. und i.c. Infektion von TMEV empfänglichen Mäusen miteinander zu vergleichen. Der Schwerpunkt lag hier auf a.) dem zeitlichen Auftreten von klinischen Symptomen sowie b.) den entzündlichen und degenerativen Veränderungen des RM und c.) der räumlichen und zeitlichen Verteilung von viruspositiven Zellen sowie der möglichen Ausbildung einer Viruspersistenz in Kombination mit dem Auftreten von PN-Läsionen.
Die zweite Fragestellung war, ob eine i.s. TMEV-Infektion von resistenten B6-Mäusen zu vergleichbaren klinischen Symptomen und pathohistologischen Veränderungen wie bei empfänglichen Mäusen führt. Hierfür wurden B6-Mäuse i.s. mit einem schwach- neurovirulenten TMEV-Stamm (BeAn) infiziert. Im Fokus der Untersuchungen stand a.) der klinische Verlauf sowie der Verlust der Motorkoordination, b.) die räumliche und zeitliche Verteilung von TMEV-positiven Zellen sowie c.) das Auftreten entzündlicher, demyelinisierender und degenerativer RM- und PN-Läsionen im Vergleich zu den bei empfänglichen SJL-Mäusen auftretenden Veränderungen.
Eine i.s. TMEV-Infektion empfänglicher SJL-Mäuse führte ab Tag 11 nach Infektion (days post infection; dpi) zu klinischen Symptomen sowie ab Tag 14 zu einer Verschlechterung der Motorkoordination, was circa 12 Wochen früher war, als nach i.c.
Infektion (98 dpi). RM-Läsionen in Form von Entzündung, Demyelinisierung und axonaler Degeneration traten ungefähr 6 Wochen früher auf als nach i.c. Infektion (14 dpi vs. 56 dpi). Nach i.s. Infektion konnte darüber hinaus eine zunehmende Anzahl an viruspositiven Zellen an allen untersuchten Zeitpunkten im RM nachgewiesen werden, was für das Vorliegen einer Viruspersistenz spricht. Interessanterweise konnte TMEV auch innerhalb der PN nachgewiesen werden (ab 7 dpi), was nach i.c. Infektion einen nur sehr seltenen Befund darstellt. Neben dem Virusnachweis wurde auch eine Schädigung der PN nachgewiesen.
Eine i.s. TMEV-Infektion resistenter B6-Mäuse führte ebenfalls zu einem frühen Auftreten klinischer Symptome und einer Verschlechterung der Motorkoordination (11 dpi und 7 dpi), wie nach i.s. TMEV-Infektion von SJL-Mäusen beobachtet. Die entsprechenden entzündlichen (ab 3 dpi), demyelinisierenden (14 dpi) und degenerativen (14 dpi) Läsionen innerhalb der weißen Substanz des RM stimmten mit denen überein, die bei SJL-Mäusen nach i.s. Infektion beobachtet wurden. Zu allen untersuchten Zeitpunkten konnte im RM eine geringe Anzahl an viruspositiven Zellen nachgewiesen werden. Darüber hinaus wurde TMEV gelegentlich in den PN von B6-
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Mäusen (7 dpi und 14 dpi) nachgewiesen. Signifikante PN-Läsionen, die durch Vakuolisierung, Entzündung, axonale Schädigung und Demyelinisierung gekennzeichnet waren, lagen ab 14 dpi vor.
Zusammenfassend kann gesagt werden, dass eine i.s. TMEV-Infektion sowohl von empfänglichen SJL- als auch von resistenten B6-Mäusen zu TMEV-IDD ähnlichen 1) klinischen Symptomen und 2) RM-läsionen, einschließlich Entzündung, Demyelinisierung und axonaler Schädigung führt, jedoch innerhalb einer kürzeren Zeitspanne. Darüber hinaus wurde das Entstehen einer 3) peripheren Neuropathie beobachtet, welche durch Entzündung, Demyelinisierung und axonale Schädigung gekennzeichnet war. Dieses Modell ermöglicht somit eine zeit- und kosteneffiziente Untersuchung von TMEV-IDD im RM empfänglicher Mäuse. Darüber hinaus ermöglicht es die Untersuchung von TMEV-induzierten RM-Läsionen in resistenten B6-Mäusen. Da nach i.s. TMEV Infektion sowohl bei empfänglichen als auch bei resistenten Mausstämmen PN-Läsionen entstehen, kann dieses Modell für die Untersuchung von PN-Läsionen beim Menschen, wie sie z.B. bei verschiedenen Subtypen des Guillain-Barré-Syndrom auftreten, verwendet werden. Abschließend kann festgehalten werden, dass die i.s. TMEV-Infektion empfänglicher und resistenter Mäuse ein vielversprechendes Modell für die Untersuchung demyelinisierender Erkrankungen sowohl im zentralen als auch im peripheren Nervensystem darstellt.
INTRODUCTION
9 Chapter 3 Introduction
3.1 Theiler’s murine encephalomyelitis virus (TMEV)
Theiler’s murine encephalomyelitis virus (TMEV) is named after its discoverer – Max Theiler, who characterized the virus in the 1930s (SON et al., 2008; THEILER, 1937).
TMEV is a member of the genus Cardiovirus (LINDNER et al., 2019), which, alongside with Encephalomyocarditis virus (EMCV), belongs to the family of Picornaviridae (LIANG et al., 2008; LIPTON et al., 2006; THEILER and GARD, 1940). TMEV has a small host range including mice, rats and certain species of voles with the feral house mouse (Mus musculus) being the most common natural host. On the other hand, although sharing > 50% of amino acid identity with TMEV, EMCV predominantly infects rats. Moreover, EMCV shows a much wider host range compared to TMEV, also including avian and invertebrate species (TESH and WALLACE, 1978) as well as other mammals, like pigs and humans (BREWER et al., 2001; CZECHOWICZ et al., 2011;
OBERSTE et al., 2009). Unlike EMCV, which is considered to have a zoonotic potential, TMEV represents no human pathogen (CAROCCI and BAKKALI-KASSIMI, 2012;
DRESCHER and SOSNOWSKA, 2008; MODICA et al., 2016). TMEV strains can be subclassified into two major subgroups according to their neurovirulent potential. The high-neurovirulent George David VII (GDVII) subgroup comprises GDVII and FA strains while the low-neurovirulent Theiler’s original (TO) subgroup consists of Daniel’s (DA), BeAn 8386 (BeAn), TO, WW, and Yale strains (DANIELS et al., 1952; LIPTON and MELVOLD, 1984; ROZHON et al., 1983; WROBLEWSKA et al., 1977). TMEV is a common enteric pathogen in mice with low morbidity in immunocompetent mice.
Naturally occurring infections are transmitted via the fecal-oral route and usually lead to an asymptomatic infection with virus replication in the gastrointestinal mucosa (GERHAUSER et al., 2019; ROZENGURT and SANCHEZ, 1992). Consequently, TMEV tolerates a relatively wide range of pH (3-9.5) and remains infective even under acidic condition in the stomach (SON et al., 2008). However, naturally occurring spread from the intestine to the central nervous system (CNS), is a rarely observed event, with an incidence of 0.2-1 ‰ in colony-bred mice (LIPTON et al., 2006; MODICA et al., 2016; OLITSKY, 1939; THEILER, 1937). Moreover, the mechanism how TMEV spreads from the intestine to the CNS still remains unclear. A conceivable scenario how the virus enters the CNS is that following an initial phase of replication within the gastrointestinal tract, the virus spreads to local lymphoid tissue. Afterwards, TMEV
INTRODUCTION
10
might enter the blood stream during a viremic phase and the brain via infected monocytes crossing the blood-brain-barrier (BBB) or endothelial cells enabling virus entry (CHRISTOPHI and MASSA, 2009; KOYUNCU et al., 2013; TSUNODA and FUJINAMI, 2010; VILLARREAL et al., 2006). Other factors possibly contributing to TMEV CNS infection include stress (MI et al., 2006). TMEV has been shown to infect different cell types including neurons, astrocytes, oligodendrocytes and microglia/macrophages (ANDERSON et al., 2000; CARPENTIER et al., 2008;
JELACHICH et al., 1999; JIN et al., 2007; LIPTON et al., 1995; LIU et al., 1967;
STROOP et al., 1981). In addition, TMEV is transferred via axonal transport (KREUTZER et al., 2012; MARTINAT et al., 1999) which might enable the virus to enter the CNS following an enteric infection (LIBBEY et al., 2001; VILLARREAL et al., 2006). Experimental intracerebral (i.c.) infection of susceptible mouse strains with low- neurovirulent TMEV strains represents a useful animal model for the investigation of the progressive forms of multiple sclerosis (MS; GERHAUSER et al., 2019; LEITZEN et al., 2019).
3.2 TMEV structure and overview upon mouse strains susceptible/resistant to persistent TMEV infection
TMEV is a non-enveloped virion (~30 nm in diameter) consisting of an icosahedral capsid and a core of ~8100 nucleotides of single-stranded RNA of positive-polarity (OBUCHI and OHARA, 1998; OHARA et al., 1988; PEVEAR et al., 1987). One open reading frame (ORF) codes for a polyprotein (2303 amino acids, 256 kDa) which is cleaved into 12 proteins, including the structural proteins of the virus capsid as well as the Leader protein (L; BRAHIC et al., 2005; LIANG et al., 2008). In addition, an out-of- frame protein, L* (156 amino acids, 18kDa,) represents an additional protein, which plays an important role for persistence of TMEV strains (BRAHIC et al., 2005;
GHADGE et al., 1998; OBUCHI et al., 1999; OKERE and KABA, 2000; SORGELOOS et al., 2013; STAVROU et al., 2010; VAN EYLL and MICHIELS, 2002). The L protein interferes with the host immune system, especially production of type-I interferon (BRAHIC et al., 2005; RICOUR et al., 2009; SCHOGGINS and RICE, 2011; STAVROU et al., 2010). L* facilitates the infection of macrophages and also shows anti-apoptotic effect in this cell type, which - at the end - facilitates virus persistence (BRAHIC et al.,
INTRODUCTION
11
2005; SORGELOOS et al., 2013). The structure of TMEV and the cell tropisms of high- and low-neurovirulent subgroups are visualized in figure 1.
However, susceptibility/resistance of mice for developing TMEV- induced demyelinating disease (TMEV-IDD) is not simply determined by virus genetics but also depends on mouse genetics, sex and age (TSUNODA and FUJINAMI, 1996). Although all mouse strains develop an acute encephalitis following i.c. TMEV infection, only few mouse strains show virus persistence in the CNS associated with inflammation, demyelination and axonal damage (BRAHIC et al., 2005). Multiple host genes are involved in TMEV persistence by modulating the immune response including the H-2D gene (AZOULAY-CAYLA et al., 2001; OLESZAK et al., 2004; RODRIGUEZ et al., 1986). Regarding the degree of susceptibility to developing TMEV-IDD, mouse strains can be divided into 3 major categories: highly (SJL/J, FVB/N, SWR, RIII, PL/J, DBA/2 and NZW) and intermediately susceptible strains (AKR, C3H, CBA and C57BR ) as well as resistant strains including C57BL/6 (B6), C57BL/10, C57/L, 129/J and BALB/C (DAL CANTO et al., 1995; JIN et al., 2015; LIPTON and DAL CANTO, 1979a). Among
Figure 1. Structure of TMEV and genetically controlled susceptibility of mice (modified according to LIPTON et al., 2008).
Single proteins, derived from the large polyprotein, are involved in different tasks. Capsid proteins (VP1 – VP4) form the virus capsid, while proteins 2B, C and 3A – D are involved in genome replication. L inhibits interferon-I production while L* is encoded by an alternative open reading frame and allows infection of macrophages. Pro: protease; Pol: polymerase; Tmevd: Theiler’s murine encephalomyelitis virus demyelination; Tmevp: Theiler’s murine encephalomyelitis virus persistence; IRES: internal ribosome entry site.
INTRODUCTION
12
the mouse strains, SJL mice represent the most susceptible and therefore most frequently used strain for TMEV infection. SJL mice reliably and reproducibly develop TMEV-IDD after i.c. infection with low-neurovirulent TMEV strains (ALLNOCH et al., 2019; HANSMANN et al., 2019; BEGOLKA et al., 2001; LIPTON and DAL CANTO, 1979a; ULRICH et al., 2008). TMEV-IDD is a well characterized animal model for studying demyelinating diseases like the progressive forms of MS (OLESZAK et al., 2004). B6 mice, which do not develop TMEV-IDD after i.c. infection, can be used for studying mechanisms of viral clearance. Moreover, B6 mice i.c. infected with low- neurovirulent TMEV strains develop acute as well as chronic seizures enabling investigators to study the pathogenesis of virus-induced epileptogenesis (ANJUM et al., 2018; BANKSTAHL et al., 2012; BRÖER et al., 2016; KÄUFER et al., 2018;
LIBBEY and FUJINAMI, 2011; VEZZANI et al., 2016; WALTL et al., 2018).
3.3 Different routes of experimental TMEV infection in mice
In addition to host genetics, age and sex, the chosen route of infection has a profound impact on the development and progression of the disease. Over the last decades, a variety of different routes of TMEV-infection, mostly triggering different disease patterns, were established. An overview about the different application sites and possible CNS infection routes, as well as the arising experimental models characterized are summarized in table 1 and figure 2.
INTRODUCTION
13 Table 1. Routes of TMEV infection in different mouse strains triggering different disease patterns RouteMouse strainsVirus strainsCardinal symptoms/diseaseBrain lesionSC lesionHistopathology in CNS/PNSReferences i.m.g, i.m.l, i.v., i.p. CBAGDVIIParalysisn.d.√ Myelitis/meningomyelitis and GM neuronal degenerationVILLARREAL et al. (2006) i.m.gSWGDVII/FA Paralysis, intense acute myositis×√ Significant myelitis in lumbar SCRUSTIGIANand PAPPENHEIMER (1949) i.m.gSWFV/4727 (TO)Paralysis, none/mild myositis×√ Brain stemlesion: inflammation and ganglia degeneration
RUSTIGIANand PAPPENHEIMER (1949) i.p.CD1,SJL, BALB/cDAMyositis, myelitisx√ Progressive inflammatory in WMGOMEZ et al. (1996) i.p.SWR/J, CH3DAMyositis, myocarditis, myelitis, x√ Progressive inflammatory in WMGOMEZ et al. (1996) i.p.B6J, DBA/1J, ABY/SnJDAMyositis, myocarditisxxAbsence of significant changeGOMEZ et al. (1996) i.ne.h, i.ne.iCBA, SJLGDVIIparalysisn.d.√ TMEV transmitted to SCVILLARREAL et al. (2006) MARTINAT et al. (1999) i.ne.iFVDDAn.d.n.d.n.d.Demyelination and inflammation in PN
DRESCHERandTRACY (2007a) i.f. CBAGDVIIparalysisn.d.√ MeningomyelitisandGM neuronal degenerationVILLARREAL et al. (2006) i.c., i.na. CBA, SWGDVII/FAAcute encephalitis√ √ Inflammationand GM neuronal degeneration
VILLARREAL et al. (2006) THEILER and GARD (1940) i.c. B6GVDII/DAAcute/chronic seizure√ xHippocampal lesions: neuron loss and apoptosis, atrophyBRÖER et al. (2016) i.c. SJL,CBA, CH3/HeDA/BeAnBiphasic: flaccid paralysisin chronic late phase√ √ Inflammation, demyelination and gliosis in WM of SC
LIPTONandDALCANTO (1979a) LIPTON and MELVOLD (1984) i.c. AKR, DBA, BALB/cDALackofchroniclate phasen.d.√ Absence of significant changeLIPTONandDALCANTO (1979a) LIPTON and MELVOLD (1984) i.m.g: intramuscular (M. gastrocnemius); i.m.l: intramuscular (M. lingualis proprius); i.v.: intravenous; i.p.: intraperitoneal; i.na.: intranasal; i.ne.h: intranerval (N. hypoglossus); i.ne.i: intranerval (N. ischiadicus); i.f.: intra-footpad; i.c.: intracerebral; GDVII: George David VII; TO: Theiler’s original; DA: Daniel’s; SC: spinal cord; CNS: central nervous system; PNS: peripheral nervous system; WM: white matter; GM: gray matter; n.d.: not declared
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3.3.1. Intramuscular TMEV infection (gastrocnemius and tongue)
Intramuscular TMEV infection has been shown to cause a local inflammation after injection in the gastrocnemius muscle of mice (RUSTIGIAN and PAPPENHEIMER, 1949). Infected animals show an acute myositis of variable degree, depending on the virus strain used and the amount of virus injected (RUSTIGIAN and PAPPENHEIMER, 1949). Lesions include a necrosis of muscle fibres associated with paralysis, progressing from unilateral to bilateral weakness of the hindlimbs and spreading to the
Figure 2. Experimental TMEV-injection site and possible routes of TMEV access to the CNS (modified according to LUDLOW et al., 2016).
i. Virus dissemination after intranasal instillation in mucosa (M) with consecutive infection of olfactory sensory neurons (OSN) within the olfactory epithelium (OE), followed by anterograde axonal transport passing cribriform plate (CP) to the olfactory bulb (OB) and with possible spread to the CNS – along the fibres of mitral cells (MC) composing the olfactory tract (OT); ii. Virus dissemination after infection of peripheral organs (skeletal muscle (SM) and footpad), with consecutive virus spread via motoric fibres to the anterior horn of spinal cord (SC), or after primary infection of peripheral nerves (sciatic nerve; hypoglossal nerve), with final spread to the CNS. MN: motor neuron; iii. Virus dissemination following intraperitoneal infection and consecutive entrance of the blood stream or direct intravenous (caudal vein) infection. During a viremic phase, virus might enter the CNS using infected monocytes crossing the blood-brain-barrier (BBB) including glia limitans (GL), or might use endothelial cells (EC) of blood vessels (BV) enabling virus entry.
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forelimbs, indicating a spread via the inferior spinal cord (SC; RUSTIGIAN and PAPPENHEIMER, 1949; VILLARREAL et al., 2006). In part, the emergence of SC lesions could be detected (RUSTIGIAN and PAPPENHEIMER, 1949). A direct injection of GDVII strains into the lingual muscle of CBA mice resulted in paralysis of the tongue (VILLARREAL et al., 2006).
3.3.2. Intraperitoneal TMEV infection
Intraperitoneal (i.p.) infection with the DA strain of TMEV of different mouse strains including ABY/SnJ, BALB/c, C3H, B6, CD-1, DAB/1J, SJL/J, SWR/J resulted in a myositis of variable degree depending on the age and genetic background of the mice (GOMEZ et al., 1996; SATO et al., 2014). Interestingly, some mouse strains (e.g.
ABY/Sn, C3H, B6, DAB/1, SWR) also developed a myocarditis with necrosis of muscle fibres. No evidence of brain pathology has been shown, but development of SC lesions (myelitis) instead of cardiac lesions could be detected in CD-1, SJL/J, BALB/c, SWR/J strains (GOMEZ et al., 1996). TMEV-induced myocarditis was established as animal model for virus triggered myocarditis in C3H mice. In this model cardiac pathology can be divided into three consecutive phases. During the acute phase, an initial innate immune response towards viral infection and replication takes place. This is followed by a second phase of viral clearance, epitope spreading and therefore autoimmunity, responsible for myocardial damage. The third and chronic phase comprises cardiac remodelling and fibrosis with subsequent dilated cardiomyopathy (GERHAUSER et al., 2019; SATO et al., 2014; TSUNODA et al., 2016). B6 and DBA/1 mice only show mild myocarditis without cardiac fibrosis (phase III) (SATO et al., 2014). An i.p. injection of the high-neurovirulent GDVII strain of CBA mice resulted in a paralysis associated with meningomyelitis and neuronal degeneration in gray matter (GM). The route of virus entry to the CNS was hypothesized to occur via the abdominal sympathetic trunk, bloodstream or invasion via the peripheral nervous system (PNS; GERHAUSER et al., 2019; SATO et al., 2014; TSUNODA et al., 2016; VILLARREAL et al., 2006). Virus infection represents the most common cause of myocarditis in humans and is thought to be a major cause of sudden death, also in young adults (FABRE and SHEPPARD, 2006; LIU and MASON, 2001; TSUNODA et al., 2016). The aforementioned, EMCV, closely related to TMEV, is also known to induce cardiac inflammation and necrosis in
INTRODUCTION
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pigs (CAROCCI and BAKKALI-KASSIMI, 2012; GELMETTI et al., 2006; PSYCHAS et al., 2001).
3.3.3. Intranasal TMEV infection
Several neurotropic viruses, like rabies or influenza cause inflammation in the CNS of mice following intranasal infection (BELSER et al., 2015; KELLY and STRICK, 2000;
VAN RIEL et al., 2015). However, after intranasal instillation of mice with TMEV, a considerably lower number of animals become paralyzed, compared to i.c. infection (THEILER, 1937). It was confirmed that only high doses (100,000x minimal infective dose of i.c. infection) of high-neurovirulent strains (GDVII / FA) cause CNS infection comparable to the one seen after i.c. infection (THEILER and GARD, 1940).
Intranasally infected mice developed a mostly fatal encephalitis with only few surviving animals, surviving mice later showed signs of paralysis (THEILER and GARD, 1940).
Accordingly, intranasal instillation does not represent a suitable infection route for investigating TMEV-IDD since this route only sporadically leads to CNS pathology and paralysis.
3.3.4. Intranerval TMEV infection (N. hypoglossus; N. ischiadicus)
To substantiate the thesis that TMEV travels along motoric nerve fibres, a direct injection of a high-neurovirulent TMEV strain (GDVII) into the N. hypoglossus was performed. Infected mice showed paralysis of the tongue or forelimbs but no combination of both (VILLARREAL et al., 2006). However, viral infections do not only utilize the PNS for trafficking and spread, they are also able to cause direct damage to the nerves (PFISTER, 1999; SUSALKA and PFISTER, 2000). There are various virus- induced demyelinating diseases primarily affecting the PNS, like Marek’s disease in chicken or some subtypes of Guillain-Barré syndrome (GBS) in humans (ARSTILA et al., 1971; DRESCHER and TRACY, 2007b; HUGHES et al., 2016; LAMPERT et al., 1977; LAMPERT, 1978). After direct injection of the low-neurovirulent strain (DA) into the sciatic nerves of mice, TMEV was detected within the nerval tissue and replicating virus could also be harvested. Moreover, an infiltration of macrophages and T cells as well as a demyelination of nerve fibres was found. Virus spread to the SC occurred one week after infection (DRESCHER and TRACY, 2007b).
INTRODUCTION
17 3.3.5. Intra-footpad TMEV infection
Another infection route used for investigating axonal transport and spread of high- neurovirulent TMEV (GDVII strain) is intra-footpad (i.f.) inoculation. Infected mice showed paralysis of the inoculated leg and subsequent involvement of the contralateral side (VILLARREAL et al., 2006). Virus spread was observed from the footpad to the ipsilateral sciatic nerve, the inferior SC, followed by superior SC. This findings indicate that TMEV travels alongside the nerve fibres innervating musculature and skin of the footpad (MARTINAT et al., 1999). Obtained results from i.f. injection coincided with those obtained after intramuscular injection into the gastrocnemius muscle, also entailing paralysis of forelimbs. SC lesions characterized by inflammation (meningomyelitis; perivascular cuffs) as well as neuronal degeneration (necrosis) could be observed. The neuronal loss consistently corresponded to the manifestation of clinical disease in paralyzed animals (VILLARREAL et al., 2006).
3.3.6. Intravenous TMEV infection
Intravenous (i.v.) infection of mice with high-neurovirulent TMEV strains (GDVII) in CBA mice resulted in a high number of mice showing paralysis of hind- and/or forelimbs.
The distribution of paralysis was correlated with the development of SC lesions consisting of inflammation and neuronal degeneration (VILLARREAL et al., 2006). I.v.
injection of the DA strain of TMEV has been shown to stimulate an antiviral immune response in SJL mice, but does not efficiently lead to an infection of the CNS (TSUNODA et al., 2005; TSUNODA et al., 2007).
3.3.7. Intracerebral TMEV infection
I.c. TMEV infection of young adult, susceptible mice with low-neurovirulent TMEV strains leads to the development of TMEV-IDD (GERHAUSER et al., 2019). TMEV strains of the GDVII group cause a fatal encephalitis with lysis of neurons following i.c.
infection regardless of the mouse strain (AUBERT and BRAHIC, 1995; DAL CANTO and LIPTON, 1980; THEILER and GARD, 1940). TMEV strains of the TO group cause a biphasic disease following i.c. infection, consisting of an acute polioencephalomyelitis which is followed by chronic progressive inflammation and demyelination in the SC, associated with virus persistence (DEPAULA-SILVA et al., 2017; GERHAUSER et al., 2019; LIPTON, 1975; LIU et al., 1967; MECHA et al., 2013;
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OLESZAK et al., 2004). Clinical signs during the acute phase are frequently mild and reversible (LIPTON, 1975). The late phase of TMEV-IDD constitutes a well-known animal model for the investigation of the pathogenesis of demyelinating diseases like MS (DAL CANTO et al., 1996; RODRIGUEZ et al., 1987). Mechanisms leading to the development of autoimmune processes against oligodendrocytes in TMEV-IDD include an epitope spread as well as molecular mimicry between viral and myelin epitopes (DONATI, 2020; KATZ-LEVY et al., 2000; MILLER et al., 1997; OLSON and MILLER, 2005). I.c. infection of resistant mouse strain (e.g. B6) results in an acute polioencephalomyelitis including hippocampal lesions with neuronal loss and infiltration of T lymphocytes and macrophages as well as microgliosis (GERHAUSER et al., 2019). However, B6 mice eliminate low-neurovirulent TMEV strains from the CNS approximately until four weeks post infection (LINDSLEY and RODRIGUEZ, 1989;
NJENGA et al., 1997).
3.3.8. Intraspinal TMEV infection
An intraspinal (i.s.) TMEV infection model investigating the early events of virus- induced demyelination in the SC is described using a DA strain of TMEV in SJL mice (DRESCHER and TRACY, 2007a). In this model the authors described the replication of TMEV at the injection site along with a demyelination (DRESCHER and TRACY, 2007a). The data of this study indicate, that the initial phase of polioencephalitis is not mandatory for investigating TMEV-induced demyelinating lesions.
3.4. Hypothesis and Aims
It seems that besides numerous variables (sex, age, virus and mouse strain used), the route of infection has a strong effect on clinical signs, virus spread, neuroinfectivity and organotropism. However, a direct comparison of i.c. and i.s. infection neither in SJL nor other mice has been conducted so far. The hypothesis of this thesis was, that i.s.
TMEV infection represents a suitable model for studying demyelinating SC as well as PN lesions in mice with a susceptible genetic background.
Therefore, the first aim was to directly compare the occurrence and course of SC pathology in susceptible mice after i.c. and i.s. TMEV infection. Special emphasis was given to the time course, inflammatory infiltrates, the emergence of demyelinating
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lesions and establishment of virus persistence. During a second experiment, i.s. TMEV infection of resistant B6 mice was implemented. The aim of this second study was to investigate whether direct injection of TMEV into the SC of resistant mice could induce demyelinating lesions comparable to those seen in SJL mice after i.s. infection, by circumventing the missing establishment of virus persistence after i.c. infection.
MANUSCRIPT 1: Comparison of TMEV-induced SC and PN lesions following i.c. and i.s. infections
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Chapter 4 Comparison of Theiler's murine encephalomyelitis virus induced spinal cord and peripheral nerve lesions following intracerebral and intraspinal infection
Wen Jin, Eva Leitzen, Sandra Goebbels, Klaus-Armin Nave, Wolfgang Baumgärtner, Florian Hansmann
Abstract
Hallmarks of Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD) include spinal cord (SC) inflammation, demyelination and axonal damage occurring approximately 5–8 weeks after classical intracerebral (i.c.) infection.
The aim of this study was to elucidate the consequences of intraspinal (i.s.) TMEV infection and a direct comparison of classical i.c. and intraspinal infection. Swiss Jim Lambert (SJL)-mice were i.s. infected with the BeAn strain of TMEV. Clinical investigations including a scoring system and rotarod analysis were performed on a regular basis. Necropsies were performed at 3, 7, 14, 28 and 63 days post infection (dpi) following i.s. and at 4, 7, 14, 28, 56, 98, 147 and 196 dpi following i.c. infection.
Serial sections of formalin-fixed, paraffin-embedded SC and peripheral nerves (PN) were investigated using hematoxylin and eosin (HE) and immunohistochemistry. I.s.
infected mice developed clinical signs and a deterioration of motor coordination approximately 12 weeks earlier than i.c. infected animals. SC inflammation, demyelination and axonal damage occurred approximately 6 weeks earlier in i.s.
infected animals. Interestingly, i.s. infected mice developed PN lesions, characterized by vacuolation, inflammation, demyelination and axonal damage, which was not seen following i.c. infection. The i.s. infection model offers the advantage of a significantly earlier onset of clinical signs, inflammatory and demyelinating SC lesions and additionally enables the investigation of virus-mediated PN lesions.
Int J Mol Sci. 2019: Oct 16;20(20):5134.
https://www.mdpi.com DOI: 10.3390/ijms20205134.
MANUSCRIPT 2: Virus-triggered spinal cord and peripheral nerve lesions
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Chapter 5 Virus-triggered spinal cord demyelination is followed by a peripheral neuropathy resembling features of Guilain-Barré Syndrome
Eva Leitzen, Barbara Raddatz, Wen Jin, Sandra Goebbels, Klaus-Amin Nave, Wolfgang Baumgärtner, Florian Hansmann
Abstract
Theiler’s murine encephalomyelitis virus (TMEV)-induces a demyelinating disease in the spinal cord (SC) of susceptible but not in resistant (B6) mouse strains. The aim of the present study was to induce SC demyelination and a peripheral neuropathy in resistant mice by switching the infection site from cerebrum to SC. B6 mice were intraspinally inoculated with TMEV. Infected mice showed clinical signs starting at 7 days post infection (dpi). Histopathology revealed a mononuclear myelitis, centred on the injection site at 3 dpi with subsequent antero- and retrograde spread, accompanied by demyelination and axonal damage within the SC. Virus protein was detected in the SC at all time points. SC inflammation decreased until the end of the investigation period (28 dpi). Concurrent with the amelioration of SC inflammation, the emergence of a peripheral neuropathy, characterized by axonal damage, demyelination and macrophage infiltration, contributing to persistent clinical sings, was observed.
Intraspinal TMEV infection of resistant mice induced inflammation, demyelination and delayed viral clearance in the spinal cord and more interestingly, subsequent, virus- triggered inflammation and degeneration within the PN associated with dramatic and progressive clinical signs. The lesions observed in the PN resemble important features of Guillain-Barré syndrome, especially of acute motor/motor-sensory axonal forms.
Sci Rep. 2019 Mar 14;9(1):4588.
www.nature.com
DOI: 10.1038/s41598-019-40964-1.
DISCUSSION
25 Chapter 6 Discussion
6.1 Intracerebral versus intraspinal TMEV-infection of susceptible mice Amongst various routes of experimental TMEV infection, i.c. infection of susceptible mouse strains with TO virus strains represents an excellent animal model for studying virus-induced demyelinating diseases of the CNS (DAL CANTO et al., 1996; LIPTON and DAL CANTO, 1979b; MECHA et al., 2013; TSUNODA and FUJINAMI, 1996;
TSUNODA et al., 2016). The present study was conducted to test the hypothesis whether i.s. infection leads to the establishment of virus persistence and a subsequent demyelination within the SC of susceptible mice, comparable to that seen in TMEV- IDD following i.c. infection. Previous studies showed that the thoracic SC is the most affected SC segment compared to the cervical and lumbar segments following i.c.
infection (LIPTON and DAL CANTO, 1979a). Therefore, thoracic SC was selected for direct i.s. TMEV infection in mice. A direct injection into thoracic SC was thought to result in an earlier onset of lesions due to the circumvention of the initial phase of encephalitis after i.c. injection. Previous studies investigating different TMEV infection localizations including footpad, skeletal muscle and peripheral nerve demonstrated a virus spread, especially via the PNS to the SC (DRESCHER and TRACY, 2007b;
MARTINAT et al., 1999; RUSTIGIAN and PAPPENHEIMER, 1949; VILLARREAL et al., 2006). Therefore, it was hypothesized, that switching the injection site from cerebrum to SC will result in a virus spread from the SC to the PNS.
In the present study i.s. TMEV infection induced similar clinical signs as described for mice in TMEV-IDD, characterized by flaccid limb paralysis and an impairment of motor coordination. SC lesions consisted of mononuclear inflammation and demyelination, consistent with morphological findings during TMEV-IDD following i.c. infection.
However, the time span between TMEV infection and the occurrence of clinical signs as well as histopathological lesions including demyelination was significantly shorter following i.s. infection compared to i.c. infection. In detail, clinical investigation of i.s.
infected mice revealed a significantly elevated clinical score and a deterioration of motor performance starting at 11 dpi and 14 dpi, respectively. Comparable findings in TMEV-IDD following i.c. infection occurred approximately 12 weeks later (starting at 98 dpi). Accordingly, an approximately 6 weeks shorter (14 dpi vs. 56 dpi) time span before onset of SC inflammation, demyelination and axonal damage was observed in i.s. compared to i.c. infected mice. This significantly shorter time span was most likely
