characterization of schmallenberg virus-induced pathology in
TRANSCRIPT
ISBN 978-3-86345-154-7
Verlag: Deutsche Veterinärmedizinische Gesellschaft Service GmbH35392 Gießen · Friedrichstraße 17 · Tel. 0641 / 24466 · Fax: 0641 / 25375
E-Mail: [email protected] · Internet: www.dvg.de
Vanessa Herder
Hannover 2013
Department of PathologyUniversity of Veterinary Medicine
Characterization of Schmallenbergvirus-induced pathology in abortedand neonatal ruminants
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1. Auflage 2013
© 2013 by Verlag: Deutsche Veterinärmedizinische Gesellschaft Service GmbH, Gießen
Printed in Germany
ISBN 978-3-86345-154-7
Verlag: DVG Service GmbH Friedrichstraße 17
35392 Gießen 0641/24466 [email protected] www.dvg.de
University of Veterinary Medicine Hannover
Characterization of Schmallenberg virus-induced pathology in aborted and neonatal
ruminants
Thesis
Submitted in partial fulfillment of the requirements for the degree
-DOCTOR OF VETERINARY MEDICINE- Doctor medicinae veterinariae
(Dr. med. vet.)
by
Vanessa Herder
Hameln
Hannover 2013
Academic supervision: Prof. Dr. Wolfgang Baumgärtner, PhD/Ohio State Univ.
Department of Pathology,
University of Veterinary Medicine Hannover
1. Referee: Prof. Dr. Wolfgang Baumgärtner, PhD/Ohio State Univ.
Department of Pathology,
University of Veterinary Medicine Hannover
2. Referee: Prof. Dr. Ludwig Haas
Department of Virology
University of Veterinary Medicine Hannover
Day of oral examination: 16th May 2013
Parts of this thesis have been published:
Herder V*, Wohlsein P*, Peters M*, Hansmann F, Baumgärtner W (2012) Salient
lesions in domestic ruminants infected with the emerging so-called Schmallenberg
virus in Germany. Veterinary Pathology 49: 588-591.
*authors contributed equally to this work
Herder V, Hansmann F, Wohlsein P, Peters M, Varela M, Palmarini M, Baumgärtner
W (2013) Immunophenotyping of inflammatory cells associated with Schmallenberg
virus infection of the central nervous system of ruminants. PLoS One 8: e62939.
Hahn K*, Habierski A*, Herder V*, Wohlsein P, Peters M, Hansmann F, Baumgärtner
W (2013) Schmallenberg virus in central nervous system of ruminants. Emerging
Infectious Diseases 19: 154-155.
*authors contributed equally to this work
Varela M, Schnettler E, Caporale M, Murgia C, Barry G, McFarlane M, McGregor E,
Piras IM, Shaw A, Lamm C, Janowicz A, Beer M, Glass M, Herder V, Hahn K,
Baumgärtner W, Kohl A, Palmarini M (2013) Schmallenberg virus pathogenesis,
tropism and interaction with the innate immune system of the host. PLoS Pathogens
9: e1003133.
To my family
Pflicht eines Jeden ist es aber, das Material, welches er auf seinen Wegen findet, bei Zeiten herbeizutragen, damit man nicht falschen Planen folge bei der Aufrichtung, damit man so bald als möglich wisse, wo der ursprüngliche Plan
beizubehalten, wo er zu erweitern, wo er einzuschränken sei. Carl Vogt, 1842
Table of contents
i
Chapter 1 Aims .................................................................................... 1
Chapter 2 Introduction ........................................................................ 3 2.1. History of Schmallenberg virus .................................................................. 3
2.1.1. The Schmallenberg virus ........................................................................................... 5 2.1.2. Spread of Schmallenberg virus .................................................................................. 6 2.1.3. Transmission of Schmallenberg virus ...................................................................... 10 2.1.4. Relevance of Schmallenberg virus infection within the ruminant population ........... 11 2.1.5. Models for Schmallenberg virus ............................................................................... 12 2.1.6. Diagnostic approaches to detect Schmallenberg virus ............................................ 13 2.1.7. Comparison of Schmallenberg virus with other teratogenic viruses ........................ 14
2.2. Malformations of the central nervous system in neonates ................... 18 2.2.1. Hydrocephalus internus (syn. internal hydrocephalus) ............................................ 18 2.2.2. Hydranencephaly ..................................................................................................... 20 2.2.3. Porencephaly ........................................................................................................... 21 2.2.4. Cerebellar hypoplasia .............................................................................................. 22 2.2.5. Micromyelia .............................................................................................................. 24 2.2.6. Multicystic encephalopathy ...................................................................................... 25
Chapter 3 Salient Lesions in Domestic Ruminants Infected With the Emerging So-called Schmallenberg virus in Germany ........................................................................... 27
Chapter 4 Immunophenotyping of inflammatory cells associated with Schmallenberg virus infection of the central nervous system ............................................................... 29
Chapter 5 Schmallenberg Virus in Central Nervous System of Ruminants ........................................................................ 31
Chapter 6 Schmallenberg Virus Pathogenesis, Tropism and Interaction with the Innate Immune System of the Host ........................................................................................... 33
Chapter 7 Discussion ....................................................................... 35
Chapter 8 Conclusions ..................................................................... 41
Chapter 9 Summary .......................................................................... 43
Chapter 10 Zusammenfassung .......................................................... 45
Chapter 11 References ....................................................................... 49
Chapter 12 Acknowledgements ......................................................... 61
List of abbreviations
iii
List of abbreviations
I. Lateral ventricle A Austria AKV Akabane virus Aq Aquaeductus mesencephali B Belgium BLAST Basic Local Alignment Search Tool BTV Bluetongue virus C. Culicoides CAHS Congenital arthrogryposis-hydranencephaly syndrome CD Cluster of differentiation CH Switzerland CNPase 2', 3'-cyclic nucleotide 3'-phosphodiesterase CNS Central nervous system CSF Cerebrospinal fluid CZ Czech Republic DN Denmark DNA Deoxyribonucleic acid ESP Spain EST Estonia F France FLI Friedrich-Löffler-Institut, Federal Research Institute for Animal Health FN Finland GFAP Glial fibrillary acidic protein IL Interleukin IR/N-IR Ireland and North-Ireland IT Italy L Large (segment) LX Luxembourg M Medium (segment) MBP Myelin basic protein mRNA Messenger ribonucleic acid N Norway NL The Netherlands P Pineal gland PCR Polymerase chain reaction PL Poland PLP Proteolipid protein RNA Ribonucleic acid RNS Ribonukleinsäure S Small (segment) SW Sweden SBV Schmallenberg virus SISPA Sequence-independent, single-primer amplification UK United Kingdom
List of tables and figures
iv
List of tables and figures
Table 1: Schmallenberg virus in comparison to other teratogenic viruses .... 15-17
Figure 1: The city of Schmallenberg in Germany ................................................... 3
Figure 2: Spread of Schmallenberg virus in Germany ........................................... 6
Figure 3: Distribution of Schmallenberg virus outbreaks in Europe ....................... 7
Figure 4: Hydrocephalus internus ........................................................................ 19
Figure 5: Hydranencephaly.................................................................................. 20
Figure 6: Porencephaly ....................................................................................... 21
Figure 7: Cerebellar hypoplasia ........................................................................... 22
Figure 8: Micromyelia .......................................................................................... 24
Figure 9: Multicystic encephalopathy ................................................................... 25
Chapter 1 Aims
1
Chapter 1 Aims Schmallenberg virus (SBV) is a new and emerging disease that was identified for the
first time in Germany in November 2011 (HOFFMANN et al. 2012). The disease
characterized by diarrhea, fever, reduced milk yields and weight loss in adult
ruminants was observed firstly in The Netherlands in the summer 2011 and was
therefore called “Holland-Seuche” (FRÜNDT 2012, MUSKENS et al. 2012). The
offspring, like sheep lambs, goat kids and calves showed malformations of the
skeletal and central nervous system (CNS; ANONYMUS 2012e, GARIGLIANY et al.
2012c). Due to the fact, that Schmallenberg virus infection is a new disease in
Germany and other European countries (TARLINTON et al. 2012), the aim of the
present study was to systematically investigate gross and histopathological lesions in
naturally SBV infected aborted and neonatal ruminants. In addition,
immunophenotypical findings with special focus on the CNS were described in detail.
Furthermore, this investigation aimed to shed light on the pathogenesis of this new
disease using naturally infected animals. Moreover, species-specific pathological
similarities and differences between aborted and neonatal ruminants were analyzed.
This study intended to identify typical SBV-induced pathological changes to improve
diagnostic approaches, especially in combination with molecular identification of SBV
RNA. Therefore, macroscopical findings were determined and analyzed by
identification of malformations in diseased neonates. In addition, formalin-fixed tissue
samples, with special emphasis on the CNS of aborted and neonatal sheep lambs,
goat kids and calves originating from North-Rhine Westphalia, were investigated
using hematoxylin-eosin staining to define typical histopathological findings in brain
and spinal cord. Additionally, a detailed immunophenotyping was performed to
identify the type of lesions in the CNS. Therefore, immune cells were characterized
and distinguished by immunohistochemistry using antibodies detecting CD3-positive
T cells, CD79α-positive B cells and CD68-positive phagocytic cells. In order to detect
astrogliosis and myelin damage, astrocytes and oligodendrocytes were identified
using cell type specific markers, like glial fibrillary acidic protein and myelin basic
protein, respectively. Beside this, special stainings were applied to show axonal
damage, hemosiderosis and mineralization in the CNS. In addition, a polyclonal
Chapter 1 Aims
2
antibody detecting the nucleoprotein of SBV was used to identify anatomical regions,
which were primarily infected by the virus. These results were correlated with
malformations and inflammation within the CNS in naturally with SBV-infected
aborted and neonatal ruminants.
Based on the hypothesis, that Schmallenberg virus is genetically related to other
well-known viruses of the Bunyaviridae family, like Akabane virus (AKV), results were
compared to infections caused by other arthropod-borne and/or teratogenic viruses.
These results may have implications for a better understanding of the pathogenesis
of naturally occurring SBV-infection in aborted and neonatal ruminants and give
insights into the pathology of this new disease on the macroscopical and
histopathological level and its relationship to virus distribution.
Chapter 2 Introduction
3
Chapter 2 Introduction 2.1. History of Schmallenberg virus
Since autumn 2011, a new emerging arthropod-borne Orthobunyavirus, the so-called
Schmallenberg virus (SBV), was detected in Europe (HOFFMANN et al. 2012). SBV
was named after the town Schmallenberg in western Germany, because the first
identification of the virus succeeded in samples of cattle housed next to this town
(HOFFMANN et al. 2012). Schmallenberg is a town in North Rhine-Westphalia with
25,000 inhabitants located in the ‘Hochsauerlandkreis’ (Figure 1).
Figure 1: The city Schmallenberg is marked in red in the map of Germany. For orientation other cities are depicted: Hamburg (HH), Bremen (HB), Hannover (H), Berlin (B), Cologne (K), Frankfurt/Main (F), Stuttgart (S) and Munich (M).
The discovery of SBV is exceptional in a way, that the virus was identified by
metagenomic analysis (HOFFMANN et al. 2012). A very good definition of what
Chapter 2 Introduction
4
metagenomics are, is given by CHEN et al. (2005): ‘Metagenomics is the
amplification of modern genomics techniques to the study of communities of
microbial organisms directly in their natural environments, bypassing the need for
isolation and lab cultivation of individual species’. Originally, pooled blood samples of
cows suffering from an undefined disease were used to exclude other infectious
diseases, such as pestiviruses, herpes viruses, bluetongue virus etc., however, all
attempts to identify the etiology failed (HOFFMANN et al. 2012). Thereafter, RNA
and DNA were directly isolated from pooled blood plasma samples. Applying
metagenomic analysis, probes were sequenced randomly for unknown microbes
(EISEN 2007). This random strategy allowed the detection of the novel pathogen.
The obtained genome fragments were compared to sequences in the ‘Basic Local
Alignment Search Tool’ (BLAST) database. Results revealed that the new virus is a
novel orthobunyavirus (HOFFMANN et al. 2012). Since this time point the virus was
named Schmallenberg virus.
Chapter 2 Introduction
5
2.1.1. The Schmallenberg virus
SBV belongs to the family Bunyaviridae and the genus Bunyavirus. It is a negative-
sense single-stranded RNA virus and the genome possesses three segments
(tripartite). The virion consists of a large (L), medium (M) and small (S) RNA genome
segment, which are highly conserved in this genus (SCHMALJOHN et al. 2007). The
L segment codes for the viral RNA-dependent RNA polymerase (L polymerase
protein), the M segment for envelope glycoproteins G1 and G2 as well as the NSm, a
non-structural protein and the S segment for the nucleoprotein N and NSs (ELLIOTT
et al. 2012, SCHMALJOHN et al. 2007). The full genome sequence of SBV is
provided under the GenBank accession number HE649912 and has a length of 6864
base pairs (HOFFMANN et al. 2012). Detailed molecular analyses and the
comparison of SBV genome with similar viruses revealed, that SBV is most likely an
ancestor of the Shamonda virus (GOLLER et al. 2012). Sequence analyses
suggested that SBV belongs to the species Sathuperi virus and does not represent a
reassortant (GOLLER et al. 2012). On the contrary, a comparison of the genomic
RNA of SBV with Sathuperi and Shamonda viruses indicated that all viruses belong
to the genus Orthobunyavirus and that SBV originated from a re-assortment of
Sathuperi and Shamonda viruses (YANASE et al. 2012). Based on a DNase SISPA-
next generation sequencing approach it was possible to show, that the SBV initially
identified by Hoffmann et al. (2012) showed differences in genome segments
compared to virus isolates from Belgium (ROSSEEL et al. 2012). These varieties of
the SBV genome indicate sequence divergence in the disease outbreak (ROSSEEL
et al. 2012).
Chapter 2 Introduction
6
2.1.2. Spread of Schmallenberg virus
SBV was firstly detected in The Netherlands and Germany (HOFFMANN et al. 2012,
MUSKENS et al. 2012, VAN DEN BROM et al. 2012). The spread of SBV occurred
from the west to east, north and south in Germany (Figure 2).
Figure 2: These maps of Germany show animal holdings affected by Schmallenberg virus (SBV) infections. SBV spreads from west to east, north and south. Blue circles depicted cattle farms, red circles show sheep holdings and in green goat farms are shown. Information of the left picture was from January 2012 and the right picture displayed affected herds one year later (January 2013). Reprinted with kind permission of the Friedrich-Löffler-Institute, Federal Research Institute for Animal Health (FLI).
Until March 2013 the following countries reported SBV infections: Germany
(HERDER et al. 2012, HOFFMANN et al. 2012), The Netherlands (MUSKENS et al.
2012), Ireland including Northern Ireland and Wales (ANONYMUS 2012b,
ANONYMUS 2012c, BRADSHAW et al. 2012), Poland (KABA et al. 2013, LARSKA
Chapter 2 Introduction
7
et al. 2013), France (DOMINGUEZ et al. 2012), Belgium (GARIGLIANY et al. 2012c),
United Kingdom, Italy, Spain, Luxembourg, Denmark, Austria, Switzerland (BEER et
al. 2012), Norway, Sweden, Finland, Estonia, Czech Republic (http://www.fli.bund.de;
24.02.2013; Figure 3).
Figure 3: The distribution of Schmallenberg virus in Europe. Germany is highlighted in blue with the city Schmallenberg as a red circle. Other countries which reported Schmallenberg virus infections include Norway (N), Finland (FN), Sweden (SW), Denmark (DN), The Netherlands (NL), Poland (PL), Czech Republic (CZ), Estonia (EST), Ireland and North-Ireland (IR/N-IR), United Kingdom (UK), Belgium (B), Luxembourg (LX), France (F), Switzerland (CH), Austria (A), Italy (IT) and Spain (ESP).
Until now, it is not known, how exactly SBV came to Germany, however parallels
were made to the epidemiology of Bluetongue virus (BTV), which emerged firstly in
Europe in 2006 and is also transmitted via arthropods (MACLACHLAN et al. 2009).
In general, climate change is suggested to be the most important factor for the
occurrence of arthropod-borne virus-infections in Europe (GOULD et al. 2006).
Possible routes of SBV entry to Europe are insects and/or animals in aircrafts or
Chapter 2 Introduction
8
imports of cut flowers from Africa (KUPFERSCHMIDT 2012). In addition, vertical
transmission, over-wintering, persistent infection in the mammalian or arthropod host
as well as the ‘nonviremic transmission between co-feeding arthropods’ represent
important survival strategies of arthropod-borne viruses, like BTV and SBV to allow
the virus to be maintained in new endemic areas (GOULD et al. 2006).
Clinical signs of SBV infection were firstly detected between August and September
2011 in the adult dairy population in The Netherlands (MUSKENS et al. 2012).
Veterinary practitioners reported that affected animals showed decreased milk yield,
diarrhea as well as fever. All attempts to find the etiologic agent failed (MUSKENS et
al. 2012). Clinical signs in adult ruminants were usually limited to a period of three
weeks and affected animals recovered completely (GIBBENS 2012). SBV-induced
malformations termed ‘congenital arthrogryposis and hydranencephaly syndrome’
(CAHS) were observed in offspring in November and December 2011 (VAN DEN
BROM et al. 2012). Occurrence of multiple malformations due to SBV infections were
firstly reported in sheep farms. Interestingly, typical clinical signs similar to those in
adult cattle were not detected in adult sheep (VAN DEN BROM et al. 2012).
Pregnant sheep suffered from dystocia and malformed lambs were usually born at
term (VAN DEN BROM et al. 2012).
Investigations on the seroprevalence of SBV in The Netherlands revealed highest
antibody values in the central-eastern parts of the country (ELBERS et al. 2012).
Applying a virus neutralization test, a seroprevalence of 72.5% in 1123 serum
samples was found during the time period between November 2011 and January
2012 (ELBERS et al. 2012). Due to the fact, that SBV-specific antibodies did not
occur in a specific age class of virus-positive animals, it was assumed that the virus
newly arrived in The Netherlands (ELBERS et al. 2012). Retrospective investigations
in cattle were also performed and serum samples tested for SBV antibodies lacked
positive results between spring 2010 and spring 2011 in Belgium (GARIGLIANY et al.
2012a). Suspicious cases of SBV were observed since July 2011 in Belgium
(MARTINELLE et al. 2012). A serological survey in roe and red deer revealed that all
blood samples (n=299) were negative for SBV antibodies in Belgium in 2010. One
Chapter 2 Introduction
9
year later the seroprevalence was 43.1% (n=225) indicating that the virus spread
rapidly in a rather short time since late summer 2011 (LINDEN et al. 2012).
Interestingly, no malformations of the skeletal and central nervous system in newborn
animals belonging to the sero-converted cervid population were detected in this
Belgian study (LINDEN et al. 2012). Furthermore, it was shown, that alpacas also
possessed antibodies against SBV (ANONYMUS 2012a, JACK et al. 2012).
Interestingly, 10 tested alpacas showed higher antibody titers compared to sheep
and cattle, however no associated clinical signs were reported (JACK et al. 2012). In
Belgium, 98.03% of sheep population was seropositive for SBV between November
2011 and April 2012, indicating, that most sheep herds had contact to the virus
(MEROC et al. 2013a). A similar investigation was performed for cattle in Belgium.
Results revealed a seroprevalence of 99.76% between January and March 2012 in
cattle (MEROC et al. 2013b). These results indicated that sheep and cattle
experienced a similar exposure rate to SBV. Furthermore, it is assumed that animals
showing antibodies against SBV should have a protective immunity against SBV
(MEROC et al. 2013b). In addition, the age of animals is significantly positively
correlated with the presence of SBV antibodies in cattle herds (MEROC et al. 2013b).
One explanation for this unexpected observation could be that SBV already
circulated in the population before summer 2011 (MEROC et al. 2013b).
Interestingly, antibodies against SBV were detected for the first time in July 2012 in
Poland in three different goat herds (KABA et al. 2013). The SBV genome in cattle
was identified for the first time in autumn 2012 in Poland (LARSKA et al. 2013). The
occurrence of SBV in cattle and Culicoides spp. was associated with the import of 2
meat bulls from France in this area (LARSKA et al. 2013).
Chapter 2 Introduction
10
2.1.3. Transmission of Schmallenberg virus
The rapid spread of SBV is supposed to be caused by a wide range of different
midges involved in virus transmission (ELBERS et al. 2013). Members of the genus
Orthobunyavirus comprise viruses infecting humans and animals and therefore, the
potential of SBV to infect humans was investigated. Serum samples of 301 people,
who were related to ruminant farms (e.g. farmers, veterinarians) were tested for the
presence of SBV antibodies. In these farms, neonatal ruminants with typical
malformations were observed (REUSKEN et al. 2012). Until now, there is no
indication that SBV is transmissible to humans (DUCOMBLE et al. 2012, REUSKEN
et al. 2012). None of the investigated participants displayed evidence of SBV
infection neither by serology nor based on clinical data (DUCOMBLE et al. 2012,
REUSKEN et al. 2012).
Midges including Culicoides (C.) scoticus, C. chiopterus and C. obsoletus sensu
stricto were collected in the field and tested positive for SBV in Belgium (ELBERS et
al. 2013). In addition, the species C. dewulfi and C. obsoletus complex were also
identified by PCR to harbor SBV specific nucleotides in Belgium (DE REGGE et al.
2012). SBV RNA was also detected in C. obsoletus in Denmark in 2011
(RASMUSSEN et al. 2012). Interestingly, in one study the heads of the midges were
isolated and tested for SBV. The detection of SBV in the heads of insects indicated
that the virus reached the salivary glands and is not only present in the midges due
to ingestion of blood from viremic ruminants. Thus, replication of SBV in the insects
was assumed (DE REGGE et al. 2012). Vertical transmission of SBV from the
infected dam to offspring resulting in various pathological findings is also described
(GARIGLIANY et al. 2012a, GARIGLIANY et al. 2012c, VAN DEN BROM et al.
2012). One study showed a decreasing prevalence of seropositive calves of infected
cows during gravidity (GARIGLIANY et al. 2012a). The highest rates of seropositive
calves were detected between 7 and 9 weeks of gestation with 28.4% decreasing to
19.2% from week 12 to 16. This indicated a decreased rate of SBV passing the
placentomes from the mother to the fetus (GARIGLIANY et al. 2012a). In addition,
SBV nucleotides were also detected by RT-PCR in the semen of cattle. However, the
finding that one bull had intermittent SBV-specific nucleotides in the semen requires
Chapter 2 Introduction
11
further investigations (personal communication; Institute of Diagnostic Virology of the
Federal Research Institute for Animal Health, Dr. Martin Beer and Dr. Bernd
Hoffmann, FLI, Insel Riems).
2.1.4. Relevance of Schmallenberg virus infection within the ruminant population
In Germany, 1165 (0.69%*) bovine herds were tested positive for SBV showing the
highest prevalence compared to other ruminants. Sheep and goat farms were
positive in 954 and 49 cases, respectively (http://www.fli.bund.de/; 24.2.2013). In
general, it was shown that SBV infection in meat sheep herds caused increased
rates of abortion, malformations, dystocia and lamb mortality (SAEGERMAN et al.
2013). Furthermore, the rate of fertility was reduced (SAEGERMAN et al. 2013).
Abortion, stillbirth and/or malformation due to SBV infection are assumed to be low
and therefore, offspring losses represent between 2 to 5% (ANONYMUS 2013).
Interestingly, herds with a synchronized oestrus displayed up to 50% of the offspring
to be deformed or stillborn (ANONYMUS 2013). Due to the fact that sheep farming
represents a small section in the agricultural industry, the overall economic loss due
to SBV-induced losses is supposed to be limited in Germany (CONRATHS et al.
2013). Applying a questionnaire-based study, veterinarian practitioners treating
cattle, sheep and goats were asked regarding the clinical and economic loss due to
SBV infections in Belgium (MARTINELLE et al. 2012). Practitioners evaluated the
treatment cost due to SBV infections per animal ranging from 40 to 200 Euros
(MARTINELLE et al. 2012). Data describing the impact of SBV infection upon sheep
holdings were evaluated in France (DOMINGUEZ et al. 2012). Results obtained from
363 flocks consisting of 64,548 animals revealed that 85% of sheep lambs were born
healthy.
* Data of absolute numbers of cattle herds in Germany obtained from ‘Tiergesundheitsjahresbericht 2011’ (GALL et al. 2011). For sheep and goat farms only absolute numbers of animals were published, therefore no values for herd numbers were available.
Chapter 2 Introduction
12
Thirteen percent of sheep lambs died until 12 hours postpartum and 10% displayed
malformations (DOMINGUEZ et al. 2012). In general, the impact of SBV upon the
animal population and associated economic losses are still discussed controversially.
It was mentioned that SBV is ‘a non-notifiable low-impact disease for which there are
no appropriate control measures’ necessary (SIMMONS 2012). On the other hand, it
was stated, that ‘SBV poses a serious threat to naïve populations of ruminant
livestock in Europe’ (GOLLER et al. 2012). Despite low numbers of ruminant herds
affected by SBV, the European Food Safety Authority (EFSA) expected that the virus
will spread to the south and east of Europe in 2013 (ANONYMUS 2012d).
2.1.5. Models for Schmallenberg virus
In order to establish a reliable in vivo-model to study SBV pathogenesis, a SBV field
strain passaged in a cattle and a SBV strain, cultured maximum 4 times in baby
hamster kidney (BHK) cells, were used as infectious models (WERNIKE et al. 2012).
The authors summarized, that in vivo-passage of SBV in cattle is superior to in vitro-
cultivation. Despite small animal numbers and a high inter-group variability, they
suggested using ‘cattle-derived infectious serum’ for a valuable SBV in vivo animal
model. A high level of infectivity and better replication rates of SBV in vivo compared
to in vitro-passaged virus was described (WERNIKE et al. 2012). One in vitro-study
showed that SBV is able to produce virus-induced small interference RNAs in cells
derived from Culicoides spp. (SCHNETTLER et al. 2013). This indicated an
exogenous antiviral RNA interference pathway in mosquitos (SCHNETTLER et al.
2013). Genetically modified viruses are often used to investigate the pathogenesis of
new viruses. The destruction of the non-structural NS protein of SBV caused
interferon production in infected cells in vitro (ELLIOTT et al. 2012, MOLLOY 2013).
This indicated a general mechanism of the NS protein of orthobunyaviruses to block
interferon production in the host (ELLIOTT et al. 2012). Furthermore, the deletion of
the non-structural NS protein of SBV resulted in reduced virulence of SBV in mice.
Attenuated virulence occurred due to a lack of blocking interferon synthesis in SBV-
infected cells (MOLLOY 2013, VARELA et al. 2013).
Chapter 2 Introduction
13
2.1.6. Diagnostic approaches to detect Schmallenberg virus
Since its first identification, several new approaches for a direct or indirect detection
of SBV were developed. SBV-induced pathology cannot be differentiated from
infections with AKV (HÖPER et al. 2012). Therefore, identification of the virus on the
molecular level, e.g. with PCR, is crucial for a final etiological diagnosis (HOFFMANN
et al. 2012). Appropriate samples for the detection of viral nucleotides from diseased
animals include external placental fluid, umbilical cord, cerebrum, and spinal cord
(BILK et al. 2012). The CNS is accessible in any animal submitted for necropsy even
in cases lacking placental fluid and umbilical cord remnants (BILK et al. 2012).
Spleen, cartilage, placental fluid from the stomach, and meconium rarely revealed a
positive PCR result in SBV-infected animals and therefore these samples are not
suitable for virus detection (BILK et al. 2012). Besides described PCR methods
(HOFFMANN et al. 2012), also commercially available indirect enzyme linked
immunosorbent assays (ELISA) may be used. They showed highly sensitive and
robust results (BREARD et al. 2013). Testing milk for the presence of SBV antibodies
is also a possible screening method to detect SBV infection in dairy herds. Individual
milk samples from cows were used and also bulk milk samples represented a tool to
detect exposure to the virus (HUMPHRIES 2012). Furthermore, a virus neutralization
test displaying a specificity of >99% and a sensitivity of approximately 100% was
developed (LOEFFEN et al. 2012). This test is useful as screening method and also
for the determination of antibody levels (LOEFFEN et al. 2012). In an
immunohistochemical approach an antibody detecting the Tinaroo virus, a
subspecies of the AKV belonging to the genus Bunyavirus, was used to detect
Schmallenberg virus protein in neurons. The cross reaction of the anti-Tinaroo
antibody was confirmed by a positive PCR result detecting SBV in an infected calf
(PEPERKAMP et al. 2012).
Chapter 2 Introduction
14
2.1.7. Comparison of Schmallenberg virus with other teratogenic viruses
Table 1 lists possible etiological differentials to SBV infection. The described
histopathological lesions are not characteristic for a particular virus and the etiology
has to be determined with other methods, e.g. molecular techniques like PCR.
Further epidemiological information as well as the geographical area (e.g. Asia,
Europe, and USA) of disease occurrence has to be considered. The table
summarizes the findings in naturally diseased animals and results of experimental
infections.
Cha
pter
2 In
trod
uctio
n 15
Tabl
e 1:
O
verv
iew
on
viru
s in
fect
ions
rep
rese
ntin
g po
ssib
le e
tiolo
gic
diffe
rent
ials
to a
Sch
mal
lenb
erg
viru
s in
fect
ion.
Pat
holo
gy
and
clin
ical
sig
ns a
fter
natu
ral t
rans
mis
sion
or
expe
rimen
tal i
nfec
tion,
sus
cept
ible
spe
cies
and
tra
nsm
issi
on m
ode
of
viru
s in
fect
ions
are
sum
mar
ized
for n
eona
tes
and
adul
t ani
mal
s.
Viru
s ta
xono
my
Dis
ease
nam
e R
efer
ence
s G
ross
find
ings
H
isto
logy
Sp
ecie
s Tr
ansm
issi
on
Schm
alle
nber
g vi
rus
Orth
obun
yavi
rus,
B
unya
virid
ae
(DE
RE
GG
E e
t al.
2012
, E
LBE
RS
et a
l. 20
13,
GA
RIG
LIA
NY
et a
l. 20
12b,
20
12c,
HE
RD
ER
et a
l. 20
12, H
OFF
MA
NN
et a
l. 20
12, Y
AN
AS
E e
t al.
2012
)
Neo
nate
s:
Arth
rogr
ypos
is,
verte
bral
m
alfo
rmat
ions
, br
achy
gnat
hia
infe
rior,
hydr
an-
and
pore
ncep
haly
, in
tern
al
hydr
ocep
halu
s,
cere
bella
r hy
popl
asia
, m
icro
mye
lia
Adu
lt co
ws:
Dia
rrhea
, fev
er, r
educ
ed m
ilk y
ield
Neo
nate
s:
Lym
pho-
hist
iocy
tic
men
ingo
ence
phal
omye
litis
, glio
sis,
mus
cula
r hy
popl
asia
A
dult
anim
als:
Not
repo
rted
She
ep,
goat
s,
cattl
e, b
ison
s
Arth
ropo
d bo
rne
viru
s:
mos
quito
s an
d m
idge
s:
e.
g.
C.
obso
letu
s, C
. de
wul
fi,
C.
scot
icus
, C
. ch
iopt
erus
; sem
en
Aka
bane
viru
s
Orth
obun
yavi
rus,
B
unya
virid
ae
Enz
ootic
bov
ine
arth
rogr
ypos
is a
nd
hydr
anen
ceph
aly;
co
ngen
ital a
rthro
gryp
osis
-hy
dran
ence
phal
y sy
ndro
me
(CA
HS
)
(BR
EN
NE
R e
t al.
2004
, C
HA
RLE
S 1
994,
HU
AN
G
et a
l. 20
03, K
AM
ATA
et a
l. 20
09, K
ON
NO
et a
l. 19
82,
KO
NO
et a
l. 20
08,
KU
RO
GI e
t al.
1977
, LE
E
et a
l. 20
02, P
AR
SO
NS
ON
et
al.
1988
, 198
1b, S
T.
GE
OR
GE
et a
l. 20
04)
Neo
nate
s:
Arth
rogr
ypos
is,
verte
bral
m
alfo
rmat
ions
, hy
dran
- an
d po
renc
epha
ly,
brai
n ag
enes
is,
lung
hy
popl
asia
, cys
tic s
eptu
m p
ellu
cidu
m
Adu
lt an
imal
s:
Sub
clin
ical
vi
rem
ia
in
rum
inan
ts;
expe
rimen
tally
infe
cted
goa
ts w
ithou
t clin
ical
sig
ns
Neo
nate
s:
Non
-sup
pura
tive
ence
phal
omye
litis
, pe
rivas
cula
r cu
ffing
, ne
uron
al l
oss
in t
he s
pina
l co
rd,
mus
cula
r at
roph
y an
d de
gene
ratio
n A
dult
cow
s: L
ymph
ohis
tiocy
tic, p
eriv
ascu
lar
ence
phal
omye
litis
, gl
iosi
s,
neur
onop
hagi
a,
neur
onal
los
s; p
ons
and
med
ulla
obl
onga
ta
ofte
n af
fect
ed
Cat
tle,
hors
es,
donk
eys,
sh
eep,
go
ats,
ca
mel
s,
buffa
loes
, pig
s
Arth
ropo
d bo
rne
viru
s:
mos
quito
s an
d m
idge
s,
e.g.
C
. sp
., A
edes
sp.
, C. i
mic
ola
Bov
ine
viru
s di
arrh
ea
viru
s
Pes
tiviru
s,
Flav
iviri
dae
(BIN
KH
OR
ST
et a
l. 19
83,
FLO
RE
S e
t al.
2000
, H
EW
ICK
ER
-TR
AU
TWE
IN
et a
l. 19
95, 1
994,
MA
XIE
et
al. 2
007)
Neo
nate
s:
Cer
ebel
lar
hypo
plas
ia,
mum
ifica
tion,
ru
ntin
g,
mic
ro-,
por-
an
d hy
dran
ence
phal
y,
hydr
ocep
halu
s in
tern
us,
mic
roph
thal
mia
, ca
tara
ct,
brac
hygn
athi
a,
thym
ic
hypo
plas
ia,
hypo
trich
osis
, al
opec
ia,
pulm
onar
y hy
popl
asia
, cy
stic
se
ptum
pe
lluci
dum
A
dult
anim
als:
Fe
ver,
sudd
en
deat
h,
diar
rhea
, pn
eum
onia
, th
rom
bocy
tope
nic
synd
rom
e, e
rosi
on a
nd
ulce
ratio
n in
the
alim
enta
ry tr
act,
coro
nitis
Neo
nate
s:
Per
ivas
cula
r no
n-su
ppur
ativ
e m
enin
geal
in
filtra
tion,
re
duct
ion
of
the
corti
cal c
ell l
ayer
s, d
egen
erat
ive
chan
ges
in
and
hete
roto
pia
of
Pur
kinj
e ce
lls,
hypo
mye
linog
enes
is,
retin
al
dege
nera
tion,
op
tic
neur
itis,
m
yoca
rditi
s,
astro
glio
sis,
le
ukoe
ncep
halo
mal
acia
A
dult
anim
als:
V
ascu
lar
necr
osis
in
m
esen
teric
ar
terio
les,
ly
mph
atic
de
plet
ion,
he
rnia
tion
of P
eyer
’s p
atch
es,
eros
ions
and
ul
cera
tion
in
the
alim
enta
ry
tract
an
d se
cond
ary
bact
eria
l inf
ectio
ns
Cat
tle,
shee
p,
goat
s, p
igs
Exc
retio
ns,
inha
latio
n,
inge
stio
n,
sem
en,
cont
amin
ated
em
bryo
tra
nsfe
r flu
id
Blu
eton
gue
viru
s
Orb
iviru
s,
Reo
virid
ae
(JA
UN
IAU
X e
t al.
2008
, M
AC
LAC
HLA
N e
t al.
2009
, M
AX
IE e
t al.
2007
, W
OU
DA
et a
l. 20
08)
Neo
nate
s: P
or-
and
hydr
anen
ceph
aly,
hyd
roce
phal
us
inte
rnus
, ce
rebe
llar
dysg
enes
is,
runt
ing,
“d
umm
y la
mbs
”, cy
stic
sep
tum
pel
luci
dum
A
dult
anim
als:
H
emor
rhag
e an
d ul
cers
in
th
e al
imen
tary
tra
ct,
cong
este
d or
cy
anot
ic
tong
ue,
skel
etal
an
d ca
rdia
c m
uscl
e ne
cros
is,
coro
nitis
, la
min
itis,
hem
orrh
age
at th
e ba
se o
f pul
mon
ary
arte
ry,
lung
ed
ema,
pe
ricar
dial
, pl
eura
l an
d ab
dom
inal
ef
fusi
on, s
ubcu
tane
ous
and
inte
rmus
cula
r ede
ma
Neo
nate
s: N
ecro
tizin
g m
enin
goen
ceph
aliti
s,
inte
rstit
ial
pneu
mon
ia,
mon
onuc
lear
cel
ls i
n ki
dney
and
live
r A
dult
anim
als:
E
ndot
helia
l da
mag
e,
mic
rova
scul
ar
necr
osis
, ed
ema,
he
mor
rhag
es, e
pith
elia
l nec
rosi
s
She
ep,
wild
ru
min
ants
, ca
mel
ids,
ca
ttle,
E
uras
ian
lynx
Arth
ropo
d bo
rne
viru
s:
C. s
p., C
. im
icol
a
Cha
pter
2 In
trod
uctio
n 16
Viru
s ta
xono
my
Dis
ease
nam
e R
efer
ence
s G
ross
find
ings
H
isto
logy
Sp
ecie
s Tr
ansm
issi
on
Bor
der d
isea
se v
irus
Pes
tiviru
s,
Flav
iviri
dae
Bor
der d
isea
se;
“hai
ry s
hake
r”,
“fuzz
y” la
mbs
(BR
AU
N e
t al.
2002
, M
AX
IE e
t al.
2007
, N
ETT
LETO
N e
t al.
1998
, P
HYS
ICK
-SH
EA
RD
et a
l. 19
80)
Neo
nate
s: E
mbr
yoni
c de
ath,
mum
mifi
catio
n, f
leec
e an
d m
andi
bula
r ab
norm
aliti
es,
runt
ing,
ce
rebe
llar
hypo
- an
d dy
spla
sia,
m
icro
-, po
r- an
d hy
dran
ence
phal
y,
leuk
oenc
epha
lom
alac
ia,
mic
rom
yelia
, st
arva
tion,
ca
rdia
c ab
norm
aliti
es,
arth
rogr
ypos
is,
kyph
osco
liosi
s,
cyst
ic
sept
um
pellu
cidu
m, t
hym
us h
ypop
lasi
a A
dult
anim
als:
Abo
rtion
, stil
lbirt
h
Neo
nate
s:
Dys
- an
d hy
pom
yelin
atio
n,
nodu
lar
peria
rterit
is
ofte
n de
tect
ed
in
the
CN
S
Adu
lt an
imal
s: N
ecro
tizin
g pl
acen
titis
with
ne
utro
phili
c in
filtra
tion
She
ep,
goat
s,
pigs
, cat
tle
Ora
l, co
njuc
tival
, in
trana
sal,
geni
tal,
sem
en
Cac
he v
alle
y vi
rus
Bun
yam
wer
a se
rogr
oup
Orth
obun
yavi
rus,
B
unya
virid
ae
(ED
WA
RD
S e
t al.
1989
, H
OLD
EN
et a
l. 19
59,
MA
XIE
et a
l. 20
07)
Neo
nate
s:
Arth
rogr
ypos
is,
hydr
ocep
halu
s,
hydr
an-,
por-
an
d m
icro
ceph
aly,
ve
rtebr
al
mal
form
atio
ns,
cere
bella
r hyp
opla
sia,
mic
rom
yelia
A
dult
anim
als:
Ofte
n su
bclin
ical
infe
ctio
ns
Neo
nate
s: N
ecro
sis
and
loss
of n
euro
pil a
nd
mot
or n
euro
ns,
myo
sitis
, po
orly
dev
elop
ed
myo
cyte
s A
dult
anim
als:
Not
repo
rted
She
ep,
deer
ca
ribou
, pi
gs,
hors
es,
cattl
e,
racc
oons
, fo
xes,
hum
ans
Arth
ropo
d bo
rne
viru
s:
Cul
icoi
des
sp.,
Cul
iset
a sp
., A
noph
eles
sp.
Chu
zan
viru
s P
alya
m s
erog
roup
Orb
iviru
s,
Reo
virid
ae
(MA
XIE
et a
l. 20
07, M
IUR
A
et a
l. 19
90)
Neo
nate
s:
Hyd
rane
ncep
haly
, ce
rebe
llar
hypo
plas
ia,
hydr
ocep
halu
s, m
icro
ceph
aly
Adu
lt an
imal
s:
Dur
ing
vire
mia
no
cl
inic
al
sign
s,
leuk
open
ia
Neo
nate
s: N
ot re
porte
d A
dult
anim
als:
Not
repo
rted
Cat
tle
Arth
ropo
d bo
rne
viru
s:
Cul
icoi
des
oxys
tom
a
Cla
ssic
al s
win
e fe
ver
viru
s
Pes
tiviru
s,
Flav
iviri
dae
H
og c
hole
ra
(ELB
ER
S e
t al.
2003
, M
AX
IE e
t al.
2007
, S
AN
CH
EZ-
CO
RD
ON
et a
l. 20
03)
Neo
nate
s:
Mum
mifi
catio
n,
runt
ing,
hy
potri
chos
is,
still
birth
, pu
lmon
ary
hypo
plas
ia,
pulm
onar
y ar
tery
m
alfo
rmat
ion,
ar
thro
gryp
osis
, ce
rebe
llar
hypo
plas
ia,
mic
roce
phal
y,
defe
ctiv
e m
yelin
atio
n,
anas
arca
, “c
onge
nita
l tre
mor
” A
dult
anim
als:
Acu
te, s
ubac
ute
and
chro
nic
form
s as
w
ell
as
a la
te
onse
t fo
rm;
hem
orrh
agic
di
athe
sis,
pe
tech
iae,
typi
cal p
inpo
int h
emor
rhag
es in
the
kidn
ey,
sple
nic
infa
rctio
n, “b
utto
n ul
cers
” in
the
gast
roin
test
inal
tra
ct,
pneu
mon
ia,
conj
unct
iviti
s, s
plen
omeg
aly,
sm
all
thym
us, d
iarr
hea
Neo
nate
s:
Nec
rotiz
ing
and
neut
roph
ilic
vasc
uliti
s (p
anar
terit
is)
in
CN
S,
inte
stin
e,
skin
, ly
mph
oid
depl
etio
n,
endo
thel
ial
dege
nera
tion,
val
vula
r fib
rosi
s, p
orta
l fib
rosi
s (li
ver),
in
ters
titia
l pn
eum
onia
, ne
uron
al
dege
nera
tion,
nec
rosi
s of
pan
crea
tic is
land
s A
dult
anim
als:
Dis
sem
inat
ed i
ntra
vasc
ular
co
agul
atio
n,
necr
otiz
ing
and
neut
roph
ilic
vasc
uliti
s (p
anar
terit
is),
lym
phoi
d de
plet
ion,
m
ucos
al
and
epith
elia
l ne
cros
is,
edem
a,
hem
orrh
ages
Pig
s;
cattl
e,
shee
p,
goat
s su
scep
tible
, bu
t no
clin
ical
si
gns
Oro
nasa
lly
by
excr
etio
ns,
inge
stio
n,
arth
ropo
ds
(e.g
. H
aem
atop
inus
sui
s)
Rift
val
ley
feve
r viru
s
Phl
ebov
irus,
B
unya
virid
ae
(ALI
et a
l. 20
12, C
OE
TZE
R
1982
, 197
7, M
AX
IE e
t al.
2007
, RIP
PY
et a
l. 19
92)
Neo
nate
s:
Intra
-ute
rine
feta
l de
ath,
ar
thro
gryp
osis
, hy
dran
ence
phal
y
Adu
lt an
imal
s: O
ften
asym
ptom
atic
; su
dden
dea
th,
hem
orrh
ages
in
va
rious
or
gans
, di
arrh
ea,
nasa
l di
scha
rge
Neo
nate
s:
Lym
phoh
istio
cytic
m
enin
goen
ceph
alom
yelit
is, h
epat
ic n
ecro
sis,
ac
idop
hilic
in
tranu
clea
r, fil
amen
tous
in
clus
ions
, ch
oles
tasi
s,
dege
nera
tion
of
lym
phoc
ytes
, he
art
mus
cle
and
rena
l tu
bule
s, v
esse
l dam
age,
lym
phat
ic d
eple
tion,
he
mor
rhag
es
Adu
lt an
imal
s: H
epat
ic n
ecro
sis,
aci
doph
ilic
intra
nucl
ear,
filam
ento
us i
nclu
sion
s, v
esse
l da
mag
e, ly
mph
atic
dep
letio
n, h
emor
rhag
es
She
ep,
cattl
e,
goat
s, c
amel
s,
hum
ans
Arth
ropo
d bo
rne
viru
s:
Aed
es s
p., C
ulex
sp.
Cha
pter
2 In
trod
uctio
n 17
Viru
s ta
xono
my
Dis
ease
nam
e R
efer
ence
s G
ross
find
ings
H
isto
logy
Sp
ecie
s Tr
ansm
issi
on
Wes
sels
bron
viru
s
Flav
iviru
s,
Flav
iviri
dae
(CO
ETZ
ER
et a
l. 19
77,
1979
, 197
8, M
AX
IE e
t al.
2007
, MU
SH
I et a
l. 19
98)
Neo
nate
s:
Mum
mifi
catio
n,
hydr
anen
ceph
aly,
ar
thro
gryp
osis
, hy
drop
s am
nii,
pore
ncep
haly
, ce
rebe
llar
hypo
plas
ia,
brac
hygn
athi
a,
jaun
dice
, he
mor
rhag
es, l
ymph
aden
opat
hy, h
epat
omeg
aly
Adu
lt an
imal
s: N
o ty
pica
l sig
ns re
porte
d
Neo
nate
s:
Non
-sup
pura
tive
men
ingo
-en
ceph
alom
yelit
is;
eosi
noph
ilic,
int
ranu
clea
r in
clus
ions
in h
epat
ocyt
es; l
iver
: deg
ener
atio
n an
d ne
cros
is,
Kup
ffer
cell
hype
rpla
sia,
he
patit
is,
hype
rpla
sia
and
infla
mm
atio
n of
bi
le d
ucts
A
dult
anim
als:
Not
repo
rted
She
ep,
goat
s,
cattl
e, h
uman
s A
rthro
pod
born
e vi
rus:
A
edes
sp.
Ain
o vi
rus
S
huni
ser
ogro
up
Orth
obun
yavi
rus,
B
unya
virid
ae
Con
geni
tal
Arth
rogr
ypos
is-
Hyd
rane
ncep
haly
S
yndr
ome
(CA
HS
)
(MA
XIE
et a
l. 20
07, N
OD
A
et a
l. 19
98, T
SU
DA
et a
l. 20
04, U
CH
INU
NO
et a
l. 19
98)
Neo
nate
s:
Hyd
rane
ncep
haly
, ar
thro
gryp
osis
, ce
rebe
llar h
ypop
lasi
a A
dult
anim
als:
Nec
rotiz
ing
plac
entit
is w
ith e
dem
a,
kidn
ey w
ith t
ubul
ar n
ecro
sis
and
lym
phop
lasm
acyt
ic
infla
mm
atio
n,
non-
supp
urat
ive
men
ingi
tis,
inte
rstit
ial
pneu
mon
ia
Neo
nate
s:
Nec
rotiz
ing
ence
phal
opat
hy,
non-
supp
urat
ive
ence
phal
omye
litis
, pe
rivas
cula
r cu
ffing
w
ith
lym
phoc
ytes
, ne
uron
al m
iner
aliz
atio
n A
dult
anim
als:
Not
repo
rted
Cat
tle, s
heep
Arth
ropo
d bo
rne
viru
s:
Cul
icoi
des
brev
itars
is,
Cul
ex
trita
enio
rhyn
chus
Chapter 2 Introduction
18
2.2. Malformations of the central nervous system in neonates
Selected complex CNS malformations such as hydrocephalus internus,
hydranencephaly, porencephaly, cerebellar hypoplasia, micromyelia and multicystic
encephalopathy that may be noticed during SBV infection are detailed in the
following pages. Furthermore, definitions of these CNS deformities are provided and
discussed.
2.2.1. Hydrocephalus internus (syn. internal hydrocephalus)
In the normal brain, the lateral ventricles represent thin, slit-like spaces and the
aquaeductus mesencephali possesses a lumen with slight amounts of cerebrospinal
fluid (CSF; Figure 4A). A hydrocephalus internus (Figure 4B) is defined as an
‘abnormal accumulation of CSF in the ventricular system of the cranial cavity’
(MAXIE et al. 2007). This has to be distinguished from an external hydrocephalus
(Figure 4C), in which the CSF is located in the arachnoid space. In cases of
increased amounts of fluid in the ventricular system and the arachnoid space (Figure
4D), the term communicating hydrocephalus is used (MAXIE et al. 2007, ZACHARY
2007). The term non-communicating hydrocephalus indicates, that the CSF is unable
to leave the ventricle system due to an obstruction causing enlargement of ventricles
(ZACHARY 2007). Hydrocephalus can also be defined based on the mechanisms,
which cause extension of the ventricles. In this case, hydrocephalus is ‘an increase in
volume of CSF’ due to compensation or obstruction (SUMMERS et al. 1995). A loss
of brain tissue due to tissue destruction or a developmental impairment represents a
compensatory mechanism. An obstructive hydrocephalus shows accumulation of
CSF due to a lack of drainage of the fluid (SUMMERS et al. 1995).
Chapter 2 Introduction
19
Figure 4: Schematic overview on the different forms of hydrocephalus. Each picture displays a coronal brain section showing the skull (brown line), subarachnoidal space (red line in A and B or red area in C and D), cerebral cortex, hippocampus, glandula pinealis (P), both lateral ventricles (I.), aquaeductus mesencephali (aq) and mesencephalon. A normal brain with small, slit-like lateral ventricles (I.), a small aquaeductus mesencephali (aq) and a thin subarachnoidal space (red line) between skull and cerebral cortex (A). The hydrocephalus internus is characterized by dilation of the lateral ventricles (I.) and of the aquaeductus mesencephali (aq), the subarachnoidal space is unaffected (red line; B). The extended subarachnoidal space (red area between skull and cerebral cortex) represents a typical feature for the external hydrocephalus (C). In the communicating hydrocephalus the subarachnoidal space, lateral ventricles (I.) and/or the aquaeductus mesencephali (aq) are widened at the same time (D).
Chapter 2 Introduction
20
2.2.2. Hydranencephaly
Hydranencephaly can be differentiated from an internal hydrocephalus by a
‘complete or almost complete absence of cerebral hemispheres, leaving only
membraneous sacs filled with CSF and enclosed by meninges’ (MAXIE et al. 2007).
The cerebral cortex is severely damaged and grossly it is impossible to distinguish
between gray and white matter (Figure 5). In some cases, this cerebral sac consists
only of astroglia, pia mater and blood vessels while neural tissue is lacking
(SUMMERS et al. 1995). Furthermore, this malformation displays a ‘full-thickness
necrosis of the hemispheres’ (MAXIE et al. 2007). In addition, ‘remaining neopallium,
which is a thin, nearly transparent membrane that collapses on the underlying brain
tissue when the brain is removed’ represents another definition of hydranencephaly
(SUMMERS et al. 1995). Despite severe lesions in the cortex, hippocampus and
brain stem may remain intact (SUMMERS et al. 1995). For hydranencephaly the term
‘fluid-filled bubble-like hemispheres’ is also used (CRUVEILHIER 1829, HARDING et
al. 1997).
Figure 5: Schematic drawings of coronal CNS sections comparing a normal brain displaying thin lateral ventricles (I.) and normal developed cerebral cortex (A) with hydranencephaly, characterized by severely distended lateral ventricles, only remnants of the cerebral cortex, and a small hippocampus and mesencephalon (B).
Chapter 2 Introduction
21
2.2.3. Porencephaly
Pores (syn. cystic cavities) in the CNS are randomly distributed, often bilaterally
occurring and usually multiple fluid-filled cavities of various sizes (SUMMERS et al.
1995). Cystic cavities in the cerebrum can ‘communicate with the lateral ventricle or
subarachnoidal space’ (SUMMERS et al. 1995). Porencephaly is usually present in
the white matter of both hemispheres, primarily affecting the temporal lobes (Figure
6; MAXIE et al. 2007). According to Heschl (1861; as cited by Harding et al. 1997),
an appropriate definition of porencephaly is a ‘hemispheric defect originating during
fetal life and antedating the acquisition of a mature astroglial response or completion
of convolutional development’ (HARDING et al. 1997, HESCHL 1861).
Figure 6: This picture shows typical features of a porencephaly in a Schmallenberg virus-infected sheep lamb. The temporal lobe displays a cystic cavity located in the white matter (asterisk). This pore does not communicate with the lateral ventricle (I.) or the subarachnoidal space. CNPase-immunohistochemistry, bar, 5 mm.
Chapter 2 Introduction
22
2.2.4. Cerebellar hypoplasia
The occurrence of an abnormal small cerebellum can be due to a) hypoplasia,
b) abiotrophy and c) atrophy. A hypoplastic cerebellum is characterized by a size
reduction in comparison to the cerebrum (Figure 7). Hypoplasia in general describes
an abnormal smallness of an organ in association with reduced cell numbers
(BEINEKE et al. 2011). Cerebellar hypoplasia occurs congenitally and is common in
domestic animals (SUMMERS et al. 1995, ZACHARY 2007). This malformation is
either genetically determined in special breeds, like beef shorthorns, Arabian horses
etc. or occurs due to a diaplacentar infection with various viruses in domestic animals
(MAXIE et al. 2007). The normal cerebellum weight (cerebellum only) ranged
between 10 and 12% of the total brain weight and smaller values indicate
malformations, like cerebellar hypoplasia or abiotrophy (SCHATZBERG et al. 2003).
Among others, the feline panleukopenia virus is able to cause cerebellar hypoplasia
in kittens and an infection with bovine virus diarrhea and Border disease virus causes
this malformation in calves (AEFFNER et al. 2006, HEWICKER-TRAUTWEIN et al.
1995, TOPLU et al. 2011).
Figure 7: Brain of a Schmallenberg virus-infected calf displaying cerebellar hypoplasia.
Chapter 2 Introduction
23
The term abiotrophy of the cerebellum is a ‘premature senescence of formed nervous
tissue as disturbance of a normal development’ (MAXIE et al. 2007) and ‘describes
an intrinsic degeneration of the Purkinje neurons as primary lesion’ (DE LAHUNTA et
al. 2009). In addition, neurons ‘die prematurely as a result of some intrinsic
genetically determined abnormality within the cell’s metabolic system’ (DE LAHUNTA
et al. 2009). Cerebellar abiotrophy occurs in dogs (Australian Kelpies), horses
(Arabian) and rabbits (BRAULT et al. 2011, SATO et al. 2012, SHEARMAN et al.
2011). Atrophies are defined as a reduction of cell size or a reduction of cell number
(numeric atrophy) and can be associated with loss of functionality (BEINEKE et al.
2011). Cerebellar atrophy is characterized by a degeneration of Purkinje and Golgi
cells while the size of the organ is macroscopical normal (MAXIE et al. 2007).
Chapter 2 Introduction
24
2.2.5. Micromyelia
The term micromyelia describes an abnormal small spinal cord with a reduced
diameter (REUTER 2004). The normally developed spinal cord displays a centrally
located, symmetrically gray matter showing a butterfly-morphology, which is
surrounded by the white matter. The malformed spinal cord displaying micromyelia in
naturally SBV-infected animals may lack large areas of the gray matter and has only
few individual neurons, multifocally and randomly distributed within the white matter
(Figure 8).
Figure 8: Schematic drawings of spinal cord cross sections. The normal spinal cord contains the gray matter in the centre (green area), white matter in the periphery (blue area) and the central canal in the middle (white circle; A). In micromyelia, the diameter of the spinal cord may be reduced and the gray matter can be nearly absent. Furthermore, only few individual neurons can be randomly located within the white matter (green neurons; B) of naturally SBV-infected animals.
Chapter 2 Introduction
25
2.2.6. Multicystic encephalopathy
Similar to the occurrence of multiple porencephalies, the ‘multicystic encephalopathy’
in humans is characterized by a ‘sponge-like arrangement of myriad glial-lined cysts
intersected by thin gliovascular strands enmeshing collections of lipid-containing
macrophages in the white and deep cortical layers of both hemispheres’ (HARDING
et al. 1997). In humans, this entity occurs in the third trimester during pregnancy and
is associated with hypoxic-ischemic insults in infants (GARTEN et al. 2007,
HARDING et al. 1997). Due to SBV infection in aborted and neonatal sheep lambs
some of the animals displayed a similar morphology with multicystic lesions in the
CNS (own observations; Figure 9).
Figure 9: SBV infection caused multiple cystic cavities in the CNS of some naturally infected animals. This malformation resembled the human entity ‘multicystic encephalopathy’ which is characterized by cysts with a sponge-like arrangement in macroscopy (A) and histopathology (B). Coronal brain section of the brain of a SBV-infected sheep lamb.
Chapter 3 Salient lesions of Schmallenberg virus infection
27
Chapter 3 Salient Lesions in Domestic Ruminants Infected With the Emerging So-called Schmallenberg virus in Germany
V. Herder, P. Wohlsein, M. Peters, F. Hansmann, W. Baumgärtner
Abstract The so-called Schmallenberg virus (SBV), first detected in a German town of the
same name in October 2011, is a novel emerging orthobunyavirus in Europe causing
malformations and severe economic loss in ruminants. This report describes lesions
in 40 sheep, 2 goats, and 16 cattle naturally infected with SBV as determined by real-
time quantitative reverse transcription polymerase chain reaction. The most common
macroscopic changes were arthrogryposis, vertebral malformations, brachygnathia
inferior, and malformations of the central nervous system, including
hydranencephaly, porencephaly, hydrocephalus, cerebellar hypoplasia, and
micromyelia. Histologic lesions included lymphohistiocytic meningoencephalomyelitis
in some cases, glial nodules mainly in the mesencephalon and hippocampus of
lambs and goats, and neuronal degeneration and necrosis mainly in the brain stem of
calves. Micromyelia was characterized by a loss of gray and white matter, with few
neurons remaining in the ventral horn in calves. The skeletal muscles had myofibrillar
hypoplasia in lambs and calves. The lesions of SBV-associated abortion and
perinatal death are similar to those attributed to Akabane virus and other viruses in
the Simbu group of bunyaviruses.
Veterinary Pathology 2012: 49(4) 588-591
http://vet.sagepub.com/
DOI: 10.1177/0300985812447831
Chapter 4 Immunophenotyping of Schmallenberg virus-induced CNS lesions
29
Chapter 4 Immunophenotyping of inflammatory cells associated with Schmallenberg virus infection of the central nervous system
V. Herder, F. Hansmann, P. Wohlsein, M. Peters, M. Varela, M. Palmarini, W. Baumgärtner Abstract Schmallenberg virus (SBV) is a recently discovered Bunyavirus associated mainly with abortions,
stillbirths and malformations of the skeletal and central nervous system (CNS) in newborn ruminants.
In this study, a detailed immunophenotyping of the inflammatory cells of the CNS of affected animals
was carried out in order to increase our understanding of SBV pathogenesis. A total of 82 SBV-
polymerase chain reaction (PCR) positive neonatal ruminants (46 sheep lambs, 34 calves and 2 goat
kids) were investigated for the presence of inflammation in the brain and spinal cord. The study
focused on 15 out of 82 animals (18.3%) showing inflammation in the CNS. All 15 neonates displayed
lymphohistiocytic meningoencephalomyelitis affecting most frequently the mesencephalon and the
parietal and temporal lobes. The majority of infiltrating cells were CD3-positive T cells, followed by
CD79α-positive B cells and CD68-positive microglia/macrophages. Malformations like pore- and
hydranencephaly, frequently found in the temporal lobe, showed associated demyelination and axonal
loss. SBV antigen was detected in 37 out of 82 (45.1%) neonatal brains by immunohistochemistry. In
particular, SBV antigen was found in 93.3% (14 out of 15 ruminants) and 32.8% (22 out of 67
ruminants) of animals with and without encephalitis, respectively. Highest amounts of virus-protein
expression levels were found in the temporal lobe. Our findings suggest that: (i) different brain regions
display differential susceptibility to SBV infection; (ii) inflammatory cells in the CNS are found only in a
minority of virus infected animals; (iii) malformations occur in association with and without
inflammation in the CNS; and (iv) viral antigen is strongly associated with the presence of
inflammation in naturally infected animals. Further studies are required to explore the cell tropism and
pathogenesis of SBV infection in ruminants.
PLoS One 8, e62939
http://www.plosone.org/
DOI: 10.1371/journal.pone.0062939
Chapter 5 Detection of Schmallenberg virus by in situ-hybridization
31
Chapter 5 Schmallenberg Virus in Central Nervous System of Ruminants
K. Hahn, A. Habierski, V. Herder, P. Wohlsein, M. Peters, F. Hansmann, W. Baumgärtner
Emerging Infectious Diseases 2013 Jan; 19(1):154-155
http://wwwnc.cdc.gov/eid/
DOI: http://dx.doi.org/10.3201/eid1901.120764
Chapter 6 Pathogenesis of Schmallenberg virus
33
Chapter 6 Schmallenberg Virus Pathogenesis, Tropism and Interaction with the Innate Immune System of the Host
M. Varela, E. Schnettler, M. Caporale, C. Murgia, G. Barry, M. McFarlane, E. McGregor, I. M. Piras, A. Shaw, C. Lamm, A. Janowicz, M. Beer, M. Glass, V. Herder, K. Hahn, W. Baumgärtner, A. Kohl, M. Palmarini
Abstract
Schmallenberg virus (SBV) is an emerging orthobunyavirus of ruminants associated with outbreaks of
congenital malformations in aborted and stillborn animals. Since its discovery in November 2011, SBV
has spread very rapidly to many European countries. Here, we developed molecular and serological
tools, and an experimental in vivo model as a platform to study SBV pathogenesis, tropism and virus-
host cell interactions. Using a synthetic biology approach, we developed a reverse genetics system for
the rapid rescue and genetic manipulation of SBV. We showed that SBV has a wide tropism in cell
culture and "synthetic" SBV replicates in vitro as efficiently as wild type virus. We developed an
experimental mouse model to study SBV infection and showed that this virus replicates abundantly in
neurons where it causes cerebral malacia and vacuolation of the cerebral cortex. These virus-induced
acute lesions are useful in understanding the progression from vacuolation to porencephaly and
extensive tissue destruction, often observed in aborted lambs and calves in naturally occurring
Schmallenberg cases. Indeed, we detected high levels of SBV antigens in the neurons of the gray
matter of brain and spinal cord of naturally affected lambs and calves, suggesting that muscular
hypoplasia observed in SBV-infected lambs is mostly secondary to central nervous system damage.
Finally, we investigated the molecular determinants of SBV virulence. Interestingly, we found a
biological SBV clone that after passage in cell culture displays increased virulence in mice. We also
found that a SBV deletion mutant of the non-structural NSs protein (SBVΔNSs) is less virulent in mice
than wild type SBV. Attenuation of SBV virulence depends on the inability of SBVΔNSs to block IFN
synthesis in virus infected cells. In conclusion, this work provides a useful experimental framework to
study the biology and pathogenesis of SBV.
PLOS Pathogens 2013 Jan;9(1):e1003133.
http://www.plospathogens.org/
DOI: 10.1371/journal.ppat.1003133
Chapter 7 Discussion
35
Chapter 7 Discussion SBV was the first simbu serogroup member of orthobunyaviruses detected in
Germany (CONRATHS et al. 2013, GOULD et al. 2006). The virus was identified by
metagenomic analysis in serum samples of adult cows displaying non-specific clinical
signs in North-Rhine Westphalia in 2011 (HOFFMANN et al. 2012). Subsequently
several cattle, sheep and goat farms in Germany, The Netherlands and other
European countries reported SBV-infected animals. Infection with SBV was
associated with multiple malformations in neonates and aborted fetuses comparable
to AKV-induced lesions in offspring.
The aim of the present thesis was to characterize pathological findings of naturally
SBV-infected animals with special emphasis on the CNS. SBV shows a tropism to
the CNS, which was demonstrated by various methods like PCR, in situ-hybridization
and immunohistochemistry (BILK et al. 2012, DE REGGE et al. 2013, HAHN et al.
2013, HERDER et al. 2013). A PCR-based study revealed that SBV-specific RNA
was found in highest amounts in brain stem and to a lesser extent in the cerebrum
and cerebellum (DE REGGE et al. 2013). However, another survey showed that
placental fluid and the umbilical cord represent more sensitive tissues to detect SBV-
specific nucleotides in affected neonates (BILK et al. 2012). These authors also
demonstrated, that cerebrum and spinal cord represent the second best tissues to
confirm the etiologic diagnosis (BILK et al. 2012). In situ-hybridization on formalin-
fixed tissues detected SBV RNA most frequently in the cerebrum, cerebellum, brain
stem, medulla oblongata and spinal cord, while peripheral organs like kidney and
spleen were negative (HAHN et al. 2013). Based on the morphology of positive cells,
neurons seem to be the primary target cell-type in the CNS detected by in situ-
hybridization at least in the late stage of the disease (HAHN et al. 2013). Similarly,
SBV-positive cells displaying axon- and dendrite-like processes in formalin-fixed,
paraffin-embedded CNS tissue have been detected using immunohistochemistry
(HERDER et al. 2013).
A detailed investigation of the topography of SBV positive cells in the CNS revealed
highest numbers of virus positive cells in temporal and parietal lobes as well as in the
Chapter 7 Discussion
36
mesencephalon (HERDER et al. 2013). In the mesencephalon virus protein was
associated with inflammation and in the temporal and parietal lobes a high virus load
was found in combination with malformations like porencephaly. Interestingly, virus
distribution in brains with and without encephalitis was similar, however, animals
displaying inflammation showed SBV-antigen in a higher percentage (93.3%)
compared to animals without inflammation (32.8%; HERDER et al. 2013).
Pathogenetically, demonstration of SBV in the temporal and parietal lobes indicates
tissue destruction by virus-induced cytolysis resulting in malformations like
porencephaly (SCHMALJOHN et al. 2007). Furthermore, virus-induced CNS damage
was associated with hemosiderosis and mineralization indicating liquefactive
necrosis with consecutive hemorrhage in the early phase possibly due to a SBV-
induced vascular disruption (HERDER et al. 2013). A virus-induced vasculitis, as also
described as a typical pathomechanism for Akabane and bluetongue virus-infection
has to be considered also for SBV infections (HERDER et al. 2013, HUANG et al.
2003, VERCAUTEREN et al. 2008). Hemosiderosis is interpreted as residuum of
hemorrhages in areas of necrosis while calcium deposits could represent dystrophic
mineralization due to virus-induced cell injury (HERDER et al. 2013). The possible
presence of SBV-induced CNS hemorrhages and necrosis in the early phase of field
infections were substantiated by results of a SBV animal model (VARELA et al.
2013). Experimentally SBV-infected mice display hemorrhages and necrosis of the
CNS parenchyma in the acute phase after intracerebral infection (VARELA et al.
2013). Until now, it is not known how and when these processes occur in naturally
occurring SBV-infection. However, parallels to AKV-infections exist. Diaplacentar
infections with AKV also caused CNS malformations in neonates characterized by
necrosis, neuronal loss, hemosiderosis and mineralization (KAMATA et al. 2009,
KONNO et al. 1982, ST. GEORGE et al. 2004, SUMMERS et al. 1995). These
findings suggest a similar pathogenesis for orthobunyavirus-induced porencephaly as
described for AKV. It is supposed that pore size increases over time after infection,
occurs bilaterally and can progress to multicystic lesions or hydranencephaly in
severe cases (MAXIE et al. 2007). Occurrence of multicystic lesions in the CNS of
humans is termed ‘multicystic encephalopathy’ and caused by hypoxia (GARTEN et
Chapter 7 Discussion
37
al. 2007, HARDING et al. 1997). Until now, this term is not used for similar lesions in
animals. However, SBV infection in neonates also leads to multicystic lesions in the
CNS and thus, it is suggested to use this term also for animals (HERDER et al.
2013). In addition, it is postulated that cavity size is positively correlated with an early
time point of infection during pregnancy (MAXIE et al. 2007).
Depending on the time point of infection during pregnancy, CNS pathology of
teratogenic viruses varies. Similar to AKV infection, it is suggested that transplacental
SBV infection at early stages of gestation causes hydranencephaly due to a
widespread loss of neuronal tissue. Infection at later time points (end of the 3rd
trimester) results in a more focal necrosis of the CNS characterized by porencephaly
(MAXIE et al. 2007). Studies with AKV showed that the virus crosses the placenta
after viremia and replicates in fetal cells of the central nervous system. The virus
prefers rapidly diving cells and causes damage to neurons (ST. GEORGE et al.
2004). An AKV infection between day 28 - 56 and 74 - 150 of gestation in sheep
lambs and calves resulted in malformations, respectively. These specific time periods
represent teratogenic determination phases in these animals (CONRATHS et al.
2013, PARSONSON et al. 1977, 1988). A similar pathogenesis is suspected for SBV
infections (CONRATHS et al. 2012, 2013).
In peripheral organs like muscle, placenta, eye, heart, aorta, lung, trachea, liver,
kidney, spleen, small and large intestine, mesenteric and pulmonary lymph nodes,
thymus, adrenal gland, testis, and uterus no viral RNA was detected by in situ-
hybridization (HAHN et al. 2013). These results emphasized CNS tropism of SBV
and suggest that skeletal muscle hypoplasia is a secondary event without direct
virus-induced cell loss in the muscle. As described for AKV, virus-induced neuronal
damage in the brain and spinal cord could be responsible for a disturbed
development of muscle fibers due to a lack of innervation resulting in hypoplasia (ST.
GEORGE et al. 2004). This abnormal muscle development could predispose for
arthrogryposis. In this context, the occurrence of micromyelia, a loss of neurons and
the almost complete absence of gray matter in SBV-infected neonates seems to play
an important role in developing muscle hypoplasia (VARELA et al. 2013). In addition,
Chapter 7 Discussion
38
cerebellar hypoplasia occurs often in naturally SBV-infected neonates and is
characterized by a loss of neurons in the molecular layer with reduced amounts of
Purkinje neurons (HERDER et al. 2012). Neuronal loss in the cerebellum is also
supposed to be virus-induced due to cell destruction.
SBV-infected animals often display multiple malformations in the brain (HERDER et
al. 2013, 2012). In various cases porencephaly is associated with internal
hydrocephalus and cerebellar hypoplasia. Possible mechanisms for development of
internal hydrocephalus include a) disturbed liquor drainage due to inflammation or
malformations (obstruction) or b) a loss of CNS parenchyma due to SBV-induced
tissue destruction (ex vacuo). Besides CNS malformations, arthrogryposis,
brachygnathia inferior, deformities of the vertebral column (torticollis, kyphosis,
lordosis or scoliosis) also occur frequently in aborted and neonatal sheep lambs,
calves and goat kids (HERDER et al. 2012). The detected malformations in SBV-
infected neonates represent also typical deformities of infections with other
arthropod-borne and/or teratogenic viruses of the same or different virus families
(COETZER et al. 1977, KONNO et al. 1982). Arthrogryposis is characterized by
deformed extremities with hypoplastic skeletal muscles causing abnormal flexion of
the legs. This malformation is characteristic for many teratogenic viruses occurring
worldwide, especially following AKV and Aino virus infections (PARSONSON et al.
1977, TSUDA et al. 2004). Occurrence of arthrogryposis in combination with
hydranencephaly in neonates is termed ‘congenital arthrogryposis and
hydranencephaly syndrome’ (CAHS). AKV, Aino virus and SBV belong to the family
of Bunyaviridae and similar gross lesions in infected neonates indicate a related
pathogenesis. Interestingly, besides AKV, Aino virus and SBV, Rift valley fever and
Wesselsbron viruses also cause arthrogryposis and hydranencephaly in
diaplacentarly infected neonates (COETZER 1982, COETZER et al. 1979, HERDER
et al. 2012, KONNO et al. 1982, TSUDA et al. 2004). Until now, SBV lacks unique
and specific pathological features, which allow an easy discrimination from other
viruses of the Bunyaviridae family or teratogenic viruses causing arthrogryposis and
malformations of the CNS, like Wesselsbron virus. An identification of the causative
agent is required by immunohistochemistry, in situ-hybridization or PCR for a
Chapter 7 Discussion
39
definitive diagnosis (HAHN et al. 2013, HERDER et al. 2013, 2012, HOFFMANN et
al. 2012, VARELA et al. 2013). In some aborted and neonatal ruminants with
malformations originating from endemic areas it was not possible to detect SBV-
specific nucleotides by real time RT-PCR (DE REGGE et al. 2013). Until now, it is
unclear, whether the virus was cleared from the fetuses or whether remaining viral
RNA yields were below the detection level (DE REGGE et al. 2013). For AKV it is
described, that the fetus is able to eliminate the virus in the second part of gestation.
Whether this phenomenon also plays a role in SBV has to be investigated in further
studies (PARSONSON et al. 1981a, POEL 2012). Therefore, epidemiological data in
combination with pathological findings should also be considered for the diagnosis.
Histopathology of the CNS revealed that lymphohistiocytic, perivascular accentuated
meningoencephalomyelitis occurred only rarely (18.3%) in naturally SBV-infected
animals. In general, inflammation in the CNS is less often observed than grossly
detectable malformations like arthrogryposis and hydranencephaly (HAHN et al.
2013, HERDER et al. 2013, 2012). Interestingly, encephalitis was rarely found in
calves (2.9 %) compared to sheep lambs. The latter showed higher percentage of
inflammation in the CNS (28.3%; HERDER et al. 2013). Pathology of goat kids was
similar to sheep lambs, but it has to be considered that only few goats were included
in the present study. Besides a non-suppurative meningoencephalomyelitis, glial
nodules, an astro- and microgliosis was also present in the brains of naturally SBV-
infected aborted and neonatal ruminants (HERDER et al. 2012). The vast majority of
inflammatory cells were CD3-positive T cells, to a lesser extend CD68-positive
microglia/macrophages and CD79α-positive B cells were found. These inflammatory
cells were detected in the perivascular space and the parenchyma most prominently
within the mesencephalon, temporal and parietal lobes (HERDER et al. 2013). In
areas without por- and hydranencephaly, single chromatolytic neurons and neuronal
necrosis can be detected and was interpreted as virus-induced neuronal damage
(HERDER et al. 2012).
Histopathological characteristics of por- and hydranencephaly consisted of various
degrees of demyelination, axonal damage and loss as well as astrogliosis, Gitter cell
Chapter 7 Discussion
40
formation and cortical atrophy next to the cavity (HERDER et al. 2013).
Demyelination and axonal pathology is suggested to be a secondary event due to
SBV infection interpreted as a ‘by-stander demyelination’ (HERDER et al. 2013,
SEEHUSEN et al. 2010, TSUNODA et al. 2002). Interestingly, porencephaly due to
naturally occurring SBV infection, which is associated with demyelination and axonal
damage, can be detected with and without inflammation. The occurrence of
demyelination and axonal damage in combination with malformations seems to be an
independent process from inflammation. The presence of inflammation in SBV-
infected aborted and neonatal ruminants is probably related to the time point of
infection and therefore to the immune status of the fetus (HERDER et al. 2013).
Thus, occurrence of encephalitis in naturally infected animals is determined by the
fetal development of the immune response. In calves and sheep lambs, the immune
system develops after 41 and 19 days post conception, respectively (TIZARD 2013).
Therefore, an SBV infection after this time period might trigger an anti-viral immune
response leading to inflammation and encephalitis. However, further experimental
investigations are needed to give insights into the pathogenesis of the development
of the inflammatory immune response during SBV infection.
Chapter 8 Conclusions
41
Chapter 8 Conclusions Results of this thesis revealed, that pathological findings of naturally occurring SBV
infections in aborted and neonatal ruminants are characterized by multiple skeletal
and CNS malformations. Deformities in the CNS comprise por- and,
hydranencephaly as well as cerebellar hypoplasia, internal hydrocephalus and
micromyelia. Furthermore, these lesions are often associated with arthrogryposis and
deformities of the vertebral column. Histopathology revealed an infrequent
occurrence of lymphohistiocytic meningoencephalomyelitis despite prominent gross
lesions. Demyelination, axonal damage, astrogliosis, Gitter cell formation and cortical
atrophy were associated with high amounts of SBV protein indicating that virus
infection resulted in damage of the CNS tissue substantiating CNS tropism of SBV.
Described pathological findings in SBV-infected neonates are similar to those of
other members of the Bunyaviridae family like AKV or Aino virus. Therefore, the term
‘arthrogryposis and hydranencephaly-syndrome’ appropriately describes the
spectrum of SBV-induced pathology in neonates. In general, encephalitis is an
infrequent event in SBV infection, and sheep lambs show a higher prevalence of
inflammatory changes in the brain compared to calves. However, the exact
mechanism of inflammation development in SBV-infected animals has to be
investigated in further studies. The present study describes the pathology of this new
disease in Europe and compares these findings with other well-known diseases.
Similarities between pathological findings in SBV-infected aborted and neonatal
ruminants and lesions caused by other teratogenic and/or arthropod-borne viruses
require demonstration of the etiologic agent to confirm the specific etiology by
immunohistochemistry, in situ-hybridization or PCR.
Chapter 9 Summary
43
Chapter 9 Summary Characterization of Schmallenberg virus-induced pathology in aborted and neonatal ruminants
Vanessa Herder
Schmallenberg virus (SBV) is a novel Orthobunyavirus causing mild clinical signs in
cows and is responsible for malformations in aborted and neonatal ruminants in
Europe. SBV belongs to the family Bunyaviridae and is transmitted by biting midges.
This new virus was identified for the first time by metagenomic analysis in blood
samples of cows next to the German city Schmallenberg in North-Rhine Westphalia
in November 2011. Since then the virus spread to several European countries. The
aim of the present thesis was to characterize the pathology of this new disease in
naturally infected sheep lambs, goat kids and calves with special focus on the central
nervous system (CNS). Therefore, gross findings of SBV-positive, stillborn, aborted
neonates or animals dying in the perinatal period originating from North-Rhine
Westphalia were determined. In addition, histopathological CNS lesions were
investigated, immunophenotyped and virus distribution was studied. Subsequently,
results were compared to lesions described for closely related viruses, like AKV.
Gross examination of SBV-infected aborted and neonatal ruminants frequently
revealed arthrogryposis, brachygnathia inferior and deformities of the vertebral
column. The CNS of affected animals often showed malformations like internal
hydrocephalus, por- and hydranencephaly and cerebellar hypoplasia. Calves and
sheep lambs also displayed micromyelia. The prevalence of CNS inflammation in
naturally infected animals was low, namely 2.9% in bovine and 28.3% in ovine cases.
The inflammation was characterized by a lymphohistiocytic, perivascular accentuated
meningoencephalomyelitis. Immunophenotyping revealed that CD3-positive T cells
outnumbered CD68-positive microglia/macrophages and CD79α-positive B cells in
brain and spinal cord. Mesencephalon, temporal and parietal lobes were the most
frequently affected brain regions by inflammation indicating that these are suitable
areas for diagnostic purposes. In addition, neuronal necrosis, gliosis and glial
Chapter 9 Summary
44
nodules were diffusely present in naturally SBV-infected aborted and neonatal
ruminants. Typical histopathological findings adjacent to porencephaly were cortical
atrophy with demyelination, axonal loss, astrogliosis, Gitter cell formation,
mineralization and hemosiderosis. These findings were interpreted as secondary
events due to a SBV-induced cell injury. Iron deposition in areas of tissue destruction
was interpreted as remnants of hemorrhages and von Kossa-positive material
putatively represents dystrophic mineralization as a consequence of virus-induced
tissue destruction.
Inflamed areas in the mesencephalon and malformed temporal and parietal lobes
contained the highest SBV protein levels as shown by immunohistochemistry. These
findings indicate that viral antigen may trigger the inflammation in the mesencephalon
leading to tissue loss and porencephaly in the cerebral cortex. Interestingly, the
development of inflammation seemed to develop independently from formation of
deformities in the CNS. However, its mechanism needs to be investigated in further
studies. It has to be considered, that the time point of infection and the immune
status of the fetus play important roles in the development of inflammation in the
CNS.
Summarized, pathological findings of the naturally-occurring SBV-infection in aborted
and neonatal ruminants showed an analogy to lesions caused by other viruses of the
Bunyaviridae family in terms of gross findings like arthrogryposis, por- and
hydranencephaly, cerebellar hypoplasia and deformities of the vertebral column.
Similarly to Akabane and Aino virus, SBV is transmitted by insects, affects sheep,
cattle and goats and caused mainly pathology in offspring. Therefore, the term
‘arthrogryposis and hydranencephaly syndrome’ is also appropriate for SBV-infected
animals with malformations and this virus has to be added to the list of teratogenic
viruses in Europe.
Chapter 10 Zusammenfassung
45
Chapter 10 Zusammenfassung Charakterisierung von Schmallenberg-Virus-induzierten pathologischen Veränderungen bei abortierten und neonatalen Wiederkäuern
Vanessa Herder
Bei dem Schmallenberg-Virus (SBV) handelt es sich um ein neues, in Europa
auftretendes Orthobunyavirus, das milde klinische Symptome bei adulten Kühen
hervorruft und Missbildungen bei abortierten und neugeborenen Wiederkäuern
verursacht. Das SBV gehört zur Familie der Bunyaviridae und wird durch
blutsaugende Insekten übertragen. Zum ersten Mal wurde das Virus im November
2011 mittels Metagenomanalyse in Blutproben von infizierten, adulten Kühen
nachgewiesen, die aus Nordrhein-Westfalen nahe der Stadt Schmallenberg
(Deutschland) stammten. Seit dieser Zeit hat sich das Virus großflächig über viele
Länder Europas ausgebreitet. Ziel der vorliegenden Arbeit war es, die Pathologie
dieser neuen Erkrankung in neugeborenen, natürlich infizierten Schaf- und
Ziegenlämmern sowie Kälbern zu charakterisieren. Bei dieser Untersuchung standen
insbesondere die Veränderungen des zentralen Nervensystems (ZNS) im
Vordergrund. Hierfür wurden die makroskopischen Veränderungen von SBV-positiv
getesteten und aus Nordrhein-Westfalen stammenden Wiederkäuern, die abortiert,
totgeboren oder perinatal verstorben waren, untersucht. Darüber hinaus wurde eine
immunhistologische Phänotypisierung der ZNS-Läsionen durchgeführt und die
Verteilung des Virus im ZNS bestimmt. Die Ergebnisse dieser Studie wurden mit
Veränderungen von nahe verwandten Viren, wie dem Akabane- oder Aino virus und
anderen teratogenen Viren, verglichen.
Die Ergebnisse der makroskopischen Untersuchung von SBV-infizierten, neonatalen
Wiederkäuern ergaben, dass Arthrogrypose, Brachygnathia inferior und
Verkrümmungen der Wirbelsäule häufige Missbildungen darstellen. Im ZNS traten
als häufige Malformationen ein Hydrocephalus internus, eine Por- und
Hydranenzephalie sowie eine Kleinhirnhypoplasie auf. Bei Kälbern und
Schaflämmern wurde zusätzlich eine Mikromyelie diagnostiziert.
Chapter 10 Zusammenfassung
46
Insgesamt ist die Prävalenz einer Entzündung des ZNS bei natürlich mit SBV-
infizierten Aborten und Neonaten gering. Sie kommt bei Kälbern in 2,9% und bei
Lämmern in 28,3% der Fälle vor. Die Entzündung des ZNS stellt sich als eine
lymphohistiozytäre, perivaskulär-akzentuierte Meningoenzephalomyelitis dar. Durch
die Immunphänotypisierung der Entzündungszellen wurde festgestellt, dass es sich
in erster Linie um CD3-positive T-Zellen handelt, in geringerem Maße fanden sich
CD68-positive Mikroglia/Makrophagen und CD79α-positive B-Zellen in Gehirn und
Rückenmark. Das Mesenzephalon sowie der Temporal- und Parietallappen stellten
die am häufigsten von einer Entzündung betroffenen Gehirnregionen dar. Darüber
hinaus fanden sich im gesamten ZNS von natürlich-infizierten, abortierten und
neonatalen Wiederkäuern neuronale Nekrosen, Gliose und Glia-Knötchen. Aus
diesem Grund handelt es sich beim Mesenzephalon, dem Temporal- und
Parietallappen, aus diagnostischer Sicht, um repräsentative Gewebeproben zur
Detektion einer SBV-induzierten Enzephalitis.
Die histopathologische Untersuchung des ZNS im Bereich von Missbildungen, wie
einer Porenzephalie, zeigte eine kortikale Atrophie mit Demyelinisierung,
Axonopathie, Gitterzellinfiltrationen sowie Mineralisierungen und eine Hämosiderose
als typische Veränderungen. Diese Befunde gehen vermutlich auf den SBV-
induzierten Gewebeuntergang zurück. Als Folge von länger zurück liegenden
Blutungen wurde der Nachweis von Eisen in Bereichen von Gewebszerstörung
interpretiert. Granula bestehend aus von Kossa-positivem Material stellen
wahrscheinlich eine dystrophische Verkalkung aufgrund einer virus-induzierten
Zerstörung des ZNS-Parenchyms dar.
Die höchsten Mengen an Virusprotein wurden immunhistologisch im Mesenzephalon
sowie im Temporal- und Parietallappen mit Entzündung und Missbildungen
nachgewiesen. Diese Befunde könnten darauf hinweisen, dass virale Antigene die
Entzündung im Mesenzephalon auslösen und die Bildung einer Porenzephalie durch
einen virus-induzierten Gewebeuntergang im Großhirn verursachen. Weiterhin wurde
festgestellt, dass Missbildungen wie eine Porenzephalie im ZNS auch ohne
Entzündung auftreten können; daher wird vermutet, dass es eine weitere,
Chapter 10 Zusammenfassung
47
unabhängige Ursache für die Entzündung geben muss. Dies sollte in
weiterführenden Studien untersucht werden. Dabei sollte allerdings berücksichtigt
werden, dass dem Zeitpunkt der Infektion und dem Immunstatus des Fetus eine
besondere Rolle bei der Entwicklung einer Entzündung im ZNS zukommen.
Zusammenfassend kann gesagt werden, dass die pathologischen Befunde der
natürlich-vorkommenden SBV-Infektion abortierter und neonataler Wiederkäuer eine
hohe Analogie zu Läsionen bei anderen Viruserkrankungen aus der Familie der
Bunyaviridae aufweisen. Dabei sind insbesondere die Missbildungen, wie
Arthrogrypose, Verkrümmungen der Wirbelsäule, eine Hydran- und Porenzephalie
sowie die Kleinhirnhypoplasie, zu nennen. Darüber hinaus weisen die Tatsachen,
dass SBV durch blutsaugende Insekten übertragen wird, Erkrankungen bei Schafen,
Kühen und Ziegen auslöst und Missbildungen in der Nachkommen-Generation
verursacht, darauf hin, dass Parallelen zur Pathogenese des Akabane- und Aino
virus bestehen. Aus diesen Gründen kann der Begriff „Arthrogrypose-und
Hydranenzephalie-Syndrom“ auch bei SBV-positiven Feten mit Missbildungen
angewendet werden. Dementsprechend sollte das Virus differentialdiagnostisch in
die Liste der teratogenen Viren in Europa eingefügt werden.
Chapter 11 References
49
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Chapter 12 Acknowledgements
61
Chapter 12 Acknowledgements I am grateful to Prof. Dr. Wolfgang Baumgärtner, Ph.D. entrusting me with the
Schmallenberg project and providing an outstanding supervision.
I like to show my gratitude to Dr. Martin Peters and Dr. Peter Wohlsein for excellent
cooperation and for providing the samples for the study.
I thank Prof. Dr. Massimo Palmarini and Dr. Mariana Varela for friendly, productive
and successful cooperation and also for providing the SBV-antibody for the study.
I want to thank the members of the ‘Schmallenberg-group’: Ingo G., Frauke, André,
Kerstin and Malin - especially Ingo G. for carefully proof-reading my thesis.
Special thanks to Prof. Dr. Andreas Beineke for endless patience and his perfect
combination of competence and friendliness.
I thank Dirk for his constructive criticism and scanning several histoslides.
It is a pleasure to thank Caro, Petra, Bettina, Kerstin, Claudia, Danuta and Kuli for
their excellent assistance and support in the lab.
I would like to thank Jogi, Annika and Dahlia for the joyful time in our office paradise.
Especially Max for his endless patience and thoughtfulness.
Many thanks to all other members of the Department of Pathology who supported my
work directly or indirectly.
I express my sincere thanks to Jan and Marc for friendship, which starts in Giessen
in 2001.
Thanks, Florian.
My deepest gratitude to my family.
ISBN 978-3-86345-154-7
Verlag: Deutsche Veterinärmedizinische Gesellschaft Service GmbH35392 Gießen · Friedrichstraße 17 · Tel. 0641 / 24466 · Fax: 0641 / 25375
E-Mail: [email protected] · Internet: www.dvg.de
Vanessa Herder
Hannover 2013
Department of PathologyUniversity of Veterinary Medicine
Characterization of Schmallenbergvirus-induced pathology in abortedand neonatal ruminants