ISBN 978-3-86345-154-7
Verlag: Deutsche Veterinärmedizinische Gesellschaft Service GmbH 35392 Gießen · Friedrichstraße 17 · Tel. 0641 / 24466 · Fax: 0641 / 25375
E-Mail: info@dvg.de · Internet: www.dvg.de
Vanessa Herder
Hannover 2013
Department of Pathology
University of Veterinary Medicine
virus-induced pathology in aborted
and neonatal ruminants
Deutschen Nationalbibliografie;
Detaillierte bibliografische Daten sind im Internet über http://dnb.ddb.de abrufbar.
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 info@dvg.de www.dvg.de
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
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
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
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
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
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
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
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.
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).
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
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
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
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).
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
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.
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).
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).
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.
Chapter 2 Introduction
15
Table 1: Overview on virus infections representing possible etiologic differentials to a Schmallenberg virus infection. Pathology and clinical signs after natural transmission or experimental infection, susceptible species and transmission mode of virus infections are summarized for neonates and adult animals. axonomy ame References Gross findingsHistology SpeciesTransmissi rg virus irus,
(DE REGGE et al. 2012, ELBERS et al. 2013, GARIGLIANY et al. 2012b, 2012c, HERDER et al. 2012, HOFFMANN et al. 2012, YANASE et al. 2012)
Neonates:Arthrogryposis, vertebral malformations, brachygnathia inferior, hydran- and porencephaly, internal hydrocephalus, cerebellar hypoplasia, micromyelia Adult cows: Diarrhea, fever, reduced milk yield
Neonates:Lympho-histiocytic meningoencephalomyelitis, gliosis, muscular hypoplasia Adult animals: Not reported
Sheep, goats, cattle, bisons
Arthropod bo mosquitos and midges: e. obsoletus, C. d C. scoticus chiopterus; s irus irus, ine is and ephaly; arthrogryposis- ephaly AHS)
(BRENNER et al. 2004, CHARLES 1994, HUANG et al. 2003, KAMATA et al. 2009, KONNO et al. 1982, KONO et al. 2008, KUROGI et al. 1977, LEE et al. 2002, PARSONSON et al. 1988, 1981b, ST. GEORGE et al. 2004)
Neonates:Arthrogryposis, vertebral malformations, hydran- and porencephaly, brain agenesis, lung hypoplasia, cystic septum pellucidum Adult animals: Subclinical viremia in ruminants; experimentally infected goats without clinical signs
Neonates:Non-suppurative encephalomyelitis, perivascularcuffing, neuronal loss in the spinal cord, muscular atrophy and degeneration Adult cows: Lymphohistiocytic, perivascular encephalomyelitis, gliosis, neuronophagia, neuronal loss; pons and medulla oblongata often affected
Cattle, horses, donkeys, sheep, goats, camels, buffaloes, pigs
Arthropod bo mosquitos and midges, e.g. Aedes sp., C. i us diarrhea e
(BINKHORST et al. 1983, FLORES et al. 2000, HEWICKER-TRAUTWEIN et al. 1995, 1994, MAXIE et al. 2007)
Neonates:Cerebellar hypoplasia, mumification, runting, micro-, por- and hydranencephaly, hydrocephalus internus, microphthalmia, cataract, brachygnathia, thymic hypoplasia, hypotrichosis, alopecia, pulmonary hypoplasia, cystic septum pellucidum Adult animals:Fever, sudden death, diarrhea, pneumonia, thrombocytopenic syndrome, erosion and ulceration in the alimentary tract, coronitis
Neonates:Perivascular non-suppurative meningeal infiltration, reduction of the cortical cell layers, degenerative changes in and heterotopia of Purkinje cells, hypomyelinogenesis, retinal degeneration, optic neuritis, myocarditis, astrogliosis, leukoencephalomalacia Adult animals:Vascular necrosis in mesenteric arterioles, lymphatic depletion, herniation of Peyer’s patches, erosions and ulceration in the alimentary tract and secondary bacterial infections
Cattle, sheep, goats, pigs
Excretions, i ingestion, s contaminated em transfer fluid e virus(JAUNIAUX et al. 2008, MACLACHLAN et al. 2009, MAXIE et al. 2007, WOUDA et al. 2008)
Neonates: Por- and hydranencephaly, hydrocephalus internus, cerebellar dysgenesis, runting, “dummy lambs”, cystic septum pellucidum Adult animals:Hemorrhage and ulcers in the alimentary tract, congested or cyanotic tongue, skeletaland cardiacmuscle necrosis, coronitis, laminitis, hemorrhage at the base of pulmonary artery, lung edema, pericardial, pleuraland abdominal effusion, subcutaneous and intermuscular edema
Neonates: Necrotizing meningoencephalitis, interstitial pneumonia, mononuclear cells in kidney and liver Adult animals:Endothelial damage, microvascularnecrosis, edema, hemorrhages, epithelial necrosis
Sheep, wild ruminants, camelids, cattle, Eurasian lynx
Arthropod bo C. sp., C. imic
Chapter 2 Introduction 16axonomy ame References Gross findingsHistology SpeciesTransmissi irus e e; er”, bs
(BRAUN et al. 2002, MAXIE et al. 2007, NETTLETON et al. 1998, PHYSICK-SHEARD et al. 1980)
Neonates: Embryonic death, mummification, fleece and mandibularabnormalities, runting, cerebellar hypo-and dysplasia, micro-, por- and hydranencephaly, leukoencephalomalacia, micromyelia, starvation, cardiacabnormalities, arthrogryposis, kyphoscoliosis, cystic septum pellucidum, thymus hypoplasia Adult animals: Abortion, stillbirth
Neonates:Dys- and hypomyelination, nodular periarteritis often detected in the CNS Adult animals: Necrotizing placentitis with neutrophilic infiltration
Sheep, goats, pigs, cattle Oral, c intranasal, semen y virus a serogroup irus, (EDWARDS et al. 1989, HOLDEN et al. 1959, MAXIE et al. 2007)
Neonates:Arthrogryposis, hydrocephalus, hydran-, por- and microcephaly, vertebral malformations, cerebellar hypoplasia, micromyelia Adult animals: Often subclinical infections
Neonates: Necrosis and loss of neuropil and motor neurons, myositis, poorly developed myocytes Adult animals: Not reported
Sheep, deer caribou, pigs, horses, cattle, raccoons, foxes, humans
Arthropod bo Culicoides s Culiseta s Anopheles sp. irus ogroup(MAXIE et al. 2007, MIURA et al. 1990)
Neonates:Hydranencephaly, cerebellar hypoplasia, hydrocephalus, microcephaly Adult animals:During viremia no clinicalsigns, leukopenia
Neonates: Not reported Adult animals: Not reportedCattle Arthropod bo Culicoides ox wine fever a
(ELBERS et al. 2003, MAXIE et al. 2007, SANCHEZ-CORDON et al. 2003)
Neonates:Mummification, runting, hypotrichosis, stillbirth, pulmonary hypoplasia, pulmonary artery malformation, arthrogryposis, cerebellar hypoplasia, microcephaly,defective myelination, anasarca, “congenital tremor” Adult animals: Acute, subacute and chronic forms as well as a late onset form; hemorrhagic diathesis, petechiae, typical pinpoint hemorrhages in the kidney, splenic infarction, “button ulcers” in the gastrointestinal tract, pneumonia, conjunctivitis, splenomegaly, small thymus, diarrhea
Neonates:Necrotizing and neutrophilic vasculitis (panarteritis) in CNS, intestine, skin, lymphoid depletion, endothelial degeneration, valvular fibrosis, portal fibrosis (liver), interstitial pneumonia, neuronal degeneration, necrosis of pancreatic islands Adult animals: Disseminated intravascular coagulation, necrotizing and neutrophilic vasculitis (panarteritis), lymphoid depletion, mucosal and epithelial necrosis, edema, hemorrhages
Pigs; cattle, sheep, goats susceptible, but no clinical signs
Oronasally b excretions, i arthropods Haematopinus ever virus , (ALI et al. 2012, COETZER 1982, 1977, MAXIE et al. 2007, RIPPY et al. 1992)
Neonates:Intra-uterine fetal death, arthrogryposis, hydranencephaly Adult animals: Often asymptomatic; sudden death, hemorrhages in various organs, diarrhea, nasal discharge
Neonates:Lymphohistiocytic meningoencephalomyelitis, hepatic necrosis, acidophilic intranuclear, filamentous inclusions, cholestasis, degeneration of lymphocytes, heart muscle and renal tubules, vessel damage, lymphatic depletion, hemorrhages Adult animals: Hepatic necrosis, acidophilic intranuclear, filamentous inclusions, vessel damage, lymphatic depletion, hemorrhages
Sheep, cattle, goats, camels, humansArthropod bo Aedes sp., Cul
