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
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-SBV-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
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,
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
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
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.