**1. Pathogenesis**

The knowledge of Schmallenberg virus (SBV) pathogeny will allow to achieve a better understanding of its most obvious expression, reflected in their striking lesional pattern; however, until now, the pathogenic features of SBV are not well understood. Current assumptions about SBV pathogenesis in ruminants are frequently based on findings described for Akabane virus (AKAV), which, similar to SBV, causes in aborted or stillborn neonatal ruminants, arthrogryposis and hydranencephaly syndrome (AHS) [1].

The tropism of SBV to the central nervous system (CNS) is already well described in the literature, associated with several congenital malformations, being the most notorious in the

brain [2]. However, taking into consideration the results obtained by Balseiro et al. [3], regarding the presence of the virus in several tissues of naturally infected calves, SBV could also have tropism to other organs and tissues, namely muscle, skeletal and cardiac, spleen, placenta and umbilical cord [3].

As referred, SBV infection during gestational period affects the foetus, through a vertical transplacental transmission. The development of teratogenic defects and its severity is directly related with gestational time (GT) at the moment of infection and, consequently, neuronal cells development in the CNS, which are the SBV target [2, 3].

The severity of lesions in the CNS is also dependent of the foetal immune system development, characterized by cellular population of the thymus and lymph nodes and the production of antibodies, which in bovines occurs between days 40 and 175 of GT and in the lamb starts 19 days post gestation and lasts until 115 days after conception and depends also of the foetal CNS vulnerability (well defined in the cow as the period between days 60 and 180 of GT) [1, 2].

Thus, the severity of lesions in the brain and spinal cord depends on the complexity of the interaction between foetal neurogenesis, immunocompetency, virulence of the viral strain, intensity and time of infection [1–3].

If maternal infection occurs in early gestational period, before the foetus becomes immunocompetent (between the 90th and the 180th day of GT in cow; between the 25th and 50th day in sheep), severe dysplastic CNS lesions, including a distinctive micromyelia, without evidence of inflammation are described, as well as abortion, stillborn and congenital malformations, with no detectable viral RNA and antigen [2, 4, 5].

Infection in the first month of gestation could probably cause embryonic death and subsequent resorption or abortion, only proven by repeat oestrus and matings, observed by farmers and shepherds [4].

If maternal infection occurs in the late gestational period (after 180th day of GT in cow; after 50th day in sheep), inflammation—nonsuppurative polioencephalomyelitis (lymphohistiocytic) or meningoencephalomyelitis—can be present due to viral antigen recognition by the foetal immune system, with detectable viral RNA and antigen [1, 2, 4, 5].

Probably due to the rapid organogenesis consequent to the shorter GT in sheep (GT of 150– 155 days), brain and spinal cord malformation are more severe in lambs than in calves [5]. As well as most viral infections, transplacental transmission could not result in notorious placentitis and most malformed newborns are stillborn at term [2].

#### **1.1. CNS congenital malformations**

The association of Schmallenberg virus with a range of congenital malformations, mostly in the CNS, is well known [2]. The brain is the most frequent target, but cerebellar and spinal cord defects are also referred by several authors [5]. The predominant malformations observed in the CNS of both lambs and calves are hydranencephaly, porencephaly, hydrocephalus, microencephaly, cerebellar hypoplasia and mild-to-marked dysplasia of the cerebellum, brain stem and spinal cord [4, 6].

According to Peperkamp et al. [5], defects occurring in the cerebrum might range from slight to moderate dysplasia, including microencephaly, porencephaly, hydrocephalus and lack of gyri (lissencephaly), to fully developed hydranencephaly.

Porencephaly refers to cystic fluid-filled cavities in the brain tissue, which communicate with the ventricular system, and is usually described as the cavitation of the cerebral hemispheres. One study performed by Herder et al. [1] revealed that the temporal and parietal lobes are more frequently affected, usually in a bilateral-symmetrical matter. Furthermore, porencephaly might be present in both temporal and parietal lobes, in a condition named multicystic encephalopathy.

Hydranencephaly is characterized by the destruction of brain hemispheres, many times with a complete or almost complete replacement of the cortex by cerebrospinal fluid, surrounded by a thin, almost transparent, membranous sac.

Cerebellar hypoplasia is one of the most frequently referred malformations in the CNS of SBVinfected animals, and, both in lambs and in calves, various degrees of cerebellar dysplasia might be present, even in animals presenting a normal cerebrum [5].

Morphologic alterations of the spinal cord are observed as a decrease in the cross-sectional area of the spinal cord, or micromyelia. This malformation is characterized by neuronal loss in the ventral horns, and is apparently positively correlated with the magnitude of musculoskeletal deformities [5].

Histologically, the described malformations might be accompanied by the presence or absence of inflammation, including encephalomyelitis, lymphohistiocytic meningoencephalomyelitis and glial nodules. Microscopic lesions include rarefaction and cavitation, degeneration, necrosis and loss of neurons, as well as mild to severe, diffuse astrogliosis and/or microgliosis [1, 7].

#### **1.2. Other lesions (musculoskeletal)**

brain [2]. However, taking into consideration the results obtained by Balseiro et al. [3], regarding the presence of the virus in several tissues of naturally infected calves, SBV could also have tropism to other organs and tissues, namely muscle, skeletal and cardiac, spleen,

58 Epidemiology of Communicable and Non-Communicable Diseases - Attributes of Lifestyle and Nature on Humankind

As referred, SBV infection during gestational period affects the foetus, through a vertical transplacental transmission. The development of teratogenic defects and its severity is directly related with gestational time (GT) at the moment of infection and, consequently, neuronal cells

The severity of lesions in the CNS is also dependent of the foetal immune system development, characterized by cellular population of the thymus and lymph nodes and the production of antibodies, which in bovines occurs between days 40 and 175 of GT and in the lamb starts 19 days post gestation and lasts until 115 days after conception and depends also of the foetal CNS vulnerability (well defined in the cow as the period between days 60 and 180 of GT) [1, 2]. Thus, the severity of lesions in the brain and spinal cord depends on the complexity of the interaction between foetal neurogenesis, immunocompetency, virulence of the viral strain,

If maternal infection occurs in early gestational period, before the foetus becomes immunocompetent (between the 90th and the 180th day of GT in cow; between the 25th and 50th day in sheep), severe dysplastic CNS lesions, including a distinctive micromyelia, without evidence of inflammation are described, as well as abortion, stillborn and congenital malformations,

Infection in the first month of gestation could probably cause embryonic death and subsequent resorption or abortion, only proven by repeat oestrus and matings, observed by farmers and

If maternal infection occurs in the late gestational period (after 180th day of GT in cow; after 50th day in sheep), inflammation—nonsuppurative polioencephalomyelitis (lymphohistiocytic) or meningoencephalomyelitis—can be present due to viral antigen recognition by the

Probably due to the rapid organogenesis consequent to the shorter GT in sheep (GT of 150– 155 days), brain and spinal cord malformation are more severe in lambs than in calves [5]. As well as most viral infections, transplacental transmission could not result in notorious

The association of Schmallenberg virus with a range of congenital malformations, mostly in the CNS, is well known [2]. The brain is the most frequent target, but cerebellar and spinal cord defects are also referred by several authors [5]. The predominant malformations observed in the CNS of both lambs and calves are hydranencephaly, porencephaly, hydrocephalus, microencephaly, cerebellar hypoplasia and mild-to-marked dysplasia of the cerebellum, brain

foetal immune system, with detectable viral RNA and antigen [1, 2, 4, 5].

placentitis and most malformed newborns are stillborn at term [2].

placenta and umbilical cord [3].

intensity and time of infection [1–3].

**1.1. CNS congenital malformations**

stem and spinal cord [4, 6].

shepherds [4].

with no detectable viral RNA and antigen [2, 4, 5].

development in the CNS, which are the SBV target [2, 3].

Malformed newborns presented underweight due to the underdevelopment of body mass, skeletal muscles and variable severity of malformations. Frequently, bones undergo normal development but vertebral malformations and arthrogryposis occur due to the imbalance in foetal muscular activity, secondary to the loss of neurons in the brain and spinal cord and demyelination, affecting descending tracts in the ventral spinal cord white matter, ventral horn motor neurons in the spinal cord grey matter and ventral spinal nerve roots [2, 4, 8].

The lack of innervation, by lower motor neuron, to the skeletal muscle motor units prevents its normal development, resulting in muscular hypoplasia. Neuronal bilateral loss in cervical and lumbar intumescences causes bilateral arthrogryposis, and unilateral neuronal loss causes unilateral arthrogryposis [4, 5].

This neuronal loss in spinal cord could be grossly visible in severely affected cases, as a small dorsoventral flattened spinal cord, designated by micromyelia and, consequently, results in denervation of the axial and appendicular musculature with the failure of normal skeletal muscle development, resulting in the lesions of vertebral column malformations and arthrogryposis [2].

**Figure 1.** Newborn lamb with 6 days revealing prognathism and kyphosis.

Therefore, vertebral column malformations, namely torticollis, scoliosis and kyphosis (**Figure 1**) of the thoracic vertebral column, mostly combined with arthrogryposis are consequent to dysplastic CNS lesions and represent the most visible gross lesions [2]. Scoliosis, kyphoscoliosis and kyphosis without torticollis are less frequent and vertebral column malformations without arthrogryposis are rare (**Table 1**) [2].


**Table 1.** Most visible gross lesions.

**Figure 2.** Stillborn lambs with severe arthrogryposis multiplex congenital affecting the four limbs.

The most frequent lesion observed in the limbs is the arthrogryposis multiplex congenital affecting the four limbs, symmetric and bilaterally (**Figures 2**–**4**), often accompanied by vertebral column malformations, as referred. Arthrogryposis could range from different severities, affecting only the forelimbs (uni- or bilaterally) or only the hindlimbs (uni- or bilaterally; rarely) (**Table 2**) [2, 5].

**Figure 3.** Arthrogryposis. Note the severe curving of joints.

muscle development, resulting in the lesions of vertebral column malformations and arthrog-

60 Epidemiology of Communicable and Non-Communicable Diseases - Attributes of Lifestyle and Nature on Humankind

Therefore, vertebral column malformations, namely torticollis, scoliosis and kyphosis (**Figure 1**) of the thoracic vertebral column, mostly combined with arthrogryposis are consequent to dysplastic CNS lesions and represent the most visible gross lesions [2]. Scoliosis, kyphoscoliosis and kyphosis without torticollis are less frequent and vertebral column malformations

**Torticollis Scoliosis Kyphosis Torticollis**

**Figure 1.** Newborn lamb with 6 days revealing prognathism and kyphosis.

**Frequency Arthrogryposis Vertebral column malformations**

Less frequent **x x**

Frequent **x x x x x**

Rare **x x x x**

**Figure 2.** Stillborn lambs with severe arthrogryposis multiplex congenital affecting the four limbs.

The most frequent lesion observed in the limbs is the arthrogryposis multiplex congenital affecting the four limbs, symmetric and bilaterally (**Figures 2**–**4**), often accompanied by vertebral column malformations, as referred. Arthrogryposis could range from different

without arthrogryposis are rare (**Table 1**) [2].

**Table 1.** Most visible gross lesions.

ryposis [2].

**Figure 4.** Newborn kid with three revealing arthrogryposis affecting only forelimbs bilaterally.


**Table 2.** Frequency of arthrogryposis.

The histopathology of skeletal muscles of arthrogrypotic limbs revealed hypoplasia of striated muscular tissue (severe reduction of muscle diameter) and myofibrillar hypoplasia, reflecting the extent of dysplastic spinal cord with neuronal loss in the cervical and lumbar intumescences [2, 4, 5]. Other musculoskeletal histopathologic lesions described include atrophy and loss of skeletal muscle mass (especially in the limbs and neck) (**Figure 5**), with the presence of fibrous or adipose tissue, as well as the depletion of fat deposits [5, 9].

**Figure 5.** Newborn kid with 6 days revealing the loss of skeletal muscle mass.

**Figure 6.** Newborn kid with 3 days revealing brachygnathia.

**Figure 7.** Stillborn lamb with prognathism.

Abnormal flattened skull with cranial vault reduced by thickened frontal, parietal and occipital bones was described [5]. Flattened ribcage could also be present, consequently to malformations of the thoracic vertebral column, as well as lordosis of the thoracolumbar part of the vertebral column [2].

Lesions of brachygnathia (**Figure 6**) and prognathism (**Figure 7**) are described in the consulted literature [5, 9].

Peperkamp et al. [5] reported hypoplasia of the lung lobes in ruminants affected with thoracic malformation but in other organs analysed, such as urinary bladder, thyroid gland, liver, spleen, heart muscle and vessels, uterus, ovaries, testes, peripheral nerves, placenta, oesophagus, abomasum, small and large intestine, pancreas, adrenal glands, celiac ganglion, trachea, skin, tongue and adipose tissue, no significant histopathological lesions were present [5, 9].

#### **1.3. Characterization of inflammatory infiltrates**

**Figure 5.** Newborn kid with 6 days revealing the loss of skeletal muscle mass.

62 Epidemiology of Communicable and Non-Communicable Diseases - Attributes of Lifestyle and Nature on Humankind

**Figure 6.** Newborn kid with 3 days revealing brachygnathia.

**Figure 7.** Stillborn lamb with prognathism.

Inflammatory changes of the brain and meninges are more frequent in small ruminants than in calves, and are characterized by lymphohistiocytic perivascular cuffs with parenchymal T cells, B cells and microglia/macrophages. The CD3-positive T cells are the dominant cellular group. CD79α-positive B cells are also found, but on a lesser extent and CD68-positive microglia/macrophages are less often detected [1].

#### **1.4. Challenges in differential diagnosis**

Taking into account the most prominent lesion of CNS congenital malformations, in ruminants, the first and most important challenge in the differential diagnosis (DD) is ruling out other viral-induced congenital malformations in ruminants, including bovine virus diarrhoea virus (BVDV), Border disease virus (BDV), blue tongue virus (BTV), Akabane virus, Aino virus (AV) and Cache Valley virus (CVV) [2, 5].

Also, molecular and serological diagnosis becomes more difficult when the infection of SBV occurs in early gestational period (aggravated in cows where the total GT is larger than sheep) for having more time to develop an effective immune response (foetal lambs have its immune system completely immunocompetent at mid-gestation, at the 90th day of GT) and performs the viral clearance in seropositive offspring, which could make them negative to the virus [4, 5]. Moreover, malformations in CNS and musculoskeletal system are frequently caused by teratogenic and genetic defects, as well as a significant number of inherited congenital syndromes that closely resemble SBV lesions, such as arthrogryposis multiplex congenital syndrome, arachnomelia, both with arthrogryposis [2]. Also, rare non-viral congenital syndromes can occur, such as congenital myoclonus, Perosomus elumbis, Cyclopia and none fragility described in bovine.

Unlike clinical signs, discrete, non-specific and variable, the lesions described above have a dramatic and showy expression, could be easily recognized by clinicians but none are pathognomonic for SBV, despite its great diagnostic value (**Table 3**). It is then extremely difficult to obtain a precise and definitive aetiological diagnosis [2, 5].


**Table 3.** The value of congenital malformations in the DD of viral-induced congenital malformations.

Therefore, definitive diagnosis requires systematic necropsies, with the examination of the brain and spinal cord, which could be done in the field but the ideal practice would be the recourse to a specialty laboratory to obtain diagnostic accuracy and specificity namely the pathology laboratory, once the diagnosis could not be exclusively done based only in gross lesions, even if arthrogryposis is present [2, 10] and require histopathology of the entire CNS whose collection should be complete, which is labour intensive and requires specific material to be performed (**Figure 8**).

**Figure 8.** Complete collection of CNS of a newborn kid with 3 days.

Pathological diagnosis must be complemented by molecular genetics, serological and virological diagnosis [2, 3].

Dissemination of information about the disease, especially in the moment of the entrance into a given country, is crucial to alert professionals about the emergence of outbreaks, the data registration for each suspect or compatible case being crucial, as proposed in **Table 4**. In this registration, it is mandatory to include the date of occurrence, season and geographic region information, to relate with vector activity, since as in the winter, with too low temperatures for the vector activity, SBV may be soon ruled out [2].


**Table 4.** Data registration case identification proposal.

Biological fresh samples collected should include abortion material, foetus, placenta, umbilical cord (**Figure 9**) and maternal blood samples but, at the local, photos must be taken and description of gross lesions must be done, as proposed in **Tables 5** and **6**.

**Figure 9.** Complete abortion material collection, including foetus, placenta, umbilical cord of a stillborn lamb.



**Table 6.** CNS malformations registration.

**SBV BTV BVDV**

(less common in cow)

**Spinal cord lesions with arthrogryposis** Yes No No Yes in late GT **Vertebral malformations** Yes No No No (rare)

64 Epidemiology of Communicable and Non-Communicable Diseases - Attributes of Lifestyle and Nature on Humankind

Therefore, definitive diagnosis requires systematic necropsies, with the examination of the brain and spinal cord, which could be done in the field but the ideal practice would be the recourse to a specialty laboratory to obtain diagnostic accuracy and specificity namely the pathology laboratory, once the diagnosis could not be exclusively done based only in gross lesions, even if arthrogryposis is present [2, 10] and require histopathology of the entire CNS whose collection should be complete, which is labour intensive and requires specific material

Pathological diagnosis must be complemented by molecular genetics, serological and viro-

Dissemination of information about the disease, especially in the moment of the entrance into a given country, is crucial to alert professionals about the emergence of outbreaks, the data registration for each suspect or compatible case being crucial, as proposed in **Table 4**. In this registration, it is mandatory to include the date of occurrence, season and geographic region information, to relate with vector activity, since as in the winter, with too low temperatures for

**Species Race Gender Abortion Perinatal**

**Age of pregnancy** **death**

**Age (days)**

**Stillborn**

**Table 3.** The value of congenital malformations in the DD of viral-induced congenital malformations.

**Cerebral defects** Yes or no

**Figure 8.** Complete collection of CNS of a newborn kid with 3 days.

the vector activity, SBV may be soon ruled out [2].

**Table 4.** Data registration case identification proposal.

**Parish Municipality Earmark**

**mother**

to be performed (**Figure 8**).

logical diagnosis [2, 3].

**Herd code**

**Collection date (season)**

**Case 1**

**and BDV**

Yes Yes Yes in early GT

**AKAV**

However, the shipment of biological fresh samples represents one of the greatest challenges in the diagnosis of SVB, once abortion is very common and the abortion products are found in the field, already in an advanced state of putrefaction and autolysis (**Figures 8** and **9**).
