Pathogenesis of the infection with SBV: hypotheses drawn from experimental infection in domestic ruminants

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Hypotheses on the pathogenesis of the lesions in fetuses and newborns

Previous works on CVV and AKAV have brought pieces of information about the pathogenesis of the viral-induced lesions. Given that SBV is phylogenetically close to these viruses and causes very similar clinical signs and lesions, it is likely they are involved in common mechanisms of disease. By analogy with these virus, it is thus possible to formulate hypotheses regarding the pathogenesis of the lesions induced by SBV.

Factors influencing the development of lesions in fetuses and newborns

From the literature, both the age of the conceptus (i.e., the embryo or fetus with its placenta) and the inoculum have an impact on the development of lesions in fetuses and newborns.

Influence of the age of the conceptus at the time of infection

In cattle, an experimental infection with AKAV showed a sequential development of lesions depending on the stage of pregnancy at which the cow was infected. Hydranencephaly and porencephaly developed in fetuses after infection between 76 and 104 dg. Arthrogryposis developed in fetuses infected later, when infection took place between 103 and 174 dg. No lesions were found in fetuses born from cows infected before 76 dg (Kirkland et al., 1988). As the authors suggested, the absence of fetal lesions before 76 dg may be explained by immaturity of the placentomes until this day, with subsequent isolation and protection of the conceptus from the virus (Kirkland et al., 1988). However, they could not monitor the early embryonic development, thus they could not exclude embryonic mortality when infection took place before 76 dg (Kirkland et al., 1988).
Similarly, in sheep, the lesions that developed seem to depend on the age of the conceptus at the time of infection with AKAV. A study from Japan showed that, when experimental infection occurred between 29 and 45 dg, newborns displayed arthrogryposis and hydranencephaly. After inoculation between 30 and 70 dg, abnormal newborns were either weak, with or without porencephaly, or dwarf; there were also stillborn lambs. However, if the inoculation took place between 90 and 100 dg, all the newborns were normal (Hashiguchi et al., 1979). Another experimental infection of pregnant ewes with AKAV showed that brain lesions (necrosis and gliosis) could still occur after inoculation at 90 dg (Narita et al., 1979).
One study showed that the ovine fetuses infected transplacentally could produce neutralizing antibodies against AKAV as soon as 64 dg (Hashiguchi et al., 1979). The development of the immune system in the fetus may inhibit viral progression and subsequent lesions; therefore it may be one reason for the lack of lesions at late stages of gestation. However, the immune system activity may not be the only factor responsible for this lack of lesions. An experimental inoculation of AKAV intraperitoneally in ovine fetuses at 120 dg resulted in a strong immune response, yet the fetuses showed damage in brain and skeletal muscle (McClure et al., 1988). The authors suggested that the stage of maturity of the target-organs may be of greater importance in determining its susceptibility to virus-induced damage than the fetal immune response (McClure et al., 1988).
The susceptibility window of goat fetuses to AKAV infection has been partially described. An experimental infection of a pregnant goat by AKAV at 40 dg resulted in one malformed fetus but a normal twin fetus. The malformed fetus displayed hydranencephaly, arthrogryposis, scoliosis, and torticollis (Kurogi et al., 1977). Another experimental infection at 30 and 60 dg led to degeneration and necrosis in the skeletal muscle and/or myositis in all fetuses at 10 dpi. Only the fetuses infected at 60 dg showed also non-suppurative encephalitis (Konno and Nakagawa, 1982).
In summary, the susceptibility of the growing embryo or fetus to AKAV infection may depend on:
– The maturity of the placentomes: if the infection of the pregnant female takes place before maturity; the conceptus may be protected from viral invasion;
– The stage of maturity of the target-organs: with increasing maturity, the target organs may be no more susceptible to the virus;
– The stage of development of the fetal immune system: with increasing development, the immune system could inhibit the progression of the virus and the virus-induced damage.
By analogy with AKAV, hypotheses can be drawn on the effects of SBV infection in pregnant females depending on the stage of gestation. Figure 5 shows the putative consequences of SBV infection in pregnant ewes and goats.
Figure 5. Hypothetic consequences of SBV infection in pregnant goats and ewes depending on the stage of gestation.
By analogy with data from experimental infection with AKAV (Hashiguchi et al., 1979; Konno and Nakagawa, 1982; Narita et al., 1979). AI: artificial insemination; CNS: central nervous system; dg: day of gestation; SKM: skeletal muscle.

Influence of the inoculum

Several teams have performed AKAV inoculations in pregnant ewes and their results sometimes differed. As an example, one study showed that lesions in fetuses occurred only if the inoculation had taken place between 30 and 36 dg but not between 38 and 82 dg (Parsonson et al., 1977), while another study showed fetal lesions after inoculation between 29 and 70 dg (Hashiguchi et al., 1979). In these cases, the protocols were similar regarding the route of inoculation (intravenous), but they differed in the strains used as well as in the number of passages and the host species used for passage. The authors of the second study hypothesize that the difference in results could rely upon the difference in virulence between the two inocula, depending on the viral strain and the history of passages (Hashiguchi et al., 1979). Moreover, the viral load of the inoculum could also exert an influence on the severity of the damage in fetal tissues.

Mechanisms involved in the development of lesions at the tissue level

Microscopic lesions and correlation with tissular and cellular tropism

In one study, pregnant ewes were inoculated at 35 dg with CVV and were sequentially slaughtered between 42 and 63 dg. The results suggest two hypotheses about the mechanism of lesion formation.
First, this study shows a progression of the microscopic lesions in the fetuses along with the course of infection between 7 and 28 dpi. Between 7 and 10 dpi, the brain displayed necrosis in matrix and intermediate zones of the cerebral cortex and brainstem; necrosis was also noticed in the dorsal horns of the spinal cord and in the skeletal muscle, without vascular lesions. At 14 dpi, hydrocephalus ex vacuo was noticed, and myositis with mononuclear cells and granulocytes was seen in skeletal muscle. Then, between 21 dpi and 28 dpi, the histological lesions were hydrocephalus ex vacuo, myositis with muscle hypoplasia, and micromyelia (Hoffmann et al., 2012a). This sequential progression suggests that hydrocephalus ex vacuo, and probably hydranencephaly, proceed from necrosis of progenitor cells in the brain.
Second, the microscopic lesions were associated with the presence of CVV in the affected tissues, i.e. the skeletal muscle and the CNS. Viral RNA and antigen, as determined by ISH and IHC, were associated with necrotic foci in muscle, brain and spinal cord. The target cells were progenitor cells in the periventricular area in the brain, and myofibers in the muscle; positive cells were also found in the spinal cord but their nature was not identified (Hoffmann et al., 2012a). This association between lesions, viral RNA and viral antigen are suggestive of a direct effect of the virus in the aforementioned tissues.
The pathogenesis of skeletal muscle hypoplasia (and the associated arthrogryposis) is a matter of debate. In calves with arthrogryposis, a relationship has been described between the lesions observed in spinal cord and the side(s) affected by arthrogryposis: bilateral depletion of the ventral horn neurons was associated to bilateral arthrogryposis while unilateral depletion of these neurons was associated to unilateral arthrogryposis (Kirkland et al., 1988). Depletion of ventral horn neurons has also been described in SBV-infected animals (Herder et al., 2012); as a consequence denervation may occur in skeletal muscle, followed by failure of muscle development (Seehusen et al., 2014).
However, muscular lesions have been found in fetuses devoid of spinal cord lesions. Ten days after intravenous inoculation of AKAV to pregnant goats at 60 dg, fetuses displayed degeneration and necrosis in skeletal muscle, but no lesions were observed in spinal cord (Konno and Nakagawa, 1982). These results suggests that AKAV may directly damage skeletal muscle.
Finally, the muscular hypoplasia could result both from damage to the myofibers as well as from motor neuron loss, as hypothesized for CVV (Hoffmann et al., 2012a).

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Insights in cellular and molecular consequences of SBV infection

To study the pathogenesis of SBV in the brain, a mouse model of SBV infection was developed with NIH-Swiss mice. Intracerebral inoculation resulted in death and severe brain lesions with malacia and hemorrhage in the cerebral cortex, multifocal vacuolation in the white matter of the cerebrum, as well as lymphocytic perivascular cuffing in the grey matter. These lesions were associated with SBV antigen in neurons (Varela et al., 2013), which is in favor of a direct role for SBV in inducing this range of lesions. In addition, activated caspase-3 staining in the brain has been later associated with SBV intracerebral infection of NIH-Swiss mice (Barry et al., 2014), suggesting apoptosis may be one mechanism leading to the SBV-induced degenerative lesions in the brain.
To explore whether the viral protein NSs of SBV was a virulence factor, a NSs deletion mutant (SBVΔNSs) was produced by reverse genetics. Its virulence was then tested in NIH-Swiss mice by intracerebral inoculation. The SBVΔNSs showed an attenuated phenotype, characterized by a delay in the time of mice death in comparison to the wild type SBV. This defined SBV NSs as virulence factor (Varela et al., 2013).
In vitro, SBVΔNSs was able to induce the production of IFN in several cell lines while wild type SBV was not, showing that SBV NSs inhibits the IFN response of the host (Varela et al., 2013). SBV NSs has also the ability to induce the degradation of the RPB1 subunit of RNA polymerase II in vitro, thus inhibiting transcription and protein synthesis. The inhibition of the IFN response may be a consequence of this global inhibition of transcription (Barry et al., 2014). Besides, a transcriptomic study showed that, in vitro, SBV NSs elicits a shutdown in the expression of genes involved in innate immunity. Nevertheless, this shutdown is incomplete as a few antiviral genes were still induced during SBV infection (Blomström et al., 2015).

Table of contents :

1 Introduction
2 State of the Art
2.1 Discovery of a new virus
2.1.1 History
2.1.2 Structure
2.1.3 Phylogeny
2.2 Epidemiology
2.2.1 Susceptible species
2.2.2 Transmission
2.2.3 Geographical repartition
2.2.4 Risk factors
2.3 Clinical signs and lesions in affected animals
2.3.1 Non-pregnant adults
2.3.2 Pregnant females and their offspring
2.3.3 Hypotheses on the pathogenesis of the lesions in fetuses and newborns
2.4 Impact on livestock farming, on wild ruminants
2.5 Diagnostics and preventive measures
2.5.1 Diagnostics
2.5.2 Preventive measures
3 Aims of the Thesis
4 Experiments
4.1 Pathogenesis in domestic ruminants
4.1.1 Infection of adult sheep (published paper)
4.1.2 Infection of adult, non-pregnant goats
4.1.3 Infection of adult goats around the time of insemination and during pregnancy
4.2 Circulation in wild and exotic ruminants
4.2.1 Free-ranging wild ruminants in France
4.2.2 Wild and exotic ruminants kept in zoos in France and the Netherlands
5 Discussion
5.1 Pathogenesis of the infection with SBV: hypotheses drawn from experimental infection in domestic ruminants
5.1.1 Pathogenesis of the infection in males and non-pregnant females
5.1.2 Pathogenesis of the infection in pregnant females
5.2 Rapid and broad dissemination of the virus among wild and exotic ruminants
5.2.1 A quick spread in many species and in various ecosystems
5.2.2 Wild ruminants: do they play a role in SBV dissemination in domestic ruminants?
5.3 Consequences in the field: impact and preventive measures
5.3.1 Domestic ruminants
5.3.2 Wild and exotic ruminants
6 Future directions
6.1 Pathogenesis of the infection in goats
6.2 SBV circulation in wild and exotic ruminants
7 Conclusion
8 References
9 Appendix


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