Immunohistochemical insights into Saffold virus infection of the brain of juvenile AG129 mice
© The Author(s). 2016
Received: 27 April 2016
Accepted: 21 November 2016
Published: 25 November 2016
Saffold Virus (SAFV) is a human cardiovirus that is suspected of causing infection of the central nervous system (CNS) in children. While recent animal studies have started to elucidate the pathogenesis of SAFV, very little is known about the mechanisms behind it.
In this study, we attempted to elucidate some of the mechanisms of the pathogenesis of SAFV in the brain of a juvenile mouse model by using immunohistochemical methods.
We first showed that SAFV is able to infect both neuronal and glial cells in the brain of 2 week-old AG129 mice. We then showed that SAFV is able to induce apoptosis in both neuronal and glial cells in the brain. Lastly, we showed that SAFV infection does not show any signs of gross demyelination in the brain.
Overall, our results provide important insights into the mechanisms of SAFV in the brain.
Saffold Virus (SAFV) is a novel human cardiovirus identified in 2007 from the stool sample of a child with a fever of unknown origin . Phylogenetic analysis revealed that SAFV is closely related to Theiler’s murine encephalomyelitis virus (TMEV) [2, 3], a virus well studied for its neurovirulence . Since then, SAFV have been isolated in children from multiple continents [1, 5–11] and have been shown to be highly prevalent in humans . Recently, SAFV was found in the cerebrospinal fluid (CSF) of a previously healthy child that experienced sudden death , and in stool samples of children with non-polio acute flaccid paralysis , suggesting that the virus might be able to cause serious invasive infection of the central nervous system (CNS) of children . TMEV, a virus similar to SAFV, is divided into two strains based on their neurovirulence after intracerebral inoculation ; the GDVII strain causes acute fatal poliomyelitis, killing all infected mice within 2 weeks, while DA strain causes milder encephalomyelitis, which then progresses to persistent infection and progressive demyelination reminiscent of multiple sclerosis . It has been suggested that infection and apoptosis of neurons are responsible for fatal outcomes, while persistence in and subsequent apoptosis of glial cells such as oligodendrocytes is responsible for persistent demyelination , suggesting that the cell type that the virus infects and persist in hugely determines the outcome of infection.
In this short study, we wanted to examine the infectivity of SAFV in the juvenile (2 week old) brain by studying the cell types it infects. We further attempted to elucidate the effects of the infection. In order to do that, we used AG129 mice which we have previously shown to be a good model for SAFV infection in the CNS . We used previously isolated genotype 3 SAFV  as a model of SAFV infection due to its high prevalence . Infectious clone used in this study was obtained as previously described in Tan et al. . We then studied brain tissue of infected animals using immunohistochemical methods to elucidate some of the mechanisms involved in the pathogenesis of SAFV. Brain tissue used in this study were either from AG129 mice (B and K Universal, North Humberside, UK), or FVB/NTac mice (Haplotype H2q) (InVivos Pte Ltd. Singapore). All animal experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Temasek Life Sciences Laboratory, Singapore (IACUC approval number TLL-14-025). Two week-old AG129 mice (n = 6) and 3–4 week old AG129 mice (n = 6) were inoculated i.p. with 5 × 105 TCID50/mouse of SAFV. Mice were sacrificed on day 6 or 35 (days post infection) dpi as described below. FVB/NTac mice (n = 6) were inoculated intracranial (I.C.) with 5 × 105 TCID50/mouse of SAFV. To obtain brains, AG129 mice were anesthetized intraperitoneal (i.p.) with Ketamine (100 mg/kg)/Xylazine (20 mg/kg) and transcardially perfused with 50 ml phosphate-buffered saline (PBS) followed by 100 ml 4% paraformaldehyde (PFA) in PBS. The mice were then decapitated and the brains and spinal cord were removed and post-fixed in 4% PFA overnight at 4 °C. Brains were then transferred to a solution of 20% sucrose in PBS and stored overnight at 4 °C before freezing over liquid nitrogen and stored at −80 °C. The brains were then embedded in Shandon M-1 Embedding Matrix (Thermo, Kalamazoo, MI, USA) and sectioned through a cryostat (16 um).
Information about SAFV infection has increased in the past few years, with data concentrated on respiratory and gastrointestinal tract infections , however data informing CNS infection remains relatively low, despite its importance shown by clinical samples of SAFV found in children with neurological symptoms [6, 11, 12]. In this short paper, we examined SAFV infection of the brain in an animal model and presented important new insights to the neuropathogenesis of SAFV. We showed that SAFV is able to infect both neuronal and glial cells in the developing brain of a mouse model, causing apoptosis in both. We however showed no gross demyelination in the brains of the infected animals. We hence provided important insights, extending the findings of our previous paper , and provided a strong basis on which the mechanisms of neuropathogenesis of SAFV can be further examined.
This work was supported by Temasek Lifesciences Laboratory. We thank Kabilan and Govindarajan for their assistance with the maintenance of the mouse strains. We thank Chua Kaw Bing for their generous gift of reagents.
SZKT and MP designed the study. SZKT performed the experiments. SZKT, MP analyzed the data. SZKT wrote the paper and MP reviewed the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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