Efficient control of Japanese encephalitis virus in the central nervous system of infected pigs occurs in the absence of a pronounced inflammatory immune response

Abstract

Background Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia. JEV infection of mice and humans can lead to an uncontrolled inflammatory response in the central nervous system (CNS), resulting in a detrimental outcome. Pigs act as important amplification and reservoir hosts, and JEV infection of pigs is mostly subclinical. Information on virus spread in the CNS and immune responses controlling JEV infection in the CNS of pigs, however remains scarce. Methods Nine-week-old pigs were inoculated intranasal or intradermal with a relevant dose of 105 TCID50 of JEV genotype 3 Nakayama strain. Clinical signs were assessed daily, and viral spread was followed by RT-qPCR. mRNA expression profiles were determined to study immune responses in the CNS. Results Besides a delay of 2 days to reach the peak viremia upon intranasal compared to intradermal inoculation, the overall virus spread via both inoculation routes was highly similar. JEV appearance in lymphoid and visceral organs was in line with a blood-borne JEV dissemination. JEV showed a particular tropism to the CNS but without the induction of neurological signs. JEV entry in the CNS probably occurred via different hematogenous and neuronal pathways, but replication in the brain was mostly efficiently suppressed and associated with a type I IFN-independent activation of OAS1 expression. In the olfactory bulb and thalamus, where JEV replication was not completely controlled by this mechanism, a short but strong induction of chemokine gene expression was detected. An increased IFNy expression was simultaneously observed, probably originating from infiltrating T cells, correlating with a fast suppression of JEV replication. The chemokine response was however not associated with the induction of a strong inflammatory response, nor was an induction of the NLRP3 inflammasome observed. Conclusions These findings indicate that an adequate antiviral response and an attenuated inflammatory response contribute to a favorable outcome of JEV infection in pigs and help to explain the limited neurological disease compared to other hosts. We show that the NLRP3 inflammasome, a key mediator of neurologic disease in mice, is not upregulated in pigs, further supporting its important role in JEV infections.

Japanese Encephalitis Virus Persistence in Porcine Tonsils Is Associated With a Weak Induction of the Innate Immune Response, an Absence of IFNγ mRNA Expression, and a Decreased Frequency of CD4+CD8+ Double-Positive T Cells

“…After having studied the local viral replication, cytokine mRNA expression, and evolution of immune cell frequency in the tonsils, we studied the induction of the peripheral adaptive immune response upon JEV infection. In our previous publication reporting on the same pigs from the first in vivo experiment, we already showed that first JEV specific antibodies could be detected between 5 and 7 days post infection onwards (Redant et al, 2020). These results were confirmed in the pigs from the second experiment.…”

“…cDNA Synthesis and Preamplification cDNA synthesis was done as previously described (Verpoest et al, 2018;Redant et al, 2020). Following the manufacturer's instructions, extracted RNA was treated with Turbo DNase (Thermo Fisher) to eliminate genomic DNA.…”

“…Primers and probes for three reference genes (ACTB, GADPH, and HPRT1) and targets of interest [IFNa, IFNb, IFNg, TNFa, IL1a, IL2, (Kumar et al, 2004;Turtle et al, 2016;Redant et al, 2020). If possible, a duplex qPCR was performed using 6-carboxyfluorescein (FAM)-and Hexachloro-fluorescein (HEX)-labeled probes.…”

Neutrophil Recruitment and Participation in Severe Diseases Caused by Flavivirus Infection

“…Given the high degree of identity between encephalitic flaviviruses, antibody cross-protective immunity is observed. Brain damage and neuronal loss during JEV infections are associated with increased inflammation marked by increased levels of TNF-α, IL-1β, CCL5, CCL2 and NO [ 99 , 100 ]. JEV infection in humans and mice leads to cellular infiltration in the brain and spleen with major increases in neutrophil counts in the periphery and in the CSF.…”

In Vitro Infection Dynamics of Japanese Encephalitis Virus in Established Porcine Cell Lines

“…Controlled in vivo challenge models remain the gold standard for investigating the complexities of viral pathogenesis, and recent studies have provided valuable insights toward a better understanding of JEV infections in swine [24,25,27,[35][36][37]. However, host and virus factors associated with JEV pathogenesis remain poorly understood and advances in treatment and prevention in swine have been hindered by a lack of thorough understanding of the viral replication cycle and host-virus molecular interactions.…”