The Mechanism Of Astrocyte Activation During PHEV Infection

Porcine hemagglutinating encephalomyelitis virus（PHEV）is a typical neurotropic coronavirus,which mainly invades in the Central nervous system（CNS）and leads to death in infected animals.In the process of virus infection,the specific role and mechanism of the innate immune response of CNS remain to be further studied.Astrocytes are the most abundant type of cells in CNS,which are responsible for neurotransmitter transport and recycling,ion homeostasis,energy metabolism,infection defense and maintenance of blood-brain barrier integrity,etc.Astrocytes can be activated during the infection of various neurophagoviruses,such as vesicular stomatitis virus（VSV）,herpes simplex virus（HSV）,etc.Activated astrocytes can play an antiviral role by regulating the innate immune function.However,whether PHEV infection causes the activation of astrocytes and the role of activated astrocytes has not been reported.Astrocytes that play a role in innate immunity in the central nervous system.To clarify whether astrocytes are activated in the CNS of mice following PHEV infection and to investigate its mechanism.We prepared a mouse model of PHEV infection,BSCs and primary astrocytes.3-week-old BALB/c mice were inoculated with 30μL PHEV(TCID₅₀ was 10^4.2/0.1m L)or equivalent PBS（0.01 M,p H 7.4）by intranasal route and GFAP was examed by using q RT-PCR,Western Blotting and indirect immunofluorescence（IF）at different time points after PHEV infection in this study.The results showed that the expression level of GFAP was significantly increased 3 days after inoculation of PHEV compared with control mice,and the expression level of GFAP increased gradually over time.IF results further confirmed the activation of astrocytes in PHEV infected mice,characterized by increased cell number and concentrated distribution,as well as cell hypertrophy and proliferation.The results indicated that PHEV infection could induce the activation of astrocytes in the brain of mice.We found an increase in GFAP staining at 24h after infected PHEV in BSCs,using indirect immunofluorescence,demonstrating that the brain’s intrinsic factors are sufficient to activate astrocytes and that this process can be independent of any peripheral immune cells.In vitro and in vivo experiments confirmed activation of astrocytes during PHEV infection.BALB/c mice inoculated nasally with PHEV showed no co-localization between GFAP and PHEV antigen,and primary astrocytes inoculated with PHEV also showed the same results.In vivo and in vitro experiments demonstrated that PHEV could not infect astrocytes,and the activation of astrocytes was not caused by direct viral infection.Mouse primary astrocytes were treated with the homogenate supernatant of PHEV-infected mouse brain tissue.The expression of GFAP was significantly increased by western blot.The expression of GFAP in primary astrocytes could not be increased by the supernatant of brain tissue homogenate of infected mice after treatment with anti-IFN-β.These results suggest that interferonβresults in the activation of astrocytes during Porcine hemagglutinative encephalomyelitis.The expression of GFAP in primary astrocytes of mice was significantly increased after the addition of exogenous interferonβ.This study showed that astrocytes were activated during PHEV infection,and the activation could be mediated by interferonβ.In conclusion,this study confirmed that PHEV infection can activate astrocytes in mouse CNS via interferonβ,These results may lay a foundation for further research on the mechanism of PHEV-induced inflammatory injury of the nervous system.