A Study Of The Role Of PGRN In The Process Of PHEV Infection In Mice

Porcine hemagglutinating encephalomyelitis is an acute,contact transmissible disease of pigs caused by the porcine hemagglutinating encephalomyelitis virus（PHEV）.Clinically diseased animals experience screaming,β-convulsions,and other symptoms.Studies have confirmed that members of diverseβ-coronavirus genera including SARS-Cov-2 utilize lysosomes for replication.Previous work found that PHEV,a member of the genus neurotropic coronavirus,also hijacks lysosomes to release progeny virions and downregulates the expression of the lysosomal protein progranulin（PGRN）.But the role of PGRN during PHEV replication is not clear.To investigate the role of PGRN in the pathogenic process of PHEV,in this study,we inoculated mice via the nose drops route with PHEV,using PHEV infected PGRN knockout mice(PGRN^-/-mice)and their control mice（WTmice）as a model,and found that when WTmice eventually died from neurological symptoms such as significant wasting,convulsions,hind limb clasping,PGRN^-/-mice did not develop obvious neurological symptoms;Using Western blot,RT-PCR,and IFA assays,we found that compared with PHEV infected WTmice,PHEV infection of PGRN^-/-mice resulted in a highly significant reduction in viral content and distribution in the brain,with only a weak viral signal in the olfactory bulb;Histopathological studies revealed that the morphology of neurons in the brain of PHEV infected PGRN^-/-mice showed little change compared with that without prior exposure,but the neurons in the brain of PHEV infected WTmice were swollen and fragmented,and the neurons underwent degeneration and necrosis.In addition,nasal tissues from both groups of mice were collected at 6 DPI after virus inoculation,and the presence of viral signals at the nasal mucosa in both groups of mice was found by IFA assay,implying that PGRN deletion blocks the penetration of PHEV into the mouse CNS via the nose dropping route.Cytokine changes were measured by ELISA in the serum of the two groups of mice after venom exposure,and levels of IFN-γand TNF-αwere increased in the serum of PGRN^-/-mice compared with those in the serum of PHEV infected WTmice.Showed that PGRN deletion could block PHEV transnasal route to invade CNS in mice by regulating body immune response.PHEV infection of mice will invade the CNS of mice and cause the death of mice.In order to further explore the effect of PGRN deletion on the invasion of the CNS by PHEV,these two groups of mice were inoculated with PHEV brain.Western blot,RT-PCR and IFA detection showed that compared with PHEV infected WT mice,the content and distribution of virus in the brain of PHEV infected pgrn-/-mice were significantly reduced;The expression of TDP-43 in the brain of PHEV infected pgrn-/-mice was significantly higher than that of WT infected mice.However,the changes of cytokines in the brain and serum of the two groups of mice were detected.Compared with WT mice infected with PHEV,TNF in the brain of pgrn-/-mice infected with PHEV-α、IFN-α、IL-1βThe expression of Bcl-2 was significantly increased;TNF in serum-αThe level was significantly increased,indicating that PGRN deletion may inhibit the replication of PHEV in the CNS by regulating the body’s immune response.In addition,the colocalization of virions with lysosomes of nerve cells in the brain of the two groups of mice did not change after PHEV infection by IFA detection,but the expression of mature CTSD in lysosomes of brain tissue of PHEV infected WT mice was up-regulated compared with that of PHEV infected pgrn-/-mice,suggesting that PGRN deletion would mediate lysosomal function and inhibit the replication of PHEV in the CNS.In conclusion,this study is the first to use PHEV to infect PGRN^-/-mice in vivo through different routes,confirming that PGRN plays an important role in the replication and dissemination of PHEV,and that PGRN deletion may inhibit PHEV infection in mice by leading to relevant immune responses and lysosomal dysfunction.This provides a research basis for uncovering the mechanism of action of the lysosomal protein PGRN during PHEV replication and dissemination,as well as a reference basis for the development of potential anti PHEV drugs targeting lysosomes.