Pathways Of PHEV Invasion Into The CNS And Mechanisms Of Endoplasmic Reticulum Stress Mediated Neuronal Antiviral Response

Porcine hemagglutinating encephalomyelitis virus（PHEV）,a member of the genus betacoronavirus,is one of the six coronaviruses known to infect pigs.Like other coronaviruses of the same genus,such as SARS-Co V,MERS-Co V,and SARS-Co V-2,PHEV infects upper respiratory tract in adult pigs casing influenza-like symptoms such as coughing and sneezing.However,PHEV is typically neurotropic and transmits along the peripheral nerve to the central nervous system（CNS）,causing obvious neurological symptoms and neurodegenerative changes,including neuron degeneration,neuronal axon growth dysplasia,dendritic spines unstable formation and neurite irregular expansion.CNS invasion and nerve damage are important causes of high morbidity and mortality in infected pigs.However,the pathways of PHEV invasion into the host CNS remain unclear.In this study,a mouse model was established by intranasal inoculation of PHEV to mimic respiratory tract infection.All mice died within 6 days post-infection（dpi）,and obvious neurological symptoms such as arching,screaming,paralysis of hind limbs were observed on 4 dpi.The viral antigen was mainly distributed in the brain and spinal cord,indicating that PHEV had obvious neurotropic characteristics.In the brain,neurons were the target cells of PHEV.Consistently,obvious neurodegenerative pathological changes,including perivascular sheath,neuronal degeneration,glial nodules,neuron loss,synaptic number and synaptic protein expression decrease in different brain regions were observed.The clinical symptoms and pathological changes of infected mice were similar to those of infected piglets,suggesting that mice could be used as an ideal animal model for studying nerve injury caused by PHEV.The distribution of the virus in the brain of mice inoculated with PHEV was detected to clarify the pathways of PHEV invasion into the CNS.At 3 dpi,the virus mainly distributed in the olfactory bulb,the olfactory cortex and the piriform lobe.Therefore,the olfactory nerve may play an important role in the PHEV neuroinvasion.The co-localization of PHEV and OMP-labeled olfactory neurons was observed in the olfactory region of the nasal mucosa of infected mice,and the virus positive signal was distributed along the olfactory nerve,indicating that PHEV could reach the olfactory bulb through the olfactory nerve.Destruction of olfactory nerve endings in the nasal mucosa by ZnSO₄ and Triton X-100 significantly delayed the time of death in mice,further demonstrating that olfactory nerve can serve as a pathway for PHEV CNS invasion.However,neither drug had a significant effect on final mortality or viral load in brain tissue,suggesting that other neuroinvasive pathways may be involved.The virus distribution in the brain tissues of mice at different time points after ZnSO₄ treatment was detected.Different from the virus signal first detected in olfactory bulb before,the virus antigen first appeared in the brain stem after drug treatment and spread to the whole CNS in the end.Immunofluorescence showed that viral antigens were distributed along the trigeminal nerve,and PHEV was co-localized with trigeminal ganglion neurons.These results suggest that the trigeminal nerve may serve as another pathway for CNS invasion of PHEV.In the nasal cavity,PHEV was mainly distributed in the respiratory and olfactory regions.PHEV infection resulted in epithelial detachment and inflammatory cell infiltration in the respiratory and olfactory regions.In the olfactory bulb,PHEV was mainly distributed in the mitral cell layer,and the positive signal of PHEV could also be detected in other layers.PHEV infection caused obvious inflammatory reaction and abnormal neuronal development in the olfactory bulb.Behavioral experiments showed that PHEV infection could lead to olfactory and taste dysfunction.Compared with other cells,neurons have more abundant endoplasmic reticulum（ER）,which can be seen under light microscope.In this study,we found that PHEV activated ERS and UPR-related IRE1,PERK,and ATF6 signaling pathways in vivo and in vitro.Further study found that ERS negatively regulate viral replication.RNA interference,specific inhibitor treatment and overexpression were used to study the effects of ERS pathways to viral inhibition.The results showed that the IRE1 signaling pathway did not participate in the replication process of PHEV,while the activation of ATF6 signaling pathway promoted viral replication.Only the activated PERK signaling pathway could inhibit the virus replication.Activated PERK phosphorylates the eukaryotic translation initiation factor（e IF2α）,leading to antiviral action.We then examined several other e IF2αphosphorylated kinases,showing that in addition to PERK,the double stranded RNA dependent protein kinase（PKR）could also phosphorylate e IF2αto perform a similar function.Finally,we investigated the antiviral mechanisms of the PERK/PKR-e IF2αsignaling pathway.It is known that e IF2αplays an important role in protein translation initiation as a translation initiation factor,and phosphorylated e IF2αcan inhibit translation initiation and lead to protein synthesis stagnation.First,we examined the total protein translation level of PHEV-infected cells,and demonstrated that phosphorylation of e IF2αinhibited PHEV replication by reducing the total protein translation.Meanwhile,phosphorylated e IF2αcan also significantly inhibit PHEV replication by promoting the stress granules（SG）formation.In conclusion,after intranasal infection with PHEV in mice,the virus infects the olfactory and respiratory mucosa in the nasal cavity and invades the CNS along the olfactory and trigeminal nerve,causing nerve damage.Infected neurons triggered ERS,and activated all three UPR branches,with the activated PERK pathway predominantly accounting for PHEV replication inhibition.Moreover,the PKR-e IF2αpathway was also activated by ds RNA following PHEV infection.PERK and PKR acted collaboratively to suppress PHEV replication by inhibiting protein translation and facilitating SG formation.The results of this study could discover the roles of neural ER Stress during PHEV infection and provide a new theoretical basis for in-depth analysis of PHEV pathogenesis.