Transferrin Receptor 1 Is A Co-receptor That Assists Transmissible Gastroenteritis Virus And Porcine Epidemic Diarrhea Virus Entry Into Porcine Intestinal Epithelium

Porcine transmissible gastroenteritis and Porcine epidemic diarrhea are caused by porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus respectively,which can cause piglets severe vomiting,diarrhea,dehydration,acute intestinal villus atrophy and high mortality rate characteristics.TGEV and PEDV all can affect different species and age of pigs,especially harm to the newborn piglets(morbidity and mortality rates as high as 100%),the fattening pig carry the virus but not sick,but continuous detoxification,slow growth,low immunity,thus secondary infection with other pathogens,resulting in a huge economic loss in pig industry.TGEV and PEDV mainly infect the intestinal epithelial cells,but the mechanism of viral invasion are not clear.Transferrin receptor 1 plays an important role in clathrin mediating other virus invasion process.The first step of viral invasion is the virus-specific receptor binding on the surface of the susceptible cell membrane.Besides,a growing number of studies have found that multiple receptors may be required to participate in the process of virus invading host cells.To provide a theoretical basis for the prevention and control of TGEV and PEDV infection,we explore new receptor of TGEV invasion and the mechanism of PEDV infection by using porcine jejunum epithelial cells(IPEC-J2)and newborn piglets as the infection model.This study is divided into the following two parts:1.Transferrin receptor 1 is a co-receptor that assists transmissible gastroenteritis virus entry into porcine intestinal epitheliumTransmissible gastroenteritis virus(TGEV),the etiologic agent of transmissible gastroenteritis,infects swine of all ages,causing vomiting and diarrhea.In newborn piglets,the mortality rate is near 100%.Intestinal epithelial cells are the primary target cells of TGEV.Transferrin receptor 1(TfR1),which is highly expressed in piglets with anemia,may play a role in TGEV infection.However,the underlying mechanisms of TGEV invasion remain largely unknown.Results:Our study investigated the possibility that TfR1 can serve as a receptor for TGEV infection and enable the invasion and replication of TGEV.We observed that TGEV infection promoted TfR1 internalization,clustering,and co-localization with TfR1 early in infection,while TfR1 expression was significantly downregulated as TGEV infection proceeded.TGEV infection and replication were inhibited by occluding TfR1 with antibodies or by decreasing TfR1 expression.TGEV infection increased in TGEV-susceptible ST or IPEC-J2 cell lines and TGEV-resistant Caco-2 cells when porcine TfR1 was overexpressed.Finally,we found that the TGEV S1 protein interacts with the extracellular region of TfR1,and that pre-incubating TGEV with a protein fragment containing the extracellular region of TfR1 blocked viral infection.Conclusions:Our results support the hypothesis that TfR1 is an additional receptor for TGEV and assists TGEV invasion and replication.2.Intracellular iron levels affect the susceptibility of newborn piglets to TfR1 mediated PEDV infectionPorcine epidemic diarrhea virus(PEDV)mainly infects the intestinal epithelial cells of newborn piglets causing acute,severe atrophic enteritis.The underlying mechanisms of PEDV infection and the reasons why newborn piglets are more susceptible than older pigs remain incompletely understood.Iron deficiency is common in newborn piglets.Here we found that high levels of transferrin receptor 1(TfR1)distributed in the apical tissue of the intestinal villi of newborns,and intracellular iron levels influence the susceptibility of newborn piglets to PEDV.We showed that iron deficiency induced by deferoxamine(DFO,an iron chelating agent)promotes PEDV infection while iron accumulation induced by ferric ammonium citrate(FAC,an iron supplement)impairs PEDV infection in vitro and in vivo.In addition,PEDV infection was inhibited by occluding TfR1 with antibodies or decreasing TfR1 expression.Additionally,PEDV infection was increased in PEDV-resistant Caco-2 and HEK 293T cells over-expressed porcine TfR1.Mechanistically,the PEDV S1 protein interacts with the extracellular region of TfR1 during PEDV entry,promotes TfR1 re-localization and clustering,then activates TfR1 tyrosine phosphorylation mediated by Src kinase,and heightens the internalization of TfR1,thereby promoting PEDV entry.Taken together,these data suggest that the higher expression of TfR1 in the apical tissue of the intestinal villi caused by iron deficiency,accounts for newborn piglets being acutely susceptible to PEDV.