Analysis Of The Differences Of The Entry Pathway Between PRRSV Strain HuN4 And Vaccine Strain HuN4-F112

Porcine Reproductive and Respiratory Syndrome(PRRS),which caused by Porcine Reproductive and Respiratory Syndrome virus(PRRSV)is considered as the most important infectious disease to the pig industry.Immunization is considered as an effective way to control PRRS worldwide.Live attenuated vaccine which obtained by continuous generation on Marc-145 cells was considered as the most effective vaccine,the generation on allogenic cell line resulting huge biological difference compaired with their parents strains including growth characteristic,cellular adaptation and may also in the first step in cell entry.Our previous results also showed that PRRSV field strain and vaccine strain might use different pathway entry the cell.In this study,the HP-PRRSV wild strain HuN4 and vaccine strain HuN4 were selected as the virus model,and the main pathway for virus entry cell were studied.The results are summarized as follows:1.PRRSV entry Marc-145 and PAM cells through clathrin-mediated endocytosis pathwayPAM and Marc-145 cells were pretreated with CPZ,a specific inhibitor of clathrin,then cells were infected with either HuN4-F5 or HuN4-F112,the virion in the cells were measured by RT-PCR,IFA and Western-blot.None significant difference was found between CPZ pretreated and control groups in the binding stage neither on Marc-145 cells nor on PAMs.The results of viral copy,IFA and Western-blot showed that after pretreated by CPZ,virus entry was reduced dramaticly on Marc-145 and PAMs compared with control group.Then siRNA of clathrin heavy chain were used to interfere the expression of clathrin,the decrease viral copy and protein expression of interfere group was observed compared with control group.These results indicated that PRRSV entry PAM and Marc-145 via clathrin-mediated endocytosis pathway.2.PRRSV do not use macropinocytosis pathway during cell entrance,and action played different role during cell entrance of different PRRSV strainsNa~+/H~+exchange is the key characteristic of macropinocytosis.Amiloride(EIPA)is the specific inhibitors of Na~+/H~+exchange.PAM and Marc-145 cells were pretreated with EIPA then infected with HuN4-F5 and HuN4-F112,the results of viral copy,IFA and Western-blot showed that there was no significant difference between EIPA pretreated group and control group.Cytochalasin D(Cyt D)was widely used to inhibited actin polymerzation.After pretreatment with Cyt D,virus adsorption was not affected,but differences were found between HuN4-F5 and HuN4-F112on Marc-145 and PAM cells.On Marc-145 cells,after pretreated wtih Cyt D,significant lower viral copy and protein expression were found in HuN4-F5 pretreat group compaired with control group,no significant differenc was found in HuN4-F112 test groups.On Marc-145 cell,after pretreated wtih Cyt D,significant lower viral copy and protein expression were found in HuN4-F112 pretreat group compaired with control group,no significant differenc was found in HuN4-F5 test groups.These results showed that PRRSV do not utilize macropinocytosis pathway entry Marc-145 and PAM cells.Instead,actin is important in PRRSV entry and the usage of actin on Mrac-145 and PAM cells is the main differenct of PRRSV field strain HuN4-F5 and vaccine strain HuN4-F112 during the cell entrance of infection.3.Depletion of cellular cholesterol inhibits PRRSV enter Marc-145 and PAM cellsLipid rasfts were considered to influence PRRSV entry Marc-145 cells,and Methyl-?-cyclodextrin (M?CD)was widely used to expend cholesterol which was a important component of lipid rafts.Our results showed that the expenditure of cholesterol was not only impact virus entry Marc-145 cells but also reduce virus entry on PAM cells.These results showed that PRRSV entry is lipid raft-associated on both Marc-145 and PAM cells.These reults revealed the differences during cell entrance of PRRSV field strain and vaccine strain,all the experimental data could be useful for the future research of PRRSV attenuation.