Construction Of Coronaviruses Pseudotype System And Its Application In Receptor Recognition

Coronaviruses(Co Vs) is widespread in nature causing serious gastrointestinal and respiratory system diseases with gastrointestinal, respiratory or neurological tropism. Receptor binding is the first step of viral infection of host cells and is one of the essential factors of infection and pathogenicity. Viral entry into cells is dependent on the interaction between spike protein and receptor. Figuring out the mechanisms of receptor recognition during the viral entry provides a clue for antiviral drug development.In our research, we use lentivirus pseudotype system and recombinate vesicular stomatitis virus pseudotype system with its glycoprotein(G) gene deleted as a tool to creat VSV-ΔG-S pseudoviruses and analyze the efficiency of invasion of coronaviruses. To further study the cell tropisms of transmissible gastroenteritis virus(TGEV) and porcine epidemic diarrhea virus(PEDV), they were used to infect different cell lines. Overall, our study provides a new sight of the mechanisms in viral entry. Main content are listed as follows:1. Construction of VSV-ΔG pseudotypes and VSV-ΔG-S recombinant pseudotyped virusesTo utilize the VSV infectious clone system, then G was replaced by green fluorescent protein(GFP) in VSV genome. VSV-ΔG pseudotypes can assembly other glycosylated proteins to creat the recombinant virus. First, 293 T cells were transfected with Co V-S plasmids respectively. After 24 h, the supernatant of transfected cells containing pseudotyped viruses were harvested. Different cell lines were infected with these pseudoviruses, and analyze the entry efficiency of different recombinant VSV-ΔG-S. Compared with the positive control, the packing efficiency of VSV-ΔG-S pseudoviruses is weaker, so lentivirus system was used to study.2. Analysis the entry efficiency of different coronaviruses with lentivirus systemLentivirus vector was developed based on HIV-1. Coronavirus S protein expression plasmids were transferred into 293 T cells with HIV-1 vector expressing plasmid(p NL4-3 Luc R-E-). Pseudoviruses are used to infect different cell lines. The results showed that the efficiency of SARS-Co V-S pseudoviruses infection was the highest, MHV-S and HCo V-229E-S pseudoviruses were higher, TGEV-S and PEDV-S pseudoviruses were similar and high, HCo V-OC43-S, BCo V-S and IBV-S pseudoviruses were low and different.TGEV-S and PEDV-S pseudoviruses were used to infect PK-15, Huh-7, Vero-CCL-81 and MDCK cells. The results showed that TGEV-S and PEDV-S pseudoviruses could enter into PK-15, Huh-7 and Vero-CCL-81 cells, but not enter into MDCK cells.3. Research on PEDV and TGEV cell tropismsThe live PEDV and TGEV virus were used to infect pig kidney cells(PK-15), human liver cells(Huh-7), monkey kidney cells(Vero-CCL-81) and canine kidney cells(MDCK) at a multiplicity of infection with the value of 0.5. Trypsin(10 μg/m L) was added to the cell culture medium to facilitate live virus infection. Cells infected with PEDV or TGEV were detected at 24 h after post-inoculation by indirect immunofluorescence assay. The results showed that pig kidney cells and monkey kidney cells were infected by PEDV and TGEV. Human liver cells were infected by PEDV, which indicated potential cross-spicies risk of human infection.