Establishment Of An Assay Based On Pseudotype Virus For Detection Of Neutralizing Antibodies Against Classical Swine Fever And Localization Of E2 Protein In The Infected Cells

Classical swine fever (CSF) caused by the Classical swine fever virus (CSFV) is an important contagious and fatal pig disease with widespread economic losses. Vaccination is of vital importance as an effective way to the prevention of CSFV infection, so it is essential to establish a stable and effective neutralizing antibody detection to test the CSF vaccine.Pseudotype CSFV (VSVâ–³G*CSF) refers to the process in which the enveloped protein gene (G) was replaced by the green fluorescent protein (GFP) gene and VSVâ–³G* was formed, containing a new reporter gene. CSFV envelope protein from external sources can reassemble with VSVâ–³G* and integrate into a one-off infectious VSV (Vesicular Stomatitis Virus) recombinant virus: VSVâ–³G*CSF. VSVâ–³G*CSF contains the genetic material of VSV and the capsule membrane integrated with the character of the infection of CSFV envelope protein. This kind of the inconsistency between genotype and phenotype is called pseudo-type, with such features of CSFV is called pseudotype virus of CSFV.In this study, we constructed a recombinant eukaryotic expression plasmid pCAGG-E012 to express all of CSFV envelope proteins Erns, E1 and E2 by cis. According to the bovine viral diarrhea virus envelope glycoprotein E2 and to partially delete the different fragments by using the biology software to compare and analysis. The result of the expression indicted that after the cis expression of all CSFV envelope glycoproteins based on the recombinant eukaryotic expression plasmid pCAGG-E012, the titer of VSVâ–³G*CSF is from 10² to 10³Â·mL^-1, and the infection can be neutralized with the positive serum anti CSFV.The traditional methods for detecting CSF antibody like ELISA and indirect immunofluorescence assay have a series of flaws in urgent need of improvement, such as time-consuming, imprecise and low specificity. In order to improve the efficiency of neutralizing antibody detection and biological safety, and then to study the mechanism of CSFV infection, this study developed a replication-defective VSV/CSFV pseudovirus (VSVâ–³G*CSF), as a model of CSFV infection to replace the wild-type CSFV virus as a novel neutralization assay to make the neutralization antibody detection process more convenient and accurate. The study shows that 200 pfu of VSVâ–³G*CSFV could be neutralized by the standard positive CSF serum which is diluted 150 times. Using this method to test 10 serum samples, 7 were divided into positive and 3 were divided into negative, which have case detection rate of 90% compliance with that of commercial kit. Therefore, the method can be used as a new method in CSF antibody detection and has good application prospects.Few reports are known for pathogenic mechanism of CSFV, some specific factors could significantly affect the results of CSFV infection, such as amino acid mutations in CSFV structural protein and changes of the major antigens E2 glycosylation site affect the virulence of CSFV and the combination of virus and target cells. The interaction of virus and cell is poorly understood, especially on the CSFV infection leading to host cell protein expression and biological function.In this study, E2 protein locations on CSFV infected cells at different time point were discussed. CSFV infected PK-15 cells were detected by laser positioning confocal microscopy at 10, 20, 35 and 60 min, indicating that during the process of infected CSFV, E2 protein was attached to the cell surface of the infected cells, then entered the cell gradually, stayed in the cytoplasm, E2 do not enter the nucleus, so we can draw a conclusion that E2 protein played an important role in CSFV attachment and entry. This study provides a technical support for further study of CSFV infection mechanism.Although the preparation of pseudo-type virus costs enormously, and it is difficult to achieve large-scale production at the same time, the titer of VSVâ–³G*CSFV virus is lower than that of the wild type CSFV, it can be used as an alternative to the wild-type CSFV in neutralizing antibody detection, making the detection of CSFV antibodies more rapid, direct-viewing and secure, just because of its good bio-security and its ease of carrying the reporter gene for qualitative or quantitative analysis. On the other hand, pseudo-type virus VSVâ–³G*CSFV can be used as a delivery tool of E2 gene to study the specific location of E2 protein in CSF infected cells at different time points, which can dynamically show the process that E2 protein from the adsorption to entry and the relationship between the infection dose and infection time. This result may be helpful for elucidating the CSFV infection mechanism.