Screening And Identification Of Encephalomyocarditis Virus Cellular Receptors

Encephalomyocarditis virus (EMCV) is a non enveloped single strand RNA virus, and it is an important pathogen of zoonosis. It distributes worldwide and can infect many kinds of animals, namely has a broad host spectrum. Human infections have also been reported.Virus infected host cells is caused by viruses and cell receptor protein interactions. Although there are many researches on EMCV receptor, the main receptor of EMCV has not been determined at present. EMCV receptor research in China is still blank. Therefore, it has an important significance to study EMCV receptor in diagnosis and control of myocarditis.In this experiment, inoculated EMCV in different sources of animal and insect cells, results showed that besides wapiti kidney cells and sf9cells did not appear CPE, other cells appeared apparently CPE. The total RNA was transcripted into cDNA by reverse transcription as PCR template, in addition to the sf9cytotoxic PCR amplification without strip, other cytotoxic PCR amplified EMCV3D gene878bp. Determination of the cytotoxic TCID50valued ranged from10’425to10’8, sf9cytotoxic TCID50cannot be calculated. Therefore, the EMCV host range is very wide. In in vitro study revealed that inoculated EMCV on wapiti kidney cells did not appear CPE but the virus multiplication; sf9cells were not sensitive to EMCV.Based on this, combined virus overlay protein blot with MS analysis,14proteins were found. Through bioinformatics analysis of this14proteins, we hypothesized that annexin A2, moesin, ezrin and radixin can be used as candidate cell receptors of EMCV.Annexin A2was chosen for further study. Via annexin A2antibody competition on blocking virus infected cells, found that EMCV infection with annexin A2antibody processed cells can delay24h appeared CPE. This declared that annexin A2antibody partially blocked the effect of EMCV infected host cells. Then, we use the gene engineering technique to construct the annexin A2eukaryotic expression vector pEGFP-C1-A2, and transfected into sf9cells, then inoculated with EMCV. The results showed that annexin A2protein can be expressed in sf9cells, small amount of sf9cells appeared CPE after EMCV inoculation. It further validated the annexin A2can mediate EMCV into sf9cells.In conclusion, we found EMCV host range was very wide, annexin A2may be one of the cellular receptor for EMCV. This research lays the foundation for the further study of EMCV receptor.