Isolation, Identification Of Porcine Alveolar Epithelial Type Ⅱ Cell And Its Infection With HP-PRRSV

Porcine reproductive and respiratory syndrome virus (PRRSV) is a enveloped,single-stranded positive-strand RNA virus, which caused porcine reproductive and respiratorydisorder. In2006, a highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV) strain was isolated from a number of swine farms in more than10provincesand cities, which was characterized by discontinue missing of30amino acids in NSP2, andcan cause high fever, high morbidity, and high mortality in pigs of all ages. The diseasequickly spread throughout the country, seriously endangered the pig industry and the socialeconomy. Therefore, understanding the pathogenic mechanism of highly pathogenic porcinereproductive and respiratory syndrome virus is of great importance.The pigs are the only natural host to PRRSV. PRRSV mainly causes respiratory injury inpiglets, and the virus mainly distributes in immune organs and lung. Cells that can be infectedwith PRRSV mainly include macrophages and lymphocytes. The more active these cells, themore serious damage to the body.PRRSV also causes serious injury of lungs, while alveolarepithelial type Ⅱcells (AECⅡ) are regarded as stem cell of the alveolar epithelium, andplays an important role in the normal cell renewal and damage repair process. To invstigatethe relationship of lung damage caused by PRRSV and the damage repair function of PAECⅡ.We isolated and purified PAECⅡ first. Then we detected whether there were any receptorsnecessary for PRRSV infection. PAECⅡwere infected with HP-PRRSV, and the morphologychanges at different time points after infection were observed. These results lay the foundationfor the future experiments and provide practical guidance for prevention, diagnosis, treatmentand drug development. The results were as follows:(1) Bronchoalveolar lavage and enzyme digestion were used to isolate porcine alveolarepithelial typeⅡcells from30days old healthy piglets, and then the cells were purified byusing porcine IgG coated with dishes. The characteristics and purity of these cells wereidentified by electronic microscopy and alkaline phosphatase staining. Results showed thatmixed enzyme digestion combining with IgG adsorption produced PAECⅡ of rather highpurity. The special structure of PAECⅡ, lamellar bodies and microvilli, were observed byelectronic microscopy, suggesting that the cells we isolated could be used in the followingresearch. （2）First, RT-PCR was applied to detect the CD163receptor on PAECⅡ which wasnecessary for PRRSV infection. Results showed that PAECⅡ had the receptor CD163,suggesting the possibility of PRRSV infection. Then, the PAECⅡ was incubated with HuN4strain of PRRSV. The CPE was observed at0h,12h,24h and48h post infection(hpi).Cellsgathered together at the beginning of growth; at12hpi, cell mass gathered more obviously; at24hpi, part of cells showed death and more irregular configuration, and the nucleus becameprotruding; at48hpi, most of cells showed death, dispersion and cell disruption. At last, weused RT-PCR technique to detect PRRSV N gene in the cells, and indirectimmunofluorescence method to detect the PRRSV N protein in cells, with expression ofPRRSV receptors CD163and vimentin detected in PAECⅡ after PRRSV infection. Theseresults showed that both PRRSV N gene and N protein can be detected in the cell afterinfection with HPPRRSV of HuN4strain, and confirmed that PRRSV can infect PAECⅡand can replicate and proliferate within the cell as well, suggesting that PAECⅡ could beused as another model of PRRSV pathogenicity investigation.