Detection Of Relative Expression Of PRRSV’s Four Receptors In Laiwu Black Pigs Of Major Tissues

Porcine Reproductive and Respiratory Syndrome is caused by Porcine Reproductive andRespiratory Syndrome virus, with reproductive disorders (miscarriage, stillbirth, weak child,mummy foetus) in breeding sows and respiratory symptoms and a high mortality rate inpiglets as the main characteristics of this infectious diseases. The disease was first recognizedin America in1987, and the PRRSV was isolated as the causative agent from the alveolarmacrophages in The Netherlands in1990. Subsequently, the disease has been reported inGermany, Spain, Britain, France, Denmark and other countries. The disease was firstconfirmed existing in China with indirect immunofluorescence method in1996. PRRSV isdivided into two distinct types, the European and North American types, and North Americantype is the main type in China.In order to detect the proliferation of PRRSV in local breed pigs,15Laiwu black pigs,being PRRSV negative comfirmed by RT-PCR and ELISA, were randomly divided into twogroups,10pigs were inoculated with1mL PRRSV solution (104TCID50/mL) per pig by nasalinoculation, and5pigs were inoculated with1mL PBS solution as control. We observed andrecorded the clinical symptoms of infected pigs. Two PRRSV-infected pigs andPRRSV-uninfected one were killed at3,7,14,21and28days post infection (DPI)respectively. At the same time, the pathological changes of infected pigs were observed. Then,the tissuses, including spleen, lung and the main peripheral lymphoid nodes (submandibularlymph nodes, inguinal lymph nodes and mesenteric lymph nodes) of the test pig lets, werecollected and frozen.For detected the proliferation of PRRSV in vivo of laiwu black pigs, TaqMan probe wassynthesized, and an absolute quantitive PCR method for detection of PRRSV was established.The standard curve equation was y=-3.1843x+39.241867, correlation coefficient R2=0.99.The quantitive PCR method had good specificity, reproducibility, high sensitivity, and minimum10copies of the virus could be detected. The sensitivity of the method was10to100times higher than the average PCR method. The viral loaded of PRRSV in these tissueswere tested by the quantitative RT-PCR method respectively. The results showed that PRRSVwas positive in infection group pigs, whereas was negative in control group pigs. In infectiongroup pigs, lower viral copies of PRRSV were detected at3DAI, higher viral copies ofPRRSV appeared at7to21DAI, and the viral copies of PRRSV decreased significantly at28DAI. The viral copies of PRRSV in tonsils and different lymph nodes were significantlyhigher than that in other tissues, which indicted that PRRSV had a distinct lymph tissuetropism of in Laiwu Black pigs.The Fluorescence quantitative RT-PCR methods for detecting the relative expression ofPRRSV’s receptors, including NMHCII-A, sialoadhesin, CD163and vimentin, wereestablished. The relative expression of PRRSV’s receptors in these issues were tested at3dpi,7dpi,14dpi,21dpi and28dpi. The results showed that the relative expression of receptorsincreased or decreased at different time in difference tissues. For example, the relativeexpression of PRRSV’s receptor increased in spleen at14dpi, while the relative expression ofPRRSV’s receptor decreased in amygdala at14dpi. The result indicated that the relativeexpression of PRRSV’s receptors have no obvious rule with different change trend in vivo.The situation might be leaded by the limited number of the experimental animals.