Using mutation PCR, we cloned the target gene containing 421-480nt (141-160aa) and 598-639nt (200-213aa) of VP1 gene of foot and mouth disease virus (FMDV) into the deleted region (508-532aa) of Nsp2 gene of a highly pathogenic porcine reproductive and respiratory syndrome virus derived vaccine strain (HuN4-F112) that was used as vector. The recombinant cDNA was in vitro transcribed followed by transfection of BHK-21 cells for 36 h. Then, the supernatant of the cell culture was continuously seeded to monolayer of MARC-145 cells for recovery of the recombinant virus. CPE was obviously visible after a couple of passages in the seeded MARC-145, and the rescued virus (designated as rPRRSV-F112-O/VP1ep) was identified by Mlu I digestion, sequencing and immunofluorescence assay. Meanwhile, expression of inserted FMDV epitopes was also detected by indirect immunofluorescence assay with polyclonal antibodies against VP1 protein of FMDV. The analysis of biological characteristics shows that the titer of the rescued recombinant PRRSV (TCID50 = -log10(-6.75)/0.1 mL) was similar to its direct parental virus rHuN4-F112-delta508-532, but higher than rHuN4-F112.
Animals ; Antigens, Viral ; immunology ; Base Sequence ; Capsid Proteins ; immunology ; Cell Line ; Cysteine Endopeptidases ; genetics ; Epitopes ; genetics ; Foot-and-Mouth Disease ; immunology ; prevention & control ; Foot-and-Mouth Disease Virus ; genetics ; immunology ; Molecular Sequence Data ; Mutation ; Porcine respiratory and reproductive syndrome virus ; genetics ; immunology ; Recombination, Genetic ; Swine ; Transfection ; Vaccines, Attenuated ; genetics ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; Viral Vaccines ; genetics ; immunology

Objective: To analyze the risk factors of cervical anastomotic leakage after thoracoscopic-lapacoscopic esophagectomy. Methods: 530 patients with esophageal cancer underwent thoracoscopic-lapacoscopic esophagectomy at the Cancer Hospital, Chinese Academy of Medical Sciences from Jan 2011 to Dec 2015. The demographic, surgical and clinical data of patients were retrospectively analyzed. Multivariate logistic regression was used to evaluate risk factors of cervical anastomotic leakage in these patients. Results: A total of 530 patients undergoing thoracoscopic-lapacoscopic esophagectomy were enrolled in this study. There were 421 males and 109 females. The mean age was (59.40±8.08) years old, and 91 patients with cervical anastomotic leakage. Sigle factor analysis revealed that the risk grading by American Society of Aneshesiologists, previous history of chest surgery, respiratory comorbidity, diffusion capacity for carbon monoxide of the lung, operation time, anastomosis, average days of postoperative hospitalization, death within 30 days after surgery, respiratory complications, pleural effusion or empyema, and poor healing of the incision were statistically associated with cervical anastomotic leakage (all P<0.05). Multivariate analysis showed that previous history of chest surgery, hepatic insufficiency, manual anastomosis, prolonged postoperative hospitalization, and poor healing of the incision were independent risk factors for cervical anastomotic leakage after thoracoscopic-lapacoscopic esophagectomy (all P<0.05). Conclusions Previous history of chest surgery, hepatic insufficiency, poor healing of the incision, manual anastomosis and prolonged postoperative hospitalization were significantly associated with cervical anastomotic leakage after thoracoscopic-lapacoscopic esophagectomy. It′s important to strengthen perioperative nursing and surgical techniques to prevent anastomotic leakage after thoracoscopic-lapacoscopic esophagectomy.