Isolation Of Respiratory Syncytial Virus And Genetic Variation Analysis Of F Protein

PART1ANALYSIS THE DIFFERENCE BETWEEN NEST-PCR ANDVIRUS ISOLATION FOR THE DETCTION OF RSV ANDTHEIR CLINICAL FEATURESObjective：Comparison the difference between Nest-PCR (N-PCR)and virus isolation for the detection of RSV, and reveal potential clinicalfeatures of these differences.Methods: From Januray2010to Auguest2012, nasopharyngealaspirates were collected from the inpatients with respiratory infection. BothN-PCR and virus culture were performed to detect RSV. Clinicalinformations were collected for statistic analysis.Results: There were1143cases totally. For N-PCR,458were RSVpositive (total positive rat:40.1%;31.7%for RSV A;7.7%for RSV B;0.7%for both RSV A and RSV B). For virus isolation,204were positive(17.8%). Comparison the results of N-PCR and virus isolation showed:165cases were positive (P+I+) and646cases were negative (P-I-) by both methods (identity was70.1%) and the most difference group (P+I-) wasN-PCR positive but virus isolation negative group (293,25.6%). Whencompared to P-I-group, the clinical features of P+I-group were as follows:younger; longer hospital stays; remarkable season distribution (with peak inwinter and lowest in summer); lower percentage of fever; higherpercentage of cough, wheezing, dyspnea, severe pneumonia and respiratoryfailure; all these differences were statistically significant（P<0.05），thesemanifestations were the clinical features of RSV infection. When comparedto P+I+group, the P+I-group were with longer symptom duration beforeadmitted to hospital (P=0.005) and with lower percentage of wheezing(P=0.009).Conclusion: The differences between N-PCR and virus isolation forthe detection of RSV were related with duration of symptoms prior tohospitalized. As the time pass by, the viral load would decrease and thenresult in lower positive rate of RSV isolation.The clinical features of P+I-group were also associated with lower viral load because of longer durationof symptoms prior to hospitalized. Both sensibility and specificity is wellfor the detection of RSV by N-PCR. PART2ASSESSMENT OF RESPIRATORY SYNCYTIAL VIRUSFUSION PROTEIN AND THE CAPACITY OFPALIVIZUMAB NEUTRALIZE TO CLINICAL STRAINSBackground: Respiratory syncytial virus (RSV) causes respiratorytract infection, particularly acute lower respiratory tract infection (ALRTI)in early childhood. The RSV fusion protein (F protein) is an importantsurface protein, and the target of both cytotoxic T lymphocytes (CTL) andneutralizing antibody; The only licensed drug which used to preventchildren with high risk factors from RSV infection palivizumab also bind toF protein; thus, study the genetic diversity of the RSV F protein. may beuseful for vaccine research.Object: To study genetic diversity of RSV F protein, mutations locatedin T cell epitopes and the binding ability of palivizumab to clinical strainscollected in China.Methods: A total of1800nasopharyngeal aspirates (NPAs) ofhospitalized children with ALRTI were collected for virus isolation betweenJune2009and March2012. The NPAs were used for RSV isolation, and thenribonucleic acid (RNA) was extracted from the RSV clinical strains. cDNAwas synthesized, the full F gene were amplification by polymerase chainreaction (PCR) and then sequenced. Phylogenetic analysis and the study of mutations located in CTL epitopes were performed by MEGA software.Western blot and micro-neutralization were carried out to evaluate thebinding ability of palivizumab to RSV clinical strains.Results: There were333RSV-positive cases (277cases of RSV A,55of RSV B, and one with both RSV A and RSV B), accounting for18.5%oftotal cases.130clinical strains (107of RSV A,23of RSV B) were selectedfor F gene sequencing. Phylogenetic analysis revealed that the F gene ishighly conservative, with significant amino acid changes at residues16,25,45,102,122,124,209, and447; Mutations at histocompatability leukocyteantigen (HLA)-restricted CTL epitopes were also observed. Variations inRSV A F protein at the palivizumab binding site276(N→S) increasedbetween2009and2012and became predominant. Western blot analysis andmicroneutralization data showed substitution at residues276(N→S) in RSVA that did not cause resistance to palivizumab.Conclusion: The RSV F gene is geographically and temporallyconserved with certain mutations. These data could be helpful for thedevelopment of vaccines against RSV infection.