Objective To investigate the mucosal immunity of L1 virus-like particles ( VLPs) of human papillomavirus ( HPV) types 16 and 18 plus JY adjuvant by intranasal immunization in cynomolgus. Methods Cynomolgus were immunized with low and high dosage of HPV types 16 and 18 L1 VLP with JY adjuvant for 3 times by intranasal route at weeks 0, 4 and 8, respectively, using PBS as control. Subsequently, vaginal secretion, oral secretion and nasal secretion were collected at weeks 0, 2, 4, 6, 8 and 16, respectively, and determined for mucosal immunity by ELISA.Results HPV-L1-specific sIgA antibodies were detected in all secretions, including oral, nasal and vaginal ones, the concentrations of sIgA antibody induced were much higher than those in PBS control group.There was no significant difference ( P>0.05) in sIgA antibody levels among cynomolgus vaccinated with low and high dosage of L1 VLP, as was between oral and nasal secretion ( P >0.05 ) , However, the concentrations of sIgA antibody in vaginal secretion were significant higher than those in oral and nasal secretion, differences were statistically significant ( P<0.01) .Conclusions Following intranasal immunization in cynomolgus, HPV types 16 and 18 L1 VLP with JY adjuvant can effectively induce sIgA antibody in vaginal secretion, and vaginal sIgA antibody concentrations were much higher than those in oral and nasal secretion.

Objective To evaluate the effect of JY adjuvant,which is composed of IL-2 and chitosan,on the immune response for mucosal immunization with human papillomavirus types 16 and 18 L1 virus-like particles (HPV16 + 18 L1 VLP).Methods Mice were immunized three times with HPV16 + 18 L1 VLP in the presence or absence of JY adjuvant by intramuscular and intranasal routes,respectively.Subsequently,experiments were undertaken to detect serum IgG antibody,neutralizing antibody and respiratory tract washes sIgA antibody titers and cellular immune response.Results Following intranasal immunization,serum IgG antibody titers were much higher in HPV16 + 18 L1 VLP with JY adjuvant group than that in VLP without adjuvant group (P ＜ 0.01),after intramuscular immunization,serum IgG titers induced by VLP with or without JY adjuvant were the same; following intramuscular immunization,neutralizing antibody titers induced by adjuvant-containing HPV16 + 18 L1 VLP were higher than those by adjuvant-free VLP,following intranasal immunization,only serum neutralizing antibody was detected in adjuvant-containing VLP group; after intranasal immunization,lung washes sIgA concentration were much higher in HPV16 + 18 L1 VLP with JY adjuvant group than that in VLP without adjuvant group (P ＜ 0.05) ; following intranasal and intramuscular immunization,respectively,the number of spot forming cells were much higher in HPV16 + 18 L1 VLP with JY adjuvant group than that in VLP without adjuvant group (P ＜ 0.01,P ＜ 0.05,respectively).Conclusion JY adjuvant enhanced the cellular,humoral and mocosal immunities induced with HPV16 + 18 L1 VLP by intranasal route,while showed no significant influence of the adjuvant was seen in the group immunized by intramuscular route.

Objective We develop a rapid,specific,sensitive tandardized SOP.And initial application for 200 nasopharyngeal aspirates of children with related pathogens of acute respiratory tract infections in BeiJing area.Methods To developed nested PCR and TaqMan probe real-time fluorescence quantitative PCR method for detection of KIPyV and WUPyV'S gene,and then the sequences of gene fragments are analyzed.Evalution of two assays from 200 nasopharyngeal aspirates.Results In this study,sensitivity of TaqMan probe real time fluorescent quantitative PCR assay was higher than one of nested PCR (500 copies/μl),and both assays did not show any positive amplification in detetion of other respiratory virus.Coefficient of varience of KIPyV and WUPyV are less than 2.9％ and 1.95％ respectively in the repeatability detection.The detection rates of KIPyV,and WUPyV were 1.5％ and 8％ in nested PCR assay and 12％ and 14％ in real time Fluorescent quantitative PCR assay respectively.Conclusion This study established good sensitivity and reproducibility,high specificity and rapid method for detection nucleic acid of these polyomaviruses that have good prospects on the clinical application.

Objective:To study the sequence characteristics and genetic variation of a human respiratory syncytial virus (HRSV) subtype A genome in Beijing.Methods:The genomic RNA of HRSV from nasopharyngeal aspirate samples was sequenced and obtained a whole genome sequence of HRSV A subtype. The phylogenetic tree was constructed with reference sequences of other HRSV strains. The major proteins were compared and single nucleotide polymorphism analyzed. In addition, the N-glycosylationsites of F and G protein were predicted.Results:Phylogenetic tree and homology analysis results suggest that the HRSV strain (RSVA/Beijing-China/2017) was the A subtype ON1 genotype. Nucleotide and amino acid variation analysis showed that G protein, F protein and L protein had some substitutions. Analysis of amino acid variation sites showed that amino acid substitution (L142S) occurred at position 142 of G protein. For F protein, there were two substitutions, which were S105N in the P27 peptide (110-136aa) and C69Y in the antigen sites ? (62-69 aa and 196-210 aa). The prediction of N-glycosylation sites revealed that there were 5 N-glycosylation sites of F protein and 4 N-glycosylation sites of G protein in this strain.Conclusions:The HRSV strain obtained in Beijing belongs to A subtype ON1 genotype. The G, F and L proteins have large variations, and 22 amino acid substitutions have occurred in the G and F proteins.

Objective:To investigate the epidemiological characteristics of human bocavirus 1 (HBoV1) and to analyze the genetic variation.Methods:A total of 2 848 nasopharyngeal aspirate (NPAs) specimens were collected from hospitalized children with acute respiratory tract infections (ARTI) in Beijing Friendship Hospital from April 2017 to March 2019, and HBoV1 was detected by quantitative real-time PCR. Epidemiological analysis was carried out based on the clinical information of the patients. The nested PCR method was used to amplify the NP1 and VP1 genes of HBoV1 for homology analysis. Maximum clade credibility tree (MCC tree) and genetic polymorphism map were constructed to analyze the time evolution of HBoV1 VP1.Results:HBoV1 was detected in 90(3.16%) of 2 848 NPAs, most (93.33%, 84/90) HBoV1-positive cases were among children <5 years of age. HBoV1 could be detected throughout the year with a higher prevalence 7.23% (18/249) in October. Of the 90 HBoV1-infected cases, the main clinical symptoms were fever and cough, 44(48.89%) were co-infected with other respiratory viruses; 55 NP1 sequences and 47 VP1 sequences were obtained by nested PCR amplification, phylogenetic analysis showed that the nucleotide homology was 98.9%~-100% and 99.1%~-100%, respectively. MCC tree showed that the HBoV1 VP1 gene sequence obtained in this study appeared in two adjacent clades, the gene evolution was stable.Conclusions:HBoV1 is one of the common viruses that cause respiratory infection among children in Beijing. HBoV1 genetic evolution is relatively stable, but it still needs to be monitored continuously.

Objective:To construct 2019 novel coronavirus (2019-nCoV) 614D and 614G pseudovirus by HIV lentivirus packaging system and explore their biological specificity.Methods:The recombinant expression plasmids pCDNA3.1-614D and pCDNA3.1-614G were transiently cotransfected with psPAX2 and pLenti CMV Puro LUC into 293T cells respectively. After 72 hours, the supernatant was collected and ultracentrifuged with 20% sucrose cushion. The titer, morphology, protein expression and neutralizing activity of pseudovirus were determined.Results:S protein specific fluorescence was detected by indirect immunofluorescence test, Western blot analysis showed S protein was expressed, and the spike of pseudovirus was observed under transmission electron microscope. The titers of pseudovirus 614D and 614G were 1.12×10 4 and 2.52×10 4 TCID 50/ml, respectively. The pseudovirus 614D and 614G could be neutralized by S rabbit polyclonal antibody, indicating that the pseudovirus has high specificity. Conclusions:In this study, 2019-nCoV 614D and 614G pseudovirus was successfully constructed, which laid the foundation for the establishment of in vitro neutralizing antibody detection platform based on pseudovirus.

Objective:To analyze antiviral activity against respiratory syncytial virus (RSV) of interferon (IFN)-α2b and IFN-λ1 on Hep2 cells and human airway epithelial (HAE) cells.Methods:IFN-α2b or IFN-λ1 was incubated with Hep2 cells after RSV infection, and 48 hours later, the cytopathic effect was observed, the viral load was determined using real time/reverse transcription quantitative polymerase chain reaction (RT qPCR), RSV F protein expression was detected using immunofluorescence, and cell survival rate was detected using crystal violet. HAE cells were incubated with IFN-α2b or IFN-λ1 for 24 hours, and then HAE were challenged with RSV. The viral load in the culture supernatant was determined on days 1-7 using RT qPCR, RSV F protein was determined with immunofluorescence and the viral titers in the culture supernatant was detected on day 7 by plaque assay.Results:In Hep2 cells, the CPE of the treatment groups (IFN-α2b and IFN-λ1) was alleviated compared to the virus control group, and the CPE of the high concentration group was lighter than that of the low concentration group. Different concentrations of IFN-α2b and IFN-λ1 could significantly reduce the viral load of RSV ( P<0.001), and the viral load of the high concentration group was significantly lower than that of the low concentration group ( P<0.001). In addition, IFN-α2b and IFN-λ1 could reduce the RSV F protein expression after RSV infection and improve cell survival rate. In HAE cells, IFN-α2b and IFN-λ1 could inhibit RSV virus replication, reduce virus titers ( P<0.001) and reduce RSV F protein expression. Conclusions:IFN-α2b and IFN-λ1 both showed great antiviral activity against RSV in Hep2 and HAE cells, providing data reference for the study of interferon against respiratory viruses.