To construct a high-titer Nipah pseudovirus packaging system using the HIV lentivirus backbone vector and establish a safe neutralization assay for Nipah pseudovirus in biosafety level 2 facilities. Nipah virus (NiV) fusion protein (F) and glycoprotein (G) recombinant expression plasmids, psPAX2, and pLenti CMV Puro LUC (w168-1) were transiently transfected into 293T cells for 72 h for the generation of a NiV pseudovirus. The neutralization ability of Nipah virus F and G protein antibodies was assessed using the pseudovirus. A NiV pseudovirus was constructed using 293T cells. The ideal mass ratio of plasmid psPAX2: w168-1: F: G for transfection was determined to be 4:4:1:1. The specificity of recombinant F and G protein expression was indicated by indirect immunofluorescence and western blotting. The pseudovirus particles showed obvious spikes under a transmission electron microscope. The NiV pseudovirus titer was 4.73 × 105 median tissue culture infective dose per mL, and the pseudovirus could be effectively neutralized by polyclonal antibodies specifically targeting the F and G proteins respectively. A NiV pseudovirus was successfully generated using HIV vector systems, and was used as a platform for a safe and reliable pseudovirus-based neutralizing assay that can be performed in biosafety level 2 facilities.

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.

Background Human adenoviruse (HAdV) is a major pathogen of paediatric respiratory tract infections (RTIs). Mutation or recombination of HAdV genes may cause changes in its pathogenicity and transmission. We described the epidemiology and genotypic diversity of HAdV in hospitalized children with RTIs in Beijing, China. Methods Nasopharyngeal aspirates were collected from hospitalized children with RTIs from April 2018 to March 2019. HAdVs were detected by a quantitative real-time PCR, and the hexon gene was used for phylogenetic analysis. Results Among 1572 samples, 90 (5.72%) were HAdV-positive. The HAdV detection rate was highest in November and July. Among HAdV-positive children, 61.11% (55/90) were co-infected with other respiratory viruses, the most common of which were human respiratory syncytial virus and human rhinovirus. The main diagnosis was bronchopneumonia, most patient have cough and fever. Children with a high viral load were more likely to have a high fever ( P = 0.041) and elevated WBC count ( P = 0.000). Of 55 HAdV-positive specimens, HAdV-B (63.64%), HAdV-C (27.27%), and HAdV-E (9.09%) were main epidemic species. Phylogenetic analysis indicated that hexon sequences of three samples were on the same branch with the recombinant HAdV strain (CBJ113), which was circulating in Beijing since 2016. Conclusion The HAdV-B3 and HAdV-B7 are the main epidemic strains in Beijing, and the recombinant HAdV-C strain CBJ113 has formed an epidemic trend.