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 isolate and culture WU polyomavirus (WUPyV), and to analyze the genome-wide evolutionary patterns, homology and population dynamics.Methods:Real-time quantitative PCR was used to detect the nasopharyngeal aspirate samples of hospitalized children with respiratory tract infection in Beijing Friendship Hospital during 2020 to 2022. Primary human airway epithelial cells cultured at the air-liquid interface were used to isolate and culture WUPyV. Whole genome sequence of the isolated strain was obtained by Sanger sequencing. For phylogenetic and evolutionary dynamics analysis, the whole genome was compared with the published whole genome sequences in GenBank database.Results:The detection rate of WUPyV was 4.7% (31/659) during 2020 to 2022, and a clinical strain BJ0593 of WUPyV type Ⅲc was successfully isolated. The homology of the whole genome and gene fragments of WUPyV was high. The average evolutionary rate of VP2 gene was about 1.256×10 -4 substitution/site every year, and the population dynamics of WUPyV tended to be flat in the last decade. Conclusions:This study successfully isolated a clinical WUPyV type Ⅲ strain for the first time, which provided the basis for further investigation on the molecular evolution and pathogenicity of WUPyV.

BackgroundThis study aims to described the epidemiology and genotypic diversity of Human metapneumovirus (HMPV) and the impact of SARS-CoV-2 on the prevalence of HMPV in hospitalized children with Acute respiratory tract infections (ARTIs) in Beijing, China.MethodsFrom April 2018 to March 2019 and from September 2020 to August 2021, nasopharyngeal aspirates (NPAs) from hospitalized children with ARTIs in Beijing were collected and subjected to real-time polymerase chain reaction tests for HMPV. Then genotyping, detection of 15 common respiratory viruses and clinical characteristics were analyzed on HMPV positive samples.Results7.9% (124/1572) enrolled pediatric patients were identified as having HMPV infection, and the majority of children under the age of 5 (78.2%, 92/124), From April 2018 to March 2019. The detection rate of HMPV in spring and winter is significantly higher than that in summer and autumn. The co-infection rate were 37.1% (46/124), the most common co-infected virus were parainfluenza virus type 3 (HPIV-3). The main diagnosis of HMPV infection was pneumonia (92.7%,115/124), most patient have cough and fever. Of 78 HMPV-positive specimens, A2b (82.1%,64/78) were the main epidemic subtypes. Hospitalized children with HMPV genotype A infection had a higher viral load compared to genotype B. During the COVID-19 outbreak, Among 232 samples, only 4 cases were HMPV-positive. After statistical test, the detection rate of HMPV during the COVID-19 pandemic has decreased significantly compared with that before the epidemic (p = 0.001).ConclusionsHMPV is an important cause of ARTIs in children under 5 years old. The epidemic peak is generally in winter and spring, and the A2b subtype is the most common. However, under the prevention and control of the COVID-19 pandemic, the HMPV infection of hospitalized children with ARTIs has decreased significantly.

Abstract Background Among hospitalized children suffering from community-acquired pneumonia, Mycoplasma pneumoniae (MP) is one of the most common pathogens. MP often exists as a co-infection with bacteria or viruses, which can exacerbate the clinical symptoms. We investigated the pathogen spectrum in MP-positive and MP-negative samples from hospitalized children with respiratory tract infections in Beijing, China. Method This study included 1038 samples of nasopharyngeal aspirates obtained between April, 2017 and March, 2018 from hospitalized children under 6 years of age with respiratory tract infections. To explore the impact of MP infection on the composition of the pathogen spectrum, 185 nasopharyngeal aspirates (83 MP-positive/102 MP-negative) were randomly selected for next-generation sequencing and comprehensive metagenomics analysis. Real-time PCR was used to detect and verify common respiratory viruses. Results Of the 1038 samples, 454 (43.7%) were infected with MP. In children < 6 years of age, the MP infection rate gradually increased with age, with the highest rate of 74.2% in 5–6-year-olds. The results of metagenomics analysis revealed 11 human, animal and plant virus families, and bacteriophages, including common respiratory viruses, enteroviruses and anelloviruses. The virus family with the highest number of reads in both MP-positive and MP-negative samples was the Pneumoviridae, and the number of reads for human respiratory syncytial virus (HRSV) in MP-positive samples was higher than that in MP-negative samples. Among the 83 MP-positive samples, 47 (56.63%) were co-infected with viruses, the most common of which was influenza virus (IFV). The durations of hospitalization and fever were higher in patients with MP co-infection than MP single infection, but the difference was not statistically significant. Conclusion The viral family with the highest number of reads in both groups was Pneumoviridae, and the number of reads matched to HRSV in MP-positive samples was much higher than MP-negative samples. Co-infection of MP and IFV infection were the most cases.

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.