Objective:To analyze the protective effect of a respiratory syncytial virus (RSV) vaccine based on bacterial outer membrane vesicle (OMV) in mice.Methods:The pre-fusion protein (preF) of RSV was linked to the surface of OMV through the transmembrane protein cytolysin A (ClyA) to form the nanovaccine OMV-preF. The morphological characteristics of OMV and OMV-preF were observed under a transmission electron microscope. OMV-preF was intramuscularly injected into BALB/c mice and the elicited humoral and cellular immune responses were evaluated. The protective effect of OMV-preF was assessed by challenging the immunized mice with RSV Long strain. One-way analysis of variance and Tukey test were used for statistical analysis.Results:The results showed that preF was stably expressed in OMV, and both OMV-preF and OMV exhibited a double-layer vesicle structures under the microscope. OMV-preF could significantly activate the cellular and humoral immune responses in mice, causing a significant increase in CD8 + T cells and CD19 + B cells as well as a significant increase in the serum level of specific IgG. The neutralizing antibodies produced in the immunized mice could significantly inhibit the replication of RSV Long strain in vivo. Conclusions:The nanovaccine OMV-preF can induce high-level humoral and cellular immune responses, and the antibodies produced following immunization can effectively inhibit viral replication. This study provides a new strategy for RSV subunit vaccines.

Objective:To analyze the epidemiological and clinical characteristics of Mycoplasma pneumoniae ( Mp) infection among hospitalized children with acute respiratory tract infections (ARTIs) in a single center in Beijing and provide reference for the prevention and treatment of Mp infection. Methods:Nasopharyngeal aspirate (NPA) samples of hospitalized children with ARTIs were collected from Beijing Friendship Hospital during two periods: from April 2018 to March 2019 and from September 2020 to August 2022. qPCR was used to detect Mp nucleic acids, and for Mp-positive samples, the mixed infections with 15 common respiratory viruses were detected. Statistical analysis was conducted using Chi-square test, Fisher′s exact test, and independent samples t-test. Results:From April 2018 to March 2019, 1 572 NPA samples were collected, with 104 positive for Mp (6.62%). From September 2020 to August 2022, 622 samples were collected, with 22 Mp-positive samples (3.54%). There was statistically significant difference in the positive rates between the two time periods ( P<0.05). From April 2018 to March 2019, the positive rate of Mp was higher in children aged ≥5 years than in those <5 years [13.03% (46/353) vs 4.76% (58/1 219), P<0.05]; the positive rates in summer (9.54%, 35/367) and autumn (7.93%, 33/416) were higher than those in spring (3.03%, 11/363) and winter (5.87%, 25/426), with statistically significant differences ( P<0.05); co-infections with other respiratory viruses were detected in 42 out of the 104 Mp-positive cases (40.38%), primarily with human rhinovirus (35.71%, 15/42) or human coronavirus NL63 (19.05%, 8/42). From September 2020 to August 2022, Mp infections mainly occurred in children aged ≥5 years [72.73% (16/22)], and co-infections with other respiratory viruses were detected in four cases (18.18%, 4/22). The Mp-infected children were mainly diagnosed with pneumonia, and there was no significant difference in clinical symptoms between Mp-infected patients with or without viral coinfection. Conclusions:The positive rate of Mp among hospitalized children with ARTIs in Beijing from September 2020 to August 2022 is significantly lower than that observed from April 2018 to March 2019. Mp is an important cause of ARTIs in children, especially in patients aged ≥5 years. Mp infection is often accompanied by viral co-infections, with high incidence in summer and autumn.

Objective:To analyze the protective effect of a respiratory syncytial virus (RSV) vaccine based on bacterial outer membrane vesicle (OMV) in mice.Methods:The pre-fusion protein (preF) of RSV was linked to the surface of OMV through the transmembrane protein cytolysin A (ClyA) to form the nanovaccine OMV-preF. The morphological characteristics of OMV and OMV-preF were observed under a transmission electron microscope. OMV-preF was intramuscularly injected into BALB/c mice and the elicited humoral and cellular immune responses were evaluated. The protective effect of OMV-preF was assessed by challenging the immunized mice with RSV Long strain. One-way analysis of variance and Tukey test were used for statistical analysis.Results:The results showed that preF was stably expressed in OMV, and both OMV-preF and OMV exhibited a double-layer vesicle structures under the microscope. OMV-preF could significantly activate the cellular and humoral immune responses in mice, causing a significant increase in CD8 + T cells and CD19 + B cells as well as a significant increase in the serum level of specific IgG. The neutralizing antibodies produced in the immunized mice could significantly inhibit the replication of RSV Long strain in vivo. Conclusions:The nanovaccine OMV-preF can induce high-level humoral and cellular immune responses, and the antibodies produced following immunization can effectively inhibit viral replication. This study provides a new strategy for RSV subunit vaccines.

Objective:To analyze the epidemiological and clinical characteristics of Mycoplasma pneumoniae ( Mp) infection among hospitalized children with acute respiratory tract infections (ARTIs) in a single center in Beijing and provide reference for the prevention and treatment of Mp infection. Methods:Nasopharyngeal aspirate (NPA) samples of hospitalized children with ARTIs were collected from Beijing Friendship Hospital during two periods: from April 2018 to March 2019 and from September 2020 to August 2022. qPCR was used to detect Mp nucleic acids, and for Mp-positive samples, the mixed infections with 15 common respiratory viruses were detected. Statistical analysis was conducted using Chi-square test, Fisher′s exact test, and independent samples t-test. Results:From April 2018 to March 2019, 1 572 NPA samples were collected, with 104 positive for Mp (6.62%). From September 2020 to August 2022, 622 samples were collected, with 22 Mp-positive samples (3.54%). There was statistically significant difference in the positive rates between the two time periods ( P<0.05). From April 2018 to March 2019, the positive rate of Mp was higher in children aged ≥5 years than in those <5 years [13.03% (46/353) vs 4.76% (58/1 219), P<0.05]; the positive rates in summer (9.54%, 35/367) and autumn (7.93%, 33/416) were higher than those in spring (3.03%, 11/363) and winter (5.87%, 25/426), with statistically significant differences ( P<0.05); co-infections with other respiratory viruses were detected in 42 out of the 104 Mp-positive cases (40.38%), primarily with human rhinovirus (35.71%, 15/42) or human coronavirus NL63 (19.05%, 8/42). From September 2020 to August 2022, Mp infections mainly occurred in children aged ≥5 years [72.73% (16/22)], and co-infections with other respiratory viruses were detected in four cases (18.18%, 4/22). The Mp-infected children were mainly diagnosed with pneumonia, and there was no significant difference in clinical symptoms between Mp-infected patients with or without viral coinfection. Conclusions:The positive rate of Mp among hospitalized children with ARTIs in Beijing from September 2020 to August 2022 is significantly lower than that observed from April 2018 to March 2019. Mp is an important cause of ARTIs in children, especially in patients aged ≥5 years. Mp infection is often accompanied by viral co-infections, with high incidence in summer and autumn.

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.

Objective:To explore the feasibility of bibliometric analysis of research papers on human metapneumovirus based on Web of Science database.Methods:The human metapneumovirus (HMPV) causes a serious disease burden worldwide. This article used bibliometric analysis method to search for papers using the keyword " metapneumovirus" , and searched for HMPV papers published from 2001 to 2023 in the Web of Science database. Statistical analysis of the distribution of papers on HMPV by year, country, journal, research institution, author, etc., in order to understand the current research status and development trends of HMPV in the international community.Results:A total of 3 282 papers were retrieved, of which 97% were in English. HMPV was first reported in 2001, and since then, research papers have been increasing year by year. The United States has the highest number of published papers, with China, the United Kingdom, France, and the Netherlands ranked 2nd, 3rd, 4th, and 5th respectively. The field of virology-general had the highest number of papers. In terms of research institution distribution, Vanderbilt University in the United States has published 135 papers, ranked the first. The journal which had the highest number of published papers was JOURNAL OF MEDICAL VIROLOGY, with a total of 143 papers. The author Williams JV of Vanderbilt University in the United States has published 92 papers, indicating its high international status in the field of HMPV research.Conclusions:Among the retrieved HMPV related papers, research institutions and universities in European and American countries have published more papers.

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.

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 establish a quick and convenient method for detecting human adenoviruses (Human adenoviruses, HAdV) based on immunochromatographic assay (ICA).Methods:Two antibody clones, 3C11 and 7E6 were found to bind to all tested HAdVs and then subsequently processed into ICA. The specificity and sensitivity were evaluated using representative strains of the respiratory HAdV types, including HAdV-1, 2, 3, 4, 5, 6, 7, 10 and a gastroenteric type HAdV-41 together with the original throat swabs of 10 HAdV patients confirmed by nuclear acid testing (NAT).Results:The ICA exhibited high specificity to HAdVs and its detection limitation ranged from 0.16 to 10 3 half tissue culture infectious dose (TCID 50)/ml for different types of HAdVs. All clinic samples with successful virus isolation tested by this ICA showed positive result . Conclusions:The ICA developed in the present study will be suitable for HAdVs screening in clinic setting, especially for those of respiratory types.