BACKGROUND:Acute respiratory tract infections (ARTI) remain a major public health burden in children globally. Nevertheless, regional data regarding postpandemic pathogen shifts, coinfection profiles, and risk factors associated with severe pediatric ARTI in Hangzhou, China, remain scarce. METHODS:A retrospective observational study was conducted on 6701 hospitalized pediatric patients (0-18 years) with ARTI in Hangzhou, China, from July 2023 to June 2025. Eleven respiratory pathogens were detected using fluorescence PCR-capillary electrophoresis. Univariate and multivariable logistic regression analyses, alongside three machine learning models, were employed to identify risk factors. Age-stratified and coinfection analyses were also performed. RESULTS:Rhinovirus (RV), Mycoplasma pneumoniae (Mp), and respiratory syncytial virus (RSV) were the most prevalent pathogens. The coinfection network was centered on RV, Mp, and adenovirus, with RV-Mp being the most common dual infection. Mixed infections were associated with severe illness (4.3% vs. 3.5% in single infections vs. 1.6% in pathogen-negative cases). Mp, bocavirus (Boca), and RV infections, along with reduced percentages of lymphocytes, monocytes, and eosinophils, were independent risk factors associated with severe illness, whereas older age was a protective factor. Logistic regression demonstrated the best predictive performance. CONCLUSIONS:This study elucidates the dynamics of pediatric ARTI pathogens and coinfection characteristics 2 years postpandemic in Hangzhou, China. It identifies key risk factors associated with severe pediatric ARTI, providing essential evidence to inform age-specific clinical prevention, targeted interventions, and public health strategies for pediatric ARTI in the region.

Non‐pharmaceutical interventions (NPIs) implemented to control SARS‐CoV‐2 have significantly influenced the activity of respiratory pathogens. This study investigated epidemiological changes among hospitalized patients with respiratory syncytial virus (RSV) before (2017–2019) and during (2020–2022) the COVID‐19 pandemic in Hangzhou, China. We also examined viral load distribution across demographic and temporal variables. Nasopharyngeal swabs were collected and RSV loads were quantified using reverse transcriptase polymerase chain reaction (RT‐qPCR). RSV epidemic characteristics, seasonal dynamics, and viral load distributions were compared between pre‐ and pandemic years. General linear models were employed to assess associations between viral loads and age. Among 19 742 cases, 1576 and 2092 tested positive during the pre‐ and pandemic years, respectively. From February to July 2020, the implementation of NPIs led to the cessation of RSV circulation. However, after these measures were relaxed, RSV cases resurged over two consecutive seasons during the pandemic, notably affecting older children compared to those in the pre‐pandemic years (1.00 years, IQR: 0.50–2.00 vs. 0.58 years, IQR: 0.27–1.00, p < 0.001). Specifically, in 2021–2022, an off‐season resurgence of RSV began earlier (mid‐June), lasted longer (40 weeks), and involved more positive cases (1238 cases) than both 2020–2021 and pre‐pandemic years. Viral load distribution demonstrated a clear age‐related relationship in both pre‐ and pandemic years, with younger children consistently showing higher viral loads, independently of gender and season (all p‐values for trends <0.001). These findings highlight the impact of NPIs on RSV epidemiology and underscore the need to prioritize RSV infection prevention in younger children from the perspective of viral load.

The burden and characteristics of respiratory viral infections in children hospitalized for acute respiratory tract infections (ARTIs) during the post-COVID-19 pandemic era are unclear. We analyzed the epidemiological and clinical characteristics of pediatric patients hospitalized with common respiratory virus infections before and after relaxation of non-pharmaceutical interventions in Hangzhou, China and evaluated the diagnostic value of the six-panel respiratory pathogen detection system. Six types of respiratory viruses were detected in respiratory samples from children with suspected ARTIs by multiplex real-time quantitative polymerase chain reaction (RT-qPCR). Changes in virus detection rates and epidemiological and clinical characteristics, obtained from electronic health records, were analyzed. Binary logistic regression was used to identify respiratory tract infections risk factors. Multiplex RT-qPCR and targeted next-generation sequencing results were compared in random samples. Among the 11,056 pediatric samples, 3228 tested positive for one or more of six common respiratory pathogens. RSV and PIV-3 detection rates differed significantly across age groups (both P < 0.001), and were more common in younger children. PIV-1 was more common in infants, toddlers, and preschoolers than in school-age children (P < 0.001). FluB was predominantly detected in school-age children (P < 0.001). RSV-, ADV-, and PIV-1-positivity rates were higher in 2022 than in 2023. Seasonal viral patterns differed across years. RSV (OR 9.156. 95

Hand, foot, and mouth disease (HFMD) is most frequently caused by several serotypes of human enterovirus (EV) including Enterovirus 71 (EV-A71), coxsackievirus A16 (CV-A16), or other types of EV. The aim of this study was to determine the epidemiological characteristics of HFMD and to describe the epidemiologic characteristics of HFMD among severe and mild cases. We collected 4760 HFMD cases in Hangzhou from 2016 to 2018. Specimens from these cases were collected and tested for EV-A71, CV-A16, CV-A6, CV-A10, CV-A2, and CV-A5 by reverse transcriptase polymerase chain reaction. From 2016 to 2018, the prevalence of HFMD was seasonal each year. Among the 4760 probable HFMD cases, 3559 cases were confirmed (74.8%), including 426 cases of EV-A71 infections (8.9%), 249 cases of CV-A16 infections (5.2%), and 2884 cases of other EV infections (60.6%). The percentage of other EV infections was more than 80%, which increased year by year. Random selection of samples for detection of other EV infections in 2017 and 2018, among the 1297 cases, showed there were 835 (64.4%) cases of CV-A6 infections, 177 (13.6%) cases of CV-A10 infections, 100 (7.7%) cases of CV-A2 infections, 40 (3.1%) cases of CV-A5 infections, 3 (0.02 %) cases of mixed infections, and 11.0% untyped EV infections. Preschool children were still the primary population susceptible to HFMD. In severe cases, EV-A71 infection was the main cause. Characterizing the epidemiology and the relationship between severe and common cases of HFMD would provide relevant evidences for the prevention and treatment of HFMD.

OBJECTIVE:To detect the anti-enterovirus 71 (EV71) IgM level in cerebrospinal fluid (CSF) of children with severe hand, foot and mouth disease (HFMD) induced by EV71 and then analyze the relationships among the IgM antibody levels, CSF routine examination and patients' clinical features, and thus to evaluate the clinical significance of anti-EV71 IgM as a new indicator for early diagnosis of children with severe HFMD induced by EV71.METHOD:A total of 294 laboratory-confirmed cases of children with severe HFMD infected with EV71 were enrolled into the research group from March 2014 to June 2014, consisting of 53 fatal cases and 241 severe cases, and their CSF samples underwent enzyme-linked immunosorbent assay (ELISA) for anti-EV71 IgM levels, CSF routine and biochemical tests. Forty-one cases of children with severe HFMD induced by other enteroviruses were collected as antibody-testing control group during the same period.RESULT:In the research group, the total positive rate of anti-EV71 IgM in 294 CSF samples of children with severe HFMD infected by EV71 was 60.2% (177/294); the positive rate of anti-EV71 IgM in the fatal HFMD subgroup was 62.3% (33/53); the positive rate of anti-EV71 IgM in the severe HFMD subgroup was 59.8% (144/241). In the control group, the results of CSF anti-EV71 IgM tests were all negative (0/41). In the research group, patients in antibody-positive subgroup (2.5±1.2) years old were younger than those in antibody-negative subgroup (2.9±1.1) years old (t=2.595, P=0.010). And within the antibody-positive subgroup, the patients ((1.9±0.7) years old) with fatal type disease were younger than those ((2.6±1.2) years old) with severe type disease (t=3.150, P=0.002). The CSF nucleated cells count and positive rates (105 (56,180) ×10(6) /L; 97.7% (173/177)) in antibody-positive subgroup were higher than those (62(30,150) ×10(6) /L; 83.8% (98/117)) in antibody-negative subgroup (Z=3.663, P=0.000; χ(2)=19.089, P=0.000). In antibody-positive subgroup, the percentage of monocytes (57±25)% was higher than that of polykaryocytes (43±25)%. In antibody-negative subgroup, the percentage of monocytes (50±26)% was close to that of polykaryocytes (50±26)%. In the antibody-positive subgroup, the ratio of the patients with nucleated cells count higher than 100×10(6)/L in fatal type group and severe type group was 69.7% (23/33) and 47.2% (68/144) respectively (χ(2)=5.429, P=0.02). The CSF protein quantity and positive rates in antibody-positive subgroup were higher than those in antibody-negative subgroup (Z=2.158, P=0.031; χ(2)=5.921, P=0.015).CONCLUSIONS:The anti-EV71 IgM levels in CSF can serve as an important indicator for early diagnosis of children with severe HFMD induced by EV71. And the anti-EV71 IgM levels in CSF correlated to the CSF nucleated cells count and classification and CSF protein quantity. In the antibody-positive subgroup, the higher the nucleated cell count or the younger the age, the higher the possibility of patients to develop into fatal cases.

ABSTRACT Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two most common etiological agents responsible for the epidemics of hand, foot, and mouth disease (HFMD), a childhood illness with occasional severe neurological complications. A number of vaccine candidates against EV71 or CA16 have been reported; however, no vaccine is currently available for clinical use. Here, we generated a secreted version of EV71 and CA16 virus-like particles (VLPs) using a baculovirus-insect cell expression system and reconstructed the three-dimensional (3D) structures of both VLPs by cryo-electron microscopy (cryo-EM) single-particle analysis at 5.2-Å and 5.5-Å resolutions, respectively. The reconstruction results showed that the cryo-EM structures of EV71 and CA16 VLPs highly resemble the recently published crystal structures for EV71 natural empty particles and CA16 135S-like expanded particles, respectively. Our cryo-EM analysis also revealed that the majority of previously identified linear neutralizing epitopes are well preserved on the surface of EV71 and CA16 VLPs. In addition, both VLPs were able to induce efficiently neutralizing antibodies against various strains of EV71 and CA16 viruses in mouse immunization. These studies provide a structural basis for the development of insect cell-expressed VLP vaccines and for a potential bivalent VLP vaccine against both EV71- and CA16-associated HFMD. IMPORTANCE The recent outbreaks of hand, foot, and mouth disease (HFMD) in the Asia Pacific region spurred the search for effective vaccines against EV71 and CA16 viruses, the two most common etiological agents responsible for HFMD. In this paper, we show that secreted versions of EV71 and CA16 VLPs generated in the baculovirus-insect cell expression system highly resemble the crystal structures of their viral conterparts and that the majority of previously identified linear neutralizing epitopes are well preserved on the VLP surfaces. In addition, the generated VLPs can efficiently induce neutralizing antibodies against various strains of EV71 and CA16 viruses in mouse immunization. These studies provide a structural basis for the development of insect cell-expressed VLP vaccines and for a potential bivalent VLP vaccine against both EV71- and CA16-associated HFMD.

Cryo-EM study of insect cell-expressed Enterovirus 71 and 1 Coxsackievirus A16 virus-like particles provides a structural 2 basis for vaccine development 3 4 Minqing Gong, Hongtao Zhu, Jun Zhou, Chunting Yang, Jing Feng, Xiaojun Huang, 5 Gang Ji, Honglin Xu & Ping Zhu 6 7 1. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of 8 Sciences, Beijing 100101, China 9 2. Department of Virology, National Vaccine and Serum Institute, Beijing 100024, China 10 3. Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing 11 100101, China 12 4. University of Chinese Academy of Sciences, Beijing 100049, China 13

OBJECTIVE:To investigate the epidemic characteristics of hand-foot-and-mouth disease (HFMD) in children and exposed population in Hangzhou city.METHODS:The throat swab or stool samples from children with HFMD admitted in Hangzhou Children's Hospital were collected. The HFMD pathogens were detected by real-time fluorescent quantitative PCR. The distribution of different HFMD pathogens in HFMD patients was subsequently determined. Human enteric virus type-71 (HEV71) in stool samples from subjects, who had close or general contact to 54 severe HFMD children with positive HEV71, was detected, and these contact persons were followed-up for one month. The diversity of predominant pathogens of HFMD in the area during 2011-2013 was investigated.RESULTS:In 641 HFMD children, the male/female ratio was 1.4:1 and 80.3% was 1-3 years old. HEV71 was detected in 24.3% HFMD children (156/641), while coxsackievirus group-A type-16 (CVA16) and other enteroviruses were detected in 4.7% (30/641) and 71.0% (455/641) of the cases, respectively. 75.6% (118/156) of HEV71-infected cases were diagnosed as severe HFMD cases, while those for CVA16-infected and other HFMD viruses-infected were 13.3% (4/30) and 6.2% (28/455) respectively (Χ(2)=43.28, P<0.05). HEV71 was the predominant HFMD pathogens during 2011-2012, while the predominant HFMD pathogens in 2013 were the other HFMD viruses. In the 54 close contact persons or 54 general contact persons, 9 or 10 persons were detectable for HEV71, but no clinical symptoms of HFMD were presented.CONCLUSION:There are no marked changes of epidemic seasons, favorable age and gender ratio of HFMD in Hangzhou area in 2013. The infection of HEV71 tends to cause the severe HFMD but the other enteroviruses have substituted HEV71 as the predominant pathogens of HFMD.

Vaccination is the most effective method used to reduce the morbidity and mortality of influenza infections. However, as exemplified in the current swine-origin influenza virus (S-OIV) pandemic, the global manufacturing capacity of influenza vaccines is severely limited. In the present proof-of-concept study, we combined cell substrate selection and antigen engineering with adjuvant development to design a potential pandemic influenza vaccine candidate, in which CpG oligodeoxynucleotides (CpG-ODN) plus alum was used as a composite adjuvant to enhance the immunogenicity of insect cell-expressed recombinant hemagglutinin (rHA). Our candidate vaccine was found to be effective in inducing protective humoral as well as cellular immunity in mice and able to protect the immunized mice from related influenza virus challenge. If this candidate vaccine is validated in humans, vaccine development can be started immediately after the release of the first HA sequence of any pandemic influenza virus. Moreover, given the potential of large-scale manufacturing capacity of the recombinant antigen, in combination with the antigen-sparing effect of the composite adjuvant, this technology could be an invaluable asset in the fight against pandemic influenza.

Poliovirus transmission is controlled globally through world-wide use of a live attenuated oral polio vaccine (OPV). However, the imminence of global poliovirus eradication calls for a switch to the inactivated polio vaccine (IPV). Given the limited manufacturing capacity and high cost of IPV, this switch is unlikely in most developing and undeveloped countries. Adjuvantation is an effective strategy for antigen sparing. In this study, we evaluated the adjuvanticity of CpG oligodeoxynucleotides (CpG-ODN) for an experimental IPV produced from Sabin strains of poliovirus. Our results showed that CpG-ODN, alone or in combination with alum, can significantly enhance both the humoral and cellular immune responses to IPV in mice, and, consequently, the antigen dose could be reduced substantially. Therefore, our study suggests that the global use of IPV could be facilitated by using CpG-ODN or other feasible adjuvants.