Human respiratory syncytial virus subgroups A and B outbreak in a kindergarten in Zhejiang Province, China, 2023.

Background: In May-June 2023, an unprecedented outbreak of human respiratory syncytial virus (HRSV) infections occurred in a kindergarten, Zhejiang Province, China. National, provincial, and local public health officials investigated the cause of the outbreak and instituted actions to control its spread. Methods: We interviewed patients with the respiratory symptoms by questionnaire. Respiratory samples were screened for six respiratory pathogens by real-time quantitative polymerase chain reaction (RT-PCR). The confirmed cases were further sequenced of G gene to confirm the HRSV genotype. A phylogenetic tree was reconstructed by maximum likelihood method. Results: Of the 103 children in the kindergarten, 45 were classified as suspected cases, and 25 cases were confirmed by RT-PCR. All confirmed cases were identified from half of classes. 36% (9/25) were admitted to hospital, none died. The attack rate was 53.19%. The median ages of suspected and confirmed cases were 32.7 months and 35.8 months, respectively. Nine of 27 confirmed cases lived in one community. Only two-family clusters among 88 household contacts were HRSV positive. A total of 18 of the G gene were obtained from the confirmed cases. Phylogenetic analyses revealed that 16 of the sequences belonged to the HRSV B/BA9 genotype, and the other 2 sequences belonged to the HRSV A/ON1 genotype. The school were closed on June 9 and the outbreak ended on June 15. Conclusion: These findings suggest the need for an increased awareness of HRSV coinfections outbreak in the kindergarten, when HRSV resurges in the community after COVID-19 pandemic.

Genetic and Epigenetic Host-Virus Network to Investigate Pathogenesis and Identify Biomarkers for Drug Repurposing of Human Respiratory Syncytial Virus via Real-World Two-Side RNA-Seq Data: Systems Biology and Deep-Learning Approach.

Human respiratory syncytial virus (hRSV) affects more than 33 million people each year, but there are currently no effective drugs or vaccines approved. In this study, we first constructed a candidate host-pathogen interspecies genome-wide genetic and epigenetic network (HPI-GWGEN) via big-data mining. Then, we employed reversed dynamic methods via two-side host-pathogen RNA-seq time-profile data to prune false positives in candidate HPI-GWGEN to obtain the real HPI-GWGEN. With the aid of principal-network projection and the annotation of KEGG pathways, we can extract core signaling pathways during hRSV infection to investigate the pathogenic mechanism of hRSV infection and select the corresponding significant biomarkers as drug targets, i.e., TRAF6, STAT3, IRF3, TYK2, and MAVS. Finally, in order to discover potential molecular drugs, we trained a DNN-based DTI model by drug-target interaction databases to predict candidate molecular drugs for these drug targets. After screening these candidate molecular drugs by three drug design specifications simultaneously, i.e., regulation ability, sensitivity, and toxicity. We finally selected acitretin, RS-67333, and phenformin to combine as a potential multimolecule drug for the therapeutic treatment of hRSV infection.

Research progress on vaccines against human respiratory syncytial virus / 中国生物制品学杂志

@#Human respiratory syncytial virus(hRSV)is one of the main pathogens that cause lower respiratory tract infection in infants and the elderly.hRSV genome contains 10 genes with a full length of 15 222 bp,encoding 11 proteins(9structural proteins and 2 non-structural proteins).Different proteins play different roles in the pathogenesis of hRSV.With the in-depth research on the biological and structural characteristics of hRSV,various types of hRSV vaccines have been developed,making rapid progress.For example,hRSV attenuated live vaccine hRSV ?NS2/?1313/I1314L has entered Phase II clinical trial,and hRSV subunit protein vaccine Pre-F-GCN4t has entered Phase III clinical trial.In this paper,the biological characteristics of hRSV and the types of hRSV vaccines with rapid progress are reviewed so as to provide a reference for the development of hRSV vaccines in China.

An Outbreak of Severe Neonatal Pneumonia Caused by Human Respiratory Syncytial Virus BA9 in a Postpartum Care Centre in Shenyang, China.

Human respiratory syncytial virus (HRSV) is a major pathogen of lower respiratory tract infections in children (<5 years) and older individuals, with outbreaks mainly reported among infants in hospital pediatric departments and intensive care units (ICUs). An outbreak of severe neonatal pneumonia occurred in a postpartum center in Shenyang city, China, from January to February 2021. In total, 34 respiratory samples were collected from 21 neonates and 13 nursing staff. The samples were screened for 27 pathogens using a TaqMan low-density array, and 20 samples tested positive for HRSV, including 16 neonates and 4 nursing staff samples. Among the 16 hospitalized neonates, seven were admitted to an ICU and nine to general wards. Four of the nursing staff had asymptomatic infections. To investigate the genetic characteristics of the HRSV responsible for this outbreak, the second hypervariable region (HVR2) sequences of the G gene were obtained from six neonates and two nursing staff. Phylogenetic analyses revealed that all eight sequences (SY strains) were identical, belonging to the HRSV BA9 genotype. Our findings highlight the necessity for strict hygiene and disease control measures so as to prevent cross-infection and further avoid potential outbreaks of severe infectious respiratory diseases. IMPORTANCE Human respiratory syncytial virus (HRSV) is one of the leading causes of acute lower respiratory infections (ALRI) worldwide. In this study, we first reported an outbreak of severe neonatal pneumonia caused by HRSVB BA9 at a postpartum care center in mainland China. Among 20 confirmed cases, 16 were hospitalized neonates with 7 in the ICU ward, and the other four were nursing staff with asymptomatic infections. Our findings highlighted the importance of preventing cross-infection in such postpartum centers.

Establishment and application of a lethal model of an HRSV-long variant strain in BALB/c mice.

Human respiratory syncytial virus (HRSV) is a major cause of lower respiratory tract infection in infants. The lack of ideal animal models is one of the major obstacles in evaluateing the efficacy of HRSV vaccines. In this study, HRSV-50 was obtained from Hep-2 cells at the 50th passage of the original Long strain (ATCC VR-26). BALB/c mice (6 weeks) were challenged with different titers of HRSV-50. Shockingly, all mice died after 4 days of challenge (6 × 106 PFU/mouse). Whole-genome sequencing revealed 7 amino acid mutations compared with the original Long strain. To verify whether the lethal model can be used to effectively evaluate the efficacy of HRSV candidate vaccines, we studied the protective effect of FRBD protein (Pre-F of HRSV and S receptor binding domain of SARS-CoV-2) with Adju-phos or MA103 adjuvant. All mice in the PBS group died after the HRSV-50 challenge, whereas Adju-phos provided partial protection. These results suggest that we have successfully established a lethal model of HRSV in BALB/c mice.

Molecular epidemiological study of the G protein of human respiratory syncytial virus detected in patients with acute respiratory infections in Gyeonggi Province, South Korea.

To investigate the molecular characteristics of human respiratory syncytial virus (HRSV) detected in Gyeonggi Province from 2015/16 to 2017/18, 2331 specimens from patients with sporadic acute respiratory illness and 85 specimens from four HRSV outbreaks in the postpartum care center were analyzed by real-time reverse transcription PCR. HRSVs were detected in 97 of the 2416 (4.0%) specimens, and among the positive specimens, 38 (39.2%) were identified as HRSV-A and 59 (60.8%) as HRSV-B. During the study periods, HRSV-B predominated in all seasons, except in 2016/17 during which HRSV-A predominated. Depending on the age groups, HRSV prevalence was the highest in 0- to 2-year-old patients. Comparison of noninfected subjects with HRSV-infected subjects revealed that HRSV infection more frequently resulted in fever, nasal obstruction, and wheezing, although the frequency of sore throat was low; however, comparison of the symptoms between HRSV-A- and HRSV-B-infected patients revealed no significant differences in symptoms. Phylogenetic analysis showed that all HRSV-A patients had an ON1 genotype, and all HRSV-B patients had an BA9 genotype. These results provide a valuable reference regarding the circulating pattern and molecular characterization of HRSV. Continuous monitoring will be essential to detect newly emerging HRSV genotypes.

Human Respiratory Syncytial Virus Infection in a Human T Cell Line Is Hampered at Multiple Steps.

Human respiratory syncytial virus (HRSV) is the most frequent cause of severe respiratory disease in children. The main targets of HRSV infection are epithelial cells of the respiratory tract, and the great majority of the studies regarding HRSV infection are done in respiratory cells. Recently, the interest on respiratory virus infection of lymphoid cells has been growing, but details of the interaction of HRSV with lymphoid cells remain unknown. Therefore, this study was done to assess the relationship of HRSV with A3.01 cells, a human CD4+ T cell line. Using flow cytometry and fluorescent focus assay, we found that A3.01 cells are susceptible but virtually not permissive to HRSV infection. Dequenching experiments revealed that the fusion process of HRSV in A3.01 cells was nearly abolished in comparison to HEp-2 cells, an epithelial cell lineage. Quantification of viral RNA by RT-qPCR showed that the replication of HRSV in A3.01 cells was considerably reduced. Western blot and quantitative flow cytometry analyses demonstrated that the production of HRSV proteins in A3.01 was significantly lower than in HEp-2 cells. Additionally, using fluorescence in situ hybridization, we found that the inclusion body-associated granules (IBAGs) were almost absent in HRSV inclusion bodies in A3.01 cells. We also assessed the intracellular trafficking of HRSV proteins and found that HRSV proteins colocalized partially with the secretory pathway in A3.01 cells, but these HRSV proteins and viral filaments were present only scarcely at the plasma membrane. HRSV infection of A3.01 CD4+ T cells is virtually unproductive as compared to HEp-2 cells, as a result of defects at several steps of the viral cycle: Fusion, genome replication, formation of inclusion bodies, recruitment of cellular proteins, virus assembly, and budding.

Structures of the Mononegavirales Polymerases.

Mononegavirales, known as nonsegmented negative-sense (NNS) RNA viruses, are a class of pathogenic and sometimes deadly viruses that include rabies virus (RABV), human respiratory syncytial virus (HRSV), and Ebola virus (EBOV). Unfortunately, no effective vaccines and antiviral therapeutics against many Mononegavirales are currently available. Viral polymerases have been attractive and major antiviral therapeutic targets. Therefore, Mononegavirales polymerases have been extensively investigated for their structures and functions. Mononegavirales mimic RNA synthesis of their eukaryotic counterparts by utilizing multifunctional RNA polymerases to replicate entire viral genomes and transcribe viral mRNAs from individual viral genes as well as synthesize 5' methylated cap and 3' poly(A) tail of the transcribed viral mRNAs. The catalytic subunit large protein (L) and cofactor phosphoprotein (P) constitute the Mononegavirales polymerases. In this review, we discuss the shared and unique features of RNA synthesis, the monomeric multifunctional enzyme L, and the oligomeric multimodular adapter P of Mononegavirales We outline the structural analyses of the Mononegavirales polymerases since the first structure of the vesicular stomatitis virus (VSV) L protein determined in 2015 and highlight multiple high-resolution cryo-electron microscopy (cryo-EM) structures of the polymerases of Mononegavirales, namely, VSV, RABV, HRSV, human metapneumovirus (HMPV), and human parainfluenza virus (HPIV), that have been reported in recent months (2019 to 2020). We compare the structures of those polymerases grouped by virus family, illustrate the similarities and differences among those polymerases, and reveal the potential RNA synthesis mechanisms and models of highly conserved Mononegavirales We conclude by the discussion of remaining questions, evolutionary perspectives, and future directions.

Labyrinthopeptins as virolytic inhibitors of respiratory syncytial virus cell entry.

Acute lower respiratory tract infections (ALRI) caused by respiratory syncytial virus (RSV) are associated with a severe disease burden among infants and elderly patients. Treatment options are limited. While numerous drug candidates with different viral targets are under development, the utility of RSV entry inhibitors is challenged by a low resistance barrier and by single mutations causing cross-resistance against a wide spectrum of fusion inhibitor chemotypes. We developed a cell-based screening assay for discovery of compounds inhibiting infection with primary RSV isolates. Using this system, we identified labyrinthopeptin A1 and A2 (Laby A1/A2), lantibiotics isolated from Actinomadura namibiensis, as effective RSV cell entry inhibitors with IC50s of 0.39 µM and 4.97 µM, respectively, and with favourable therapeutic index (>200 and > 20, respectively). Both molecules were active against multiple RSV strains including primary isolates and their antiviral activity against RSV was confirmed in primary human airway cells ex vivo and a murine model in vivo. Laby A1/A2 were antiviral in prophylactic and therapeutic treatment regimens and displayed synergistic activity when applied in combination with each other. Mechanistic studies showed that Laby A1/A2 exert virolytic activity likely by binding to phosphatidylethanolamine moieties within the viral membrane and by disrupting virus particle membrane integrity. Probably due to its specific mode of action, Laby A1/A2 antiviral activity was not affected by common resistance mutations to known RSV entry inhibitors. Taken together, Laby A1/A2 represent promising candidates for development as RSV inhibitors. Moreover, the cell-based screening system with primary RSV isolates described here should be useful to identify further antiviral agents.

Characteristics of Respiratory Syncytial Virus isolated from Acute Respiratory Infectious Disease in Busan

Human respiratory syncytial virus (HRSV) is known as the leading cause of respiratory tract illness in infancy and elderly children worldwide. We investigate the prevalence pattern and genetic characteristics in the second variable region G protein gene of HRSV during 5 consecutive seasons from 2010 to 2015. A total of 4,793 specimens (throat swabs) were collected from patients with acute respiratory tract. HRSV were evaluated and classified as HRSV A (n=111) or HRSV B (n=64) by real-time RT-PCR or RT-PCR. In general HRSV were detected in winter season. Coughing, fever, rhinorrhea and sputum were confirmed main symptoms in patients with HRSV. There were no significant differences in clinical characteristics or severity according to the HRSV subgroup infections. Out of 175 HRSV positive samples, 94 samples were successfully sequenced using G gene. Phylogenetic analysis revealed that 62 HRSV-A strains clustered into genotypes ON1 (n=54, 87.1%), NA1 (n=7), NA2 (n=1) and 32 HRSV-B strains clustered into three genotypes: BA4 (n=28, 87.5%), BA5 (n=2), BA6 (n=2). These results provide a better understanding of HRSV prevalence pattern and genetic characteristics.

Rapid replacement of prevailing genotype of human respiratory syncytial virus by genotype ON1 in Beijing, 2012-2014.

Human respiratory syncytial virus (HRSV) is the most common viral pathogen causing lower respiratory infections in infants and young children worldwide. HRSV ON1 genotype in subgroup A with a characteristic of a 72 nucleotide duplication in the second highly variable region of attachment glycoprotein gene, has been reported in some countries since it was first detected in clinical samples collected in Canada in 2010. In this study, 557 HRSV antigen-positive nasopharyngeal aspirates were randomly selected during 2012/2013 to 2013/2014 HRSV seasons in Beijing for subgroup typing and for ON1 genotype screening by using a PCR based method developed for easily identifying genotype ON1 out of strains of subtype A. It was found that subgroup B was dominant in the 2012/2013 season and sudden shift of subgroup dominance from B to A and rapid replacement of previously prevailing NA1 genotype by ON1 genotype occurred in the 2013/2014 season. Reversible amino acid replacement in the G protein gene was found in a new branch of ON1 genotype. The evolutionary rate of the 351 global ON1 sequences was estimated to 7.34 × 10(-3) nucleotide substitutions per site per year (95% highest probability density intervals, HPD, 5.71 × 10(-3) to 9.04 × 10(-3)), with the time of most recent common ancestor dating back to June 2009.

Genetic variation of human respiratory syncytial virus among children with fever and respiratory symptoms in Shanghai, China, from 2009 to 2012.

Human respiratory syncytial virus (HRSV) of genus Pneumovirus is one of the most common pathogens causing severe acute lower respiratory tract infection in infants and children. No information on the genotype distribution of HRSV is available in East China (e.g. Shanghai). From August 2009 to December 2012, 2407 nasopharyngeal swabs were collected from outpatient children with fever and respiratory symptoms in Shanghai. HRSV infection was determined using a multiplex RT-PCR assay. The second hypervariable region (HVR2) of G protein gene of HRSV was amplified and sequenced from HRSV positive samples. Genotypes were characterized by phylogenetic analyses. Of 2407 nasopharyngeal samples, 184 (7.6%) were tested as HRSV positive. From 160 positive subjects with sufficient nasopharyngeal samples, 69 HVR2 sequences were obtained by RT-PCR and sequencing. Three HRSV epidemic seasons were observed from August 2009 to December 2012, and an extreme outbreak of HRSV occurred in the 2009-2010 epidemic season. A genotype shift of predominant HRSV strains from B group in the 2009-2010 epidemic season to group A in the subsequent epidemic seasons was observed. Ten HRSV genotypes, including four group A genotypes NA1, NA3, NA4, and ON1, and six group B genotypes BA9, BA10, SAB4, CB1, BAc, and BA?, were detected in Shanghai. Seven genotypes (NA1, BA9-10, SAB4, CB1, BAc and BA?) were found in the 2009-2010 epidemic season. The co-circulation of multiple genotypes was associated with the extreme outbreak of HRSV among children with fever and respiratory symptoms in the 2009-2010 epidemic season.

A duplex recombinant viral nucleoprotein microbead immunoassay for simultaneous detection of seroresponses to human respiratory syncytial virus and metapneumovirus infections.

Serologic diagnosis of human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) infections has been shown to complement virus detection methods in epidemiologic studies. Enzyme immunoassays (EIAs) using cultured virus lysate antigens are often used to diagnose infection by demonstration of a ≥4-fold rises in antibody titer between acute and convalescent serum pairs. In this study, hRSV and hMPV nucleocapsid (recN) proteins were expressed in a baculovirus system and their performance compared with virus culture lysate antigen in EIAs using paired serum specimens collected from symptomatic children. The recN proteins were also used to develop a duplex assay based on the Luminex microbead-based suspension array technology, where diagnostic rises in antibody levels could be determined simultaneously at a single serum dilution. Antibody levels measured by the recN and viral lysate EIAs correlated moderately (hRSV, r(2)=0.72; hMPV, r(2)=0.76); the recN EIAs identified correctly 35 of 37 (94.6%) and 48 of 50 (96%) serum pairs showing diagnostic antibody rises by viral lysate EIAs. Purified recN proteins were then coupled to microbeads and serum pairs were tested at a single dilution on a Luminex MAGPIX(®) analyzer. The duplex recN assay identified correctly 33 of 39 (85%) and 41 of 47 (86.7%) serum pairs showing diagnostic rises to hRSV and hMPV, respectively. The recN assay permits simultaneous testing for acute hRSV and hMPV infections and offers a platform for expanded multiplexing of other respiratory virus assays.

Rapid replacement of human respiratory syncytial virus A with the ON1 genotype having 72 nucleotide duplication in G gene.

Human respiratory syncytial virus (HRSV) is the main cause of severe respiratory illness in young children and elderly people. We investigated the genetic characteristics of the circulating HRSV subgroup A (HRSV-A) to determine the distribution of genotype ON1, which has a 72-nucleotide duplication in attachment G gene. We obtained 456 HRSV-A positive samples between October 2008 and February 2013, which were subjected to sequence analysis. The first ON1 genotype was discovered in August 2011 and 273 samples were identified as ON1 up to February 2013. The prevalence of the ON1 genotype increased rapidly from 17.4% in 2011-2012 to 94.6% in 2012-2013. The mean evolutionary rate of G protein was calculated as 3.275 × 10(-3) nucleotide substitution/site/year and several positively selected sites for amino acid substitutions were located in the predicted epitope region. This basic and important information may facilitate a better understanding of HRSV epidemiology and evolution.

Novel clinical features of recurrent human respiratory syncytial virus infections.

Children and elderly individuals are often infected easily and repeatedly with human respiratory syncytial virus (HRSV); however, the features of recurrent infection in the same individual are defined poorly. To clarify the clinical significance of repeated HRSV infections in relation to subgroup epidemiology, this study performed prospective and longitudinal analyses in children with lower respiratory tract infections over 20 consecutive epidemics between 1985 and 2005 at a pediatric outpatient clinic in Kawasaki, Japan. HRSV infections were confirmed by 2 types of reverse-transcription PCR. Samples obtained from patients with repeated infections were subjected to sequence analysis and cloning analysis. A total of 1,312 lower respiratory tract infections observed in 1,010 patients were diagnosed as HRSV infections. Repeated HRSV infections occurred in 208 of the 1,010 patients. Analysis of the patients with repeated infections revealed that children were often infected multiple times even within a single short epidemic. Some patients were re-infected with strains having the same or virtually identical N gene sequences. In patients infected more than 4 times, cloning analysis revealed more frequent dual infections with both subgroups (23.8%). The HRSV-A subgroup caused subsequent homologous infections more frequently than did HRSV-B; furthermore, HRSV-A infections provided no protection from a second homologous infection. In contrast, HRSV-B infections offered significant protection against a second homologous infection. Statistical analysis revealed alleviation of symptoms with a reduced rate of dyspnoeic attacks only in the group re-infected with homologous HRSV-A strains. Thus, this study elucidates new clinical features of recurrent HRSV infection.