Within-Host Rhinovirus Evolution in Upper and Lower Respiratory Tract Highlights Capsid Variability and Mutation-Independent Compartmentalization.

BACKGROUND: Rhinovirus (RV) infections can progress from the upper (URT) to lower (LRT) respiratory tract in immunocompromised individuals, causing high rates of fatal pneumonia. Little is known about how RV evolves within hosts during infection. METHODS: We sequenced RV complete genomes from 12 hematopoietic cell transplant patients with infection for up to 190 days from both URT (nasal wash, NW) and LRT (bronchoalveolar lavage, BAL). Metagenomic and amplicon next-generation sequencing were used to track the emergence and evolution of intrahost single nucleotide variants (iSNVs). RESULTS: Identical RV intrahost populations in matched NW and BAL specimens indicated no genetic adaptation is required for RV to progress from URT to LRT. Coding iSNVs were 2.3-fold more prevalent in capsid over nonstructural genes. iSNVs modeled were significantly more likely to be found in capsid surface residues, but were not preferentially located in known RV-neutralizing antibody epitopes. Newly emergent, genotype-matched iSNV haplotypes from immunocompromised individuals in 2008-2010 could be detected in Seattle-area community RV sequences in 2020-2021. CONCLUSIONS: RV infections in immunocompromised hosts can progress from URT to LRT with no specific evolutionary requirement. Capsid proteins carry the highest variability and emergent mutations can be detected in other, including future, RV sequences.

Pan-antiviral effects of a PIKfyve inhibitor on respiratory virus infection in human nasal epithelium and mice.

Endocytosis, or internalization through endosomes, is a major cell entry mechanism used by respiratory viruses. Phosphoinositide 5-kinase (PIKfyve) is a critical enzyme for the synthesis of phosphatidylinositol (3, 5)biphosphate (PtdIns (3, 5)P2) and has been implicated in virus trafficking via the endocytic pathway. In fact, antiviral effects of PIKfyve inhibitors against SARS-CoV-2 and Ebola have been reported, but there is little evidence regarding other respiratory viruses. In this study, we demonstrated the antiviral effects of PIKfyve inhibitors on influenza virus and respiratory syncytial virus in vitro and in vivo. PIKfyve inhibitors Apilimod mesylate (AM) and YM201636 concentration-dependently inhibited several influenza strains in an MDCK cell-cytopathic assay. AM also reduced the viral load and cytokine release, while improving the cell integrity of human nasal air-liquid interface cultured epithelium infected with influenza PR8. In PR8-infected mice, AM (2 mg/mL), when intranasally treated, exhibited a significant reduction of viral load and inflammation and inhibited weight loss caused by influenza infection, with effects being similar to oral oseltamivir (10 mg/kg). In addition, AM demonstrated antiviral effects in RSV A2-infected human nasal epithelium in vitro and mouse in vivo, with an equivalent effect to that of ribavirin. AM also showed antiviral effects against human rhinovirus and seasonal coronavirus in vitro. Thus, PIKfyve is found to be involved in influenza and RSV infection, and PIKfyve inhibitor is a promising molecule for a pan-viral approach against respiratory viruses.

Identification of epidermal growth factor receptor-tyrosine kinase inhibitor targeting the VP1 pocket of human rhinovirus.

Screening a library of 1,200 preselected kinase inhibitors for anti-human rhinovirus 2 (HRV-2) activity in HeLa cells identified a class of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) as effective virus blockers. These were based on the 4-anilinoquinazoline-7-oxypiperidine scaffold, with the most potent representative AZ5385 inhibiting the virus with EC50 of 0.35 µM. Several structurally related analogs confirmed activity in the low µM range, while interestingly, other TKIs targeting EGFR lacked anti-HRV-2 activity. To further probe this lack of association between antiviral activity and EGFR inhibition, we stained infected cells with antibodies specific for activated EGFR (Y1068) and did not observe a dependency on EGFR-TK activity. Instead, consecutive passages of HRV-2 in HeLa cells in the presence of a compound and subsequent nucleotide sequence analysis of resistant viral variants identified the S181T and T210A alterations in the major capsid VP1 protein, with both residues located in the vicinity of a known hydrophobic pocket on the viral capsid. Further characterization of the antiviral effects of AZ5385 showed a modest virus-inactivating (virucidal) activity, while anti-HRV-2 activity was still evident when the inhibitor was added as late as 10 h post infection. The RNA copy/infectivity ratio of HRV-2 propagated in AZ5385 presence was substantially higher than that of control HRV indicating that the compound preferentially targeted HRV progeny virions during their maturation in infected cells. Besides HRV, the compound showed anti-respiratory syncytial virus activity, which warrants its further studies as a candidate compound against viral respiratory infections.

Prevalence and genetic diversity of human rhinovirus among patients with acute respiratory infections in China, 2012-2021.

To understand the prevalence of rhinovirus (RV) among acute respiratory infection (ARI) patients, 10-year ARI surveillance in multiple provinces of China were conducted during 2012-2021. Of 15 645 ARI patients, 1180 (7.54%) were confirmed to have RV infection and 820 (69.49%) were children under 5 years of age. RV typing was performed on the 527 VP1 gene sequences, and species A, B, and C accounted for 73.24%, 4.93%, and 21.82%, respectively. Although no significant difference in the proportions of age groups or disease severity was found between RV species, RV-C was more frequently detected in children under 5 years of age, RV-A was more frequently detected in elderly individuals (≥60), and the proportions of pneumonia in RV-A and RV-C patients were higher than those in RV-B patients. The epidemic peak of RV-A was earlier than that of RV-C. A total of 57 types of RV-A, 13 types of RV-B, and 35 types of RV-C were identified in RV-infected patients, and two uncertain RV types were also detected. The findings showed a few differences in epidemiological and clinical features between RV species in ARI patients, and RV-A and RV-C were more prevalent than RV-B.

Characterization of bacterial and viral pathogens in the respiratory tract of children with HIV-associated chronic lung disease: a case-control study.

INTRODUCTION: Chronic lung disease is a major cause of morbidity in African children with HIV infection; however, the microbial determinants of HIV-associated chronic lung disease (HCLD) remain poorly understood. We conducted a case-control study to investigate the prevalence and densities of respiratory microbes among pneumococcal conjugate vaccine (PCV)-naive children with (HCLD +) and without HCLD (HCLD-) established on antiretroviral treatment (ART). METHODS: Nasopharyngeal swabs collected from HCLD + (defined as forced-expiratory-volume/second < -1.0 without reversibility postbronchodilation) and age-, site-, and duration-of-ART-matched HCLD- participants aged between 6-19 years enrolled in Zimbabwe and Malawi (BREATHE trial-NCT02426112) were tested for 94 pneumococcal serotypes together with twelve bacteria, including Streptococcus pneumoniae (SP), Staphylococcus aureus (SA), Haemophilus influenzae (HI), Moraxella catarrhalis (MC), and eight viruses, including human rhinovirus (HRV), respiratory syncytial virus A or B, and human metapneumovirus, using nanofluidic qPCR (Standard BioTools formerly known as Fluidigm). Fisher's exact test and logistic regression analysis were used for between-group comparisons and risk factors associated with common respiratory microbes, respectively. RESULTS: A total of 345 participants (287 HCLD + , 58 HCLD-; median age, 15.5 years [IQR = 12.8-18], females, 52%) were included in the final analysis. The prevalence of SP (40%[116/287] vs. 21%[12/58], p = 0.005) and HRV (7%[21/287] vs. 0%[0/58], p = 0.032) were higher in HCLD + participants compared to HCLD- participants. Of the participants positive for SP (116 HCLD + & 12 HCLD-), 66% [85/128] had non-PCV-13 serotypes detected. Overall, PCV-13 serotypes (4, 19A, 19F: 16% [7/43] each) and NVT 13 and 21 (9% [8/85] each) predominated. The densities of HI (2 × 104 genomic equivalents [GE/ml] vs. 3 × 102 GE/ml, p = 0.006) and MC (1 × 104 GE/ml vs. 1 × 103 GE/ml, p = 0.031) were higher in HCLD + compared to HCLD-. Bacterial codetection (≥ any 2 bacteria) was higher in the HCLD + group (36% [114/287] vs. (19% [11/58]), (p = 0.014), with SP and HI codetection (HCLD + : 30% [86/287] vs. HCLD-: 12% [7/58], p = 0.005) predominating. Viruses (predominantly HRV) were detected only in HCLD + participants. Lastly, participants with a history of previous tuberculosis treatment were more likely to carry SP (adjusted odds ratio (aOR): 1.9 [1.1 -3.2], p = 0.021) or HI (aOR: 2.0 [1.2 - 3.3], p = 0.011), while those who used ART for ≥ 2 years were less likely to carry HI (aOR: 0.3 [0.1 - 0.8], p = 0.005) and MC (aOR: 0.4 [0.1 - 0.9], p = 0.039). CONCLUSION: Children with HCLD + were more likely to be colonized by SP and HRV and had higher HI and MC bacterial loads in their nasopharynx. The role of SP, HI, and HRV in the pathogenesis of CLD, including how they influence the risk of acute exacerbations, should be studied further. TRIAL REGISTRATION: The BREATHE trial (ClinicalTrials.gov Identifier: NCT02426112 , registered date: 24 April 2015).

Lactobacillus rhamnosus dampens cytokine and chemokine secretion from primary human nasal epithelial cells infected with rhinovirus.

AIMS: To investigate the effect of Lactobacillus rhamnosus on viral replication and cellular response to human rhinovirus (HRV) infection, including the secretion of antiviral and inflammatory mediators from well-differentiated nasal epithelial cells (WD-NECs). METHODS AND RESULTS: The WD-NECs from healthy adult donors (N = 6) were cultured in vitro, exposed to different strains of L. rhamnosus (D3189, D3160, or LB21), and infected with HRV (RV-A16) after 24 h. Survival and adherence capacity of L. rhamnosus in a NEC environment were confirmed using CFSE-labelled isolates, immunofluorescent staining, and confocal microscopy. Shed virus and viral replication were quantified using TCID50 assays and RT-qPCR, respectively. Cytotoxicity was measured by lactate dehydrogenase (LDH) activity. Pro-inflammatory mediators were measured by multiplex immunoassay, and interferon (IFN)-λ1/3 was measured using a standard ELISA kit. Lactobacillus rhamnosus was able to adhere to and colonize WD-NECs prior to the RV-A16 infection. Lactobacillus rhamnosus did not affect shed RV-A16, viral replication, RV-A16-induced IFN-λ1/3 production, or LDH release. Pre-exposure to L. rhamnosus, particularly D3189, reduced the secretion of RV-A16-induced pro-inflammatory mediators by WD-NECs. CONCLUSIONS: These findings demonstrate that L. rhamnosus differentially modulates RV-A16-induced innate inflammatory immune responses in primary NECs from healthy adults.

Coinfection with respiratory syncytial virus and rhinovirus increases IFN-λ1 and CXCL10 expression in human primary bronchial epithelial cells.

Acute respiratory tract infection (ARTI) is common in all age groups, especially in children and the elderly. About 85% of children who present with bronchiolitis are infected with respiratory syncytial virus (RSV); however, nearly one-third are coinfected with another respiratory virus, such as human rhinovirus (HRV). Therefore, it is necessary to explore the immune response to coinfection to better understand the molecular and cellular pathways involving virus-virus interactions that might be modulated by innate immunity and additional host cell response mechanisms. This study aims to investigate the host innate immune response against RSV-HRV coinfection compared with monoinfection. Human primary bronchial/tracheal epithelial cells (HPECs) were infected with RSV, HRV, or coinfected with both viruses, and the infected cells were collected at 48 and 72 hours. Gene expression profiles of IL-6, CCL5, TNF-α, IFN-ß, IFN-λ1, CXCL10, IL-10, IL-13, IRF3, and IRF7 were investigated using real-time quantitative PCR, which revealed that RSV-infected cells exhibited increased expression of IL-10, whereas HRV infection increased the expression of CXCL10, IL-10, and CCL5. IFN-λ1 and CXCL10 expression was significantly different between the coinfection and monoinfection groups. In conclusion, our study revealed that two important cytokines, IFN-λ1 and CXCL10, exhibited increased expression during coinfection.

Airway-delivered short-chain fatty acid acetate boosts antiviral immunity during rhinovirus infection.

BACKGROUND: Microbiota are recognized to play a major role in regulation of immunity through release of immunomodulatory metabolites such as short-chain fatty acids (SCFAs). Rhinoviruses (RVs) induce upper respiratory tract illnesses and precipitate exacerbations of asthma and chronic obstructive pulmonary disease through poorly understood mechanisms. Local interactions between SCFAs and antiviral immune responses in the respiratory tract have not been previously investigated. OBJECTIVE: We sought to investigate whether pulmonary metabolite manipulation through lung-delivered administration of SCFAs can modulate antiviral immunity to RV infection. METHODS: We studied the effects of intranasal administration of the SCFAs acetate, butyrate, and propionate on basal expression of antiviral signatures, and of acetate in a mouse model of RV infection and in RV-infected lung epithelial cell lines. We additionally assessed the effects of acetate, butyrate, and propionate on RV infection in differentiated human primary bronchial epithelial cells. RESULTS: Intranasal acetate administration induced basal upregulation of IFN-ß, an effect not observed with other SCFAs. Butyrate induced RIG-I expression. Intranasal acetate treatment of mice increased interferon-stimulated gene and IFN-λ expression during RV infection and reduced lung virus loads at 8 hours postinfection. Acetate ameliorated virus-induced proinflammatory responses with attenuated pulmonary mucin and IL-6 expression observed at day 4 and 6 postinfection. This interferon-enhancing effect of acetate was confirmed in human bronchial and alveolar epithelial cell lines. In differentiated primary bronchial epithelial cells, butyrate treatment better modulated IFN-ß and IFN-λ gene expression during RV infection. CONCLUSIONS: SCFAs augment antiviral immunity and reduce virus load and proinflammatory responses during RV infection.

Molecular Determinants of Human Rhinovirus Infection, Assembly, and Conformational Stability at Capsid Protein Interfaces.

Human rhinovirus (HRV), one of the most frequent human pathogens, is the major causative agent of common colds. HRVs also cause or exacerbate severe respiratory diseases, such as asthma or chronic obstructive pulmonary disease. Despite the biomedical and socioeconomic importance of this virus, no anti-HRV vaccines or drugs are available yet. Protein-protein interfaces in virus capsids have increasingly been recognized as promising virus-specific targets for the development of antiviral drugs. However, the specific structural elements and residues responsible for the biological functions of these extended capsid regions are largely unknown. In this study, we performed a thorough mutational analysis to determine which particular residues along the capsid interpentamer interfaces are relevant to HRV infection as well as the stage(s) in the viral cycle in which they are involved. The effect on the virion infectivity of the individual mutation to alanine of 32 interfacial residues that, together, removed most of the interpentamer interactions was analyzed. Then, a representative sample that included many of those 32 single mutants were tested for capsid and virion assembly as well as virion conformational stability. The results indicate that most of the interfacial residues, and the interactions they establish, are biologically relevant, largely because of their important roles in virion assembly and/or stability. The HRV interpentamer interface is revealed as an atypical protein-protein interface, in which infectivity-determining residues are distributed at a high density along the entire interface. Implications for a better understanding of the relationship between the molecular structure and function of HRV and the development of novel capsid interface-binding anti-HRV agents are discussed. IMPORTANCE The rising concern about the serious medical and socioeconomic consequences of respiratory infections by HRV has elicited a renewed interest in the development of anti-HRV drugs. The conversion into effective drugs of compounds identified via screening, as well as antiviral drug design, rely on the acquisition of fundamental knowledge about the targeted viral elements and their roles during specific steps of the infectious cycle. The results of this study provide a detailed view on structure-function relationships in a viral capsid protein-protein interface, a promising specific target for antiviral intervention. The high density and scattering of the interfacial residues found to be involved in HRV assembly and/or stability support the possibility that any compound designed to bind any particular site at the interface will inhibit infection by interfering with virion morphogenesis or stabilization of the functional virion conformation.

Establishment of RT-RPA-Cas12a assay for rapid and sensitive detection of human rhinovirus B.

Human rhinovirus B (HRV-B) is a major human viral pathogen that can be responsible for various kinds of infections. Due to the health risks associated with HRV-B, it is therefore crucial to explore a rapid, specific, and sensitive method for surveillance. Herein, we exploited a novel detection method for HRV-B by combining reverse-transcription recombinase polymerase amplification (RT-RPA) of nucleic acids isothermal amplification and the trans-cleavage activity of Cas12a. Our RT-RPA-Cas12a-based fluorescent assay can be completed within 35-45 min and obtain a lower detection threshold to 0.5 copies/µL of target RNA. Meanwhile, crRNA sequences without a specific protospacer adjacent motif can effectively activate the trans-cleavage activity of Cas12a. Moreover, our RT-RPA-Cas12a-based fluorescent method was examined using 30 clinical samples, and exhibited high accuracy with positive and negative predictive agreement of 90% and 100%, respectively. Taken together, a novel promising, rapid and effective RT-RPA-Cas12a-based detection method was explored and shows promising potential for on-site HRV-B infection in resource-limited settings.

Identification of two proposed novel human rhinovirus types: Bpat107 and Cpat58.

Human rhinovirus (RV) is an important viral pathogen associated with severe acute respiratory tract infection. The present study retrospectively identified RV types in hospitalized patients with severe acute respiratory infection (SARI) from October 2017 to June 2019 in Henan Province, China. Real-time PCR was used to screen pharyngeal swab samples for RV. Then, the VP1 gene sequences of RV-positive samples were amplified and sequenced with nested primer PCR; subsequently, analyses of the molecular epidemiology and genetic diversity characteristics of the RV types were performed. Seventy-three out of 1015 respiratory samples were identified as RV-positive, from which 65 complete VP1 sequences were successfully sequenced. These RVs were classified into 41 different types, including 26 RV-A types, 2 RV-B types, and 13 RV-C types. The RVs showed an obvious seasonal distribution, with peaks in summer and autumn. The epidemic peak of RV-C was later than that of RV-A. In addition, two new types of species, B and C, were proposed, Bpat107 and Cpat58, respectively. Compared with other types in the same RV species, the pairwise nucleotide p-distances of the two novel RV types were 0.262~0.402 and 0.251~0.508, respectively. This study analyzed the seasonal and genetic characteristics of RV associated with SARI cases in Henan Province, China. Two novel RV types were proposed.

Effects of treatment with corticosteroids on human rhinovirus-induced asthma exacerbations in pediatric inpatients: a prospective observational study.

BACKGROUND: Human rhinoviruses (HRVs) infection is a common cause of exacerbations in pediatric patients with asthma. However, the effects of corticosteroids on HRV-induced exacerbations in pediatric asthma are unknown. We conducted a prospective observational study to determine the viral pathogens in school-age pediatric inpatients with asthma exacerbations. We assessed the effects of maintenance inhaled corticosteroids (ICS) on the detection rates of HRV species and treatment periods of systemic corticosteroids during exacerbations on pulmonary lung function after exacerbations. METHODS: Nasopharyngeal samples and clinical information were collected from 59 patients with asthma exacerbations between April 2018 and March 2020. Pulmonary function tests were carried out 3 months after exacerbations in 18 HRV-positive patients. Changes in forced expiratory volume in 1 second (FEV1)% predicted from baseline in a stable state were compared according to the treatment periods of systemic corticosteroids. RESULTS: Fifty-four samples collected from hospitalized patients were analyzed, and viral pathogens were identified in 45 patients (83.3%) using multiplex PCR assay. HRV-A, -B, and -C were detected in 16 (29.6%), one (1.9%), and 16 (29.6%) patients, respectively. The detection rates of HRV-C were lower in the ICS-treated group compared with those in the ICS-untreated group (p = 0.01), whereas maintenance ICS treatment did not affect the detection rate for viral pathogens in total and HRV-A. Changes in FEV1% predicted in patients treated with systemic corticosteroids for 6-8 days (n = 10; median, 4.90%) were higher than those in patients treated for 3-5 days (n = 8; median, - 10.25%) (p = 0.0085). CONCLUSIONS: Maintenance ICS reduced the detection rates of HRV (mainly HRV-C) in school-age inpatients with asthma exacerbations, and the treatment periods of systemic corticosteroids during exacerbations affected lung function after HRV-induced exacerbations. The protective effects of corticosteroids on virus-induced asthma exacerbations may be dependent upon the types of viral pathogen.

Interactions Between Severe Acute Respiratory Syndrome Coronavirus 2 Replication and Major Respiratory Viruses in Human Nasal Epithelium.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), along with extensive nonpharmacological interventions, have profoundly altered the epidemiology of major respiratory viruses. Some studies have described virus-virus interactions, particularly manifested by viral interference mechanisms at different scales. However, our knowledge of the interactions between SARS-CoV-2 and other respiratory viruses remains incomplete. Here, we studied the interactions between SARS-CoV-2 and several respiratory viruses (influenza, respiratory syncytial virus, human metapneumovirus, and human rhinovirus) in a reconstituted human epithelial airway model, exploring different scenarios affecting the sequence and timing of coinfections. We show that the virus type and sequence of infections are key factors in virus-virus interactions, the primary infection having a determinant role in the immune response to the secondary infection.

Viral Interference between Respiratory Viruses.

Multiple respiratory viruses can concurrently or sequentially infect the respiratory tract and lead to virus‒virus interactions. Infection by a first virus could enhance or reduce infection and replication of a second virus, resulting in positive (additive or synergistic) or negative (antagonistic) interaction. The concept of viral interference has been demonstrated at the cellular, host, and population levels. The mechanisms involved in viral interference have been evaluated in differentiated airway epithelial cells and in animal models susceptible to the respiratory viruses of interest. A likely mechanism is the interferon response that could confer a temporary nonspecific immunity to the host. During the coronavirus disease pandemic, nonpharmacologic interventions have prevented the circulation of most respiratory viruses. Once the sanitary restrictions are lifted, circulation of seasonal respiratory viruses is expected to resume and will offer the opportunity to study their interactions, notably with severe acute respiratory syndrome coronavirus 2.

Honeybee Iflaviruses Pack Specific tRNA Fragments from Host Cells in Their Virions.

The Picornavirales include viruses that infect vertebrates, insects, and plants. It was believed that they pack only their genomic mRNA in the particles; thus, we envisaged these viruses as excellent model systems for studies of mRNA modifications. We used LC-MS to analyze digested RNA isolated from particles of the sacbrood and deformed wing iflaviruses as well as of the echovirus 18 and rhinovirus 2 picornaviruses. Whereas in the picornavirus RNAs we detected only N6 -methyladenosine and 2'-O-methylated nucleosides, the iflavirus RNAs contained a wide range of methylated nucleosides, such as 1-methyladenosine (m1 A) and 5-methylcytidine (m5 C). Mapping of m1 A and m5 C through RNA sequencing of the SBV and DWV RNAs revealed the presence of tRNA molecules. Both modifications were detected only in tRNA. Further analysis revealed that tRNAs are present in form of 3' and 5' fragments and they are packed selectively. Moreover, these tRNAs are typically packed by other viruses.

Rhinovirus persistence during the COVID-19 pandemic-Impact on pediatric acute wheezing presentations.

Rhinoviruses have persisted throughout the COVID-19 pandemic, despite other seasonal respiratory viruses (influenza, parainfluenza, respiratory syncytial virus, adenoviruses, human metapneumovirus) being mostly suppressed by pandemic restrictions, such as masking and other forms of social distancing, especially during the national lockdown periods. Rhinoviruses, as nonenveloped viruses, are known to transmit effectively via the airborne and fomite route, which has allowed infection among children and adults to continue despite pandemic restrictions. Rhinoviruses are also known to cause and exacerbate acute wheezing episodes in children predisposed to this condition. Noninfectious causes such as air pollutants (PM2.5 , PM10 ) can also play a role. In this retrospective ecological study, we demonstrate the correlation between UK national sentinel rhinovirus surveillance, the level of airborne particulates, and the changing patterns of pediatric emergency department presentations for acute wheezing, before and during the COVID-19 pandemic (2018-2021) in a large UK teaching hospital.

Identification of an IRES within the coding region of the structural protein of human rhinovirus 16.

As an alternative mechanism for cap-dependent (m7GpppN) translation, internal ribosome entry site (IRES)-dependent translation has been observed in the 5' untranslated regions (5' UTR) and coding regions of a number of viral and eukaryotic mRNAs. In this study, a series of 5' terminal truncated structural protein genes that were fused with GFP was used to screen for potential IRESs, and IRESs were identified using a bicistronic luciferase vector or GFP expression vector possessing a hairpin structure. Our results revealed that a putative IRES was located between nt 1982 and 2281 in the VP3 coding region of the human rhinovirus 16 (HRV16) genomes. We also demonstrated that effective IRES-initiated protein expression in vitro did not occur through splicing sites or cryptic promoters. We confirmed that thapsigargin (TG), an inducer of endoplasmic reticulum stress (ERS), facilitated increased IRES activity in a dose-dependent manner. Additionally, the secondary structure of the IRES was predicted online using the RNAfold web server.

Comparison of immune response to human rhinovirus C and respiratory syncytial virus in highly differentiated human airway epithelial cells.

BACKGROUND: Human rhinovirus C (HRV-C) accounts for a large proportion of HRV-related illnesses, but the immune response to HRV-C infection has not been elucidated. Our objective was to assess the effect of HRV-C on cytokine secretion in human bronchial epithelial (HBE) cells grown at air-liquid interface (ALI) and compare it with that of respiratory syncytial virus (RSV). METHODS: HBE cells were differentiated at ALI culture and the full-length cDNA clones of HRV-C651 and HRV-C15, clinical isolates of HRV-C79 and HRV-C101, and two RSV isolates were inoculated in the HBE cells. The effect of HRV-C on cytokine secretion was assessed and compared with that of RSV. RESULTS: HRV-Cs infect and propagate in fully differentiated HBE cells and significantly increase the secretion of IFN-λ1, CCL5, IP10, IL-6, IL-8, and MCP-1. The virus loads positively correlated with the levels of the cytokines. HRV-C induced lower secretion of CCL5 (P = 0.048), IL-6 (P = 0.016), MCP-1 (P = 0.008), and IL-8 (P = 0.032), and similar secretion of IP10 (P = 0.214) and IFN-λ1 (P = 0.214) when compared with RSV. CONCLUSION: HBE ALI culture system supported HRV-C infection and propagation and HRV-C induced relatively weaker cytokine expression than RSV.

Enteroviral replication inhibition by N-Alkyl triazolopyrimidinone derivatives through a non-capsid binding mode.

Small-molecule inhibitors exhibiting broad-spectrum enteroviral inhibition by targeting viral replication proteins are highly desirable in antiviral drug discovery. We used the previously identified antiviral compound 1 as the starting material to develop a novel compound series with high efficacy against human rhinovirus (hRV). Further optimization of N-substituted triazolopyrimidinone derivatives revealed that the N-alkyl triazolopyrimidinone derivatives (2) had more potent antiviral activity against hRVs than compound 1. The new compounds showed improved selectivity index values, and compound 2c (KR-25210) displayed broad anti-hRV activity, with half-maximal effective concentration values ≤ 2 µM against all tested hRVs. In addition, 2c showed notable activity against other enteroviruses. Drug-likeness elucidation showed that 2c exhibited reasonable human and rat liver microsomal phase-I stability and safe CYP inhibition. Replication studies revealed that 2c is not a capsid inhibitor, and a time-of-addition assay indicated that 2c targets the virus replication stages.

Human rhinoviruses prevailed among children in the setting of wearing face masks in Shanghai, 2020.

BACKGROUND: Human rhinovirus (HRV) is the predominant etiological agent of the common cold in children and adults. A recent study showed that the inhibitory effect of face masks on viral shedding of HRV was less prominent than that on other respiratory viruses. Considering that most Chinese people have worn face masks in public area since the outbreak of coronavirus disease 2019, we aimed to find out whether HRV prevailed among children in 2020 and demonstrate the details of the epidemiological features of HRV under such a special circumstance. METHODS: We summarized the incidences of various respiratory virus infections in patients who visited the Children's Hospital of Fudan University during 2018-2020, and genotyped HRV positive nasopharyngeal specimens collected from 316 inpatients and 72 outpatients that visited the hospital in 2020. RESULTS: There was a major prevalence of HRV among children in the latter half of 2020, with a clear seasonality that HRV-As prevailed in summer while HRV-Cs in autumn. HRV-As were more prone to cause severe lower respiratory tract infections (LRTI), while HRV-Cs were closely associated with childhood wheezing. The predominant genotypes were A11, A28, A47, A82, A101, C40 and C43. Notably, A21, A82 and A101 took up larger proportions in severe cases than in non-severe cases. CONCLUSIONS: Our findings described a major prevalence of HRVs among children in 2020, which highlight the unique transmitting pattern of HRV and help to narrow the targets for antiviral strategies.