Breaking the Chain: Protease Inhibitors as Game Changers in Respiratory Viruses Management.

Respiratory viral infections (VRTIs) rank among the leading causes of global morbidity and mortality, affecting millions of individuals each year across all age groups. These infections are caused by various pathogens, including rhinoviruses (RVs), adenoviruses (AdVs), and coronaviruses (CoVs), which are particularly prevalent during colder seasons. Although many VRTIs are self-limiting, their frequent recurrence and potential for severe health complications highlight the critical need for effective therapeutic strategies. Viral proteases are crucial for the maturation and replication of viruses, making them promising therapeutic targets. This review explores the pivotal role of viral proteases in the lifecycle of respiratory viruses and the development of protease inhibitors as a strategic response to these infections. Recent advances in antiviral therapy have highlighted the effectiveness of protease inhibitors in curtailing the spread and severity of viral diseases, especially during the ongoing COVID-19 pandemic. It also assesses the current efforts aimed at identifying and developing inhibitors targeting key proteases from major respiratory viruses, including human RVs, AdVs, and (severe acute respiratory syndrome coronavirus-2) SARS-CoV-2. Despite the recent identification of SARS-CoV-2, within the last five years, the scientific community has devoted considerable time and resources to investigate existing drugs and develop new inhibitors targeting the virus's main protease. However, research efforts in identifying inhibitors of the proteases of RVs and AdVs are limited. Therefore, herein, it is proposed to utilize this knowledge to develop new inhibitors for the proteases of other viruses affecting the respiratory tract or to develop dual inhibitors. Finally, by detailing the mechanisms of action and therapeutic potentials of these inhibitors, this review aims to demonstrate their significant role in transforming the management of respiratory viral diseases and to offer insights into future research directions.

7-Amino-3-phenyl-2-methyl-pyrazolopyrimidine derivatives inhibit human rhinovirus replication.

Small molecules that exhibit broad-spectrum enteroviral inhibitory activity by targeting viral replication proteins are highly desired in antiviral drug discovery studies. To discover new human rhinovirus (hRV) inhibitors, we performed a high-throughput screening of 100,000 compounds from the Korea Chemical Bank library. This search led to identification of two phosphatidylinositol-4-kinase IIIß (PI4KIIIß) inhibitors having the pyrazolo-pyrimidine core structure, which display moderate anti-rhinoviral activity along with mild cytotoxicity. The results of a study aimed at optimizing the activity of the hit compounds showed that the pyrazolo-pyrimidine derivative 6f exhibits the highest activity (EC50 = 0.044, 0.066, and 0.083 µM for hRV-B14, hRV-A16, and hRV-A21, respectively) and moderate toxicity (CC50 = 31.38 µM). Furthermore, 6f has broad-spectrum activities against various hRVs, coxsackieviruses and other enteroviruses, such as EV-A71, EV-D68. An assessment of kinase inhibition potencies demonstrated that 6f possesses a high and selective kinase inhibition activity against PI4KIIIß (IC50 value of 0.057 µM) and not against PI4KIIIα (>10 µM). Moreover, 6f exhibits modest hepatic stability (46.9 and 55.3 % remaining after 30 min in mouse and human liver microsomes, respectively). Finally, an in vivo study demonstrated that 6f possesses a desirable pharmacokinetic profile reflected in low systemic clearance (0.48 L∙h-1 kg-1) and modest oral bioavailability (52.4 %). Hence, 6f (KR-26549) appears to be an ideal lead for the development of new antiviral drugs.

Pathogenic Role of Human Rhinovirus Mono-Infection in Pediatric Lower Respiratory Tract Infection.

Background and objective Human rhinovirus (HRV) is one of the leading causes of pediatric respiratory tract infection with a prevalence rate of 30-50%, mostly affecting children below five years of age and causing a substantial amount of economic loss. In children, it can alone or as a co-infection, cause a wide range of symptoms from mild to life-threatening ones. With the above background, the current study was carried out to emphasize the role of HRV mono-infection in pediatric acute respiratory tract infections by correlating clinical and molecular laboratory findings. Methods This study was carried out in a tertiary care teaching hospital over a duration of four years (March 2019-October 2023). Children up to 14 years of age visiting the outpatient department or admitted to the ward with diagnoses of acute respiratory tract infections (ARTIs) were included. The clinical and laboratory data were retrieved and analyzed. A nasopharyngeal swab (NPS) or throat swab (TS) was collected and sent to the Microbiology laboratory maintaining the cold chain. Nucleic acid was extracted and subjected to multiplex real-time polymerase chain reaction (RT-PCR). Result Of the 245 samples tested for the respiratory viral pathogen, 52 samples tested positive for HRV, of which 27 had HRV mono-infection. The clinico-demographic details of these 27 patients were studied in detail. The majority of the cases (24/27; 88.8%) were less than five years of age. Fever and shortness of breath were the most consistent symptoms in all. Nineteen (19/27; 62.9%) HRV mono-infection cases had underlying co-morbidities, all requiring respiratory support. The HRV mono-infection cases either developed bronchiolitis, lower respiratory tract infection, or pneumonia. All mono-infection cases had cycle threshold value (Ct) < 25, while the Ct value of HRV was > 30 in co-infection with other viruses. Conclusion Mono-infection of HRV in under-five children with underlying comorbidities and a lesser Ct value indicates severe disease manifestation and should be dealt with more cautiously.

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).

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.

Molecular Epidemiology and Clinical Characteristics of an Outbreak on Respiratory Virus Coinfection in Gansu, China.

This study aims to analyze the epidemiological and pathogenic characteristics of an outbreak primarily caused by respiratory syncytial virus (RSV), human rhinovirus (HRV), and human metapneumovirus (HMPV) in a kindergarten and primary school. The outbreak was investigated by field epidemiological investigation, and the common respiratory pathogens were screened by RT-PCR detection technology. The attack rate of this outbreak was 63.95% (110/172). Main symptoms included cough (85.45%), sore throat (60.91%), and sneezing (60.00%). Multifactorial logistic regression analysis revealed that continuous handwashing and mouth and nose covering when sneezing were protective factors. All 15 collected throat swab specimens tested positive for viruses, with HMPV as the predominant pathogen (80.00%), followed by HRV (53.33%), and two cases of positive respiratory syncytial virus (13.33%). Among them, six samples showed coinfections of HMPV and HRV, and one had coinfections of HMPV and RSV, resulting in a coinfection rate of 46.67%. Genetic sequencing indicated that the HMPV genotype in this outbreak was A2c, and the HRV genotype was type A, resulting in a coinfection outbreak of HMPV, HRV, and RSV in schools and kindergartens, suggesting that multi-pathogen surveillance of respiratory tract infections should be strengthened.

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.

Ginsenoside Re protects rhinovirus-induced disruption of tight junction through inhibition of ROS-mediated phosphatases inactivation in human nasal epithelial cells.

Maintaining tight junction integrity significantly contributes to epithelial barrier function. If the barrier function is destroyed, the permeability of the cells increases, and the movement of the pathogens is promoted, thereby further increasing the susceptibility to secondary infection. Ginsenoside components have multiple biological activities, including antiviral effects. In this study, we examined the protective effects of ginsenoside Re against rhinovirus-induced tight junction disruption in primary human nasal epithelial cells (HNE). Incubation with human rhinovirus resulted in marked disruption of tight junction proteins (ZO-1, E-cadherin, claudin-1, and occludin) in human nasal epithelial cells. Rhinovirus-induced disruption of tight junction proteins was strongly inhibited by the treatment of cells with ginsenoside Re. Indeed, significant amounts of reactive oxygen species (ROS) have been detected in human nasal epithelial cells co-incubated with rhinovirus. Moreover, rhinovirus-induced ROS generation was markedly reduced by the ginsenoside Re. However, ginsenosides Rb1 and Rc did not inhibit tight junction disruption or ROS generation in nasal epithelial cells following incubation with rhinovirus. Furthermore, incubation with rhinovirus resulted in a marked decrease in protein phosphatase activity and an increase in protein tyrosine phosphorylation levels in nasal epithelial cells. Treatment of cells with ginsenoside Re inhibited rhinovirus-induced inactivation of phosphatases and phosphorylation of tyrosine. Our results identified ginsenoside Re as an effective compound that prevented rhinovirus-induced tight junction disruption in human nasal epithelial cells.

Microbiological profile of long COVID and associated clinical and radiological findings: a prospective cross-sectional study.

OBJECTIVE: To study the frequency of microbiological etiology of respiratory infections in patients with long COVID and their associated clinical and radiological findings. METHODS: Nasopharyngeal swabs and sputum specimens were collected from 97 patients with respiratory illness stemming from long COVID. The specimens were assessed for their microbiological profile (bacteria and virus) and their association with the overall clinical and radiological picture. RESULTS: In total, 23 (24%) patients with long COVID had viral infection (n = 12), bacterial infection (n = 9), or coinfection (n = 2). Microorganisms were detected at significantly higher rates in hospitalized patients, patients with moderate COVID-19, and patients with asthma (P < .05). Tachycardia (65%) was the most common symptom at presentation. A statistically significant number of patients with long COVID who had viral infection presented with cough and myalgia; and a statistically significant number of patients with long COVID who had bacterial infection presented with productive coughing (P < .05). Post-COVID fibrotic changes were found in 61% of cohort patients (31/51). CONCLUSION: A decreasing trend of respiratory pathogens (enveloped viruses and bacteria) was found in long COVID. An analysis including a larger group of viral- or bacterial-infected patients with long COVID is needed to obtain high-level evidence on the presenting symptoms (cough, myalgia) and their association with the underlying comorbidities and severity.

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.

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.

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.

Clinical Outcomes of Human Rhinovirus/Enterovirus Infection in Pediatric Hemopoietic Cell Transplant Patients.

BACKGROUND: Respiratory viral infections are common among pediatric transplant patients, with human rhinovirus (HRV) being the most frequent. In pediatric patients undergoing hemopoietic cell transplant (HCT), infection with HRV has been associated with progression to lower respiratory tract infection (LRTI) and adverse outcomes. We describe the clinical presentation and outcomes of HRV infection in children undergoing HCT. METHODS: Single-center retrospective study. HCT recipients who were positive for HRV/EV (HRV+) or negative for any respiratory virus (VN) by BioFire® FilmArray® panel between October 2014 and December 2017, were included. Primary outcomes were progression to LRTI, ICU admission, all-cause mortality at 3 and 6 months, and respiratory event-related mortality at 6 months. RESULTS: 227 patients (160 allogeneic HCT) were included. Of all patients, 108/227 (47.6%) were HRV+. From all HRV+, 95/108 (88%) were symptomatic and 68/107 (63.6%) of the diagnosis were made pretransplant. The median age of HRV+ was significantly lower than VN patients (5 vs 10 years). Cough and rhinorrhea were more frequently observed in HRV+ (53.7 and 60% vs 19.8 and 22.8%, respectively). No differences were found between both groups pretransplant and overall in rates progression to LRTI, ICU admission, mechanical ventilation, all-cause within 3 and 6 months, and mortality related with respiratory failure. No significant association was found between the severity of respiratory disease and the type of conditioning, type of transplant, or absolute lymphocyte count. CONCLUSIONS: HRV infection is frequently detected in HCT recipients but is not associated with severity of respiratory disease, need for intensive care unit or mortality, including those diagnosed before transplant, suggesting that delaying HCT in this scenario may not be needed. Multicenter larger studies are required to confirm these findings.

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.

Development of Human Rhinovirus RNA Reference Material Using Digital PCR.

The human rhinovirus (RV) is a positive-stranded RNA virus that causes respiratory tract diseases affecting both the upper and lower halves of the respiratory system. RV enhances its replication by concentrating RNA synthesis within a modified host membrane in an intracellular compartment. RV infections often occur alongside infections caused by other respiratory viruses, and the RV virus may remain asymptomatic for extended periods. Alongside qualitative detection, it is essential to accurately quantify RV RNA from clinical samples to explore the relationships between RV viral load, infections caused by the virus, and the resulting symptoms observed in patients. A reference material (RM) is required for quality evaluation, the performance evaluation of molecular diagnostic products, and evaluation of antiviral agents in the laboratory. The preparation process for the RM involves creating an RV RNA mixture by combining RV viral RNA with RNA storage solution and matrix. The resulting RV RNA mixture is scaled up to a volume of 25 mL, then dispensed at 100 µL per vial and stored at -80 °C. The process of measuring the stability and homogeneity of RV RMs was conducted by employing reverse transcription droplet digital polymerase chain reaction (RT-ddPCR). Digital PCR is useful for the analysis of standards and can help to improve measurement compatibility: it represents the equivalence of a series of outcomes for reference materials and samples being analyzed when a few measurement procedures are employed, enabling objective comparisons between quantitative findings obtained through various experiments. The number of copies value represents a measured result of approximately 1.6 × 105 copies/µL. The RM has about an 11% bottle-to-bottle homogeneity and shows stable results for 1 week at temperatures of 4 °C and -20 °C and for 12 months at a temperature of -80 °C. The developed RM can enhance the dependability of RV molecular tests by providing a precise reference value for the absolute copy number of a viral target gene. Additionally, it can serve as a reference for diverse studies.

Development of a Melting-Curve-Based Multiplex Real-Time PCR Assay for the Simultaneous Detection of Viruses Causing Respiratory Infection.

The prompt and accurate identification of the etiological agents of viral respiratory infections is a critical measure in mitigating outbreaks. In this study, we developed and clinically evaluated a novel melting-curve-based multiplex real-time PCR (M-m-qPCR) assay targeting the RNA-dependent RNA polymerase (RdRp) and nucleocapsid phosphoprotein N of SARS-CoV-2, the Matrix protein 2 of the Influenza A virus, the RdRp domain of the L protein from the Human Respiratory Syncytial Virus, and the polyprotein from Rhinovirus B genes. The analytical performance of the M-m-qPCR underwent assessment using in silico analysis and a panel of reference and clinical strains, encompassing viral, bacterial, and fungal pathogens, exhibiting 100% specificity. Moreover, the assay showed a detection limit of 10 copies per reaction for all targeted pathogens using the positive controls. To validate its applicability, the assay was further tested in simulated nasal fluid spiked with the viruses mentioned above, followed by validation on nasopharyngeal swabs collected from 811 individuals. Among them, 13.4% (109/811) tested positive for SARS-CoV-2, and 1.1% (9/811) tested positive for Influenza A. Notably, these results showed 100% concordance with those obtained using a commercial kit. Therefore, the M-m-qPCR exhibits great potential for the routine screening of these respiratory viral pathogens.

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.

Acute Respiratory Distress Syndrome: A Rare Manifestation of Rhinovirus Infection.

Human rhinovirus (HRV) is a common cause of respiratory infections. HRV-related lower respiratory tract infections, including community-acquired pneumonia, are seldom seen in the clinical setting, and progression to acute respiratory distress syndrome (ARDS) is even rarer. We report on a case of a young immunosuppressed host who presented to the hospital for respiratory distress. She was diagnosed with HRV-related pneumonia, which rapidly progressed to ARDS based on clinical examination. After orotracheal intubation and mechanical ventilation with a low tidal volume strategy, she made a rapid recovery. This case highlights the importance of understanding that HRV may be an etiology of community-acquired pneumonia in immunosuppressed hosts and that ARDS may be a complication of this infection. Rapid recognition and clinical suspicion are important to the care of these patients, as ARDS has a high mortality rate.

Myeloid-associated differentiation marker is associated with type 2 asthma and is upregulated by human rhinovirus infection.

Background: Human rhinoviruses are known to predispose infants to asthma development during childhood and are often associated with exacerbations in asthma patients. MYADM epithelial expression has been shown to associate with asthma severity. The goal of this study was to determine if MYADM expression patterns were altered in asthma and/or rhinovirus infection and if increased MYADM expression is associated with increased asthma-associated factors. Methods: Utilizing H1HeLa cells and differentiated primary human airway epithelial cells (AECs), we measured the expression of MYADM and inflammatory genes by qRT-PCR in the presence or absence of RV-1B infection or poly I:C treatment and with siRNA knockdown of MYADM. Expression of MYADM in the asthmatic lung was determined in the ovalbumin (ova)-challenged murine model. Results: MYADM expression was upregulated in the lungs from ova-treated mice and in particular on the subsurface vesicle membrane in airway epithelial cells. Upon infection with RV-1B, human AECs grown at an air-liquid interface had increased the MYADM expression predominantly detected in ciliated cells. We found that the presence of MYADM was required for expression of several inflammatory genes both in a resting state and after RV-1B or poly I:C treatments. Conclusions: Our studies show that in a mouse model of asthma and during RV-1B infection of primary human AECs, increased MYADM expression is observed. In the mouse model of asthma, MYADM expression was predominantly on the luminal side of airway epithelial cells. Additionally, MYADM expression was strongly associated with increases in inflammatory genes, which may contribute to more severe asthma and RV-linked asthma exacerbations.