Characterization of human respiratory syncytial virus in children with severe acute respiratory infection before and during the COVID-19 pandemic.

Objectives: Annual outbreaks of human respiratory syncytial virus (HRSV) are caused by newly introduced and locally persistent strains. During the COVID-19 pandemic, global and local circulation of HRSV significantly decreased. This study was conducted to characterize HRSV in 2018-2022 and to analyze the impact of COVID-19 on the evolution of HRSV. Design/methods: Combined oropharyngeal and nasopharyngeal swabs were collected from children hospitalized with severe acute respiratory infection at two hospitals in Zambia. The second hypervariable region of the attachment gene G was targeted for phylogenetic analysis. Results: Of 3113 specimens, 504 (16.2%) were positive for HRSV, of which 131 (26.0%) and 66 (13.1%) were identified as HRSVA and HRSVB, respectively. In early 2021, an increase in HRSV was detected, caused by multiple distinct clades of HRSVA and HRSVB. Some were newly introduced, whereas others resulted from local persistence. Conclusions: This study provides insights into the evolution of HRSV, driven by global and local circulation. The COVID-19 pandemic had a temporal impact on the evolution pattern of HRSV. Understanding the evolution of HRSV is vital for developing strategies for its control.

Rapid Detection of SARS-CoV-2 RNA Using Reverse Transcription Recombinase Polymerase Amplification (RT-RPA) with Lateral Flow for N-Protein Gene and Variant-Specific Deletion-Insertion Mutation in S-Protein Gene.

Rapid molecular testing for severe acute respiratory coronavirus 2 (SARS-CoV-2) variants may contribute to the development of public health measures, particularly in resource-limited areas. Reverse transcription recombinase polymerase amplification using a lateral flow assay (RT-RPA-LF) allows rapid RNA detection without thermal cyclers. In this study, we developed two assays to detect SARS-CoV-2 nucleocapsid (N) gene and Omicron BA.1 spike (S) gene-specific deletion-insertion mutations (del211/ins214). Both tests had a detection limit of 10 copies/µL in vitro and the detection time was approximately 35 min from incubation to detection. The sensitivities of SARS-CoV-2 (N) RT-RPA-LF by viral load categories were 100% for clinical samples with high (>9015.7 copies/µL, cycle quantification (Cq): < 25) and moderate (385.5-9015.7 copies/µL, Cq: 25-29.9) viral load, 83.3% for low (16.5-385.5 copies/µL, Cq: 30-34.9), and 14.3% for very low (<16.5 copies/µL, Cq: 35-40). The sensitivities of the Omicron BA.1 (S) RT-RPA-LF were 94.9%, 78%, 23.8%, and 0%, respectively, and the specificity against non-BA.1 SARS-CoV-2-positive samples was 96%. The assays seemed more sensitive than rapid antigen detection in moderate viral load samples. Although implementation in resource-limited settings requires additional improvements, deletion-insertion mutations were successfully detected by the RT-RPA-LF technique.

The Inhibition of Glycolysis in T Cells by a Jak Inhibitor Ameliorates the Pathogenesis of Allergic Contact Dermatitis in Mice.

Allergic contact dermatitis (ACD) and atopic dermatitis develop through delayed-type hypersensitivity reactions mediated by T cells. The development of immunomodulatory drugs, such as Jak inhibitors, would be useful for the long-term management of these diseases owing to their profile of favorable adverse effects. However, the efficacy of Jak inhibitors for ACD treatment has not been fully determined under a variety of settings. Therefore, we evaluated the effects of ruxolitinib, a Jak inhibitor for Jak1 and Jak2, using a mouse ACD model. As a result, the lower numbers of immune cells, including CD4+ T cells, CD8+ T cells, neutrophils, and possibly macrophages, as well as milder pathophysiological aspects have been observed in the inflamed skin of ACD with the administration of ruxolitinib. In addition, the treatment of differentiating T cells with ruxolitinib downregulated the level of IL-2-mediated glycolysis in vitro. Furthermore, symptoms of ACD did not develop in T-cell-specific Pgam1-deficient mice whose T cells had no glycolytic capacity. Taken together, our data suggest that the downregulation of glycolysis in T cells by ruxolitinib could be an important factor in the suppression of ACD development in mice.

Seroprevalence of four endemic human coronaviruses and, reactivity and neutralization capability against SARS-CoV-2 among children in the Philippines.

Four endemic human coronaviruses (HCoV), HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43, are closely related to SARS-CoV-2. These coronaviruses are known to infect humans living in temperate areas, including children under 5 years old; however, the seroprevalence of four HCoVs among children in tropical areas, including the Philippines, remains unclear. This study aimed to assess the prevalence of antibodies against four HCoVs and to determine the reactivity and neutralization of these antibodies against SARS-CoV-2 among children in the Philippines. A total of 315 serum samples collected from 2015 to 2018, before the emergence of SARS-CoV-2, in Biliran island, Philippines, were tested for the presence of antibodies against four HCoVs and SARS-CoV-2 using recombinant spike ectodomain proteins by IgG-enzyme-linked immunosorbent assay (ELISA). Reactivity to and neutralization of SARS-CoV-2 were also investigated. The seroprevalence of the four HCoVs was 63.8% for HCoV-229E, 71.4% for HCoV-NL63, 76.5% for HCoV-HKU1, and 83.5% for HCoV-OC43 by ELISA. Age group analysis indicated that seropositivity to all HCoVs reached 80% by 2-3 years of age. While 69/315 (21.9%) of the samples showed reactive to SARS-CoV-2, almost no neutralization against SARS-CoV-2 was detected using neutralization assay. Reactivity of antibodies against SARS-CoV-2 spike protein obtained by ELISA may not correlate with neutralization capability.

Comparison of Rhinovirus A-, B-, and C-Associated Respiratory Tract Illness Severity Based on the 5'-Untranslated Region Among Children Younger Than 5 Years.

Background: Rhinoviruses (RVs) are among the most frequently detected viruses from hospitalized children with severe acute respiratory infections, being classified into RV-A, RV-B, and RV-C (4 clades: C, GAC1, GAC2, and A2). This study aimed to compare the clinical characteristics and respiratory tract illness severity between the RV species and RV-C clades in children in primary care and hospital settings in rural communities in the Philippines. Methods: Clinical samples and information of children <5 years old in the Philippines were collected from 2014 to 2016. The samples were tested by reverse-transcription polymerase chain reaction (RT-PCR) targeting the 5'-untranslated region. PCR-positive samples were sequenced, and RV species were identified by phylogenetic analysis. Results: Overall, 3680 respiratory tract illness episodes in 1688 cohort children were documented; 713 of those were RV positive and identified as RV-A (n = 271), RV-B (n = 47), and RV-C (n = 395: C [n = 76], GAG1 [n = 172], GAG2 [n = 8], A2 [n = 138], and unidentified [n = 1]). Severe illnesses, low oxygen saturation, cough, and wheezing were more common in patients with RV-C, especially with GAC1, than in those with RV-A or RV-B. Furthermore, severe illness was significantly more common in RV-C (GAC1)-positive cases than in RV-A-positive cases (odds ratio, 2.61 [95% CI, 1.17-4.13]). Conclusions: Children infected with RV-C had more severe illnesses than children infected with RV-A and RV-B. Moreover, emerging clades of RV-C were associated with increased severity.

Practical Validation of United States Centers for Disease Control and Prevention Assays for the Detection of Human Respiratory Syncytial Virus in Pediatric Inpatients in Japan.

The World Health Organization initiated a global surveillance system for respiratory syncytial virus (RSV) in 2015, and the pilot surveillance is ongoing. The real-time RT-PCR RSV assays (Pan-RSV and duplex assays) developed by the United States Centers for Disease Control and Prevention are applied as the standard assays. To introduce these as standard assays in Japan, their practicality was evaluated using 2261 specimens obtained from pediatric inpatients in Japan, which were collected from 2018 to 2021. Although the Pan-RSV and duplex assays had similar analytical sensitivities, they yielded 630 (27.9%) and 786 (34.8%) RSV-positive specimens, respectively (p < 0.001). Although sequencing analysis showed mismatches in the reverse primer used in the Pan-RSV assay, these mismatches did not affect its analytical sensitivity. The analysis of read numbers of RSV isolates from air−liquid interface culture of human bronchial/tracheal epithelial cells showed that the duplex assay had a greater number of reads than did the Pan-RSV assay. Therefore, the duplex assay has superior detection performance compared with the Pan-RSV assay, but the two assays have similar analytical sensitivities.

Prolonged shedding of infectious viruses with haplotype switches of SARS-CoV-2 in an immunocompromised patient.

A concern has been raised that the persistent COVID-19 infection in an immunocompromised host can be the source of the SARS-CoV-2 variants. This is the case of a 61-year-old man in complete remission of a follicular lymphoma after six cycles of rituximab and bendamustine with additional two cycles of rituximab completed eight months prior to the episode of COVID-19 pneumonia. The patient's respiratory failure was long-lasting, and required mechanical ventilation until day 75. Acquired immunity tested negative throughout the observational period. The viral RNA was detectable until day 100 while the infectious virus was isolated until day 79. Seven haplotypes were identified and the non-synonymous mutations accumulated in the spike gene which included E484Q and S494P. In the management of COVID-19 cases with suppressed immune statuses, initial evaluation of existing immunity and monitoring for infectiousness throughout the clinical course including the convalescent stage may be necessary.

Incidence of lower respiratory tract infection and associated viruses in a birth cohort in the Philippines.

BACKGROUND: Lower respiratory tract infection (LRTI) is an important cause of morbidity and mortality in infants and young children. However, the etiological role of viruses and the timing of developing LRTI are not well defined. METHODS: We analyzed the data of a prospective cohort study in the Philippines as a birth cohort. We detected LRTI among children who visited healthcare facilities with respiratory symptom, and collected nasopharyngeal swabs for virus detection. We analyzed the incidence rates (IRs) and cumulative proportion of LRTI and severe LRTI by age group and each virus detected. RESULTS: A total of 350 LRTI episodes were observed from 473 child-years yielded from 419 children. The IRs of LRTI were 70.8, 70.7, and 80.8 per 100 child-years for 0-5, 6-11, and 12-23 months of age, respectively. By 12 months of age, 45% of children developed LRTI at least once. Rhinovirus and respiratory syncytial virus were the most frequently detected viruses in all age groups. However, the IRs of influenza virus were low especially at 0-5 months of age. CONCLUSIONS: We identified various patterns of age-specific IRs of LRTI and severe LRTI for different viruses, which should be considered to establish more effective interventions including vaccinations.

Serotype Identification of Human Adenoviruses Associated with Influenza-Like Illnesses in the Philippines from 2006-2012 by Microneutralization and Molecular Techniques.

OBJECTIVES: Human adenoviruses (HAdV) are known to cause a wide range of diseases including acute respiratory infections, conjunctivitis, and acute gastroenteritis. In this study, we aimed to determine the serotypes of HAdV in patients with influenza-like illness (ILI) in the Philippines from 2006-2012 and to describe the demographic and epidemiological characteristics of patients who tested positive for HAdV. METHODS: Between 2006 and 2012, the Philippine National Influenza Centre detected HAdV in 1294 samples of patients with ILI. Serotype determination was done in select samples using microneutralization, polymerase chain reaction (PCR), and sequencing methods. RESULTS: A total of 8 serotypes were identified (HAdV 1-7 and 11), with HAdV-2 (27.8%), and HAdV-3 (27.8%) being the most prevalent. The majority of HAdV infections were found in children below 5 years of age (79.9%). CONCLUSIONS: The identification of HAdV circulating serotypes may serve as guide for designing disease intervention and control strategies and will provide important information regarding the contribution of this virus to respiratory infections, particularly in children, which remain a public health burden in the Philippines.

Near-Complete Genome Sequencing of Influenza C Virus in the Philippines between 2014 and 2019.

We report 19 nearly complete genome sequences of influenza C virus isolated from clinical samples recovered from children in the Philippines between 2014 and 2019.

Complete Genome Sequences of Enterovirus D68 Clade A and D Strains in the Philippines.

Complete genome sequences were determined for 4 clade A and 12 clade D enterovirus D68 strains detected in nasopharyngeal swabs from children with acute respiratory illness in the Philippines. These sequence data will be useful for future epidemiological monitoring, including watching for viral evolution.

Risk of Transmission and Viral Shedding From the Time of Infection for Respiratory Syncytial Virus in Households.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection worldwide, but reports of temporal changes in the risk of transmission among close contacts has been scarce. This study aimed to examine an association between the viral load trajectory and transmission risk to develop a better control strategy for the disease spread. We conducted a household-based prospective cohort study in Biliran Province, the Philippines, and enrolled 451 participants to observe the development of acute respiratory infection. Including the cases found at the health-care facility, we analyzed the data of viral loads with symptom records obtained from 172 followed participants who had household member positive for RSV with a rapid test during an RSV outbreak in 2018-2019. We developed a model estimating a temporal change in the viral shedding from the infection and evaluated transmission dynamics. We found that most transmission events occurred within approximately 7 days of the household exposure, including potential presymptomatic transmissions. The inferred risk of infection among those younger than 5 years was 3.5 times higher than that of those older than 5 years. This finding suggested that the initial week after the household exposure is particularly important for preventing RSV spread.

Global burden of acute lower respiratory infection associated with human parainfluenza virus in children younger than 5 years for 2018: a systematic review and meta-analysis.

BACKGROUND: Human parainfluenza virus (hPIV) is a common virus in childhood acute lower respiratory infections (ALRI). However, no estimates have been made to quantify the global burden of hPIV in childhood ALRI. We aimed to estimate the global and regional hPIV-associated and hPIV-attributable ALRI incidence, hospital admissions, and mortality for children younger than 5 years and stratified by 0-5 months, 6-11 months, and 12-59 months of age. METHODS: We did a systematic review of hPIV-associated ALRI burden studies published between Jan 1, 1995, and Dec 31, 2020, found in MEDLINE, Embase, Global Health, Cumulative Index to Nursing and Allied Health Literature, Web of Science, Global Health Library, three Chinese databases, and Google search, and also identified a further 41 high-quality unpublished studies through an international research network. We included studies reporting community incidence of ALRI with laboratory-confirmed hPIV; hospital admission rates of ALRI or ALRI with hypoxaemia in children with laboratory-confirmed hPIV; proportions of patients with ALRI admitted to hospital with laboratory-confirmed hPIV; or in-hospital case-fatality ratios (hCFRs) of ALRI with laboratory-confirmed hPIV. We used a modified Newcastle-Ottawa Scale to assess risk of bias. We analysed incidence, hospital admission rates, and hCFRs of hPIV-associated ALRI using a generalised linear mixed model. Adjustment was made to account for the non-detection of hPIV-4. We estimated hPIV-associated ALRI cases, hospital admissions, and in-hospital deaths using adjusted incidence, hospital admission rates, and hCFRs. We estimated the overall hPIV-associated ALRI mortality (both in-hospital and out-hospital mortality) on the basis of the number of in-hospital deaths and care-seeking for child pneumonia. We estimated hPIV-attributable ALRI burden by accounting for attributable fractions for hPIV in laboratory-confirmed hPIV cases and deaths. Sensitivity analyses were done to validate the estimates of overall hPIV-associated ALRI mortality and hPIV-attributable ALRI mortality. The systematic review protocol was registered on PROSPERO (CRD42019148570). FINDINGS: 203 studies were identified, including 162 hPIV-associated ALRI burden studies and a further 41 high-quality unpublished studies. Globally in 2018, an estimated 18·8 million (uncertainty range 12·8-28·9) ALRI cases, 725 000 (433 000-1 260 000) ALRI hospital admissions, and 34 400 (16 400-73 800) ALRI deaths were attributable to hPIVs among children younger than 5 years. The age-stratified and region-stratified analyses suggested that about 61% (35% for infants aged 0-5 months and 26% for 6-11 months) of the hospital admissions and 66% (42% for infants aged 0-5 months and 24% for 6-11 months) of the in-hospital deaths were in infants, and 70% of the in-hospital deaths were in low-income and lower-middle-income countries. Between 73% and 100% (varying by outcome) of the data had a low risk in study design; the proportion was 46-65% for the adjustment for health-care use, 59-77% for patient groups excluded, 54-93% for case definition, 42-93% for sampling strategy, and 67-77% for test methods. Heterogeneity in estimates was found between studies for each outcome. INTERPRETATION: We report the first global burden estimates of hPIV-associated and hPIV-attributable ALRI in young children. Globally, approximately 13% of ALRI cases, 4-14% of ALRI hospital admissions, and 4% of childhood ALRI mortality were attributable to hPIV. These numbers indicate a potentially notable burden of hPIV in ALRI morbidity and mortality in young children. These estimates should encourage and inform investment to accelerate the development of targeted interventions. FUNDING: Bill & Melinda Gates Foundation.

Inactivation of SARS-CoV-2 by Catechins from Green Tea.

Green tea extracts effectively inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro in a dose-dependent manner. Ten-fold serially diluted solutions of catechin mixture reagent from green tea were mixed with the viral culture fluid at a volume ratio of 9:1, respectively, and incubated at room temperature for 5 min. The solution of 10 mg/mL catechin reagent reduced the viral titer by 4.2 log and 1.0 mg/mL solution by one log. Pre-infection treatment of cells with the reagent alone did not affect viral growth. In addition, cells treated with only the reagent were assayed for host cell viability using the WST-8 system, and almost no host cell damage by the treatment was observed. These findings suggested that the direct treatment of virus with the reagent before inoculation decreased the viral activity and that catechins might have the potential to suppress SARSCoV-2 infection.

Gene signature of children with severe respiratory syncytial virus infection.

BACKGROUND: The limited treatment options for children with severe respiratory syncytial virus (RSV) infection highlights the need for a comprehensive understanding of the host cellular response during infection. We aimed to identify host genes that are associated with severe RSV disease and to identify drugs that can be repurposed for the treatment of severe RSV infection. METHODS: We examined clinical data and blood samples from 37 hospitalized children (29 mild and 8 severe) with RSV infection. We tested RNA from blood samples using next-generation sequencing to profile global mRNA expression and identify cellular processes. RESULTS: Retractions, decreased breath sounds, and tachypnea were associated with disease severity. We observed upregulation of genes related to neutrophil, inflammatory response, blood coagulation, and downregulation of genes related to T cell response in children with severe RSV. Using network-based approach, 43 drugs were identified that are predicted to interact with the gene products of these differentially expressed genes. CONCLUSIONS: These results suggest that the changes in the expression pattern in the innate and adaptive immune responses may be associated with RSV clinical severity. Compounds that target these cellular processes can be repositioned as candidate drugs in the treatment of severe RSV. IMPACT: Neutrophil, inflammation, and blood coagulation genes are upregulated in children with severe RSV infection. Expression of T cell response genes are suppressed in cases of severe RSV. Genes identified in this study can contribute in understanding the pathogenesis of RSV disease severity. Drugs that target cellular processes associated with severe RSV can be repositioned as potential therapeutic options.

Epidemiological and clinical characteristics of children with acute respiratory viral infections in the Philippines: a prospective cohort study.

OBJECTIVES: Viral acute respiratory infection (ARI) remains a major global health problem, especially among children in low- and middle-income countries. The study was conducted to reveal aetiological significance of respiratory viruses among both non-hospitalized and hospitalized children. METHODS: A cohort study of children with ARI at the household, primary healthcare facility, and hospital levels was conducted alongside a hospital-based study including non-cohort children from 2014 to 2016 in the Philippines. The ARI cases were recorded at households and healthcare facilities, and a clinical investigation was performed. Nasopharyngeal swabs were collected from the symptomatic children and tested for respiratory viruses via polymerase chain reaction. Then, the association between healthcare facility utilization and viral detection was investigated. RESULTS: Overall, 18,514 ARI cases were enrolled in the cohort study, and samples were collected from 4735 of these cases. The hospital-based study detected 648 ARI cases, all of which were sampled. Rhinovirus (22.2%; 1052/4735) was most frequently detected followed by respiratory syncytial virus (12.0%; 566/4735). Enterovirus (adjusted odds ratio, 1.8; 95% confidence interval, 1.1-2.8), human metapneumovirus (2.1, 1.4-3.2), rhinovirus (2.1, 1.8-2.6), and respiratory syncytial virus (1.6, 1.2-1.9) were significantly more prevalent in the ARI cases at healthcare facilities than in those in households. Of all ARI cases, 0.6% required hospitalization while 1.8% were hospitalized among the respiratory syncytial virus-positive cases (3.8, 3.0-4.9). CONCLUSIONS: We determined the prevalence of respiratory viruses among children with ARIs at the household, primary healthcare facility, and hospital levels and the association with clinical characteristics. In particular, we discovered a significant disease burden and impact of respiratory syncytial virus infections as well as a considerable aetiological implication of rhinovirus infections.

Receptor-Binding Assays of Enterovirus D68.

Human enterovirus D68 (EV-D68) is a causative agent for acute respiratory infections and potentially central nervous system illnesses with increasing epidemiological significance. Recent studies have highlighted the role of sialic acids as a functional receptor for EV-D68 in vitro. However, further investigations are required to reveal its significance in actual infections in human.