Respiratory viral infections in bone marrow transplant patients: insights from a tertiary care hospital in Rawalpindi, Pakistan.

OBJECTIVE: To determine the incidence of respiratory viral infections in bone marrow transplant patients. METHODS: The prospective, descriptive, cross-sectional study was conducted at a tertiary care hospital in Rawalpindi, Pakistan, from September 2019 to August 2020, and comprised respiratory specimens from recipients of haematopoietic stem cell transplant. The specimens were collected in viral transport medium, and were then taken to the Department of Virology. Multiplex polymerase chain reaction was performed on the specimens to ascertain the incidence and prevalence of respiratory viruses. Data was analysed using SPSS 24. RESULTS: Of the 85 subjects, 53(62.35%) were males and 32(37.65%) were females. The overall median age was 20.0 years (interquartile range: 11.0-32.0 years). Respiratory viral infections were detected in 31(36.4%) specimens. Among them, human rhinovirus was detected in 12(38.7%) cases, respiratory syncytial virus in 5(16.1%), influenza A/H3 in 4(13%), human parainfluenza virus-1 in 3(9.7%), adenovirus in 2(6.4%), human parainfluenza virus-3 in 1(3.2%), human parainfluenza virus-4 in 1(3.2%) and human metapneumovirus in 1(3.2%) case. There were 2(6.4%) cases of co-infection. CONCLUSIONS: More than one-third recipients of haematopoietic stem cell transplant were found to have respiratory viral infections, highlighting the importance of employing multiplex respiratory polymerase chain reaction in early diagnosis and treatment of such infections.

Epidemiology of human metapneumovirus in Taiwan from 2013 to 2023.

Human metapneumovirus (HMPV) is a member of the genus Metapneumovirus in the family Pneumoviridae of the order Mononegavirales that can cause upper and lower respiratory tract disease. This retrospective study describes the epidemiology of hMPV based on community viral surveillance results from sentinel sites across Taiwan from 2013 to 2023. A total of 114 hMPV strains were isolated and analyzed to assess viral evolution through sequencing of their fusion protein genes. This study revealed that hMPV cases occur almost year-round in Taiwan, with a peak occurring during spring (March to May). Of the 114 infected patients, 68.4% were children under 4 years old. The geographical distribution of hMPV positivity was highest in Penghu County, followed by Changhua County and Hsinchu County. The clinical symptoms of hMPV infection are nonspecific, with fever (56.1%), cough (44.7%), rhinorrhea (21.1%), and sore throat (14.9%) being the most common. However, a few patients also developed severe central nervous system symptoms (1.8%) or dyspnea (0.9%). Phylogenetic analysis revealed genetic diversity among the 114 isolated hMPV strains, with the A2 lineage (57.9%) being the most frequently observed, followed by the B2 lineage (33.3%), in the Taiwanese community from 2013 to 2023. In conclusion, hMPV causes a serious acute respiratory disease in Taiwan that should not be neglected. Further epidemiological surveillance and investigations of the clinical characteristics of hMPV should be performed continually for prevention and control of this virus.

The circulating characteristics of common respiratory pathogens in Ningbo, China, both before and following the cessation of COVID-19 containment measures.

To assess the circulating characteristics of common respiratory pathogens following the complete relaxation of non-pharmaceutical interventions (NPIs) and the cessation of the dynamic zero-COVID policy. The retrospective analysis was conducted from 14,412 patients with acute respiratory infections (ARIs) from January 24, 2020, to December 31, 2023, including Influenza A virus (IFV-A), Influenza B virus (IFV-B), Respiratory Syncytial Virus (RSV), Human Rhinovirus (HRV), Human Parainfluenza Virus (HPIV), Human Metapneumovirus (HMPV), Human Coronavirus (HCoV), Human Bocavirus (HBoV), Human Adenovirus (HAdV), and Mycoplasma pneumoniae (MP). Compared with 2020-2022, Joinpoint analysis indicated a monthly increase in overall pathogen activity in 2023, rising from an average of 43.05% to an average of 68.46%. The positive rates of IFV-A, IFV-B, HMPV, HPIV, HCoV, and MP increased, while those of HRV and RSV decreased, and no differences in HAdV and HBoV. The outbreak of IFV-A and MP was observed, the positive rate of MP has surpassed pre-COVID-19 pandemic levels and the spread of RSV was interrupted by IFV-A. Infants and toddlers were primarily infected by HRV and RSV, Children and adolescents exhibited a higher prevalence of infections with MP, IFV-A, and HRV, whereas Adults and the elderly were primarily infected by IFV-A. The incidence of co-infections rose from 4.25 to 13.73%. Restricted cubic spline models showed that the susceptible age ranges for multiple pathogens expanded. These changes serve as a reminder to stay alert in the future and offer clinicians a useful guide for diagnosing and treating.

Changes in the epidemiological patterns of respiratory syncytial virus and human metapneumovirus infection among pediatric patients and their correlation with severe cases: a long-term retrospective study.

Objectives: We aim to investigate the prevalence of respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) among pediatric patients with acute respiratory tract illness (ARTI) in southern China both pre- and post-COVID-19 pandemic, as well as identify associated risk factors for severe infections. Methods: The study conducted a real-time PCR analysis on hospitalized children with ARTI from 2012 to 2023, specifically targeting RSV, hMPV, and other respiratory pathogens. Additionally, demographic data was collected during this analysis. Results: The prevalence of RSV occurs triennially, and likewise, the temporal pattern of hMPV outbreaks mirrors that of RSV. The peak infection rates of RSV and hMPV occurred during and following the implementation of COVID-19 epidemic prevention and control measures. The incidence of RSV infection exhibited bimodal peaks in 2022, while hMPV demonstrated seasonal peaks during the spring, fall, and winter periods post-COVID-19 pandemic. After the COVID-19 outbreak, there has been an upward trend in the proportion of female patients and patients aged one year and older presenting with ARTI, RSV infections, and hMPV infections. Infant (OR = 4.767, 95%CI: [3.888-5.846], p < 0.0001), presence of co-infection (OR = 0.540, 95%CI: [0.404-0.722], p < 0.0001), and existence of comorbidities (OR = 1.582, 95%CI: [1.285-1.949], p < 0.0001) was the risk ratio for the severity of RSV infection. Children infected with hMPV under the age of 1 year (OR = 0.322, 95%CI: [0.180 - 0.575], p < 0.0001), as well as those with comorbidities (OR = 8.809, 95%CI: [4.493 - 17.272], p < 0.0001), have a higher risk of developing severe illness. Conclusion: The changing epidemiological patterns have the potential to lead to widespread severe outbreaks among children, particularly those with underlying medical conditions who may experience more severe symptoms. Conducting surveillance for pneumoviridae viruses in children is an imperative measure to establish a robust foundation for future epidemic prevention and treatment strategies.

Genetic absence of PD-L1 does not restore CD8+ T cell function during respiratory virus infection and delays virus clearance.

A key mediator of T cell impairment during respiratory virus infection is the inhibitory receptor PD-1. PD-1 is induced on T cells following antigen exposure, whereas proinflammatory cytokines upregulate the ligands PD-L1 and PD-L2. Respiratory virus infection leads to upregulation of PD-L1 on airway epithelial cells, dendritic cells, and alveolar macrophages. However, the role of PD-L1 on different cell types in acute respiratory virus infections is not known. We sought to determine the role of PD-L1 on different cell types in CD8+ T cell impairment. We found that PD-L1-/- mice challenged with human metapneumovirus or influenza showed a similar level of CD8+ T cell impairment compared to wild-type (WT) mice. Moreover, virus clearance was delayed in PD-L1-/- mice compared to WT. CD8+ T cells from PD-L1-deficient mice expressed higher levels of inhibitory receptors both at baseline and after respiratory virus infection. The antibody blockade of PD-L2 failed to restore function to the impaired cells. While reciprocal bone marrow chimeras between WT and PD-L1-/- mice did not restore CD8+ T cell function after the respiratory virus challenge, mice that received the PD-L1-/- bone marrow had higher inhibitory receptor expression on CD8+ cells. This discrepancy in the inhibitory receptor expression suggests that cells of the hematopoietic compartment contribute to T cell impairment on CD8+ T cells.IMPORTANCEThe phenomenon of pulmonary CD8+ T cell impairment with diminished antiviral function occurs during acute respiratory virus infection mediated by Programmed Cell Death-1 (PD-1) signaling. Moreover, PD-1 blockade enhances T cell function to hasten viral clearance. The ligand PD-L1 is expressed in many cell types, but which cells drive lung T cell impairment is not known. We used genetic approaches to determine the contribution of PD-L1 on lung T cell impairment. We found that PD-L2 cannot compensate for the loss of PD-L1, and PD-L1-deficient mice exhibit increased expression of other inhibitory receptors. Bone marrow chimeras between PD-L1-deficient and wild-type mice indicated that hematopoietic PD-L1 expression is associated with inhibitory receptor upregulation and impairment.

Molecular characterization of avian metapneumovirus subtype C associated with hydrosalpinx fluid and egg drop syndrome in Jinding ducks.

A disease called "hydrosalpinx fluid and egg drop syndrome" (HFEDS) was observed in four flocks of Jinding ducks (Anas platyrhynchos domesticus) in Northeast China during the years 2022 to 2023. Here, we investigated the possible involvement of avian metapneumovirus (AMPV) infection. Full-length genome sequencing and sequence analysis of two AMPV strains showed that they belong to Eurasian lineage of AMPV subtype C. Based on surface glycoprotein (G) sequence comparisons, the Eurasian lineage can be divided into two sublineages (E1 and E2), and sublineage E2 is circulating in Jinding duck populations in Northeast China.

Molecular detection and genotyping of HMPV in patients with severe acute respiratory infection in India.

BACKGROUND: Human metapneumovirus (HMPV) is a common respiratory pathogen that causes respiratory tract infections. In India, HMPV has been identified as one of the leading causes of morbidity and mortality in infants and young children with respiratory tract infections. The most reported sublineages of HMPV in India are B1, B2, A2b and A2c. OBJECTIVE: A retrospective study was conducted to determine the circulating genotypes of HMPV among SARI cases from January 2016 to December 2018. MATERIALS AND METHODS: Positive throat swab samples were confirmed with real-time RT-PCR. Subsequently, these samples were analysed using semi-nested conventional RT-PCR targeting the G gene, followed by sequencing and phylogenetic analysis. Clinical data analysis was also performed using SPSS 15.0 software. RESULTS: All 20 samples from the SARI cases were classified under the A2c sublineage of HMPV. Phylogenetic analysis indicated that these strains were genetically related to those circulating in Japan, China, and Croatia. Among the samples, ten showed 111-nucleotide duplications, while the other ten had 180-nucleotide duplications. CONCLUSION: Clinical analysis showed that four cases had coinfections with other pathogens. Our extensive analysis of patient samples determined that HMPV, especially the A2c genotype, significantly contributed to SARI cases within our study population, which signifies the importance of considering HMPV as a probable aetiological agent when investigating SARI outbreaks.

Respiratory Viral Testing Rate Patterns in Young Children Attending Tertiary Care Across Western Australia: A Population-Based Birth Cohort Study.

BACKGROUND: An understanding of viral testing rates is crucial to accurately estimate the pathogen-specific hospitalisation burden. We aimed to estimate the patterns of testing for respiratory syncytial virus (RSV), influenza virus, parainfluenza virus (PIV) and human metapneumovirus (hMPV) by geographical location, age and time in children <5 years old in Western Australia. METHODS: We conducted a population-based cohort study of children born between 1 January 2010 and 31 December 2021, utilising linked administrative data incorporating birth and death records, hospitalisations and respiratory viral surveillance testing records from state-wide public pathology data. We examined within-hospital testing rates using survival analysis techniques and identified independent predictors of testing using binary logistic regression. RESULTS: Our dataset included 46,553 laboratory tests for RSV, influenza, PIV, or hMPV from 355,021 children (52.5% male). Testing rates declined in the metropolitan region over the study period (RSV testing in infants: from 242.11/1000 child-years in 2012 to 155.47/1000 child-years in 2018) and increased thereafter. Conversely, rates increased in non-metropolitan areas (e.g., RSV in Goldfields: from 364.92 in 2012 to 504.37/1000 child-years in 2021). The strongest predictors of testing were age <12 months (adjusted odds ratio [aOR] = 2.25, 95% CI 2.20-2.31), preterm birth (<32 weeks: aOR = 2.90, 95% CI 2.76-3.05) and remote residence (aOR = 0.77, 95% CI 0.73-0.81). CONCLUSION: These current testing rates highlight the potential underestimation of respiratory virus hospitalisations by routine surveillance and the need for estimation of the true burden of respiratory virus admissions.

Research note: Indirect evidence of avian Metapneumovirus circulation in broilers in Italy.

The clinical relevance of avian metapneumovirus (aMPV) is growing in the poultry sector, especially in broiler farming, where no vaccination is administered in Italy. Given the naïve status of the birds, a serological survey was conducted in a densely populated area of Northern Italy, to evaluate aMPV circulation. Seven farms were selected and sampled in summer/fall, then sampling was repeated in the following season (winter/spring) to assess a possible seasonal effect. In each farm, fifteen birds were blood sampled towards the end of the cycle and sera were analyzed with an ELISA test. Clinical signs were reported in 5 out of 7 farms, although all farms were positive at both sampling points, except for one, which was negative at the first sampling. The seroprevalence within farm ranged from 26.6% to 100%, and antibody titres appear to increase with age. No seasonality effect was evidenced, whereas a farm effect was more distinct. aMPV circulation appears wide in Northern Italian farms, with different clinical outcomes that could be modulated by intrinsic characteristics of the farms. In absence of vaccination, serological monitoring can be a useful tool for viral entrance monitoring, although sampling timing should be evaluated in order to spot seroconversion after late infections.

Seasonality of common respiratory viruses: Analysis of nationwide time-series data.

BACKGROUND AND OBJECTIVE: Understanding the seasonal behaviours of respiratory viruses is crucial for preventing infections. We evaluated the seasonality of respiratory viruses using time-series analyses. METHODS: This study analysed prospectively collected nationwide surveillance data on eight respiratory viruses, gathered from the Korean Influenza and Respiratory Surveillance System. The data were collected on a weekly basis by 52 nationwide primary healthcare institutions between 2015 and 2019. We performed Spearman correlation analyses, similarity analyses via dynamic time warping (DTW) and seasonality analyses using seasonal autoregressive integrated moving average (SARIMA). RESULTS: The prevalence of rhinovirus (RV, 23.6%-31.4%), adenovirus (AdV, 9.2%-16.6%), human coronavirus (HCoV, 3.0%-6.6%), respiratory syncytial virus (RSV, 11.7%-20.1%), influenza virus (IFV, 11.7%-21.5%), parainfluenza virus (PIV, 9.2%-12.6%), human metapneumovirus (HMPV, 5.6%-6.9%) and human bocavirus (HBoV, 5.0%-6.4%) were derived. Most of them exhibited a high positive correlation in Spearman analyses. In DTW analyses, all virus data from 2015 to 2019, except AdV, exhibited good alignments. In SARIMA, AdV and RV did not show seasonality. Other viruses showed 12-month seasonality. We describe the viruses as winter viruses (HCoV, RSV and IFV), spring/summer viruses (PIV, HBoV), a spring virus (HMPV) and all-year viruses with peak incidences during school periods (RV and AdV). CONCLUSION: This is the first study to comprehensively analyse the seasonal behaviours of the eight most common respiratory viruses using nationwide, prospectively collected, sentinel surveillance data.

SARS-CoV-2 co-detection with other respiratory pathogens-descriptive epidemiological study.

BACKGROUND: Co-detection of respiratory pathogens with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is poorly understood. This descriptive epidemiological study aimed to determine the effect of the interaction of different respiratory pathogens on clinical variables. METHODS: We retrospectively reviewed the results of comprehensive multiplex polymerase chain reaction (PCR) testing from November 2020 to March 2023 to estimate respiratory pathogen co-detection rates in Shinjuku, Tokyo. We evaluated the interactions of respiratory pathogens, particularly SARS-CoV-2, between observed and expected co-detection. We estimated the trend of co-detection with SARS-CoV-2 in terms of age and sex and applied a multiple logistic regression model adjusted for age, testing period, and sex to identify influencing factors between co-detection and single detection for each pathogen. RESULTS: Among 57,746 patients who underwent multiplex PCR testing, 10,516 (18.2%) had positive for at least one of the 22 pathogens. Additionally, 881 (1.5%) patients were confirmed to have a co-detection. SARS-CoV-2 exhibited negative interactions with adenovirus, coronavirus, human metapneumovirus, parainfluenza virus, respiratory syncytial virus, and rhino/enterovirus. SARS-CoV-2 co-detection with other pathogens occurred most frequently in patients of the youngest age group (0-4 years). A multiple logistic regression model indicated that younger age was the most influential factor for SARS-CoV-2 co-detection with other respiratory pathogens. CONCLUSION: The study highlights the prevalence of SARS-CoV-2 co-detection with other respiratory pathogens in younger age groups, necessitating further exploration of the clinical implications and severity of SARS-CoV-2 co-detection.

Viral Cytopathic Changes in Sputum Cytology in a Patient with Known Human Metapneumovirus.

INTRODUCTION: Viral cytopathic changes seen in sputum cytology have been described in association with infection by viruses such as cytomegalovirus (CMV), herpes simplex virus (HSV), adenovirus, and even measles. However, viral cytopathic changes due to human metapneumovirus (hMPV) have not yet been well described in cytology. hMPV is a relatively new entity, discovered in 2001. It is known to cause upper and lower respiratory tract infections in children, the elderly, and immunocompromised patients. CASE PRESENTATION: We describe the viral cytopathic changes seen in sputum in a 63-year-old male patient with known hMPV. These changes include multinucleation, nuclear enlargement, homogenised nuclei, basophilic nuclear inclusions with perinuclear halos, and small eosinophilic cytoplasmic inclusions. CONCLUSION: We aim to raise awareness that hMPV can cause viral cytopathic changes and to describe these cytological features, which have been elucidated in only 1 case report thus far. Distinction from other viruses with similar changes, such as HSV and CMV, is important due to their differing clinical implications.

An affordable detection system based on RT-LAMP and DNA-nanoprobes for avian metapneumovirus.

Airborne animal viral pathogens can rapidly spread and become a global threat, resulting in substantial socioeconomic and health consequences. To prevent and control potential epidemic outbreaks, accurate, fast, and affordable point-of-care (POC) tests are essential. As a proof-of-concept, we have developed a molecular system based on the loop-mediated isothermal amplification (LAMP) technique for avian metapneumovirus (aMPV) detection, an airborne communicable agent mainly infecting turkeys and chickens. For this purpose, a colorimetric system was obtained by coupling the LAMP technique with specific DNA-functionalized AuNPs (gold nanoparticles). The system was validated using 50 different samples (pharyngeal swabs and tracheal tissue) collected from aMPV-infected and non-infected chickens and turkeys. Viral detection can be achieved in about 60 min with the naked eye, with 100% specificity and 87.88% sensitivity for aMPV. In summary, this novel molecular detection system allows suitable virus testing in the field, with accuracy and limit of detection (LOD) values highly close to qRT-PCR-based diagnosis. Furthermore, this system can be easily scalable to a platform for the detection of other viruses, addressing the current gap in the availability of POC tests for viral detection in poultry farming. KEY POINTS: •aMPV diagnosis using RT-LAMP is achieved with high sensitivity and specificity. •Fifty field samples have been visualized using DNA-nanoprobe validation. •The developed system is a reliable, fast, and cost-effective option for POCT.

Lobar pneumonia due to human metapneumovirus: a case report.

Human metapneumovirus (hMPV) is a respiratory pathogen that can cause lower respiratory tract infections and pneumonia in immunocompetent adults. Pneumonia caused by hMPV is reportedly more likely to cause bronchial wall thickening and ground-glass opacity (GGO). A 44-year-old woman with no significant medical history developed fever, cough, and nausea. Computed tomography of the chest showed scattered GGOs in the right upper lobe and infiltrating shadows with air bronchograms in the left lingual and bilateral lower lobes. The patient was admitted to our hospital for further evaluation. Atypical pneumonia was suspected and lascufloxacin (LSFX) was started. Multiplex polymerase chain reaction (PCR) detected hMPV on hospital day 2 using the FilmArray Respiratory Panel 2.1. Pneumonia due to hMPV was suspected and LSFX was discontinued. The patient subsequently showed spontaneous improvement and was discharged on hospital day 6 after admission. After discharge, pneumonia continued to improve. Early detection of respiratory pathogens using multiplex PCR can help determine the appropriate treatment strategy. As hMPV can also cause lobar pneumonia, we should consider pneumonia due to hMPV in the differential diagnosis of lobar pneumonia.

Efficacious human metapneumovirus vaccine based on AI-guided engineering of a closed prefusion trimer.

The prefusion conformation of human metapneumovirus fusion protein (hMPV Pre-F) is critical for eliciting the most potent neutralizing antibodies and is the preferred immunogen for an efficacious vaccine against hMPV respiratory infections. Here we show that an additional cleavage event in the F protein allows closure and correct folding of the trimer. We therefore engineered the F protein to undergo double cleavage, which enabled screening for Pre-F stabilizing substitutions at the natively folded protomer interfaces. To identify these substitutions, we developed an AI convolutional classifier that successfully predicts complex polar interactions often overlooked by physics-based methods and visual inspection. The combination of additional processing, stabilization of interface regions and stabilization of the membrane-proximal stem, resulted in a Pre-F protein vaccine candidate without the need for a heterologous trimerization domain that exhibited high expression yields and thermostability. Cryo-EM analysis shows the complete ectodomain structure, including the stem, and a specific interaction of the newly identified cleaved C-terminus with the adjacent protomer. Importantly, the protein induces high and cross-neutralizing antibody responses resulting in near complete protection against hMPV challenge in cotton rats, making the highly stable, double-cleaved hMPV Pre-F trimer an attractive vaccine candidate.

Probenecid Inhibits Human Metapneumovirus (HMPV) Replication In Vitro and in BALB/c Mice.

Human metapneumovirus (HMPV) is an important cause of acute respiratory tract infection and causes significant morbidity and mortality. There is no specific antiviral drug to treat HMPV or vaccine to prevent HMPV. This study determined if probenecid, a host-targeting antiviral drug, had prophylactic (pre-virus) or therapeutic (post-virus) efficacy to inhibit HMPV replication in LLC-MK2 cells in vitro and in the lungs of BALB/c mice. This study showed that ≥0.5 µM probenecid significantly inhibited HMPV replication in vitro, and 2-200 mg/kg probenecid prophylaxis or treatment reduced HMPV replication in BALB/c mice.

[Genotype and epidemiological characteristics of human metapneumovirus among hospitalized cases of acute respiratory infection in children in Changchun City, Jilin Province from 2019 to 2023].

Objective: To investigate the genotype and epidemiological characteristics of human metapneumovirus (HMPV) among hospitalized cases with acute respiratory infections (ARI) in children in Changchun City, Jilin Province, China. Methods: From June 2019 to June 2023, throat swabs of ARI inpatients in Changchun Children's Hospital were collected, and their epidemiological and clinical information were also collected. Quantitative reverse transcription-PCR was used to identify HMPV-positive cases, followed by the amplification of the G gene and genetic analysis in the HMPV-positive cases. Results: A total of 3 311 children hospitalized with ARI were included in this study. Their age ranged from 0 to 17 years old, and the M (Q1, Q3) of age was 2 (1, 3) years. About 1 811 (54.70%) cases were males. A total of 167 HMPV-positive cases were detected with a positive rate of 5.04%, of which 92.81% (155/167) were children under 5 years old. The positive rate of HMPV in 2019 was 6.37% (30/471), which dropped to the lowest in 2020 (2.31%, 10/432). The HMPV-positive rate was then rebounded in 2021 (4.70%, 60/1 277) and 2022 (4.56%, 21/461), which increased to 6.87% (46/670) in 2023. The difference in HMPV-positive rate among each year was statistically significant (P<0.05). The prevalence peak of HMPV varied in different years, showing either a unimodal or bimodal distribution in one year. A total of 79 HMPV G gene sequences were obtained, of which subtype A and subtype B accounted for 48.10% and 51.90%, respectively. All of the subtype A sequences were clarified as A2c duplicated variants, and subtype B was mainly B2 genotype. Besides, subtypes A and B were prevalent alone or co-circulated in different years, and there was a subtype replacement pattern in HMPV. Conclusion: The positive rate of HMPV in hospitalized ARI cases in children is significantly different from 2019 to 2023 in Changchun City. Notably, there are certain switch patterns of HMPV subtypes A and B in different years.

Rapid and one-tube detection of human metapneumovirus using the RT-RPA and CRISPR/Cas12a.

Human metapneumovirus (HMPV) is a common pathogen that can cause acute respiratory tract infections and is prevalent worldwide. There is yet no effective vaccine or specific treatment for HMPV. Early, rapid, and accurate detection is essential to treat the disease and control the spread of infection. In this study, we created the One-tube assay by combining Reverse Transcription-Recombinase Polymerase Amplification (RT-RPA) with the CRISPR/Cas12a system. By targeting the nucleoprotein (N) gene of HMPV to design specific primers and CRISPR RNAs (crRNAs), combining RT-RPA and CRISPR/Cas12a, established the One-tube assay. Meanwhile, the reaction conditions of the One-tube assay were optimized to achieve rapid and visual detection of HMPV. This assay could detect HMPV at 1 copy/µL in 30 min, without cross-reactivity with nine other respiratory pathogens. We validated the detection performance using clinical specimens and showed that the coincidence rate was 98.53 %，compared to the quantitative reverse-transcription polymerase chain reaction. The One-tube assay reduced the detection time and simplified the manual operation, while maintaining the detection performance and providing a new platform for HMPV detection.

Mechanism and spectrum of inhibition of a 4'-cyano modified nucleotide analog against diverse RNA polymerases of prototypic respiratory RNA viruses.

The development of safe and effective broad-spectrum antivirals that target the replication machinery of respiratory viruses is of high priority in pandemic preparedness programs. Here, we studied the mechanism of action of a newly discovered nucleotide analog against diverse RNA-dependent RNA polymerases (RdRps) of prototypic respiratory viruses. GS-646939 is the active 5'-triphosphate metabolite of a 4'-cyano modified C-adenosine analog phosphoramidate prodrug GS-7682. Enzyme kinetics show that the RdRps of human rhinovirus type 16 (HRV-16) and enterovirus 71 incorporate GS-646939 with unprecedented selectivity; GS-646939 is incorporated 20-50-fold more efficiently than its natural ATP counterpart. The RdRp complex of respiratory syncytial virus and human metapneumovirus incorporate GS-646939 and ATP with similar efficiency. In contrast, influenza B RdRp shows a clear preference for ATP and human mitochondrial RNA polymerase does not show significant incorporation of GS-646939. Once incorporated into the nascent RNA strand, GS-646939 acts as a chain terminator although higher NTP concentrations can partially overcome inhibition for some polymerases. Modeling and biochemical data suggest that the 4'-modification inhibits RdRp translocation. Comparative studies with GS-443902, the active triphosphate form of the 1'-cyano modified prodrugs remdesivir and obeldesivir, reveal not only different mechanisms of inhibition, but also differences in the spectrum of inhibition of viral polymerases. In conclusion, 1'-cyano and 4'-cyano modifications of nucleotide analogs provide complementary strategies to target the polymerase of several families of respiratory RNA viruses.

Human monoclonal antibodies protect against viral-mediated pneumococcal superinfection.

Introduction: Community-acquired pneumonia (CAP) is a global health concern, with 25% of cases attributed to Streptococcus pneumoniae (Spn). Viral infections like influenza A virus (IAV), respiratory syncytial virus (RSV), and human metapneumovirus (hMPV) increase the risk of Spn, leading to severe complications due to compromised host immunity. Methods: We evaluated the efficacy of an anti-PhtD monoclonal antibody (mAb) cocktail therapy (PhtD3 + 7) in improving survival rates in three viral/bacterial coinfection models: IAV/Spn, hMPV/Spn, and RSV/Spn. Results: The PhtD3 + 7 mAb cocktail outperformed antiviral mAbs, resulting in prolonged survival. In the IAV/Spn model, it reduced bacterial titers in blood and lungs by 2-4 logs. In the hMPV/Spn model, PhtD3 + 7 provided greater protection than the hMPV-neutralizing mAb MPV467, significantly reducing bacterial titers. In the RSV/Spn model, PhtD3 + 7 offered slightly better protection than the antiviral mAb D25, uniquely decreasing bacterial titers in blood and lungs. Discussion: Given the threat of antibiotic resistance, our findings highlight the potential of anti-PhtD mAb therapy as an effective option for treating viral and secondary pneumococcal coinfections.