Graphene Multiplexed Sensor for Point-of-Need Viral Wastewater-Based Epidemiology.

Wastewater-based epidemiology (WBE) can help mitigate the spread of respiratory infections through the early detection of viruses, pathogens, and other biomarkers in human waste. The need for sample collection, shipping, and testing facilities drives up the cost of WBE and hinders its use for rapid detection and isolation in environments with small populations and in low-resource settings. Given the ubiquitousness and regular outbreaks of respiratory syncytial virus, SARS-CoV-2, and various influenza strains, there is a rising need for a low-cost and easy-to-use biosensing platform to detect these viruses locally before outbreaks can occur and monitor their progression. To this end, we have developed an easy-to-use, cost-effective, multiplexed platform able to detect viral loads in wastewater with several orders of magnitude lower limit of detection than that of mass spectrometry. This is enabled by wafer-scale production and aptamers preattached with linker molecules, producing 44 chips at once. Each chip can simultaneously detect four target analytes using 20 transistors segregated into four sets of five for each analyte to allow for immediate statistical analysis. We show our platform's ability to rapidly detect three virus proteins (SARS-CoV-2, RSV, and Influenza A) and a population normalization molecule (caffeine) in wastewater. Going forward, turning these devices into hand-held systems would enable wastewater epidemiology in low-resource settings and be instrumental for rapid, local outbreak prevention.

Assembly of respiratory syncytial virus matrix protein lattice and its coordination with fusion glycoprotein trimers.

Respiratory syncytial virus (RSV) is an enveloped, filamentous, negative-strand RNA virus that causes significant respiratory illness worldwide. RSV vaccines are available, however there is still significant need for research to support the development of vaccines and therapeutics against RSV and related Mononegavirales viruses. Individual virions vary in size, with an average diameter of ~130 nm and ranging from ~500 nm to over 10 µm in length. Though the general arrangement of structural proteins in virions is known, we use cryo-electron tomography and sub-tomogram averaging to determine the molecular organization of RSV structural proteins. We show that the peripheral membrane-associated RSV matrix (M) protein is arranged in a packed helical-like lattice of M-dimers. We report that RSV F glycoprotein is frequently observed as pairs of trimers oriented in an anti-parallel conformation to support potential interactions between trimers. Our sub-tomogram averages indicate the positioning of F-trimer pairs is correlated with the underlying M lattice. These results provide insight into RSV virion organization and may aid in the development of RSV vaccines and anti-viral targets.

[Analysis of epidemiological characteristics of respiratory pathogens in children with influenza-like illnesses in a children's hospital in Beijing from 2022 to 2023].

To investigate the status and epidemiological characteristics of respiratory pathogens infections in children with influenza-like illnesses (ILI) in Beijing Children's Hospital from 2022 to 2023. A dual amplification technique was used to detect nucleic acids of seven common respiratory pathogens, including influenza A virus (Flu A), influenza B virus (Flu B), mycoplasma pneumoniae (MP), respiratory syncytial virus (RSV), parainfluenza virus (PIV), adenovirus (ADV), and Chlamydia pneumoniae (CP), in outpatient and inpatient children (aged 0-18 years) with influenza-like symptoms who sought medical care at Beijing Children's Hospital, from January 2022 to March 2023. A total of 43 663 children were included in the study, of which 27 903 tested positive for respiratory pathogens with a total detection rate of 63.91%. Flu A had the highest detection rate of 69.93% (27 332/39 084), followed by MP about 13.22% (380/2 875). The total detection rate of RSV, PIV and ADV was 7.69% (131/1 704). Flu B had a detection rate of 0.16% (64/39 084). No CP was detected in this study. A total of 7 cases of dual infections were detected, with a detection rate of 0.41% (7/1 704). The Chi-square test was used to analyze the differences in detection rates of pathogens among different genders, age groups, and different seasons. Among the seven pathogens, only Flu A had statistically significant differences in gender (χ2=16.712, P<0.001). The detection rates of Flu A and MP showed an increasing trend with age (both P trend<0.001), while the detection rates of RSV and PIV showed a decreasing trend with age (both P trend<0.001). Flu A had its epidemic peak in winter and spring, with detection rates of 61.30% (3 907/6 374) and 77.47% (23 207/29 958) respectively; MP and PIV had higher detection rates in autumn (25.14% and 7.64% respectively); RSV showed a relatively higher detection rate in winter (8.69%); Flu B and ADV had lower detection rates throughout the study period (0.16% and 1.17% respectively). In conclusion, children with ILI in 2022-2023 were mainly infected with a single respiratory pathogen, and occasionally dual pathogen infections were observed. Among them, the detection rate of Flu A was the highest, and only Flu A showed a gender difference in detection rate. As the age of the children patients increased, the detection rate of Flu A and MP showed an increasing trend, while RSV and PIV showed a decreasing trend. The prevalence of Flu A, Flu B, MP, PIV, and RSV were seasonal.

Filling two needs with one deed: a combinatory mucosal vaccine against influenza A virus and respiratory syncytial virus.

Influenza A Virus (IAV) and Respiratory Syncytial Virus (RSV) are both responsible for millions of severe respiratory tract infections every year worldwide. Effective vaccines able to prevent transmission and severe disease, are important measures to reduce the burden for the global health system. Despite the strong systemic immune responses induced upon current parental immunizations, this vaccination strategy fails to promote a robust mucosal immune response. Here, we investigated the immunogenicity and efficacy of a mucosal adenoviral vector vaccine to tackle both pathogens simultaneously at their entry site. For this purpose, BALB/c mice were immunized intranasally with adenoviral vectors (Ad) encoding the influenza-derived proteins, hemagglutinin (HA) and nucleoprotein (NP), in combination with an Ad encoding for the RSV fusion (F) protein. The mucosal combinatory vaccine induced neutralizing antibodies as well as local IgA responses against both viruses. Moreover, the vaccine elicited pulmonary CD8+ and CD4+ tissue resident memory T cells (TRM) against the immunodominant epitopes of RSV-F and IAV-NP. Furthermore, the addition of Ad-TGFß or Ad-CCL17 as mucosal adjuvant enhanced the formation of functional CD8+ TRM responses against the conserved IAV-NP. Consequently, the combinatory vaccine not only provided protection against subsequent infections with RSV, but also against heterosubtypic challenges with pH1N1 or H3N2 strains. In conclusion, we present here a potent combinatory vaccine for mucosal applications, which provides protection against two of the most relevant respiratory viruses.

Lung ILC2s are activated in BALB/c mice born to immunized mothers despite complete protection against respiratory syncytial virus.

Activated lung ILC2s produce large quantities of IL-5 and IL-13 that contribute to eosinophilic inflammation and mucus production following respiratory syncytial virus infection (RSV). The current understanding of ILC2 activation during RSV infection, is that ILC2s are activated by alarmins, including IL-33, released from airway epithelial cells in response to viral-mediated damage. Thus, high levels of RSV neutralizing maternal antibody generated from maternal immunization would be expected to reduce IL-33 production and mitigate ILC2 activation. Here we report that lung ILC2s from mice born to RSV-immunized dams become activated despite undetectable RSV replication. We also report, for the first time, expression of activating and inhibitory Fcgamma receptors on ILC2s that are differentially expressed in offspring born to immunized versus unimmunized dams. Alternatively, ex vivo IL-33-mediated activation of ILC2s was mitigated following the addition of antibody: antigen immune complexes. Further studies are needed to confirm the role of Fcgamma receptor ligation by immune complexes as an alternative mechanism of ILC2 regulation in RSV-associated eosinophilic lung inflammation.

Respiratory Virus-Induced PARP1 Alters DC Metabolism and Antiviral Immunity Inducing Pulmonary Immunopathology.

Previous studies from our laboratory and others have established the dendritic cell (DC) as a key target of RSV that drives infection-induced pathology. Analysis of RSV-induced transcriptomic changes in RSV-infected DC revealed metabolic gene signatures suggestive of altered cellular metabolism. Reverse phase protein array (RPPA) data showed significantly increased PARP1 phosphorylation in RSV-infected DC. Real-time cell metabolic analysis demonstrated increased glycolysis in PARP1-/- DC after RSV infection, confirming a role for PARP1 in regulating DC metabolism. Our data show that enzymatic inhibition or genomic ablation of PARP1 resulted in increased ifnb1, il12, and il27 in RSV-infected DC which, together, promote a more appropriate anti-viral environment. PARP1-/- mice and PARP1-inhibitor-treated mice were protected against RSV-induced immunopathology including airway inflammation, Th2 cytokine production, and mucus hypersecretion. However, delayed treatment with PARP1 inhibitor in RSV-infected mice provided only partial protection, suggesting that PARP1 is most important during the earlier innate immune stage of RSV infection.

Contributing to the management of viral infections through simple immunosensing of the arachidonic acid serum level.

A trendsetting direct competitive-based biosensing tool has been developed and implemented for the determination of the polyunsaturated fatty acid arachidonic acid (ARA), a highly significant biological regulator with decisive roles in viral infections. The designed methodology involves a competitive reaction between the target endogenous ARA and a biotin-ARA competitor for the recognition sites of anti-ARA antibodies covalently attached to the surface of carboxylic acid-coated magnetic microbeads (HOOC-MµBs), followed by the enzymatic label of the biotin-ARA residues with streptavidin-horseradish peroxidase (Strep-HRP) conjugate. The resulting bioconjugates were magnetically trapped onto the sensing surface of disposable screen-printed carbon transducers (SPCEs) to monitor the extent of the biorecognition reaction through amperometry. The operational functioning of the exhaustively optimized and characterized immunosensing bioplatform was highly convenient for the quantitative determination of ARA in serum samples from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2-) and respiratory syncytial virus (RSV)-infected individuals in a rapid, affordable, trustful, and sensitive manner.

Coletânea de Respostas Rápidas-Turma 2: programa educacional em vigilância em saúde no enfrentamento da COVID-19 e de outras doenças virais VigiEpidemia / Collection of Rapid Responses-Class 2: educational program in health surveillance in coping with COVID-19 and other viral diseases VigiEpidemia

As Políticas Informadas por Evidências (PIE) tem ocupado uma posição de destaque nas últimas décadas. Nota-se, portanto, um aumento do estimulo e da valorização da síntese do conhecimento cientifico para apoiar o desenvolvimento de políticas públicas de saúde baseadas em evidências. No Brasil, o Ministério da Saúde institucionalizou em 2009 a Rede para Politicas Informadas por Evidencias (Evidence-Informed Policy Network ­ EVIPNet Brasil). Essa Rede foi uma criação da Organização Mundial da Saúde (OMS) para promover o uso sistemático e transparente de evidências cientificas no desenvolvimento e na implementação de políticas de saúde, proporcionando o intercâmbio de conhecimento entre gestores, pesquisadores e representantes da sociedade civil, e facilitando a formulação, a implementação de políticas e a gestão dos serviços e sistemas de saúde. Coordenada pelo Departamento de Ciência e Tecnologia da Secretaria de Ciência, Tecnologia e Inovação e do Complexo Econômico-Industrial da Saúde do Ministério da Saúde (DECIT/SCTICS/MS), a EVIPNet Brasil, integrada por Núcleos de Evidências em Saúde (NEv), oferece periodicamente cursos diversos sobre PIE, além de materiais, como diretrizes metodológicas, para auxiliar na síntese de evidências para tomada de decisão em saúde. Com o evento da pandemia da COVID-19, a busca por evidências cientificas tornou-se mais intensa. A cobrança por resposta a curto prazo marcou esse período recente, desafiando o mundo todo a aprimorar mecanismos de elaboração, tradução e disseminação de conhecimento científico. Nesse contexto, surge o VigiEpidemia, um Programa Educacional em Vigilância e Cuidado em Saúde no Enfrentamento da COVID-19 e de outras doenças virais que oferece qualificação aos trabalhadores do Sistema Único de Saúde (SUS). Por meio desse programa, foram elaborados produtos, baseados no Portfolio de Produtos do Serviço de Produção de Evidências para Apoio a Tomada de Decisão, do DECIT, que tiveram como objetivo principal estimular o uso de evidências cientificas no cotidiano dos serviços de saúde. Este e-book oferece a leitora e ao leitor 26 pesquisas criteriosamente selecionadas com conteúdo interessante produzidos por especializandos no âmbito do VigiEpidemia, dividido em duas partes: a primeira sobre COVID-19 e outras doenças virais e a segunda sobre Dengue e outras arboviroses.

Evidence-Informed Policies (EIP) have gained prominence over the past few decades. Consequently, there has been an increase in the stimulation and appreciation of the synthesis of scientific knowledge to support the development of evidence-based public health policies. In Brazil, the Ministry of Health institutionalized the Evidence-Informed Policy Network (EVIPNet Brazil) in 2009. This network was an initiative by the World Health Organization (WHO) to promote the systematic and transparent use of scientific evidence in the development and implementation of health policies, facilitating knowledge exchange among managers, researchers, and representatives of civil society, and aiding in policy formulation, implementation, and the management of health services and systems. Coordinated by the Department of Science and Technology of the Secretariat of Science, Technology, Innovation, and the Economic-Industrial Health Complex of the Ministry of Health (DECIT/SCTICS/MS), EVIPNet Brazil, consisting of Health Evidence Centers (NEv), periodically offers various courses on EIP, along with resources such as methodological guidelines to assist in the synthesis of evidence for health decision-making. With the advent of the COVID-19 pandemic, the search for scientific evidence became more intense. The demand for short-term responses marked this recent period, challenging the world to enhance mechanisms for the elaboration, translation, and dissemination of scientific knowledge. In this context, the VigiEpidemia program emerged, an Educational Program in Health Surveillance and Care in addressing COVID-19 and other viral diseases, offering qualifications to workers of the Unified Health System (SUS). Through this program, products were developed based on the DECIT's Evidence Production Service Portfolio to Support Decision Making, primarily aimed at stimulating the use of scientific evidence in the daily operations of health services. This e-book provides the reader with 26 meticulously selected research studies with interesting content produced by specialists within the scope of VigiEpidemia, divided into two parts: the first on COVID-19 and other viral diseases, and the second on Dengue and other arboviruses.

[Preparation of a mouse monoclonal antibody against the NS1 protein of respiratory syncytial virus].

The aim of this study was to prepare a mouse monoclonal antibody against the nonstructural protein 1 (NS1) of respiratory syncytial virus (RSV) to analyze its expression and distribution during transfection and infection. Additionally, we aimed to evaluate the antibody's application in immunoprecipitation assay. Firstly, the NS1 gene fragment was cloned into a prokaryotic plasmid and expressed in Escherichia coli. The resulting NS1 protein was then purified by affinity chromatography, and used to immunize the BALB/c mice. Subsequently, hybridoma cells capable of stably secreting the NS1 monoclonal antibody were selected using indirect enzyme linked immunosorbent assay (ELISA). This monoclonal antibody was employed in both indirect immunofluorescence assay (IFA) and Western blotting to analyze the expression and distribution of RSV NS1 in overexpressed and infected cells. Finally, the reliability of this monoclonal antibody was evaluated through the immunoprecipitation assay. The results showed that the RSV NS1 protein was successfully expressed and purified. Following immunization of mice with this protein, we obtained a highly specific RSV NS1 monoclonal antibody, which belonged to the IgG1 subtype with an antibody titer of 1:15 360 000. Using this monoclonal antibody, the RSV NS1 protein was identified in both transfected and infected cells. The IFA results revealed predominant distribution of NS1 in the cytoplasm and nucleus. Moreover, we confirmed that this monoclonal antibody could effectively bind specifically to NS1 protein in cell lysates, making it suitable as a capture antibody in immunoprecipitation assay. In conclusion, our study successfully achieved production of the RSV NS1 protein through a prokaryotic expression system and prepared a specific monoclonal antibody against NS1. This antibody demonstrates the ability to specifically identify the NS1 protein and can be used in the immunoprecipitation assay, thereby laying a foundation for the functional studies of the NS1 protein.

Quercetin induces itaconic acid-mediated M1/M2 alveolar macrophages polarization in respiratory syncytial virus infection.

BACKGROUND: Quercetin has received extensive attention for its therapeutic potential treating respiratory syncytial virus (RSV) infection diseases. Recent studies have highlighted quercetin's ability of suppressing alveolar macrophages (AMs)-derived lung inflammation. However, the anti-inflammatory mechanism of quercetin against RSV infection still remains elusive. PURPOSE: This study aims to elucidate the mechanism about quercetin anti-inflammatory effect on RSV infection. METHODS: BALB/c mice were intranasally infected with RSV and received quercetin (30, 60, 120 mg/kg/d) orally for 3 days. Additionally, an in vitro infection model utilizing mouse alveolar macrophages (MH-S cells) was employed to validate the proposed mechanism. RESULTS: Quercetin exhibited a downregulatory effect on glycolysis and tricarboxylic acid (TCA) cycle metabolism in RSV-infected AMs. However, it increased itaconic acid production, a metabolite derived from citrate through activating immune responsive gene 1 (IRG1), and further inhibiting succinate dehydrogenase (SDH) activity. While the suppression of SDH activity orchestrated a cascading downregulation of Hif-1α/NLRP3 signaling, ultimately causing AMs polarization from M1 to M2 phenotypes. CONCLUSION: Our study demonstrated quercetin stimulated IRG1-mediated itaconic acid anabolism and further inhibited SDH/Hif-1α/NLRP3 signaling pathway, which led to M1 to M2 polarization of AMs so as to ameliorate RSV-induced lung inflammation.

Antiviral C-geranylated flavonoids from Artocarpus communis.

Ten C-geranylated flavonoids, along with three known analogues, were isolated from the leaves of Artocarpus communis. The chemical structures of these compounds were unambiguously determined via comprehensive spectroscopic analysis, single-crystal X-ray diffraction experiments, and quantum chemical electronic circular dichroism calculations. Structurally, artocarones A-I (1-9) represent a group of unusual, highly modified C-geranylated flavonoids, in which the geranyl chain is cyclised with the ortho-hydroxy group of flavonoids to form various heterocyclic scaffolds. Notably, artocarones E and G-I (5 and 7-9) feature a 6H-benzo[c]chromene core that is hitherto undescribed in C-geranylated flavonoids. Artocarone J (10) is the first example of C-9-C-16 connected C-geranylated aurone. Meanwhile, the plausible biosynthetic pathways for these rare C-geranylated flavonoids were also proposed. Notably, compounds 1, 2, 4, 8, 11, and 12 exhibited promising in vitro inhibitory activities against respiratory syncytial virus and herpes simplex virus type 1.

Rapid and on-site wireless immunoassay of respiratory virus aerosols via hydrogel-modulated resonators.

Rapid and accurate detection of respiratory virus aerosols is highlighted for virus surveillance and infection control. Here, we report a wireless immunoassay technology for fast (within 10 min), on-site (wireless and battery-free), and sensitive (limit of detection down to fg/L) detection of virus antigens in aerosols. The wireless immunoassay leverages the immuno-responsive hydrogel-modulated radio frequency resonant sensor to capture and amplify the recognition of virus antigen, and flexible readout network to transduce the immuno bindings into electrical signals. The wireless immunoassay achieves simultaneous detection of respiratory viruses such as severe acute respiratory syndrome coronavirus 2, influenza A H1N1 virus, and respiratory syncytial virus for community infection surveillance. Direct detection of unpretreated clinical samples further demonstrates high accuracy for diagnosis of respiratory virus infection. This work provides a sensitive and accurate immunoassay technology for on-site virus detection and disease diagnosis compatible with wearable integration.

The imbalance of pulmonary Th17/Treg cells in BALB/c suckling mice infected with respiratory syncytial virus-mediated intestinal immune damage and gut microbiota changes.

The immune response induced by respiratory syncytial virus (RSV) infection is closely related to changes in the composition and function of gastrointestinal microorganisms. However, the specific mechanism remains unknown and the pulmonary-intestinal axis deserves further study. In this study, the mRNA levels of ROR-γt and Foxp3 in the lung and intestine increased first and then decreased. IL-17 and IL-22 reached the maximum on the third day after infection in the lung, and on the second day after infection in the small intestine and colon, respectively. RegⅢγ in intestinal tissue reached the maximum on the third day after RSV infection. Moreover, the genus enriched in the RSV group was Aggregatibacter, and Proteus was reduced. RSV infection not only causes Th17/Treg cell imbalance in the lungs of mice but also leads to the release of excessive IL-22 from the lungs through blood circulation which binds to IL-22 receptors on the intestinal surface, inducing RegⅢγ overexpression, impaired intestinal Th17/Treg development, and altered gut microbiota composition. Our research reveals a significant link between the pulmonary and intestinal axis after RSV infection. IMPORTANCE: RSV is the most common pathogen causing acute lower respiratory tract infections in infants and young children, but the complex interactions between the immune system and gut microbiota induced by RSV infection still requires further research. In this study, it was suggested that RSV infection in 7-day-old BALB/c suckling mice caused lung inflammation and disruption of Th17/Treg cells development, and altered the composition of gut microbiota through IL-22 induced overexpression of RegⅢγ, leading to intestinal immune injury and disruption of gut microbiota. This research reveals that IL-22 may be the link between the lung and gut. This study may provide a new insight into the intestinal symptoms caused by RSV and other respiratory viruses and the connection between the lung and gut axis, as well as new therapeutic ideas for the treatment of RSV-infected children.

Effects of WuHuTang on the function and autophagy of dendritic cells treated with exosomes induced by RSV.

ETHNOPHARMACOLOGICAL RELEVANCE: WuHuTang (WHT) is a traditional Chinese medicine compound for treating asthma, and the evidence supports that it has a good effect on acute asthma attacks in children and adults. Respiratory syncytial virus (RSV) is an important factor in the pathogenesis of acute asthma attacks, and the effect on dendritic cells is the key to its pathogenesis. Previous studies have confirmed that the pathogenesis of viruses is related to exosomes. However, there are few studies on the exosomes induced by RSV. Whether WHT can improve the changes caused by RSV-induced exosomes or not is worthy of further exploration. AIM OF THE STUDY: We aim to study the effects of RSV-induced exosomes on the function and autophagy of dendritic cells, and to observe the intervention effect of WHT serum on the above effects. METHODS: The co-culture model of exosomes derived from bone marrow mesenchymal stem cells induced by RSV (BMSCs-Exo-RSV) and dendritic cells was established, and then WHT serum was used to intervene. After 24 h of intervention, the CCK-8 method, flow cytometry, Elisa, RT-qCPR, and Western blot were used to detect the above-mentioned culture model. RESULTS: RSV-induced exosomes had certain effects on viability, apoptosis, and costimulatory molecules generation of dendritic cells. At the same time, the levels of IL-6, IL-12, TNF-α, and autophagy increased, while the levels of IL-4, IL-10, and TGF-ß decreased, and the AKT/TSC/mTOR pathway was inhibited. WHT serum could activate this pathway and reverse the above changes in dendritic cells. CONCLUSION: This study reveals that the pathogenic effect of RSV is related to the exosomes induced by RSV. The exosomes induced by RSV affect the function of dendritic cells by inhibiting the AKT/TSC/mTOR pathway, which can be activated by WHT to reverse the effects caused by RSV-induced exosomes.

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.

Anti-respiratory syncytial virus and anti-herpes simplex virus activity of chemically engineered sulfated fucans from Cystoseira indica.

Seaweed polysaccharides, particularly sulfated ones, exhibited potent antiviral activity against a wide variety of enveloped viruses, such as herpes simplex virus and respiratory viruses. Different mechanisms of action were suggested, which may range from preventing infection to intracellular antiviral activity, at different stages of the viral cycle. Herein, we generated two chemically engineered sulfated fucans (C303 and C304) from Cystoseira indica by an amalgamated extraction-sulfation procedure using chlorosulfonic acid-pyridine/N,N-dimethylformamide and sulfur trioxide-pyridine/N,N-dimethylformamide reagents, respectively. These compounds exhibited activity against HSV-1 and RSV with 50 % inhibitory concentration values in the range of 0.75-2.5 µg/mL and low cytotoxicity at concentrations up to 500 µg/mL. The antiviral activities of chemically sulfated fucans (C303 and C304) were higher than the water (C301) and CaCl2 extracted (C302) polysaccharides. Compound C303 had a (1,3)-linked fucan backbone and was branched. Sulfates were present at positions C-2, C-4, and C-2,4 of Fucp, and C-6 of Galp residues of this polymer. Compound C304 had a comparable structure but with more sulfates at C-4 of Fucp residue. Both C303 and C304 were potent antiviral candidates, acting in a dose-dependent manner on the adsorption and other intracellular stages of HSV-1 and RSV replication, in vitro.

Simultaneous detection of influenza A, B and respiratory syncytial virus in wastewater samples by one-step multiplex RT-ddPCR assay.

BACKGROUND: After the occurrence of the COVID-19 pandemic, detection of other disseminated respiratory viruses using highly sensitive molecular methods was declared essential for monitoring the spread of health-threatening viruses in communities. The development of multiplex molecular assays are essential for the simultaneous detection of such viruses even at low concentrations. In the present study, a highly sensitive and specific multiplex one-step droplet digital PCR (RT-ddPCR) assay was developed for the simultaneous detection and absolute quantification of influenza A (IAV), influenza B (IBV), respiratory syncytial virus (RSV), and beta-2-microglobulin transcript as an endogenous internal control (IC B2M). RESULTS: The assay was first evaluated for analytical sensitivity and specificity, linearity, reproducibility, and recovery rates with excellent performance characteristics and then applied to 37 wastewater samples previously evaluated with commercially available and in-house quantitative real-time reverse transcription PCR (RT-qPCR) assays. IAV was detected in 16/37 (43%), IBV in 19/37 (51%), and RSV in 10/37 (27%) of the wastewater samples. Direct comparison of the developed assay with real-time RT-qPCR assays showed statistically significant high agreement in the detection of IAV (kappa Cohen's correlation coefficient: 0.834, p = 0.001) and RSV (kappa: 0.773, p = 0.001) viruses between the two assays, while the results for the detection of IBV (kappa: 0.355, p = 0.27) showed good agreement without statistical significance. CONCLUSIONS: Overall, the developed one-step multiplex ddPCR assay is cost-effective, highly sensitive and specific, and can simultaneously detect three common respiratory viruses in the complex matrix of wastewater samples even at low concentrations. Due to its high sensitivity and resistance to PCR inhibitors, the developed assay could be further used as an early warning system for wastewater monitoring.

Detailed analysis of low temperature inactivation of respiratory syncytial virus.

Our previous findings indicated that many respiratory syncytial virus (RSV) isolates are unstable at 4 °C compared to 20 °C. Some of the strains completely lose infectivity after 24 h at 4 °C. This study analyzed the inactivation process at 4 °C using a representative strain, RSV/Sendai/851/13. After 24 h of storage at 4 °C, the virus was completely inactivated but retained its ability to attach to and to be taken into host cells. It suggested a reduced fusion ability between the viral and cellular membranes. During storage at 4 °C, the RSV fusion (F) protein underwent a conformational change and was no longer recognized by pre-fusion form-specific antibodies. When the RSV/Sendai/851/13 strain was passaged at 4 °C, a variant with an amino acid substitution, I148T, in the F protein fusion peptide was selected. Also, an amino acid change in G protein demonstrating stability at low temperatures was obtained. These results show that the inactivation of RSV at 4 °C is due to the loss of membrane fusion activity in the F protein, which cannot maintain its pre-fusion state at 4 °C.

Exacerbated lung inflammation in offspring with high maternal antibody levels following secondary RSV exposure.

Respiratory syncytial virus (RSV) is the primary cause of bronchiolitis-related hospitalizations among children under 5 years of age, with reinfection being common throughout life. Maternal vaccination has emerged as a promising strategy, delivering elevated antibody levels to newborns for immediate protection. However, limited research has explored the protective efficacy of maternal antibodies (matAbs) against secondary RSV infections in offspring. To address this gap, we employed a mouse model of maternal RSV vaccination and secondary infection of offspring to evaluate lung pathology following RSV reinfection in mice with varying levels of maternal antibody (matAb). Additionally, we aimed to investigate the potential causes of exacerbated lung inflammation in offspring with high matAb levels following secondary RSV exposure. Our findings revealed that offspring with elevated levels of maternal pre-F antibody demonstrated effective protection against lung pathology following the initial RSV infection. However, this protection was compromised upon reinfection, manifesting as heightened weight loss, exacerbated lung pathology, increased expression of RSV-A N genes, eosinophilia, enhanced IL-5, IL-13, MUC5AC, and eosinophils Major Basic Protein (MBP) production in lung tissue compared to offspring lacking matAbs. Importantly, these unexpected outcomes were not attributed to antibody-dependent enhancement (ADE) resulting from declining matAb levels over time. Notably, our findings showed a decline in secretory IgA (sIgA), mucosal IgA, and mucosal IgG levels in offspring with high matAb levels post-primary RSV challenge. We propose that this decline may be a critical factor contributing to the ineffective protection observed during secondary RSV exposure. Overall, these findings offer valuable insights into maternal vaccination against RSV, contributing to a comprehensive understanding and mitigation of potential risks associated with maternal RSV vaccination.

Intranasal respiratory syncytial virus vaccine attenuated by codon-pair deoptimization of seven open reading frames is genetically stable and elicits mucosal and systemic immunity and protection against challenge virus replication in hamsters.

Respiratory syncytial virus (RSV) is the most important viral agent of severe pediatric respiratory illness worldwide, but there is no approved pediatric vaccine. Here, we describe the development of the live-attenuated RSV vaccine candidate Min AL as well as engineered derivatives. Min AL was attenuated by codon-pair deoptimization (CPD) of seven of the 11 RSV open reading frames (ORFs) (NS1, NS2, N, P, M, SH and L; 2,073 silent nucleotide substitutions in total). Min AL replicated efficiently in vitro at the permissive temperature of 32°C but was highly temperature sensitive (shut-off temperature of 36°C). When serially passaged at increasing temperatures, Min AL retained greater temperature sensitivity compared to previous candidates with fewer CPD ORFs. However, whole-genome deep-sequencing of passaged Min AL revealed mutations throughout its genome, most commonly missense mutations in the polymerase cofactor P and anti-termination transcription factor M2-1 (the latter was not CPD). Reintroduction of selected mutations into Min AL partially rescued its replication in vitro at temperatures up to 40°C, confirming their compensatory effect. These mutations restored the accumulation of positive-sense RNAs to wild-type (wt) RSV levels, suggesting increased activity by the viral transcriptase, whereas viral protein expression, RNA replication, and virus production were only partly rescued. In hamsters, Min AL and derivatives remained highly restricted in replication in the upper and lower airways, but induced serum IgG and IgA responses to the prefusion form of F (pre F) that were comparable to those induced by wt RSV, as well as robust mucosal and systemic IgG and IgA responses against RSV G. Min AL and derivatives were fully protective against challenge virus replication. The derivatives had increased genetic stability compared to Min AL. Thus, Min AL and derivatives with selected mutations are stable, attenuated, yet highly-immunogenic RSV vaccine candidates that are available for further evaluation.