[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.

First DFT-supported point of care and novel electrochemical biosensing: Determination of yellow fever NS1 antibody in human plasma.

In our study, we developed a point of care electrochemical biosensing platform based on the functionalized cysteine-positioned gold electrode to diagnose yellow fever disease from human plasma samples. The developed platform underwent characterization through diverse methods encompassing cyclic voltammetry, electrochemical impedance spectroscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy, and density-functional theory. The capacitive interaction between yellow fever virus non-structural antigen and antibody gave a cathodic signal at approximately -260 mV, and increased in proportion to the amount of non-structural antibody. The created electrochemical biosensor has an ability to detect 96 ag/mL of the yellow fever non-structural antibody with an extensive analytical range varied from 0.1 fg/mL to 1 µg/mL. The interference effects of various substances that could be found in human plasma, and the performance of the method were examined from the point of recovery and relative standard deviation for human plasma samples; hereby, the results confirmed the unprecedented selectivity and accuracy of the proposed method.

Development of a Double-Antibody Sandwich ELISA Based on a Monoclonal Antibody against the Viral NS1 Protein for the Detection of Chicken Parvovirus.

Chicken parvovirus (ChPV) infection can cause runting-stunting syndrome (RSS) in chickens. There is currently no commercially available vaccine for controlling ChPV, and ChPV infection in chickens is widespread globally. The rapid detection of ChPV is crucial for promptly capturing epidemiological data on ChPV. Two monoclonal antibodies (mAbs), 1B12 and 2B2, against the ChPV NS1 protein were generated. A double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for detecting ChPV based on the mAb 1B12 and an anti-chicken polyclonal antibody against the ChPV NS1 protein. The detection limit for the ChPV recombinant pET32a-NS1 protein was approximately 31.2 ng/mL. A total of 192 throat and cloaca swab samples were analyzed for ChPV by the established DAS-ELISA and nested PCR methods. The concordance rate between the DAS-ELISA and the nested PCR method was 89.1%. The DAS-ELISA can detect the ChPV antigen without any cross-reaction with FAdV-4, FAdV-1, NDV, AIV, MS, CIAV, aMPV, EDSV, IBV, or AGV2. The method also has high repeatability, with a coefficient of variation (CV) of less than 5%. These findings indicate that the DAS-ELISA exhibits high accuracy, good sensitivity, and specificity, making it suitable for viral detection, field surveillance, and epidemiological studies.

Changes in the Expression of Proteins Associated with Neurodegeneration in the Brains of Mice after Infection with Influenza A Virus with Wild Type and Truncated NS1.

Influenza type A virus (IAV) infection is a major cause of morbidity and mortality during influenza epidemics. Recently, a specific link between IAV infection and neurodegenerative disease progression has been established. The non-structural NS1 protein of IAV regulates viral replication during infection and antagonizes host antiviral responses, contributing to influenza virulence. In the present study, we have prepared a mouse lung-to-lung adapted to the NS1-truncated virus (NS80ad). Transcriptome analysis of the gene expression in the lungs revealed that infection with wild-type A/WSN/33 (WSN), NS80, and NS80ad viruses resulted in different regulation of genes involved in signaling pathways associated with the cell proliferation, inflammatory response, and development of neurodegenerative diseases. NS1 protein did not influence the genes involved in the RIG-I-like receptor signaling pathway in the brains. Lethal infection with IAVs dysregulated expression of proteins associated with the development of neurodegenerative diseases (CX3CL1/Fractalkine, Coagulation factor III, and CD105/Endoglin, CD54/ICAM-1, insulin-like growth factor-binding protein (IGFBP)-2, IGFBP-5, IGFBP-6, chitinase 3-like 1 (CHI3L1), Myeloperoxidase (MPO), Osteopontin (OPN), cystatin C, and LDL R). Transcription of GATA3 mRNA was decreased, and expression of MPO was inhibited in the brain infected with NS80 and NS80ad viruses. In addition, the truncation of NS1 protein led to reduced expression of IGFBP-2, CHI3L1, MPO, and LDL-R proteins in the brains. Our results indicate that the influenza virus influences the expression of proteins involved in brain function, and this might occur mostly through the NS1 protein. These findings suggest that the abovementioned proteins represent a promising target for the development of potentially effective immunotherapy against neurodegeneration.

Exploration of comprehensive marine natural products database against dengue viral non-structural protein 1 using high-throughput computational studies.

Dengue virus (DENV) non-structural protein 1 (NS1) is a versatile quasi-protein essential for the multiplication of the virus. This study applied high-throughput virtual screening (HTVS) and molecular dynamics (MD) simulation to detect the potential marine natural compounds against the NS1 of DENV. The structure of the NS1 protein was retrieved from Protein Data Bank with (PDB ID: 4O6B). Missing residues were added using modeler software. Molecular operating environment (MOE) programme was used to prepare the protein before docking. Virtual screening was performed on PyRx software to identify natural compounds retrieved from Comprehensive Marine Natural Products Database (CMNPD) against the NS1 protein, and best-docked compounds were examined by molecular docking and molecular dynamic (MD) simulation. Out of 31,561 marine compounds, the top 10 compounds showed docking scores lesser than -8.0 kcal/mol. One of the best hit compounds, CMNPD6802, was further analyzed using MD simulation study at 100 nanoseconds and Molecular Mechanics with Generalized Born and Surface Area Solvation (MM/GBSA). Based on its total binding energy, determined using the MM/GBSA approach, CMNPD6802 was ranked first. Its pharmacokinetic properties concerning the target protein NS1 were also evaluated. The results of the MD simulation showed that CMNPD6802 remained in close contact with the protein throughout the activation period, mapped using principal component analysis. These findings suggest that CMNPD6802 could serve as an NS1 inhibitor and may be a potential candidate for treating DENV infections.Communicated by Ramaswamy H. Sarma.

Nucleotide at position 66 of NS2A in Japanese encephalitis virus is associated with the virulence and proliferation of virus.

Most wild strains of Japanese encephalitis virus (JEV) produce NS1' protein, which plays an important role in viral infection and immune escape. The G66A nucleotide mutation in NS2A gene of the wild strain SA14 prevented the ribosomal frameshift that prevented the production of NS1' protein, thus reduced the virulence. In this study, the 66th nucleotide of the NS2A gene of SA14 was mutated into A, U or C, respectively. Both the G66U and G66C mutations cause the E22D mutation of the NS2A protein. Subsequently, the expression of NS1' protein, plaque size, replication ability, and virulence to mice of the three mutant strains were examined. The results showed that the three mutant viruses could not express NS1' protein, and their proliferation ability in nerve cells and virulence to mice were significantly reduced. In addition, the SA14(G66C) was less virulent than the other two mutated viruses. Our results indicate that only when G is the 66th nucleotide of NS2A, the JEV can produce NS1' protein, which affects the virulence.

Roles of NS1 Protein in Flavivirus Pathogenesis.

Flaviviruses such as dengue, Zika, and West Nile viruses are highly concerning pathogens that pose significant risks to public health. The NS1 protein is conserved among flaviviruses and is synthesized as a part of the flavivirus polyprotein. It plays a critical role in viral replication, disease progression, and immune evasion. Post-translational modifications influence NS1's stability, secretion, antigenicity, and interactions with host factors. NS1 protein forms extensive interactions with host cellular proteins allowing it to affect vital processes such as RNA processing, gene expression regulation, and cellular homeostasis, which in turn influence viral replication, disease pathogenesis, and immune responses. NS1 acts as an immune evasion factor by delaying complement-dependent lysis of infected cells and contributes to disease pathogenesis by inducing endothelial cell damage and vascular leakage and triggering autoimmune responses. Anti-NS1 antibodies have been shown to cross-react with host endothelial cells and platelets, causing autoimmune destruction that is hypothesized to contribute to disease pathogenesis. However, in contrast, immunization of animal models with the NS1 protein confers protection against lethal challenges from flaviviruses such as dengue and Zika viruses. Understanding the multifaceted roles of NS1 in flavivirus pathogenesis is crucial for effective disease management and control. Therefore, further research into NS1 biology, including its host protein interactions and additional roles in disease pathology, is imperative for the development of strategies and therapeutics to combat flavivirus infections successfully. This Review provides an in-depth exploration of the current available knowledge on the multifaceted roles of the NS1 protein in the pathogenesis of flaviviruses.

The nonstructural protein 1 of respiratory syncytial virus hijacks host mitophagy as a novel mitophagy receptor to evade the type I IFN response in HEp-2 cells.

Mitochondria are good targets for viruses to manipulate their hosts. However, it remains obscure whether respiratory syncytial virus (RSV) target mitochondria to suppress the type I interferon (IFN) responses. Here, we show that nonstructural protein 1 (NS1) protein of RSV interacts with Tu translation elongation factor mitochondrial (TUFM), which can lead to its localization in mitochondria and finally induce TUFM-dependent mitophagy and inhibition of IFNß. Mechanically, NS1-mediated TUFM-dependent mitophagy does not depend on the PINK1-PARKIN pathway and classic mitophagy receptors. Importantly, NS1 may act as a new receptor protein to bridge mitochondria and autophagosomes by interacting with TUFM and LC3B. The LIR motif of NS1 protein is essential for its interaction with LC3B and is of great importance for its mitophagy induction and IFNß suppression. Finally, NS1-induced TUFM-dependent mitophagy was essential for its attenuated IFNß response using autophagy-deficient cells and mice. Our study provides a novel mitophagy receptor molecular and a new antiviral option by suppressing antiviral innate immune via targeting TUFM-dependent mitophagy. IMPORTANCE It is a worthy concern for us to understand virus-host interactions which affect progression and prognosis of disease. We demonstrated that the non-structural protein 1 of respiratory syncytial virus (RSV NS1) may act as a novel mitophagy receptor to induce mitophagy by binding LC3B and mitochondrial protein TUFM, and finally dampen interferon (IFN) responses induced by RIG1 and RSV infection. TUFM is beneficial for RSV replication in vivo and vitro. It is new and interesting that RSV NS1 may function as a mitophagy receptor to interact with LC3B. The LIR motif of NS1 protein is essential for its interaction with LC3B. We further confirm that RSV NS1 inhibited IFNß response and promoted RSV replication in autophagy-dependent mechanisms in vivo and vitro. Our study contributes to understanding virus-host interaction, enriching our insights into RSV pathogenic mechanism and exploiting new antiviral treatments targeting TUFM.

Label-free genosensing of dengue serotypes with an electrodeposited reduced graphene oxide-tris(bipyridine)ruthenium(II).

Constructing a label-free electrochemical transducer platform without compromising inherent biocompatibility against specific bioreceptor remains challenging, particularly probing nucleic acid hybridization at electrode interface without external redox-mediator. Here, we show that electrochemically reduced graphene oxide-tris(bipyridine)ruthenium(II) (ErGO-TBR) nanosheets electrodeposited on carbon screen printed electrode can quantify hybridization of clinically important target sequences specific to serotypes of dengue virus (DENV) non-structural 1 (NS1) protein. Different variables including deposition potential, time, and electrolytic composition were optimized for fabrication of label-free transducer platform. Structural and electrochemical properties of ErGO-TBR/SPE were comprehensively elucidated using microscopic and spectroscopic techniques. Electrochemical quartz crystal microbalance (EQCM) analysis reveals the growth of electrodeposited redox-active species on the electrode interface. Surface functional group investigations suggested that TBR deposited on the basal and edges of ErGO substrate via electrostatic and π-π interactions. Functionalization of bio-affinity layer (B) on ErGO-TBR/SPE enables better loading of probe DNA (PDNA) toward specific detection of DENV target DNA (TDNA) with an ultralow detection limit promising for clinical diagnosis. Scalable chronoamperometry-based redox-active surface growth, customizable bioactivation strategy and external mediator-less probing of nucleic acid hybridization make the present system suitable for other translational application in healthcare diagnosis.

Advanced Detection Method for Dengue NS1 Protein Using Ultrasensitive ELISA with Thio-NAD Cycling.

Dengue fever, a mosquito-borne disease in tropical and subtropical climates caused by the dengue virus (DENV), has become a major social and economic burden in recent years. However, current primary detection methods are inadequate for early diagnosis of DENV because they are either time-consuming, expensive, or require training. Non-structural protein 1 (NS1) is secreted during DENV infection and is thus considered a suitable biomarker for the development of an early detection method. In the present study, we developed a detection method for the NS1 protein based on a previously reported thio-NAD cycling ELISA (i.e., ultrasensitive ELISA) and successfully achieved a LOD of 1.152 pg/mL. The clinical diagnosis potential of the detection system was also evaluated by using 85 patient specimens, inclusive of 60 DENV-positive and 25 DENV-negative specimens confirmed by the NAAT method. The results revealed 98.3% (59/60) sensitivity and 100% (25/25) specificity, which was in almost perfect agreement with the NAAT data with a kappa coefficient of 0.972. The present study demonstrates the diagnostic potential of using an ultrasensitive ELISA as a low-cost, easy-to-use method for the detection of DENV compared with NAAT and could be of great benefit in low-income countries.

Interferon-stimulated gene 15 facilitates BTV replication through interacting with the NS1 protein.

Bluetongue virus (BTV) infection effectively activates the innate immune response, followed by the expression of interferon (IFN) and multiple interferon-stimulated genes (ISGs). ISG15 is one of the most induced ISGs, and often plays a role in inhibiting virus replication. This study aims to explore the role and specific mechanisms of ovine ISG15 (oISG15) in BTV infection. We found that the transcription level of oISG15 was upregulated in a time-dependent and BTV multiplicity of infection-dependent manner. The overexpression of exogenous oISG15 enhances BTV replication, whereas the knockdown of endogenous oISG15 inhibits BTV replication. The viral protein in wild-type oISG15-overexpressed cells and ISGylation defective oISG15-overexpressed cells have no significant differences, which indicated that oISG15 promoted BTV replication in an ISGylation-independent manner. A co-immunoprecipitation assay showed that four viral BTV proteins-VP3, VP4, VP5, and NS1-interacted with oISG15. We also found that the VP4 and NS1 proteins associated with ubiquitin via co-immunoprecipitation, and that oISG15 overexpression improved the stability of both proteins. Further results showed that the degradation of NS1 was involved in lysine 63-linked polyubiquitin. This suggested that oISG15 may interfere with NS1 degradation via the autophagy pathway. This study provides new insights on the interaction between BTV and ISG15, and enriches our understanding of the regulation and biological function of ISG15 with virus replication.

NS1 and PA-X of H1N1/09 influenza virus act in a concerted manner to manipulate the innate immune response of porcine respiratory epithelial cells.

Live-attenuated influenza A viruses (LAIV) may be superior to inactivated or subunit vaccines since they can be administered via mucosal routes to induce local immunity in the respiratory tract. In addition, LAIV are expected to trigger stronger T-cell responses that may protect against a broader range of antigen-drifted viruses. However, the development of LAIV is challenging since a proper balance between immunogenicity and safety has to be reached. In this study, we took advantage of reverse genetics to generate three LAIV based on the pandemic H1N1 2009 (pH1N1/09) virus strain: ΔPA-X, which is defective in the synthesis of the accessory PA-X protein, NS1(1-126) lacking 93 amino acids at the C-terminus of the NS1 protein, and a combination of both. Characterization of these recombinant viruses using a novel porcine bronchiolar epithelial cell line (T3) revealed that the ΔPA-X mutant replicated similar to wild type (WT) virus. However, in contrast to the parental virus the ΔPA-X mutant allowed transcription of genes involved in cell cycle progression and limits apoptosis. The NS1(1-126) mutant also replicated comparable to WT virus, but triggered the release of type I and III IFN and several chemokines and cytokines. Surprisingly, only the NS1(1-126)/ΔPA-X double mutant was significantly attenuated on T3 cells, and this was associated with enhanced transcription of genes of the innate immune system and complete absence of apoptosis induction. In conclusion, these findings indicate that NS1 and PA-X act in a concerted manner to manipulate the host cell response, which may help to develop swine LAIV vaccine with a more favorable balance of safety and immunogenicity.

Role of CIV NS1 Protein in Innate Immunity and Viral Replication.

The innate immune pathway serves as the first line of defense against viral infections and plays a crucial role in the host's immune response in clearing viruses. Prior research has indicated that the influenza A virus has developed various strategies to avoid host immune responses. Nevertheless, the role of the NS1 protein of the canine influenza virus (CIV) in the innate immune pathway remains unclear. In this study, eukaryotic plasmids of NS1, NP, PA, PB1, and PB2 were constructed, and it was found that these proteins interact with melanoma differentiation-associated gene 5 (MDA5) and antagonize the activation of IFN-ß promoters by MDA5. We selected the NS1 protein for further study and found that NS1 does not affect the interaction between the viral ribonucleoprotein (RNP) subunit and MDA5, but that it downregulates the expression of the laboratory of genetics and physiology 2 (LGP2) and retinoic acid-inducible gene-I (RIG-I) receptors in the RIG-I pathway. Additionally, NS1 was found to inhibit the expression of several antiviral proteins and cytokines, including MX dynamin like GTPase 1 (MX1), 2'-5'oligoadenylate synthetase (OAS), Signal Transducers and Activators of Transcription (STAT1), tripartite motif 25 (TRIM25), interleukin-2 (IL-2), IFN, IL-8, and IL-1ß. To further investigate the role of NS1, a recombinant H3N2 virus strain (rH3N2) and an NS1-null virus (rH3N2ΔNS1) were rescued using reverse-genetic technology. The rH3N2ΔNS1 virus exhibited lower viral titers compared to rH3N2, but had a stronger activation effect on the receptors LGP2 and RIG-I. Furthermore, when compared to rH3N2, rH3N2ΔNS1 exhibited a more pronounced activation of antiviral proteins such as MX1, OAS, STAT1, and TRIM25, as well as antiviral cytokines such as IL-6, IFN-ß, and IL-1ß. These findings suggest a new mechanism by which NS1, a nonstructural protein of CIV, facilitates innate immune signaling and provides new avenues for the development of antiviral strategies.

Identification of Zika Virus NS1-Derived Peptides with Potential Applications in Serological Tests.

Zika virus (ZIKV), a mosquito-borne pathogen, is an emerging arbovirus associated with sporadic symptomatic cases of great medical concern, particularly among pregnant women and newborns affected with neurological disorders. Serological diagnosis of ZIKV infection is still an unmet challenge due to the co-circulation of the dengue virus, which shares extensive sequence conservation of structural proteins leading to the generation of cross-reactive antibodies. In this study, we aimed to obtain tools for the development of improved serological tests for the detection of ZIKV infection. Polyclonal sera (pAb) and a monoclonal antibody (mAb 2F2) against a recombinant form of the ZIKV nonstructural protein 1 (NS1) allowed the identification of linear peptide epitopes of the NS1 protein. Based on these findings, six chemically synthesized peptides were tested both in dot blot and ELISA assays using convalescent sera collected from ZIKV-infected patients. Two of these peptides specifically detected the presence of ZIKV antibodies and proved to be candidates for the detection of ZIKV-infected subjects. The availability of these tools opens perspectives for the development of NS1-based serological tests with enhanced sensitivity regarding other flaviviruses.

Bioinformatics Analysis of Mutations Sheds Light on the Evolution of Dengue NS1 Protein With Implications in the Identification of Potential Functional and Druggable Sites.

Non-structural protein (NS1) is a 350 amino acid long conserved protein in the dengue virus. Conservation of NS1 is expected due to its importance in dengue pathogenesis. The protein is known to exist in dimeric and hexameric states. The dimeric state is involved in its interaction with host proteins and viral replication, and the hexameric state is involved in viral invasion. In this work, we performed extensive structure and sequence analysis of NS1 protein, and uncovered the role of NS1 quaternary states in its evolution. A three-dimensional modeling of unresolved loop regions in NS1 structure is performed. "Conserved" and "Variable" regions within NS1 protein were identified from sequences obtained from patient samples and the role of compensatory mutations in selecting destabilizing mutations were identified. Molecular dynamics (MD) simulations were performed to extensively study the effect of a few mutations on NS1 structure stability and compensatory mutations. Virtual saturation mutagenesis, predicting the effect of every individual amino acid substitution on NS1 stability sequentially, revealed virtual-conserved and variable sites. The increase in number of observed and virtual-conserved regions across NS1 quaternary states suggest the role of higher order structure formation in its evolutionary conservation. Our sequence and structure analysis could enable in identifying possible protein-protein interfaces and druggable sites. Virtual screening of nearly 10,000 small molecules, including FDA-approved drugs, permitted us to recognize six drug-like molecules targeting the dimeric sites. These molecules could be promising due to their stable interactions with NS1 throughout the simulation.

Hypoxia alters the immune response in mouse peritoneal macrophages infected with influenza a virus with truncated NS1 protein.

Macrophages are the most abundant cells in infected tissue and are involved in the clearing infection, and immunomodulation of the innate and adaptive immune response. NS80 virus of influenza A virus, which encodes only the first 80 aa of the NS1 protein, suppresses the immune host response and is associated with enhanced pathogenicity. Hypoxia promotes infiltration of peritoneal macrophages into the adipose tissue and production of cytokines. To understand the role of hypoxia in the regulation of immune response, macrophages were infected with A/WSN/33 (WSN) and NS80 virus, and transcriptional profiles of the RIG-I-like receptor signalling pathway and expression of cytokines were evaluated in normoxia and hypoxia. Hypoxia inhibited the proliferation of IC-21 cells, downregulated the RIG-I-like receptor signalling pathway, and inhibited transcriptional activity of IFN-α, IFN-ß, IFN-ε, and IFN-λ mRNA in infected macrophages. While transcription of IL-1ß and Casp-1 mRNAs were increased in infected macrophages in normoxia, hypoxia resulted in decreased transcription activity of IL-1ß and Casp-1 mRNAs. Hypoxia significantly affected expression of the translation factors IRF4, IFN-γ, and CXCL10 involved in regulation of immune response and polarization of the macrophages. The expression of pro-inflammatory cytokines such as sICAM-1, IL-1α, TNF-α, CCL2, CCL3, CXCL12, and M-CSF was to a large extent affected in uninfected and infected macrophages cultivated in hypoxia. The NS80 virus increased the expression of M-CSF, IL-16, CCL2, CCL3, and CXCL12, especially under hypoxia. The results show that hypoxia may play an important role in peritoneal macrophage activation, regulates the innate and adaptive immune response, changes production of pro-inflammatory cytokines, promotes macrophage polarization, and could affect the function of other immune cells.

NS2 is a key determinant of compatibility in reassortant avian influenza virus with heterologous H7N9-derived NS segment.

Influenza A viruses are common pathogens with high prevalence worldwide and potential for pandemic spread. While influenza A infections typically elicit robust cellular innate immune responses, the non-structural protein 1 (NS1) antagonizes host anti-viral responses and is critical for efficient virus replication and virulence. The avian influenza virus (AIV) H7N9 initially emerged in China in 2013 and has since crossed the avian-human barrier, causing severe disease in humans. To investigate the influence of the H7N9 NS gene (NS079) on viral replication and innate immune response, we generated several recombinant AIVs bearing various NS079 segments on the backbone of H6N1 (strain 0702). Intriguingly, the recombinant virus bearing the heterologous NS079 gene was highly attenuated compared with virus carrying the homologous NS gene (NS0702). Furthermore, we generated a NS079-0702R virus that expresses a chimeric NS gene in which part of the NS079 effector domain was replaced with the sequence from NS0702. The NS079-0702R virus exhibited significantly enhanced viral yield, approximately 100-fold more than virus bearing NS079. The high infection rate of NS079-0702R virus was reflected by strong induction of IFN and Mx expression in human A549 cells. Intriguingly, our in vitro comparative analysis suggested that the increased NS079-0702R infection capacity was independent of the ability of NS1 to interact with cellular partners, such as PKR and CPSF30. Since partial substitution of the effector domain from NS0702 altered the coding sequence of NS2, we further generated another recombinant virus with NS2 derived from H7N9. Surprisingly, the virus with H7N9-derived NS2 exhibited growth characteristics similar to NS079. Our data demonstrate that swapping NS2 components changes infection efficiency, suggesting a key role for NS2 as a determinant of viral compatibility upon reassortment. These findings warrant further investigation into the precise mechanisms by which NS2 contributes to viral replication and host immunity.1.

The lncRNA HCG4 regulates the RIG-I-mediated IFN production to suppress H1N1 swine influenza virus replication.

Influenza A virus (IAV) non-structural protein 1 (NS1) is a virulence factor that allows the virus to replicate efficiently by suppressing host innate immune responses. Previously, we demonstrated that the serine (S) at position 42 of NS1 in H1N1 swine influenza virus (SIV) is a critical residue in interferon (IFN) resistance, thus facilitating viral infections. Here, by lncRNA-seq, a total of 153 differentially expressed lncRNAs were identified, and the lncRNA HCG4 was selected due to its significantly higher expression after infection with the NS1 S42P mutant virus. Overexpression of HCG4 enhanced IFN-ß production and suppressed SIV infection, highlighting the potential antiviral activity of HCG4 against SIV. Further investigation suggested that HCG4 served as a positive feedback mediator for RIG-I signaling. It alleviated the inhibitory effect on RIG-I K63-linked ubiquitination by NS1 protein, thereby resulting in an increase in RIG-I-mediated IFN production. Taken together, our findings demonstrate that HCG4 modulates the innate immune response to SIV infection through K63-linked RIG-I ubiquitination, providing insights into the role of lncRNAs in controlling viral infections.

Severe dengue associated with Staphylococcus aureus sepsis in pediatric patients: a case series.

BACKGROUND: Staphylococcus aureus co-infection is seldom reported in children with severe dengue. METHODOLOGY: In this retrospective study, we reported five children with severe dengue and S. aureus co-infection admitted to pediatric intensive care unit (PICU) during July-December 2021. RESULTS: All children had prolonged fever, persistence of bilateral pleural effusion beyond the critical phase, thrombocytopenia and raised inflammatory markers [C-reactive protein (CRP) and procalcitonin]. S. aureus was isolated from pleural fluid (n = 2, 40%), blood (n = 2, 40%) and endotracheal aspirate (n = 1, 20%). Four children (80%) grew methicillin-sensitive S. aureus, while 1 (20%) had methicillin-resistant S. aureus. Two children (40%) had septic thromboemboli in skin, and 1 (20%) had limb cellulitis. One child required anterior thoracotomy, pericardiectomy and bilateral pleural decortication, while all other children required intercostal chest tube drainage. All children required prolonged targeted antibiotics, invasive mechanical ventilation and had prolong stay in PICU and all of them survived. CONCLUSION: In children with severe dengue, persistence of fever, persistence of pleural effusion beyond critical phase and raised CRP and procalcitonin should raise suspicion of bacterial/S. aureus co-infection.

Immuno-dominant dengue NS1 peptides as antigens for production of monoclonal antibodies.

Synthetic peptides have recently become common as antigens for antibody production. Peptides are short chains of amino acids that can be used to elicit an immune response. The immunogenicity of the peptide antigens varies depending on the length, charge, solubility, and amino acids contained in the peptide sequence. Dengue NS1 protein is an important target antigen in the early detection of dengue infection. In this study, peptides corresponding to a highly conserved region from the dengue NS1 region were designed and synthesized. Balb/C mice were immunized against each peptide and spleen cells extracted from the immunized mice were fused with NS0 murine myeloma cells. Hybridoma clones obtained from the fusions were tested against peptides using ELISA. Out of 1,830 growing clones, 28 clones produced antibodies reacting with dengue NS1 peptides. A purified monoclonal antibody reacting with all four peptides was tested for reactivity with dengue NS1 native protein using dengue-confirmed serum and urine samples. The monoclonal antibody shows significant reactivity with both serum and urine. The findings of the current research can be used to detect dengue infection using urine, which ultimately results in the prevention of dengue epidemics through painless diagnosis, following treatment, and patient management to safeguard human and economic wellness.