Cellular NS1-BP Protein Interacts with the mRNA Export Receptor NXF1 to Mediate Nuclear Export of Influenza Virus M mRNAs.

Influenza A viruses have eight genomic RNAs that are transcribed in the host cell nucleus. Two of the viral mRNAs undergo alternative splicing. The M1 mRNA encodes the matrix protein 1 (M1) and is also spliced into M2 mRNA, which encodes the proton channel matrix protein 2 (M2). Our previous studies have shown that the cellular NS1-binding protein (NS1-BP) interacts with the viral non-structural protein 1 (NS1) and M1 mRNA to promote M1 to M2 splicing. Another pool of NS1 protein binds the mRNA export receptor NXF1 (nuclear RNA export factor-1), leading to nuclear retention of cellular mRNAs. Here we show a series of biochemical and cell biological findings that suggest a model for nuclear export of M1 and M2 mRNAs despite the mRNA nuclear export inhibition imposed by the viral NS1 protein. NS1-BP competes with NS1 for NXF1 binding, allowing the recruitment of NXF1 to the M mRNAs after splicing. NXF1 then binds GANP (Germinal-center Associated Nuclear Protein), a member of the TRanscription and EXport complex (TREX)-2. Although both NS1 and NS1-BP remain in complex with GANP-NXF1, they dissociate once this complex docks at the nuclear pore complex (NPC), and the M mRNAs are translocated to the cytoplasm. Since this mRNA nuclear export pathway is key for expression of M1 and M2 proteins that function in viral intracellular trafficking and budding, these viral-host interactions are critical for influenza virus replication.

Clinical Profile of Dengue Seropositive Infection From a Tertiary Care Hospital Situated in Mysuru, South India.

Introduction Dengue, a viral infection transmitted by Aedes mosquitoes, has become a significant global health concern. Its incidence has surged dramatically over the past decades, with severe cases potentially leading to life-threatening conditions such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Despite its prevalence in tropical regions, including India, the clinical manifestations of dengue can vary widely, sometimes presenting atypically. Recent outbreaks, particularly in Northern India, underscore the urgency of understanding and managing this disease. This study focuses on the clinical and laboratory findings of hospitalized dengue fever patients from January 2022 to January 2023, aiming to provide insights for effective patient care and mortality reduction. Methods This was a prospective study at JSS (Jagadguru Sri Shivarathreeshwara) Medical College and Hospital, Mysuru, Karnataka, India (January 2022-January 2023). Blood samples from suspected dengue patients presenting acute febrile symptoms were collected. NS1 antigen and IgM antibody were detected using enzyme-linked immunosorbent assay (ELISA). Patients positive for dengue NS1 antigen and IgM antibodies were included in the study, excluding those with co-infections or comorbidities. Results A nine-month study at JSS Hospital (January 2022-January 2023) screened 1019 samples, identifying 316 dengue cases. Among these, 84.8% were dengue fever and 15.1% were DHF/DSS. Male predominance (60.1%) was noted, with peak incidence in the age groups of 11-20 years (29.11%) and 0-10 years (27.53%). Common symptoms included fever (98.1%), headache (32.91%), myalgia (40.87%), and vomiting (42.7%). Thrombocytopenia was found in 60.6% of cases. NS1 was detected in 56% of patients and IgM was positive in 20.8% of the patients. Comorbidities like type 2 diabetes mellitus (T2DM) (7.59%) and hypertension (7.27%) were observed. Among severe cases, 43.6% had platelet counts <1 lakh/cumm, and 27.5% required intravenous fluids. Seven deaths occurred, primarily in patients with comorbidities and severe dengue. Discussion and conclusion High dengue seropositivity among males (60.12%) compared to females (39.87%) was noted, possibly due to varied exposures. Patients aged 11-20 years had the highest dengue infection, with a peak in admissions during the rainy season. Thrombocytopenia (60.6%) and comorbidities like T2DM and HTN were common, with seven fatalities linked to severe dengue and comorbidities, emphasizing the need for early recognition and management to reduce mortality.

Differential expression of cytokines and elevated levels of MALAT1 - Long non-coding RNA in response to non-structural proteins of human respiratory syncytial virus.

Human Respiratory Syncytial Virus (hRSV), a prevalent respiratory pathogen affecting various age groups, can trigger prolonged and intense inflammation in humans. The severity and outcome of hRSV infection correlate with elevated levels of pro-inflammatory agents, yet the underlying reasons for this immune system overstimulation remain elusive. We focused on the impact of hRSV non-structural proteins, NS1 and NS2, on immune response within epithelial cells. Available data indicates that these proteins impair the interferon pathway. We reinforce that NS1 and NS2 induce heightened secretion of the pro-inflammatory cytokines IL-6 and CXCL8. We also indicate that hRSV non-structural proteins provoke differential gene expression of human host FosB and long non-coding RNAs (MALAT1, RP11-510N19.5). It suggests an impact of NS molecules beyond IFN pathways. Thus, new light is shed on the interplay between hRSV and host cells, uncovering unexplored avenues of viral interference, especially the NS2 role in cytokine expression and immune modulation.

SARS-CoV-2 targets ribosomal RNA biogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hinders host gene expression, curbing defenses and licensing viral protein synthesis and virulence. During SARS-CoV-2 infection, the virulence factor non-structural protein 1 (Nsp1) targets the mRNA entry channel of mature cytoplasmic ribosomes, limiting translation. We show that Nsp1 also restrains translation by targeting nucleolar ribosome biogenesis. SARS-CoV-2 infection disrupts 18S and 28S ribosomal RNA (rRNA) processing. Expression of Nsp1 recapitulates the processing defects. Nsp1 abrogates rRNA production without altering the expression of critical processing factors or nucleolar organization. Instead, Nsp1 localizes to the nucleolus, interacting with precursor-rRNA and hindering its maturation separately from the viral protein's role in restricting mature ribosomes. Thus, SARS-CoV-2 Nsp1 limits translation by targeting ribosome biogenesis and mature ribosomes. These findings revise our understanding of how SARS-CoV-2 Nsp1 controls human protein synthesis, suggesting that efforts to counter Nsp1's effect on translation should consider the protein's impact from ribosome manufacturing to mature ribosomes.

Protective Role of NS1-Specific Antibodies in the Immune Response to Dengue Virus through Antibody-Dependent Cellular Cytotoxicity.

BACKGROUND: Dengue virus (DENV) non-structural protein 1 (NS1) has multiple functions within infected cells, on the cell surface, and in secreted form, and is highly immunogenic. Immunity from previous DENV infections is known to exert both positive and negative effects on subsequent DENV infections, but the contribution of NS1-specific antibodies to these effects is incompletely understood. METHODS: We investigated the functions of NS1-specific antibodies and their significance in DENV infection. We analyzed plasma samples collected in a prospective cohort study prior to symptomatic or subclinical secondary DENV infection. We measured binding to purified recombinant NS1 protein and to NS1-expressing CEM cells, antibody-mediated NK cell activation by plate-bound NS1 protein, and antibody-dependent cellular cytotoxicity (ADCC) of NS1-expressing target cells. RESULTS: We found that antibody responses to NS1 were highly serotype-cross-reactive and that subjects who experienced subclinical DENV infection had significantly higher antibody responses to NS1 in pre-infection plasma than subjects who experienced symptomatic infection. We observed strong positive correlations between antibody binding and NK activation. CONCLUSIONS: These findings demonstrate the involvement of NS1-specific antibodies in ADCC and provide evidence for a protective effect of NS1-specific antibodies in secondary DENV infection.

Engineering recombinant replication-competent bluetongue viruses expressing reporter genes for in vitro and non-invasive in vivo studies.

Bluetongue virus (BTV) is the causative agent of the important livestock disease bluetongue (BT), which is transmitted via Culicoides bites. BT causes severe economic losses associated with its considerable impact on health and trade of animals. By reverse genetics, we have designed and rescued reporter-expressing recombinant (r)BTV expressing NanoLuc luciferase (NLuc) or Venus fluorescent protein. To generate these viruses, we custom synthesized a modified viral segment 5 encoding NS1 protein with the reporter genes located downstream and linked by the Porcine teschovirus-1 (PTV-1) 2A autoproteolytic cleavage site. Therefore, fluorescent signal or luciferase activity is only detected after virus replication and expression of non-structural proteins. Fluorescence or luminescence signals were detected in cells infected with rBTV/Venus or rBTV/NLuc, respectively. Moreover, the marking of NS2 protein confirmed that reporter genes were only expressed in BTV-infected cells. Growth kinetics of rBTV/NLuc and rBTV/Venus in Vero cells showed replication rates similar to those of wild-type and rBTV. Infectivity studies of these recombinant viruses in IFNAR(-/-) mice showed a higher lethal dose for rBTV/NLuc and rBTV/Venus than for rBTV indicating that viruses expressing the reporter genes are attenuated in vivo. Interestingly, luciferase activity was detected in the plasma of viraemic mice infected with rBTV/NLuc. Furthermore, luciferase activity quantitatively correlated with RNAemia levels of infected mice throughout the infection. In addition, we have investigated the in vivo replication and dissemination of BTV in IFNAR (-/-) mice using BTV/NLuc and non-invasive in vivo imaging systems.IMPORTANCEThe use of replication-competent viruses that encode a traceable fluorescent or luciferase reporter protein has significantly contributed to the in vitro and in vivo study of viral infections and the development of novel therapeutic approaches. In this work, we have generated rBTV that express fluorescent or luminescence proteins to track BTV infection both in vitro and in vivo. Despite the availability of vaccines, BTV and other related orbivirus are still associated with a significant impact on animal health and have important economic consequences worldwide. Our studies may contribute to the advance in orbivirus research and pave the way for the rapid development of new treatments, including vaccines.

Antiviral responses versus virus-induced cellular shutoff: a game of thrones between influenza A virus NS1 and SARS-CoV-2 Nsp1.

Following virus recognition of host cell receptors and viral particle/genome internalization, viruses replicate in the host via hijacking essential host cell machinery components to evade the provoked antiviral innate immunity against the invading pathogen. Respiratory viral infections are usually acute with the ability to activate pattern recognition receptors (PRRs) in/on host cells, resulting in the production and release of interferons (IFNs), proinflammatory cytokines, chemokines, and IFN-stimulated genes (ISGs) to reduce virus fitness and mitigate infection. Nevertheless, the game between viruses and the host is a complicated and dynamic process, in which they restrict each other via specific factors to maintain their own advantages and win this game. The primary role of the non-structural protein 1 (NS1 and Nsp1) of influenza A viruses (IAV) and the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respectively, is to control antiviral host-induced innate immune responses. This review provides a comprehensive overview of the genesis, spatial structure, viral and cellular interactors, and the mechanisms underlying the unique biological functions of IAV NS1 and SARS-CoV-2 Nsp1 in infected host cells. We also highlight the role of both non-structural proteins in modulating viral replication and pathogenicity. Eventually, and because of their important role during viral infection, we also describe their promising potential as targets for antiviral therapy and the development of live attenuated vaccines (LAV). Conclusively, both IAV NS1 and SARS-CoV-2 Nsp1 play an important role in virus-host interactions, viral replication, and pathogenesis, and pave the way to develop novel prophylactic and/or therapeutic interventions for the treatment of these important human respiratory viral pathogens.

Peptide-ligand conjugate based immunotherapeutic approach for targeted dismissal of non-structural protein 1 of dengue virus: A novel therapeutic solution for mild and severe dengue infections.

Dengue virus infection has significantly increased, with reported cases soaring from 505,430 in 2000 to 2,809,818 in 2022, emphasizing the need for effective treatments. Among the eleven structural and non-structural proteins of DENV, Non-structural protein 1 (NS1) has emerged as a promising target due to its diverse role in modulating the immune response, inducing vascular leakage, and facilitating viral replication and assembly. Monoclonal antibodies are the sole therapeutics to target NS1, but concerns about their cross-reactivity persist. Given these concerns, our study focuses on designing a novel Peptide Ligand Conjugate (PLC) as a potential alternative immunotherapeutic agent against NS1. This PLC aims to mediate the immune elimination of soluble NS1 and NS1-presenting DENV-infected host cells by pre-existing vaccine-induced immunity. By employing the High Throughput Virtual Screening (HTVS) method, QikProp analysis, and Molecular Dynamics studies, we identified three hits from Asinex Biodesigned Ligands out of 220,177 compounds that show strong binding affinity towards the monoclonal binding site of NS1 protein. After a rigorous analysis of physicochemical characteristics, antigenicity, allergenicity, and toxicity using various servers, we selected two peptides: the minimum epitopic region of the Diphtheria and Tetanus toxins as the peptide components of the PLCs. A non-cleavable, non-reactive oxime linker connected the ligand with the peptide through oxime and amide bonds. DPT vaccine is widely used in dengue-endemic countries, and it has been reported that antibodies titer against MER of Diphtheria toxin and Tetanus toxins persist lifelong in DPT-vaccinated people. Therefore, once the rationally designed PLCs bind to NS1 through the ligands, the peptide will induce an immune response against NS1 by triggering pre-existing DPT antibodies and activating memory cells. This orchestrated immune response will destroy soluble NS1 and NS1-expressing DENV-infected cells, thereby reducing the illness of severe dengue hemorrhagic fever and the DENV infection, respectively. Given the increasing demand for new therapeutics for DENV treatment, further investigation into this novel immune-therapeutic strategy may offer a new avenue for treating mild and severe dengue infections.

Seroprevalence of tick-borne encephalitis virus and vaccination coverage of tick-borne encephalitis, Sweden, 2018 to 2019.

BackgroundIn Sweden, information on seroprevalence of tick-borne encephalitis virus (TBEV) in the population, including vaccination coverage and infection, is scattered. This is largely due to the absence of a national tick-borne encephalitis (TBE) vaccination registry, scarcity of previous serological studies and use of serological methods not distinguishing between antibodies induced by vaccination and infection. Furthermore, the number of notified TBE cases in Sweden has continued to increase in recent years despite increased vaccination.AimThe aim was to estimate the TBEV seroprevalence in Sweden.MethodsIn 2018 and 2019, 2,700 serum samples from blood donors in nine Swedish regions were analysed using a serological method that can distinguish antibodies induced by vaccination from antibodies elicited by infection. The regions were chosen to reflect differences in notified TBE incidence.ResultsThe overall seroprevalence varied from 9.7% (95% confidence interval (CI): 6.6-13.6%) to 64.0% (95% CI: 58.3-69.4%) between regions. The proportion of vaccinated individuals ranged from 8.7% (95% CI: 5.8-12.6) to 57.0% (95% CI: 51.2-62.6) and of infected from 1.0% (95% CI: 0.2-3.0) to 7.0% (95% CI: 4.5-10.7). Thus, more than 160,000 and 1,600,000 individuals could have been infected by TBEV and vaccinated against TBE, respectively. The mean manifestation index was 3.1%.ConclusionA difference was observed between low- and high-incidence TBE regions, on the overall TBEV seroprevalence and when separated into vaccinated and infected individuals. The estimated incidence and manifestation index argue that a large proportion of TBEV infections are not diagnosed.

The ER-Golgi transport of influenza virus through NS1-Sec13 association during virus replication.

IMPORTANCE: Influenza A virus is a respiratory virus that can cause complications such as acute bronchitis and secondary bacterial pneumonia. Drug therapies and vaccines are available against influenza, albeit limited by drug resistance and the non-universal vaccine administration. Hence there is a need for host-targeted therapies against influenza to provide an effective alternative therapeutic target. Sec13 was identified as a novel host interactor of influenza. Endoplasmic reticulum-to-Golgi transport is an important pathway of influenza virus replication and viral export. Specifically, Sec13 has a functional role in influenza replication and virulence.

The Identification of Host Proteins That Interact with Non-Structural Proteins-1α and -1ß of Porcine Reproductive and Respiratory Syndrome Virus-1.

Porcine reproductive and respiratory syndrome viruses (PRRSV-1 and -2) are the causative agents of one of the most important infectious diseases affecting the global pig industry. Previous studies, largely focused on PRRSV-2, have shown that non-structural protein-1α (NSP1α) and NSP1ß modulate host cell responses; however, the underlying molecular mechanisms remain to be fully elucidated. Therefore, we aimed to identify novel PRRSV-1 NSP1-host protein interactions to improve our knowledge of NSP1-mediated immunomodulation. NSP1α and NSP1ß from a representative western European PRRSV-1 subtype 1 field strain (215-06) were used to screen a cDNA library generated from porcine alveolar macrophages (PAMs), the primary target cell of PRRSV, using the yeast-2-hybrid system. This identified 60 putative binding partners for NSP1α and 115 putative binding partners for NSP1ß. Of those taken forward for further investigation, 3 interactions with NSP1α and 27 with NSP1ß were confirmed. These proteins are involved in the immune response, ubiquitination, nuclear transport, or protein expression. Increasing the stringency of the system revealed NSP1α interacts more strongly with PIAS1 than PIAS2, whereas NSP1ß interacts more weakly with TAB3 and CPSF4. Our study has increased our knowledge of the PRRSV-1 NSP1α and NSP1ß interactomes, further investigation of which could provide detailed insight into PRRSV immunomodulation and aid vaccine development.

Superior efficacy of a skin-applied microprojection device for delivering a novel Zika DNA vaccine.

Zika virus (ZIKV) infections are spreading silently with limited global surveillance in at least 89 countries and territories. There is a pressing need to develop an effective vaccine suitable for equitable distribution globally. Consequently, we previously developed a proprietary DNA vaccine encoding secreted non-structural protein 1 of ZIKV (pVAX-tpaNS1) to elicit rapid protection in a T cell-dependent manner in mice. In the current study, we evaluated the stability, efficacy, and immunogenicity of delivering this DNA vaccine into the skin using a clinically effective and proprietary high-density microarray patch (HD-MAP). Dry-coating of pVAX-tpaNS1 on the HD-MAP device resulted in no loss of vaccine stability at 40°C storage over the course of 28 days. Vaccination of mice (BALB/c) with the HD-MAP-coated pVAX-tpaNS1 elicited a robust anti-NS1 IgG response in both the cervicovaginal mucosa and systemically and afforded protection against live ZIKV challenge. Furthermore, the vaccination elicited a significantly higher magnitude and broader NS1-specific T helper and cytotoxic T cell response in vivo compared with traditional needle and syringe intradermal vaccination. Overall, the study highlights distinctive immunological advantages coupled with an excellent thermostability profile of using the HD-MAP device to deliver a novel ZIKV DNA vaccine.

Vaccination against tick-borne encephalitis elicits a detectable NS1 IgG antibody response.

Vaccine-induced protection against tick-borne encephalitis virus (TBEV) is mediated by antibodies to the viral particle/envelope protein. The detection of non-structural protein 1 (NS1) specific antibodies has been suggested as a marker indicative of natural infections. However, recent work has shown that TBEV vaccines contain traces of NS1, and immunization of mice induced low amounts of NS1-specific antibodies. In this study, we investigated if vaccination induces TBEV NS1-specific antibodies in humans. Healthy army members (n = 898) were asked to fill in a questionnaire relating to flavivirus vaccination or infection, and blood samples were collected. In addition, samples of 71 suspected acute TBE cases were included. All samples were screened for the presence of TBEV NS1-specific IgG antibodies using an in-house developed ELISA. Antibodies were quantified as percent positivity in reference to a positive control. For qualitative evaluation, cut-off for positivity was defined based on the mean OD of the lower 95% of the vaccinated individuals + 3 SD. We found significantly higher NS1-specific IgG antibody titers (i.e., quantitative evaluation) in individuals having received 2, 3, or 4 or more vaccine doses than in non-vaccinated individuals. Similarly, the percentage of individuals with a positive test result (i.e., qualitative evaluation) was higher in individuals vaccinated against tick-borne encephalitis than in unvaccinated study participants. Although NS1-specific IgG titers remained at a relatively low level when compared to TBE patients, a clear distinction was not always possible. Establishing a clear cut-off point in detection systems is critical for NS1-specific antibodies to serve as a marker for distinguishing the immune response after vaccination and infection.

Structural Investigations of Interactions between the Influenza a Virus NS1 and Host Cellular Proteins.

The Influenza A virus is a continuous threat to public health that causes yearly epidemics with the ever-present threat of the virus becoming the next pandemic. Due to increasing levels of resistance, several of our previously used antivirals have been rendered useless. There is a strong need for new antivirals that are less likely to be susceptible to mutations. One strategy to achieve this goal is structure-based drug development. By understanding the minute details of protein structure, we can develop antivirals that target the most conserved, crucial regions to yield the highest chances of long-lasting success. One promising IAV target is the virulence protein non-structural protein 1 (NS1). NS1 contributes to pathogenicity through interactions with numerous host proteins, and many of the resulting complexes have been shown to be crucial for virulence. In this review, we cover the NS1-host protein complexes that have been structurally characterized to date. By bringing these structures together in one place, we aim to highlight the strength of this field for drug discovery along with the gaps that remain to be filled.

Celastrol attenuates human parvovirus B19 NS1­induced NLRP3 inflammasome activation in macrophages.

Human parvovirus B19 (B19V) has been strongly associated with a variety of inflammatory disorders, such as rheumatoid arthritis (RA), inflammatory bowel disease and systemic lupus erythematosus. Non­structural protein 1 (NS1) of B19V has been demonstrated to play essential roles in the pathological processes of B19V infection due to its regulatory properties on inflammatory cytokines. Celastrol, a quinone methide isolated from Tripterygium wilfordii, has displayed substantial potential in treating inflammatory diseases, such as psoriasis and RA. However, little is known about the effects of celastrol on B19V NS1­induced inflammation. Therefore, cell viability assay, migration assay, phagocytosis analysis, zymography assay, ELISA and immunoblotting were conducted to verify the influences of celastrol on macrophages. The present study reported the attenuating effects of celastrol on B19V NS1­induced inflammatory responses in macrophages derived from human acute monocytic leukemia cell lines, U937 and THP­1. Although the migration was not significantly decreased by celastrol in both U937 and THP­1 macrophages, significantly decreased viability, migration and phagocytosis were detected in both B19V NS1­activated U937 and THP­1 macrophages in the presence of celastrol. Additionally, celastrol significantly decreased MMP­9 activity and the levels of inflammatory cytokines, including IL­6, TNF­α and IL­1ß, in B19V NS1­activated U937 and THP­1 cells. Notably, significantly decreased levels of NLR family pyrin domain­containing 3, apoptosis­associated speck­like, caspase­1 and IL­18 proteins were observed in both B19V NS1­activated U937 and THP­1 cells in the presence of celastrol, indicating the involvement of the inflammasome pathway. To the best of our knowledge, the present study is the first to report on the attenuating effects of celastrol on B19V NS1­induced inflammatory responses in macrophages, suggesting a therapeutic role for celastrol in B19V NS1­related inflammatory diseases.

Secreted NS1 proteins of tick-borne encephalitis virus and West Nile virus block dendritic cell activation and effector functions.

The flavivirus non-structural protein 1 (NS1) is secreted from infected cells into the circulation and the serum levels correlate with disease severity. The effect of secreted NS1 (sNS1) on non-infected mammalian immune cells is largely unknown. Here, we expressed recombinant sNS1 proteins of tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) and investigated their effects on dendritic cell (DC) effector functions. Murine bone marrow-derived DCs (BMDCs) showed reduced surface expression of co-stimulatory molecules and decreased release of pro-inflammatory cytokines when treated with sNS1 of TBEV or WNV prior to poly(I:C) stimulation. Transcriptional profiles of BMDCs that were sNS1-exposed prior to poly(I:C) stimulation showed two gene clusters that were downregulated by TBEV or WNV sNS1 and that were associated with innate and adaptive immune responses. Functionally, both sNS1 proteins modulated the capacity for BMDCs to induce specific T-cell responses as indicated by reduced IFN-γ levels in both CD4+ and CD8+ T cells after BMDC co-cultivation. In human monocyte-derived DCs, poly(I:C)-induced upregulation of co-stimulatory molecules and cytokine responses were even more strongly impaired by TBEV sNS1 or WNV sNS1 pretreatment than in the murine system. Our findings indicate that exogenous flaviviral sNS1 proteins interfere with DC-mediated stimulation of T cells, which is crucial for the initiation of cell-mediated adaptive immune responses in human flavivirus infections. Collectively, our data determine soluble flaviviral NS1 as a virulence factor responsible for a dampened immune response to flavivirus infections. IMPORTANCE The effective initiation of protective host immune responses controls the outcome of infection, and dysfunctional T-cell responses have previously been associated with symptomatic human flavivirus infections. We demonstrate that secreted flavivirus NS1 proteins modulate innate immune responses of uninfected bystander cells. In particular, sNS1 markedly reduced the capacity of dendritic cells to stimulate T-cell responses upon activation. Hence, by modulating cellular host responses that are required for effective antigen presentation and initiation of adaptive immunity, sNS1 proteins may contribute to severe outcomes of flavivirus disease.

The Adjuvant Activity of BCG Cell Wall Cytoskeleton on a Dengue Virus-2 Subunit Vaccine.

The uneven immunogenicity of the attenuated tetravalent dengue vaccine has made it difficult to achieve balanced protection against all four serotypes of the dengue virus (DENV). To overcome this problem, non-replicative vaccines have come into focus, as their immunogenicity is adjustable. This approach is excellent for multivalent vaccines but commonly faces the issue of low immunogenicity. In this present study, we developed a non-replicating dengue vaccine composed of UV-inactivated dengue virus-2 (UV-DENV-2) and DENV-2 NS1-279 protein encapsidated within nanoparticles. This vaccine candidate was administered in the presence of BCG cell wall cytoskeleton (BCG-CWS) as an adjuvant. We revealed, here, that encapsidated immunogens with BCG-CWS exerted potent activities on both B and T cells and elicited Th-1/Th-2 responses in mice. This was evidenced by BCG-CWS significantly augmenting antibody-mediated complement-fixing activity, strongly stimulating the antigen-specific polyfunctional T cell responses, and activating mixed Th-1/Th-2 responses specific to DENV-2- and NS1-279 antigens. In conclusion, BCG-CWS potently adjuvanted the inactivated DENV-2 and DENV subunit immunogens. The mechanism of adjuvanticity remains unclear. This study revealed the potential use of BCG-CWS in vaccine development.

An in-silico receptor-pharmacophore based multistep molecular docking and simulation study to evaluate the inhibitory potentials against NS1 of DENV-2.

DENV-2 strain is the most fatal and infectious of the five dengue virus serotypes. The non-structural protein NS1 encoded by its genome is the most significant protein required for viral pathogenesis and replication inside the host body. Thus, targeting the NS1 protein and designing an inhibitor to limit its stability and secretion is a propitious attempt in our fight against dengue. Four novel inhibitors are designed to target the conserved cysteine residues (C55, C313, C316, and C329) and glycosylation sites (N130 and N207) of the NS1 protein in an attempt to halt the spread of the dengue infection in the host body altogether. Numerous computer-aided drug designing techniques including molecular docking, molecular dynamics simulation, virtual screening, principal component analysis, and dynamic cross-correlation matrix were employed to determine the structural and functional activity of the NS1-inhibitor complexes. From our analysis, it was evident that the extent of structural and atomic level fluctuations of the ligand-bound protein exhibited a declining trend in contrast to unbound protein which was prominently noticeable through the RMSD, RMSF, Rg, and SASA graphs. The ADMET analysis of the four ligands revealed a promising pharmacokinetics and pharmacodynamic profile, along with good bioavailability and toxicity properties. The proposed drugs when bound to the targeted cavities resulted in stable conformations in comparison to their unbound state, implying they have good affinity promising effective drug action. Thus, they can be tested in vitro and used as potential anti-dengue drugs.Communicated by Ramaswamy H. Sarma.

In-silico screening and identification of potential drug-like compounds for dengue-associated thrombocytopenia from Carica papaya leaf extracts.

Dengue virus is a mosquito-borne pathogen that causes a variety of illnesses ranging from mild fever to severe and fatal dengue haemorrhagic fever or dengue shock syndrome. One of the major clinical manifestations of severe dengue infection is thrombocytopenia. The dengue non-structural protein 1 (NS1) is the primary protein that stimulates immune cells via toll-like receptor 4 (TLR4), induces platelets, and promotes aggregation, which could result in thrombocytopenia. The leaf extracts of Carica papaya seem to have therapeutic benefits in managing thrombocytopenia associated with dengue. The present study focuses on understanding the underlying mechanism of the use of papaya leaf extracts in treating thrombocytopenia. We have identified 124 phytocompounds that are present in the papaya leaf extract. The pharmacokinetics, molecular docking, binding free energy calculations, and molecular dynamic simulations were performed to investigate the drug-like properties, binding affinities, and interaction of phytocompounds with NS1 protein as well as the interactions of NS1 with TLR4. Three phytocompounds were found to bind with the ASN130, a crucial amino acid residue in the active site of the NS1 protein. Thus, we conclude that Rutin, Myricetin 3-rhamnoside, or Kaempferol 3-(2''-rhamnosylrutinoside) may serve as promising molecules by ameliorating thrombocytopenia in dengue-infected patients by interfering the interaction of NS1 with TLR4. These molecules can serve as drugs in the management of dengue-associated thrombocytopenia after verifying their effectiveness and assessing the drug potency, through additional in-vitro assays.Communicated by Ramaswamy H. Sarma.

Expression of recombinant dengue virus type 1 non-structural protein 1 in mammalian cells and preliminary assessment of its suitability to detect human IgG antibodies elicited by viral infection.

In recent years dengue has become a rapidly growing public health problem worldwide, however, the availability of accurate and affordable diagnostic immunoassays is limited, partly due to the difficulty of producing large quantities of purified antigen. Non-structural protein 1 (NS1) has shown to be a good candidate for inclusion in diagnostic assays and for serosurveys, particularly in endemic countries as a prerequisite for vaccination. In this work the NS1 antigen derived from dengue virus type-1 (DENV1) was expressed in HEK293-T cells and purified by affinity chromatography. The recombinant protein was recovered properly folded as dimers, highly purified and with good yield (1.5 mg/L). It was applied as a serological probe in an indirect ELISA developed in this work to detect human IgG antibodies. Preliminary comparative performance values of 81.1% sensitivity and 83.0% specificity of the developed and preliminary validated iELISA, relative to a commercial kit were obtained, suggesting that the purified recombinant DENV1 NS1 antigen is suitable to detect IgG antibodies, indicative of past DENV infection.