Dengue NS1 interaction with lipids alters its pathogenic effects on monocyte derived macrophages.

BACKGROUND: While dengue NS1 antigen has been shown to be associated with disease pathogenesis in some studies, it has not been linked in other studies, with the reasons remaining unclear. NS1 antigen levels in acute dengue are often associated with increased disease severity, but there has been a wide variation in results based on past dengue infection and infecting dengue virus (DENV) serotype. As NS1 engages with many host lipids, we hypothesize that the type of NS1-lipid interactions alters its pathogenicity. METHODS: Primary human monocyte derived macrophages (MDMs) were co-cultured with NS1 alone or with HDL, LDL, LPS and/or platelet activating factor (PAF) from individuals with a history of past dengue fever (DF = 8) or dengue haemorrhagic fever (DHF = 8). IL-1ß levels were measured in culture supernatants, and gene expression analysis carried out in MDMs. Monocyte subpopulations were assessed by flow cytometry. Hierarchical cluster analysis with Euclidean distance calculations were used to differentiate clusters. Differentially expressed variables were extracted and a classifier model was developed to differentiate between past DF and DHF. RESULTS: Significantly higher levels of IL-1ß were seen in culture supernatants when NS1 was co-cultured with LDL (p = 0.01, median = 45.69 pg/ml), but lower levels when NS1 was co-cultured with HDL (p = 0.05, median = 4.617 pg/ml). MDMs of those with past DHF produced higher levels of IL-1ß when NS1 was co-cultured with PAF (p = 0.02). MDMs of individuals with past DHF, were significantly more likely to down-regulate RPLP2 gene expression when macrophages were co-cultured with either PAF alone, or NS1 combined with PAF, or NS1 combined with LDL. When NS1 was co-cultured with PAF, HDL or LDL two clusters were detected based on IL10 expression, but these did not differentiate those with past DF or DHF. CONCLUSIONS: As RPLP2 is important in DENV replication, regulating cellular stress responses and immune responses and IL-10 is associated with severe disease, it would be important to further explore how differential expression of RPLP2 and IL-10 could lead to disease pathogenesis based on NS1 and lipid interactions.

The greasy finger region of DTMUV NS1 plays an essential role in NS1 secretion and viral proliferation.

Duck Tembusu virus (DTMUV) of the Orthoflavivirus genus poses a significant threat to waterfowl aquaculture. Nonstructural protein 1 (NS1), a multifunctional glycoprotein, exists in various oligomeric forms and performs diverse functions. The greasy finger (GF) region within NS1 of other flaviviruses has been shown to be a crucial component of the hydrophobic protrusion aiding in anchoring NS1 to the endoplasmic reticulum (ER). However, detailed studies on the role of the GF region in viral proliferation in vitro and the biological properties of NS1 remain scarce. A series of recombinant DTMUV (rDTMUV) with mutations in the GF region, including NS1-F158A, G159A, F160A, G161A, V162A, L163A, F160R, multipoint mutations (GF-4M), or regional deletions (ΔGF), were rescued using a DNA-based reverse genetics system. Only 5 rDTMUV variants (G159A, F160A, G161A, V162A, and L163A) could be rescued successfully, and these mutations were found to impair replication, reduce virulence, and decrease plaque size, as shown by growth kinetics, duck embryo virulence, and plaque assays, respectively. Upon examining NS1 expression by western blot, we discovered that secreted NS1 (sNS1) presented in large quantities in the supernatant of cells infected with rDTMUV-NS1-G159A, whereas intracellular NS1 was less abundant. These mutations also impacted the primary forms and secretion rates of NS1 in cases of overexpression by western blot and indirect ELISA. Exception for F160A and G161A, which showed decreased secretion rates, all other mutations increased sNS1 expression, with the most pronounced increase observed in F158A and ΔGF, and rDTMUV with these mutations can't be rescued. Co-localization studies of NS1 with the ER demonstrated that the ΔGF mutation attenuated NS1 anchoring to the ER, thereby inhibiting its intracellular residence and promoting secretion. Although these effects vary between flaviviruses, our data reveal that the GF region of NS1 is crucial for viral proliferation and NS1 secretion.

The C-terminal amino acid motifs of NS1 protein affect the replication and virulence of naturally NS-truncated H1N1 canine influenza virus.

The vast majority of data obtained from sequence analysis of influenza A viruses (IAVs) have revealed that nonstructural 1 (NS1) proteins from H1N1 swine, H3N8 equine, H3N2 avian and the correspondent subtypes from dogs have a conserved four C-terminal amino acid motif when independent cross-species transmission occurs between these species. To test the influence of the C-terminal amino acid motifs of NS1 protein on the replication and virulence of IAVs, we systematically generated 7 recombinants, which carried naturally truncated NS1 proteins, and their last four C-terminal residues were replaced with PEQK and SEQK (for H1N1), EPEV and KPEI (for H3N8) and ESEV and ESEI (for H3N2) IAVs. Another recombinant was generated by removing the C-terminal residues by reverse genetics. Remarkably, the ESEI and KPEI motifs circulating in canines largely contributed efficient replication in cultured cells and these had enhanced virulence. In contrast, the avian ESEV motif was only responsible for high pathogenicity in mice. We examined the effects of these motifs upon interferon (IFN) induction. The 7 mutant viruses replicated in vitro in an IFN-independent manner, and the canine SEQK motif was able to induced higher levels of IFN-ß in human cell lines. These findings shed further new light on the role of the four C-terminal residues in replication and virulence of IAVs and suggest that these motifs can modulate viral replication in a species-specific manner.

Performance of Fujifilm Dengue NS1 Antigen Rapid Diagnosis Kit Compared to Quantitative Real-Time Polymerase Chain Reaction.

Dengue is a viral infection caused by the dengue virus (DENV), transmitted to humans through the bite of infected Aedes mosquitoes. About half of the world's population is now at risk of dengue, which represents a global public health concern, especially in tropical and subtropical countries. Early detection of the viral infection is crucial to manage the disease; hence, effective rapid diagnostic tests are essential. In this study, we evaluated the performance between the new Fujifilm Dengue non-structural antigen diagnosis kit (FF NS1 kit) and the SD Bioline NS1 antigen test kit (SD NS1 kit) against the quantitative real-time polymerase chain reaction (qRT-PCR) assays. The 140 acute serum samples collected from the Yangon General Hospital and Yangon Children's Hospital, Myanmar, from 2017 to 2019 were characterised by the three assays. With the qRT-PCR as the standard, the FF NS1 kit and the SD NS1 kit exhibited sensitivity of 94.3% and 88.6%, respectively, and specificity of 100% in both kits. Moreover, the positivity rates of the FF NS1 kit and the SD NS1 kit were 97.5% and 95% in primary infection and 90% and 80% in secondary infection, respectively. Our overall results suggest that the FF NS1 kit is reliable and accurate for detecting DENV infection.

Prevalence of Aleutian Mink Disease Virus (AMDV) in Free-Ranging American Mink from Biebrza and Narew National Parks (Poland)-An Epidemiological Concern.

Aleutian Mink Disease Virus (AMDV) is the causative agent of Aleutian disease (AD). This progressive and chronic disorder significantly impacts the mink breeding industry, affecting farmed and free-ranging American and European mink. This study investigated AMDV variants isolated from free-ranging American mink in northeastern Poland. Between 2018 and 2019, 26 spleen samples were collected from mink in Narew National Park (NNP) and Biebrza National Park (BNP). DNA was extracted and subjected to PCR to amplify the NS1 gene, followed by sequencing and phylogenetic analysis. The NS1 gene was detected in 50% of samples from NNP minks and in 30% of samples from BNP minks, with an overall prevalence of 42.31%; these findings align with global data and indicate serious ecological and health concerns. Ten closely related AMDV variants and one distinct variant were identified. The grouped variants exhibited high genetic homogeneity, closely related to strains found in mink from the USA, Germany, Greece, Latvia, and Poland; meanwhile, the distinct variant showed similarities to strains found in mink from Finland, Denmark, China, Poland, and Latvia, suggesting multiple infection sources. These findings, consistent with data from Polish mink farms, indicate significant genetic similarity between farmed and wild mink strains, suggesting potential bidirectional transmission. This underscores the importance of a One Health approach, emphasizing the interconnectedness of human, animal, and environmental health. Continuous surveillance and genetic studies are crucial for understanding AMDV dynamics and mitigating their impacts. Measures to reduce transmission between farmed and wild mink populations are vital for maintaining mink health and ecosystem stability.

Harnessing Natural Resources for Advancements in Dengue Virus Treatment.

Dengue fever, caused by the Dengue virus (DENV) and transmitted by Aedes aegypti mosquitoes, has become endemic in over 100 countries. Despite considerable research, there is a lack of specific drugs for clinical use against dengue. Hence, further exploration to identify anti-- dengue compounds is essential. In recent years, natural products have gained attention for their antiviral properties. Plant-based medicines are particularly appealing due to their safety and low toxicity. This review summarizes natural compounds with potential antiviral activity against DENV, highlighting their mechanisms of action. Various compounds, from traditional herbal remedies to novel plant isolates, show promise against dengue, targeting crucial viral proteins like the envelope protein, proteases, and RNA polymerase. Exploring natural sources of antiviral agents against dengue is crucial. These compounds offer hope for effective treatments and mitigating dengue's global impact.

Zika Virus NS1 Protein Detection Using Gold Nanoparticle-Assisted Dynamic Light Scattering.

The Zika virus (ZIKV) is a global health threat due to its rapid spread and severe health implications, including congenital abnormalities and neurological complications. Differentiating ZIKV from other arboviruses such as dengue virus (DENV) is crucial for effective diagnosis and treatment. This study presents the development of a biosensor for detecting the ZIKV non-structural protein 1 (NS1) using gold nanoparticles (AuNPs) functionalized with monoclonal antibodies employing dynamic light scattering (DLS). The biosensor named ZINS1-mAb-AuNP exhibited specific binding to the ZIKV NS1 protein, demonstrating high colloidal stability indicated by a hydrodynamic diameter (DH) of 140 nm, detectable via DLS. In the absence of the protein, the high ionic strength medium caused particle aggregation. This detection method showed good sensitivity and specificity, with a limit of detection (LOD) of 0.96 µg mL-1, and avoided cross-reactivity with DENV2 NS1 and SARS-CoV-2 spike proteins. The ZINS1-mAb-AuNP biosensor represents a promising tool for the early and accurate detection of ZIKV, facilitating diagnostic and treatment capabilities for arboviral infections.

Differential Proteomic Profiling at Different Phases of Dengue Infection: An Intricate Insight from Proteins to Pathogenesis.

Dengue fever is a rapidly emerging tropical disease and an important cause of morbidity in its severe form worldwide. A wide spectrum of the pathophysiology is associated with the transition of dengue fever to severe dengue, which is driven by the host immune response and might reflect in patients' proteome profile. This study aims to analyze the plasma from different phases of dengue-infected patients at two time points. A mass-spectrometry-based proteomic approach was utilized to understand the involvement of probable candidate proteins toward developing a more severe, hemorrhagic form of dengue fever. Dengue-infected hospital-admitted patients with <5 days of fever were included in this study. Patient samples from the acute phase were screened for the presence of NS1 antigen using ELISA and subjected to molecular serotyping. Dengue molecular serotype-confirmed patient samples, pairwise from acute and critical phases with healthy control were subjected to qualitative and quantitative proteomic analysis, and then pathway analysis was performed. The protein-protein interaction network between the dengue virus and host proteins was depicted in the search for proteins associated with severe dengue pathophysiology. An array of apolipoprotein, cytokines, and endothelial proteins in association with virus replication and endothelial dysfunction were validated as biomolecules involved in severe dengue pathophysiology.

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.

SLC25A12 inhibits Japanese encephalitis virus replication by interacting with the NS1 and enhancing the type I interferon response.

Japanese encephalitis virus (JEV) is a mosquito-borne, zoonotic orthoflavivirus causing human encephalitis and reproductive disorders in pigs. Cell-intrinsic antiviral restriction factors are the first line of defense that prevent a virus from establishing a productive infection, while the molecular mechanism of the virus-host interaction is still not fully understood. Our in vitro experiments demonstrated that the Solute Carrier Family 25 Member 12 (SLC25A12) interacted with the JEV nonstructural protein 1 (NS1) and inhibited JEV replication. Furthermore, we showed that knockdown or knockout of SLC25A12 promoted JEV replication, while overexpression of SLC25A12 repressed viral replication. Finally, we demonstrated that SLC25A12 increased IRF7 mRNA levels, which promoted IFN-ß expression and subsequently induced antiviral effects. Collectively, our study revealed that SLC25A12 interacted with NS1, inhibiting viral RNA synthesis and transcription and enhancing type I interferon induction for antiviral effects.

Membrane Retention of West Nile Virus NS5 Depends on NS1 or NS3 for Enzymatic Activity.

West Nile virus (WNV) nonstructural protein 5 (NS5) possesses multiple enzymatic domains essential for viral RNA replication. During infection, NS5 predominantly localizes to unique replication organelles (ROs) at the rough endoplasmic reticulum (RER), known as vesicle packets (VPs) and convoluted membranes (CMs), with a portion of NS5 accumulating in the nucleus. NS5 is a soluble protein that must be in the VP, where its enzymatic activities are required for viral RNA synthesis. However, the mechanistic processes behind the recruitment of NS5 from the cytoplasm to the RER membrane remain unclear. Here, we utilize high-resolution confocal microscopy and sucrose density gradient ultracentrifugation to investigate whether the association of NS5 with other NS proteins contributes to its membrane recruitment and retention. We demonstrate that NS1 or NS3 partially influences the NS5 association with the membrane. We further demonstrate that processed NS5 is predominantly in the cytoplasm and nucleus, indicating that the processing of NS5 from the viral polyprotein does not contribute to its membrane localization. These observations suggest that other host or viral factors, such as the enwrapment of NS5 by the RO, may also be necessary for the complete membrane retention of NS5. Therefore, studies on the inhibitors that disrupt the membrane localization of WNV NS5 are warranted for antiviral drug development.

Commercial Enzyme-Linked Immunosorbent Assay Kit Is Useful for Detection of Recombinant and Secretory Nonstructural-1 Protein Antigen of Dengue Virus.

Background: Dengue is a mosquito-borne tropical disease, caused by the Dengue virus (DENV). It has become a severe problem and is a rising threat to public health. In this study, we have evaluated commercial Merilisa i Dengue NS1 Antigen kit (Meril LifeSciences India Pvt. Ltd.) to detect recombinant dengue virus 2 NS1 antigen (rDNS1Ag) and secreted forms of NS1 antigen (sDNS1Ag). Methods: To determine the detection limit of the kit, 100 nanogram (ng) to 0.001 ng rDNS1Ag was tested. The sensitivity and specificity of the kit was determined using recombinant NS1 antigens of all serotypes of DENV and other flaviviruses. For testing sDNS1Ag, the culture supernatant of the Vero cell lines infected with DENV-2 was tested. Further, a spiking experiment was carried out to check the sensitivity of the kit to detect rDNS1Ag in the pools of Aedes aegypti mosquitoes. Results: It was observed that the kit can detect the rDNS1Ag at 1 ng concentration. The kit was sensitive to detect NS1 antigen of DENV-1, DENV-2 and DENV-3 serotypes and specific for detection of only DNS1Ag as it did not cross-react with NS1 antigen of flaviviruses. The kit was sensitive to detect rDNS1Ag in the mosquito pools as well. In addition, the kit was able to detect the sDNS1Ag in Vero cell culture supernatant. Conclusions: Overall, we observed that the Merilisa i Dengue NS1 Ag kit is sensitive and specific for the detection of DNS1Ag both in recombinant and secretory forms.

Ubiquitination of NS1 Confers Differential Adaptation of Zika Virus in Mammalian Hosts and Mosquito Vectors.

Arboviruses, transmitted by medical arthropods, pose a serious health threat worldwide. During viral infection, Post Translational Modifications (PTMs) are present on both host and viral proteins, regulating multiple processes of the viral lifecycle. In this study, a mammalian E3 ubiquitin ligase WWP2 (WW domain containing E3 ubiquitin ligase 2) is identified, which interacts with the NS1 protein of Zika virus (ZIKV) and mediates K63 and K48 ubiquitination of Lys 265 and Lys 284, respectively. WWP2-mediated NS1 ubiquitination leads to NS1 degradation via the ubiquitin-proteasome pathway, thereby inhibiting ZIKV infection in mammalian hosts. Simultaneously, it is found Su(dx), a protein highly homologous to host WWP2 in mosquitoes, is capable of ubiquitinating NS1 in mosquito cells. Unexpectedly, ubiquitination of NS1 in mosquitoes does not lead to NS1 degradation; instead, it promotes viral infection in mosquitoes. Correspondingly, the NS1 K265R mutant virus is less infectious to mosquitoes than the wild-type (WT) virus. The above results suggest that the ubiquitination of the NS1 protein confers different adaptations of ZIKV to hosts and vectors, and more importantly, this explains why NS1 K265-type strains have become predominantly endemic in nature. This study highlights the potential application in antiviral drug and vaccine development by targeting viral proteins' PTMs.

The Disorderly Nature of Caliciviruses.

An intrinsically disordered protein (IDP) or region (IDR) lacks or has little protein structure but still maintains function. This lack of structure creates flexibility and fluidity, allowing multiple protein conformations and potentially transient interactions with more than one partner. Caliciviruses are positive-sense ssRNA viruses, containing a relatively small genome of 7.6-8.6 kb and have a broad host range. Many viral proteins are known to contain IDRs, which benefit smaller viral genomes by expanding the functional proteome through the multifunctional nature of the IDR. The percentage of intrinsically disordered residues within the total proteome for each calicivirus type species can range between 8 and 23%, and IDRs have been experimentally identified in NS1-2, VPg and RdRP proteins. The IDRs within a protein are not well conserved across the genera, and whether this correlates to different activities or increased tolerance to mutations, driving virus adaptation to new selection pressures, is unknown. The function of norovirus NS1-2 has not yet been fully elucidated but includes involvement in host cell tropism, the promotion of viral spread and the suppression of host interferon-λ responses. These functions and the presence of host cell-like linear motifs that interact with host cell caspases and VAPA/B are all found or affected by the disordered region of norovirus NS1-2. The IDRs of calicivirus VPg are involved in viral transcription and translation, RNA binding, nucleotidylylation and cell cycle arrest, and the N-terminal IDR within the human norovirus RdRP could potentially drive liquid-liquid phase separation. This review identifies and summarises the IDRs of proteins within the Caliciviridae family and their importance during viral replication and subsequent host interactions.

Development and validation of a novel enzyme-linked immunosorbent assay for the differentiation of tick-borne encephalitis infections caused by different virus subtypes.

OBJECTIVES: Tick-borne encephalitis (TBE) is an infection caused by the tick-borne encephalitis virus (TBEV) that can lead to symptoms of central nervous system inflammation. There are five subtypes of TBEV, three of which - European, Siberian and Far Eastern - occur in Europe. As it is thought that different subtype infections exhibit varying clinical courses and outcomes, serological differentiation of the virus subtypes is clearly important. However, to date, this has proved difficult to achieve. METHODS: An ELISA format was developed based on TBE virus NS1 antigen against the European, Siberian and Far Eastern subtype. The three NS1 antigens were biotechnologically produced in a human cell line and used for ELISA coating. Sera from German (European subtype) and Russian (Siberian and/or Far Eastern subtypes) TBE patients with positive TBEV IgG were used to test the reactivity against these three NS1 antigens. RESULTS: Testing of 23 German and 32 Russian TBEV IgG-positive sera showed that the ELISA was able to differentiate between TBEV European subtype and TBEV Siberian and Far Eastern subtype infections. CONCLUSIONS: In geographical areas where two or more TBEV subtype infections can occur, the NS1-IgG ELISA developed here constitutes an important diagnostic tool to differentiate between European subtype infections and Siberian/Far Eastern subtype infections and to use the new assay for epidemiological studies to clarify the importance of particular subtype infections in an area. Consequently, it may help to better describe and anticipate the clinical courses and outcomes of particular TBEV subtype infections.

Microbial infection among SARS-COV-2-infected patients in a COVID-19-dedicated tertiary care hospital of Bangladesh: a cross-sectional study.

Objectives. This study aimed to determine patterns of respiratory, blood-borne and uropathogenic microbial pathogens among SARS-CoV-2-infected patients in a COVID-19-(coronavirus disease 2019) dedicated tertiary care hospital in Dhaka, Bangladesh. Design.This was a cross-sectional study. Setting. In a COVID-19-dedicated tertiary care hospital in Dhaka, Bangladesh, conducted from March to June 2021. Participants. Hospitalized individuals with COVID-19 infection regardless of age or sex. Primary and secondary outcome measures. The percentage of co-infected COVID-19 patients and the characterization of the micro-organisms responsible for co-infection served as the primary outcome measures. Finding any associations between co-infection and age, co-infection and sex and co-infection and comorbidity was the secondary outcome variable. Interventions. Not applicable. Results.Out of 79 patients, 61 % were male, and the mean age was 49.53 years. Co-infection was seen in 7.7 % of patients, out of which 5.1 % of isolates were from urine samples, followed by 2.6 % from blood. Bacteria isolated from urine were Enterococcus (2.6 %), coagulase-negative Staphylococcus (CONS) (1.3 %) and Enterobacter spp. (1.3 %). Pseudomonas spp. was the only organism isolated from blood sample. Mixed growth was found in nasopharyngeal and throat swabs, with the predominant species being Staphylococcus aureus and Streptococcus spp. At the time of data collection, 55.7 % of patients had been given antimicrobials, and 30.4 % of patients had been given a single antimicrobial. HBsAg was positive in 1.3 % of patients and none were anti-hepatitis C or dengue NS1Ag positive. Conclusion. Microbial infection has been seen to be associated with SARS-CoV-2 infections and is of great value in prescribing antimicrobials and reducing fatal outcomes of hospitalized patients.

In turkeys, unlike chickens, the non-structural NS1 protein does not play a significant role in the replication and tissue tropism of the H7N1 avian influenza virus.

The economic losses caused by high pathogenicity (HP) avian influenza viruses (AIV) in the poultry industry worldwide are enormous. Although chickens and turkeys are closely related Galliformes, turkeys are thought to be a bridging host for the adaptation of AIV from wild birds to poultry because of their high susceptibility to AIV infections. HPAIV evolve from low pathogenicity (LP) AIV after circulation in poultry through mutations in different viral proteins, including the non-structural protein (NS1), a major interferon (IFN) antagonist of AIV. At present, it is largely unknown whether the virulence determinants of HPAIV are the same in turkeys and chickens. Previously, we showed that mutations in the NS1 of HPAIV H7N1 significantly reduced viral replication in chickens in vitro and in vivo. Here, we investigated the effect of NS1 on the replication and virulence of HPAIV H7N1 in turkeys after inoculation with recombinant H7N1 carrying a naturally truncated wild-type NS1 (with 224 amino-acid "aa" in length) or an extended NS1 with 230-aa similar to the LP H7N1 ancestor. There were no significant differences in multiple-cycle viral replication or in the efficiency of NS1 in blocking IFN induction in the cell culture. Similarly, all viruses were highly virulent in turkeys and replicated at similar levels in various organs and swabs collected from the inoculated turkeys. These results suggest that NS1 does not play a role in the virulence or replication of HPAIV H7N1 in turkeys and further indicate that the genetic determinants of HPAIV differ in these two closely related galliform species.

Cross-Reactive Antibodies to the NS1 Protein of Omsk Hemorrhagic Fever Virus Are Absent in the Sera of Patients with Tick-Borne Encephalitis.

Omsk hemorrhagic fever virus (OHFV) is a member of the tick-borne encephalitis virus (TBEV) complex of the Flaviviridae family. Currently, there are no data on the cross-reactivity of antibodies to the NS1 proteins of OHFV and TBEV. Such data are of major interest for monitoring viral encephalitis of unknown etiology due to the increasing geographical distribution of OHFV. In this study, a recombinant OHFV NS1 protein was produced using the Escherichia coli expression system and purified. The recombinant OHFV NS1 protein was recognized by specific mice immune ascetic fluids to the native OHFV NS1 protein. A Western blot analysis and ELISA of the recombinant NS1 proteins of OHFV and TBEV were used to study the cross-reactivity of antibodies from immune ascites fluid obtained from OHFV-infected mice and mAbs against TBEV NS1. Anti-TBEV NS1 mouse monoclonal antibodies (mAbs) have been shown to not be cross-reactive to the OHFV NS1 protein. Sera from patients with confirmed tick-borne encephalitis (TBE) were examined by ELISA using recombinant OHFV NS1 and TBEV NS1 proteins as antigens. It was shown for the first time that cross-reactive antibodies to the OHFV NS1 protein were not detected in the sera of TBE patients, whereas the sera contained antibodies to the TBEV NS1 protein.

Multi-epitope peptide vaccines targeting dengue virus serotype 2 created via immunoinformatic analysis.

The Middle East has witnessed a greater spread of infectious Dengue viruses, with serotype 2 (DENV-2) being the most prevalent form. Through this work, multi-epitope peptide vaccines against DENV-2 that target E and nonstructural (NS1) proteins were generated through an immunoinformatic approach. MHC class I and II and LBL epitopes among NS1 and envelope E proteins sequences were predicted and their antigenicity, toxicity, and allergenicity were investigated. Studies of the population coverage denoted the high prevalence of NS1 and envelope-E epitopes among different countries where DENV-2 endemic. Further, both the CTL and HTL epitopes retrieved from NS1 epitopes exhibited high conservancies' percentages with other DENV serotypes (1, 3, and 4). Three vaccine constructs were created and the expected immune responses for the constructs were estimated using C-IMMSIM and HADDOCK (against TLR 2,3,4,5, and 7). Molecular dynamics simulation for vaccine construct 2 with TLR4 denoted high binding affinity and stability of the construct with the receptor which might foretell favorable in vivo interaction and immune responses.

Repurposing of FDA-approved drugs against oligomerization domain of dengue virus NS1 protein: a computational approach.

Dengue fever is a serious health hazard on a global scale and its primary causative agent is the dengue virus (DENV). The non-structural protein 1 (NS1) of DENV plays a pivotal role in pathogenesis. It is associated with several autoimmune events, endothelial cell apoptosis, and vascular leakage, which increase mainly during the critical phase of infection. In this study, important residues of the oligomerization domain of NS1 protein were identified by literature searches. Virtual screening has been conducted using the entire dataset of the DrugBank database and the potential small-molecule inhibitors against the NS1 protein have been chosen on the basis of binding energy values. This is succeeded by molecular dynamics (MD) simulations of the shortlisted compounds, ultimately giving rise to five compounds. These five compounds were further subjected to RAMD simulations by applying a random direction force of specific magnitude on the ligand center of mass in order to push the ligand out of the protein-binding pocket, for the quantitative estimation of their binding energy values to determine the interaction strength between protein and ligand which prevents ligand unbinding from its binding site, ultimately leading to the selection of three major compounds, DB00826 (Natamycin), DB11274 (Dihydro-alphaergocryptine), and DB11275 (Epicriptine), with the DB11274 having a role against idiopathic Parkinson's disease, and thus may have possible important roles in the prevention of dengue-associated Parkinsonism. These compounds may act as prospective drugs against dengue, by preventing the oligomerization of the NS1 protein, thereby preventing disease progression and pathogenesis.