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Crimean–Congo hemorrhagic fever virus (CCHFV) belongs to Nairoviridae family and has tripartite RNA genome. It is endemic in various countries of Asia, Africa, and Europe and is primarily transmitted by Hyalomma ticks but nosocomial transmission also been reported. Vaccines for CCHF are in early phase of clinical trial; therefore, this work is centered on identification of potential immunogenic peptide as vaccine candidates with application of different immunoinformatics approaches. Eleven conserved (>90%) peptides of CCHFV nucleoprotein were selected for CD8+ T‐cell (NetMHCpan 4.1b and NetCTLpan 1.1 server) and CD4+ T‐cell (NetMHCIIpan‐4.0 server and Tepitool) epitope prediction. Three peptides containing multiple CD8+ and CD4+ T‐cell and B‐cell epitopes were identified on basis of consensus prediction approach. Peptides displayed good antigenicity score of 0.45–0.68 and predicted to bind with diverse human leukocyte antigen (HLA) alleles. Molecular docking was performed with epitopes to HLA and HLA‐epitopes complex to T‐cell receptor (TCR). In most of the cases, docked complex of HLA‐epitope and HLA‐epitopes‐TCR have the binding energy close to respective natural bound peptide complex with HLA and TCR. Molecular dynamic simulation also revealed that HLA‐peptide complexes have minimum fluctuation and deviation than HLA‐peptide‐TCR docked over 50 ns simulation run. Considering these findings, identified peptides can serve as potential vaccine candidates for CCHFV disease. [ABSTRACT FROM AUTHOR]

Rabies is controlled by mass animal vaccination campaigns. Cats, dogs, and wild animals (e.g., bats) are large reservoirs of this virus and can pose a significant threat to the human health, especially in the developing countries. The nucleoprotein of the rabies virus is of great scientific interest since it has the potential to generate immunity in animals and can be used as for immunochemical diagnostics. The study aimed to test a large-scale expression of the rabies N protein in a prokaryotic system. The recombinant N protein was successfully expressed and purified. It was immunologically recognized by specific antibodies and was able to induce the production of specific antibodies in a mouse immunization assay. These encouraging results indicate that the recombinant N protein can be evaluated as an antigen for the development of a subunit vaccine or for a diagnostic assay. [ABSTRACT FROM AUTHOR]

Lipid droplets (LDs) are organelles involved in lipid storage, maintenance of energy homeostasis, protein sequestration, signaling events and inter-organelle interactions. Recently, LDs have been shown to favor the replication of members from different viral families, such as the Flaviviridae and Coronaviridae. In this work, we show that LDs are essential organelles for members of the Arenaviridae family. A virus-driven reduction of LD number was observed in cultures infected with Junı́n mammarenavirus (JUNV), caused in part by action of the viral nucleoprotein. Notably, we identified a new pool of nucleoprotein and viral RNA that localizes in the vicinity of LDs, suggesting that LDs play a role during the viral replication cycle. Regarding the mechanism behind LD exhaustion, we found evidence that lipophagy is involved in LD degradation with the resulting fatty acids being substrates of fatty acid β-oxidation, which fuels viral multiplication. This work highlights the importance of LDs during the replication cycle of JUNV, contributing to the knowledge of the metabolic changes these mammarenaviruses cause in their hosts. [ABSTRACT FROM AUTHOR]

Long-lasting COVID-19 (long COVID) diseases constitute a real life-changing burden for many patients around the globe and, overall, can be considered societal and economic issues. They include a variety of symptoms, such as fatigue, loss of smell (anosmia), and neurological–cognitive sequelae, such as memory loss, anxiety, brain fog, acute encephalitis, and stroke, collectively called long neuro-COVID-19 (long neuro-COVID). They also include cardiopulmonary sequelae, such as myocardial infarction, pulmonary damage, fibrosis, gastrointestinal dysregulation, renal failure, and vascular endothelial dysregulation, and the onset of new diabetes, with each symptom usually being treated individually. The main unmet challenge is to understand the mechanisms of the pathophysiologic sequelae, in particular the neurological symptoms. This mini-review presents the main mechanistic hypotheses considered to explain the multiple long neuro-COVID symptoms, namely immune dysregulation and prolonged inflammation, persistent viral reservoirs, vascular and endothelial dysfunction, and the disruption of the neurotransmitter signaling along various paths. We suggest that the nucleoprotein N of SARS-CoV-2 constitutes a "hub" between the virus and the host inflammation, immunity, and neurotransmission. [ABSTRACT FROM AUTHOR]

Rabies is controlled by mass animal vaccination campaigns. Cats, dogs, and wild animals (e.g., bats) are large reservoirs of this virus and can pose a significant threat to the human health, especially in the developing countries. The nucleoprotein of the rabies virus is of great scientific interest since it has the potential to generate immunity in animals and can be used as for immunochemical diagnostics. The study aimed to test a large-scale expression of the rabies N protein in a prokaryotic system. The recombinant N protein was successfully expressed and purified. It was immunologically recognized by specific antibodies and was able to induce the production of specific antibodies in a mouse immunization assay. These encouraging results indicate that the recombinant N protein can be evaluated as an antigen for the development of a subunit vaccine or for a diagnostic assay.

Antigen-specific T cells are an important part of antiviral responses, and modern influenza vaccines are designed to induce this mode of immunity. Live attenuated influenza vaccine (LAIV) is a potent inducer of T-cell immunity because of its ability to cause productive infection in the upper respiratory tract. Inactivated influenza vaccines (IIV) and novel vaccine candidates can also induce virus-specific T-cells when appropriate adjuvants are used. In this case, non-structural and intrinsic antigens of the master donor viruses, particularly nucleoprotein (NP), are the main targets for the development of T-cell immunity. The most commonly used donor strains for LAIVs and IIVs worldwide were derived from viruses isolated between 1933 and 1960. In this regard, the question of conservation of epitopes immunogenic for CD8⁺ T-lymphocytes (CTL-epitopes) in donor-derived NPs, i.e., the ability of cytotoxic T cells specific to the donor’s NP to recognize modern influenza A virus nucleoproteins, is relevant. The aim of the study was to evaluate the conservation of CTL-immunogenic NP epitopes of donors traditionally used to create LAIVs and IIVs. Materials and methods. Epitope NP analysis was performed for 1614 and 1767 strains of influenza A virus subtypes H1N1 and H3N2, respectively, which circulated in 2009–2023 (data from the NCBI Influenza Virus Database). Immune Epitope Database (IEDB, www.iedb.org), NetCTL’s built-in CTL-epitope prediction algorithm and NetChop proteolysis site predictor were used. CTL-epitopes were mapped to NPs of master donor viruses A/Leningrad/134/17/57 (H2N2), A/Ann Arbor/6/60 (H2N2), A/PR/8/34 (H1N1), and A/WSN/1933 (H1N1) using the CrustalO alignment algorithm in JalView 2.8.1 Software. The immunogenicity and conservation of selected epitopes were further evaluated using IEDB T-cell Immunogenicity Predictor and Epitope Conservancy Assay, respectively. Results. The majority of immunogenic CTL-epitopes of donor viruses proved to be non-conserved, i.e., not found in NPs of circulating influenza strains. Conversely, most CTL-immunogenic NP epitopes of modern viruses are absent in donor viruses and cannot be induced by vaccination with conventional vaccines. The data obtained indicate the need to actualize NP in vaccine composition by directed mutagenesis of the donor-derived NP gene or by introduction of the gene encoding NP of circulating influenza viruses into vaccine strains.

Severe fever with thrombocytopenia syndrome is an emerging viral hemorrhagic fever caused by a tick-borne bunyavirus, severe fever with thrombocytopenia syndrome virus (SFTSV), with a high case fatality. We previously found that SFTSV nucleoprotein (NP) induces macroautophagy/autophagy to facilitate virus replication. However, the role of NP in antagonizing host innate immunity remains unclear. Mitophagy, a selected form of autophagy, eliminates damaged mitochondria to maintain mitochondrial homeostasis. Here, we demonstrate that SFTSV NP triggers mitophagy to degrade MAVS (mitochondrial antiviral signaling protein), thereby blocking MAVS-mediated antiviral signaling to escape the host immune response. Mechanistically, SFTSV NP translocates to mitochondria by interacting with TUFM (Tu translation elongation factor, mitochondrial), and mediates mitochondrial sequestration into phagophores through interacting with LC3, thus inducing mitophagy. Notably, the N-terminal LC3-interacting region (LIR) motif of NP is essential for mitophagy induction. Collectively, our results demonstrated that SFTSV NP serves as a novel virulence factor, inducing TUFM-mediated mitophagy to degrade MAVS and evade the host immune response.Abbreviation: 3-MA: 3-methyladenine; ACTB: actin beta; co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI: 4ʹ,6-diamidino-2-phenylindole, dihydrochloride; DMSO: dimethyl sulfoxide; FCCP: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GFP: green fluorescent protein; HTNV: Hantan virus; IAV: influenza A virus; IFN: interferon; LAMP1: lysosomal associated membraneprotein 1; LIR: LC3-interacting region; MAP1LC3B/LC3B: microtubule associatedprotein 1 light chain 3 beta; MAVS: mitochondrial antiviral signaling protein; Mdivi-1: mitochondrial division inhibitor 1; MOI: multiplicity of infection; MT-CO2/COXII: mitochondrially encoded cytochrome C oxidase II; NP: nucleoprotein; NSs: nonstructural proteins; poly(I:C): polyinosinic:polycytidylic acid; RIGI: RNA sensor RIG-I; RLR: RIGI-like receptor; SFTSV: severe fever withthrombocytopenia syndrome virus; TCID50: 50% tissue culture infectiousdose; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20:translocase of outer mitochondrial membrane 20; TUFM: Tu translation elongationfactor, mitochondrial. [ABSTRACT FROM AUTHOR]

The relationship between pangolin-CoV and SARS-CoV-2 has been a subject of debate. Further evidence of a special relationship between the two viruses can be found by the fact that all known COVID-19 viruses have an abnormally hard outer shell (low M disorder, i.e., low content of intrinsically disordered residues in the membrane (M) protein) that so far has been found in CoVs associated with burrowing animals, such as rabbits and pangolins, in which transmission involves virus remaining in buried feces for a long time. While a hard outer shell is necessary for viral survival, a harder inner shell could also help. For this reason, the N disorder range of pangolin-CoVs, not bat-CoVs, more closely matches that of SARS-CoV-2, especially when Omicron is included. The low N disorder (i.e., low content of intrinsically disordered residues in the nucleocapsid (N) protein), first observed in pangolin-CoV-2017 and later in Omicron, is associated with attenuation according to the Shell-Disorder Model. Our experimental study revealed that pangolin-CoV-2017 and SARS-CoV-2 Omicron (XBB.1.16 subvariant) show similar attenuations with respect to viral growth and plaque formation. Subtle differences have been observed that are consistent with disorder-centric computational analysis. [ABSTRACT FROM AUTHOR]

Complement activation is considered to contribute to the pathogenesis of severe SARS-CoV-2 infection, mainly by generating potent immune effector mechanisms including a strong inflammatory response. Involvement of the lectin complement pathway, a major actor of the innate immune anti-viral defense, has been reported previously. It is initiated by recognition of the viral surface Spike glycoprotein by mannose-binding lectin (MBL), which induces activation of the MBL-associated protease MASP-2 and triggers the proteolytic complement cascade. A role for the viral nucleoprotein (N) has also been reported, through binding to MASP-2, leading to protease overactivation and potentiation of the lectin pathway. In the present study, we reinvestigated the interactions of the SARS-CoV-2 N protein, produced either in bacteria or secreted by mammalian cells, with full-length MASP-2 or its catalytic domain, in either active or proenzyme form. We could not confirm the interaction of the N protein with the catalytic domain of MASP-2 but observed N protein binding to proenzyme MASP-2. We did not find a role of the N protein in MBL-mediated activation of the lectin pathway. Finally, we showed that incubation of the N protein with MASP-2 results in proteolysis of the viral protein, an observation that requires further investigation to understand a potential functional significance in infected patients. [ABSTRACT FROM AUTHOR]

The nucleoprotein (NP) of type A influenza virus (IAV) is highly conserved across all virus strains, making it an attractive candidate antigen for universal vaccines. While various studies have explored NP-induced mucosal immunity, here we interrogated the mechanistic differences between intramuscular (IM) and intranasal (IN) delivery of a recombinant adenovirus carrying NP fused with a bifunctional CD40 ligand. Despite being less effective than IM delivery in inducing systemic cellular immune responses and antibody-dependent cellular cytotoxicity (ADCC), IN immunization elicited superior antigen-specific recall humoral and cellular response in the nasal associated lymphoid tissue (NALT) of the upper respiratory tract, the initial site of immune recognition and elimination of inhaled pathogens. IN vaccination also induced significantly stronger pulmonary T cell responses in the lower respiratory tract than IM vaccination, in particular the CD8 T cells. Moreover, blocking lymphocyte circulation abrogated IM but not IN immunization induced protection, illustrating the critical role of local memory immune response upon viral infection. Notably, the CD40-targeted nasal delivery not only improved the magnitude but also the breadth of protection, including against lethal challenge with a newly isolated highly pathogenic avian H5N1 strain. These findings are informative for the design of universal mucosal vaccines, where the predominant mode of protection is independent of neutralizing antibodies.

BackgroundSnakehead vesiculovirus (SHVV) has led to huge economic losses in snakehead aquaculture, and its pathogenic mechanisms is still not fully understood. MicroRNAs (miRNAs), as an important class of non-coding RNAs, play a key regulatory role in the process of viral infection.MethodsWe examined the effect of SHVV infection on the expression of miR-130c-5p and the effect of overexpression of miR-130c-5p on the proliferation of SHVV. Cotransfection of viral N protein and miR-130c-5p, and the effect of miR-130c-5p on the expression of N protein was detected. Meanwhile, the effect of overexpression of miR-130c-5p on the expression of various immune factors in the case of viral infection were also tested.ResultsIn this study, SHVV infection significantly upregulated the expression of miR-130c-5p in channel catfish ovary (CCO) cells in a time- and dose-dependent manner. The further research revealed that miR-130c-5p mimic significantly inhibited, while its inhibitors promoted SHVV replication. In addition, miR-130c-5p could directly target the viral mRNA of n gene, and overexpression of miR-130c-5p could significantly decrease, and conversely, downregulation of miR-130c-5p could increase the mRNA and protein expression of the viral n gene. Meanwhile, overexpression of miR-130c-5p also upregulated the expression of immune-related genes, such as nucleotide-oligomerization domain (NOD)-like receptor subfamily C3 (NLRC3), myeloid differentiation factor 88 (MyD88), nuclear factor kappa-B (NF-κB), interleukin-6 (IL-6), interleukin-22 (IL-22), and interleukin-1beta (IL-1β) in host cells.ConclusionmiR-130c-5p was upregulated in the host during SHVV infection, and the upregulated miR-130c-5p directly inhibited viral replication by targeting the n gene of SHVV and promoting viral nucleoprotein degradation. The up-regulated miR-130c-5p also activated the expression of immune-related genes such as NLRC3, MyD88, NF-κB, IL-6, IL-22, and IL-1β, which were involved in the regulation of the signaling pathways including NF-κB, MyD88, Toll-like receptor (TLR), NLR, and janus tyrosine kinase-signal converter and activator of transcription (JAK-STAT), to enhance the host's antiviral immune response, and thus indirectly inhibited the viral proliferation.

Micropterus salmoides rhabdovirus (MSRV) is one of the most serious pathogens harming M. salmoides juvenile, which had brought huge economic losses to farming industry. Studies involving candidate genes to the clinical diseases, however, are limited. In this study, the viral target and clinical manifestation of MSRV on M. salmoides juvenile were analyzed, and the protective effects of a single immune enhancer and a compound immune enhancer were evaluated. The results showed that the brain, liver, intestine and muscle of M. salmoides showed obvious lesions after infection with MSRV. The relative expression levels of nucleoprotein (N) and matrix protein (M) genes showed a trend of increasing at first and then decreasing and reached the peak in each tissue at 36 h post-infection. The mortality rate of M. salmoides was over 90% after 7 days of MSRV infection. The immune enhancers containing free nucleotides and Astragalus polysaccharide added to the diet effectively inhibited the replication of N and M genes in M. salmoides and increased the survival rate by 25% to 28%. This study provided basic data and theoretical reference for the analysis of the pathological mechanism and prevention and treatment of MSRV.

ABSTRACT The heterogeneous nuclear ribonucleoprotein (hnRNP A2B1) is a key component of the hnRNP complex involving RNA modulation in eukaryotic cells and it has also been reported to be involved in the replication of the hepatitis E virus, influenza A virus, and hepatitis B virus. However, it is not clear whether the role of the hnRNP A2B1 in viral replication is conserved among RNA viruses and what is the mechanism of hnRNP A2B1 in RNA virus replication. In this study, we first used severe fever with thrombocytopenia syndrome virus (SFTSV), a tick-borne RNA virus that causes a severe viral hemorrhagic fever as well as other RNA viruses including VSV-GFP, SeV, EV71, and ZIKV to demonstrate that knockout hnRNPA2B1 gene inhibited viral RNA replication and overexpression of hnRNP A2B1 could restore the RNA levels of all tested RNA viruses. These results suggest that hnRNPA2B1 upregulation of viral replication is conserved among RNA viruses. Next, we demonstrated that hnRNP A2B1 was translocated from the nucleus to the cytoplasm under RNA virus infection including SFTSV, VSV-GFP, SeV, EV71, and ZIKV, suggesting translocation of hnRNP A2B1 from the nucleus to the cytoplasm is crucial for RNA virus replication. We then used SFTSV as a model to demonstrate the mechanism of hnRNP A2B1 in the promotion of RNA virus replication. We found that overexpression of SFTSV nucleoprotein can also cause hnRNP A2B1 translocation from the nucleus to the cytoplasm and that the SFTSV NP interacted with the RNA recognition motif 1 domain of hnRNP A2B1. We further demonstrated that the hnRNP A2B1 interacted with the 5′ UTR of SFTSV RNA. In conclusion, we revealed that the hnRNP A2B1 upregulation of viral RNA replication is conserved among RNA viruses; the mechanism of hnRNP A2B1 in promotion of SFTSV viral RNA replication is that SFTSV NP interacted with the hnRNPA2B1 to retain it in the cytoplasm where the hnRNP A2B1 interacted with the 5′ UTR of SFTSV RNA to promote the viral RNA replication.IMPORTANCESevere fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne RNA virus with a high mortality rate of up to 30%. In this study, we first used SFTSV as a model to demonstrate that the role of hnRNPA2B1 in viral replication is conserved in SFTSV. Then we used other RNA viruses, including VSV-GFP, SeV, EV71, and ZIKV, to repeat the experiment and demonstrated the same results as SFTSV in all tested RNA viruses. By knocking out the hnRNPA2B1 gene, SFTSV RNA replication was inhibited, and overexpression of hnRNPA2B1 restored RNA levels of SFTSV and other tested RNA viruses. We revealed a novel mechanism where the SFTSV nucleoprotein interacts with hnRNPA2B1, retaining it in the cytoplasm. This interaction promotes viral RNA replication by binding to the 5′ UTR of SFTSV RNA. The findings suggest that targeting hnRNPA2B1 could be a potential strategy for developing broad-spectrum antiviral therapies, given its conserved role across different RNA viruses. This research provides significant insights into the replication mechanisms of RNA viruses and highlights potential targets for antiviral interventions.

Objective To construct a universal influenza mRNA vaccine and evaluate its immunogenicity. Methods The antigen sequence of hemagglutinin (HA), nucleoprotein (NP) and matrix protein 2 ectodomain (M2e) in influenza A/California/04/2009 was optimized.HA, NP and 3 tandem M2e (3M2e) were cloned into pcDNA3.1 vector, respectively.Then the mRNAs were synthesized by linearization, in vitro transcription, enzymatic capping and enzymatic tailing, and named as mRNA-HA, mRNA-NP and mRNA-3M2e, respectively.The protein expression of the 3 kinds of mRNAs in 293T cells was detected by immunofluorescence assay.Comb-mRNA vaccine was prepared by enveloped mRNA-HA, mRNA-NP and mRNA-3M2e with lipid nanoparticles, respectively, and the particle size and potential were identified.Twenty-eight 6-week-old female BALB/c mice (18~22 g) were randomly divided into LNP group (n=14) and Comb-mRNA group (n=14).Hemagglutination inhibition (HI) method and microneutralization (MN) test were used to evaluate the serum antibody titer induced by Comb-mRNA vaccines.The mice were infected by 5LD50 wild-type H1N1 influenza virus to evaluate the protective efficacy. Results The mRNA-HA, mRNA-NP and mRNA-3M2e were successfully constructed, and the 3 mRNAs could be expressed in 293T cells.The average size of mRNA encapsulated by lipid nanoparticles was 119.53±6.5 nm, and the average potential was-8.23±1.3 mV.The geometric mean titer (GMT) of HI and MN in the Comb-mRNA group were 179.6 and 201.6, compared with the LNP group.The ratio of IFN-γ+CD4+/CD8+T cells was increased.The Comb-mRNA group could provide protection against 5LD50 wild type influenza H1N1 virus after 2 weeks of booster immunization. Conclusion Comb-mRNA, an influenza vaccine candidate, can induce immune responses and protect mice from influenza virus challenge.

The Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne bunyavirus that causes high mortality in humans. This enveloped virus harbors two surface glycoproteins (GP), Gn and Gc, that are released by processing of a glycoprotein precursor complex whose maturation takes place in the ER and is completed through the secretion pathway. Here, we characterized the trafficking network exploited by CCHFV GPs during viral assembly, envelopment, and/or egress. We identified membrane trafficking motifs in the cytoplasmic domains (CD) of CCHFV GPs and addressed how they impact these late stages of the viral life cycle using infection and biochemical assays, and confocal microscopy in virus-producing cells. We found that several of the identified CD motifs modulate GP transport through the retrograde trafficking network, impacting envelopment and secretion of infectious particles. Finally, we identified PACS-2 as a crucial host factor contributing to CCHFV GPs trafficking required for assembly and release of viral particles.

• Influenza NP, HA, and M2e show potential for a universal vaccine, with anticipated synergistic effects. • Nasal mucosal delivery overcomes pre-existing immunity against adenovirus-based vaccines. • Combined administration of NP and HA-M2e viral vectors exhibits dose-sparing effect and safety in preclinical studies. It is necessary to develop universal vaccines that act broadly and continuously to combat regular seasonal epidemics of influenza and rare pandemics. The aim of this study was to find the optimal dose regimen for the efficacy and safety of a mixture of previously developed recombinant adenovirus-based vaccines that expressed influenza nucleoprotein, hemagglutinin, and ectodomain of matrix protein 2 (rAd/NP and rAd/HA-M2e). The vaccine efficacy and safety were measured in the immunized mice with the mixture of rAd/NP and rAd/HA-M2e intranasally or intramuscularly. The minimum dose that would be efficacious in a single intranasal administration of the vaccine mixture and cross-protective efficacy against various influenza strains were examined. In addition, the immune responses that may affect the cross-protective efficacy were measured. We found that intranasal administration is an optimal route for 107 pfu of vaccine mixture, which is effective against pre-existing immunity against adenovirus. In a study to find the minimum dose with vaccine efficacy, the 106 pfu of vaccine mixture showed higher antibody titers to the nucleoprotein than did the same dose of rAd/NP alone in the serum of immunized mice. The 106 pfu of vaccine mixture overcame the morbidity and mortality of mice against the lethal dose of pH1N1, H3N2, and H5N1 influenza infections. No noticeable side effects were observed in single and repeated toxicity studies. We found that the mucosal administration of adenovirus-based universal influenza vaccine has both efficacy and safety, and can provide cross-protection against various influenza infections even at doses lower than those previously known to be effective. [ABSTRACT FROM AUTHOR]

Influenza A viruses (IAVs) possess a segmented genome consisting of eight viral RNAs (vRNAs) associated with multiple copies of viral nucleoprotein (NP) and a viral polymerase complex. Despite the crucial role of RNA structure in IAV replication, the impact of NP binding on vRNA structure is not well understood. In this study, we employed SHAPE chemical probing to compare the structure of NS and M vRNAs of WSN IAV in various states: before the addition of NP, in complex with NP, and after the removal of NP. Comparison of the RNA structures before the addition of NP and after its removal reveals that NP, while introducing limited changes, remodels local structures in both vRNAs and long-range interactions in the NS vRNA, suggesting a potentially biologically relevant RNA chaperone activity. In contrast, NP significantly alters the structure of vRNAs in vRNA/NP complexes, though incorporating experimental data into RNA secondary structure prediction proved challenging. Finally, our results suggest that NP not only binds single-stranded RNA but also helices with interruptions, such as bulges or small internal loops, with a preference for G-poor and C/U-rich regions. [ABSTRACT FROM AUTHOR]

The increased detection of H3 C-IVA (1990.4.a) clade influenza A viruses (IAVs) in US swine in 2019 was associated with a reassortment event to acquire an H1N1pdm09 lineage nucleoprotein (pdmNP) gene, replacing a TRIG lineage NP (trigNP). We hypothesized that acquiring the pdmNP conferred a selective advantage over prior circulating H3 viruses with a trigNP. To investigate the role of NP reassortment in transmission, we identified two contemporary 1990.4.a representative strains (NC/19 and MN/18) with different evolutionary origins of the NP gene. A reverse genetics system was used to generate wild-type (wt) strains and swap the pdm and TRIG lineage NP genes, generating four viruses: wtNC/19-pdmNP, NC/19-trigNP, wtMN/18-trigNP, and MN/18-pdmNP. The pathogenicity and transmission of the four viruses were compared in pigs. All four viruses infected 10 primary pigs and transmitted to five indirect contact pigs per group. Pigs infected via contact with MN/18-pdmNP shed virus 2 days earlier than pigs infected with wtMN/18-trigNP. The inverse did not occur for wtNC/19-pdmNP and NC/19-trigNP. This suggests that pdmNP reassortment resulted in a combination of genes that improved transmission efficiency when paired with the 1990.4.a hemagglutinin (HA). This is likely a multigenic trait, as replacing the trigNP gene did not diminish the transmission of a wild-type IAV in swine. This study demonstrates how reassortment and evolutionary change of internal genes can result in more transmissible viruses that influence HA clade detection frequency. Thus, rapidly identifying novel reassortants paired with dominant hemagglutinin/neuraminidase may improve the prediction of strains to include in vaccines. [ABSTRACT FROM AUTHOR]

Complement activation is considered to contribute to the pathogenesis of severe SARS-CoV-2 infection, mainly by generating potent immune effector mechanisms including a strong inflammatory response. Involvement of the lectin complement pathway, a major actor of the innate immune anti-viral defense, has been reported previously. It is initiated by recognition of the viral surface Spike glycoprotein by mannose-binding lectin (MBL), which induces activation of the MBL-associated protease MASP-2 and triggers the proteolytic complement cascade. A role for the viral nucleoprotein (N) has also been reported, through binding to MASP-2, leading to protease overactivation and potentiation of the lectin pathway. In the present study, we reinvestigated the interactions of the SARS-CoV-2 N protein, produced either in bacteria or secreted by mammalian cells, with full-length MASP-2 or its catalytic domain, in either active or proenzyme form. We could not confirm the interaction of the N protein with the catalytic domain of MASP-2 but observed N protein binding to proenzyme MASP-2. We did not find a role of the N protein in MBL-mediated activation of the lectin pathway. Finally, we showed that incubation of the N protein with MASP-2 results in proteolysis of the viral protein, an observation that requires further investigation to understand a potential functional significance in infected patients.

ABSTRACT Cyclic GMP-AMP synthase (cGAS) is an important DNA pattern recognition receptor that senses double-stranded DNA derived from invading pathogens or self DNA in cytoplasm, leading to an antiviral interferon response. A tick-borne Bunyavirus, severe fever with thrombocytopenia syndrome virus (SFTSV), is an RNA virus that causes a severe emerging viral hemorrhagic fever in Asia with a high case fatality rate of up to 30%. However, it is unclear whether cGAS interacts with SFTSV infection. In this study, we found that SFTSV infection upregulated cGAS RNA transcription and protein expression, indicating that cGAS is an important innate immune response against SFTSV infection. The mechanism of cGAS recognizing SFTSV is by cGAS interacting with misplaced mitochondrial DNA in the cytoplasm. Depletion of mitochondrial DNA significantly inhibited cGAS activation under SFTSV infection. Strikingly, we found that SFTSV nucleoprotein (N) induced cGAS degradation in a dose-dependent manner. Mechanically, N interacted with the 161-382 domain of cGAS and linked the cGAS to LC3. The cGAS-N-LC3 trimer was targeted to N-induced autophagy, and the cGAS was degraded in autolysosome. Taken together, our study discovered a novel antagonistic mechanism of RNA viruses, SFTSV is able to suppress the cGAS-dependent antiviral innate immune responses through N-hijacking cGAS into N-induced autophagy. Our results indicated that SFTSV N is an important virulence factor of SFTSV in mediating host antiviral immune responses.IMPORTANCESevere fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne RNA virus that is widespread in East and Southeast Asian countries with a high fatality rate of up to 30%. Up to now, many cytoplasmic pattern recognition receptors, such as RIG-I, MDA5, and SAFA, have been reported to recognize SFTSV genomic RNA and trigger interferon-dependent antiviral responses. However, current knowledge is not clear whether SFTSV can be recognized by DNA sensor cyclic GMP-AMP synthase (cGAS). Our study demonstrated that cGAS could recognize SFTSV infection via ectopic mitochondrial DNA, and the activated cGAS-stimulator of interferon genes signaling pathway could significantly inhibit SFTSV replication. Importantly, we further uncovered a novel mechanism of SFTSV to inhibit innate immune responses by the degradation of cGAS. cGAS was degraded in N-induced autophagy. Collectively, this study illustrated a novel virulence factor of SFTSV to suppress innate immune responses through autophagy-dependent cGAS degradation.

The highly pathogenic arenavirus, Junín virus (JUNV), expresses three truncated alternative isoforms of its nucleoprotein (NP), i.e., NP53kD, NP47kD, and NP40kD. While both NP47kD and NP40kD have been previously shown to be products of caspase cleavage, here, we show that expression of the third isoform NP53kD is due to alternative in-frame translation from M80. Based on this information, we were able to generate recombinant JUNVs lacking each of these isoforms. Infection with these mutants revealed that, while all three isoforms contribute to the efficient control of caspase activation, NP40kD plays the predominant role. In contrast to full-length NP (i.e., NP65kD), which is localized to inclusion bodies, where viral RNA synthesis takes place, the loss of portions of the N-terminal coiled-coil region in these isoforms leads to a diffuse cytoplasmic distribution and a loss of function in viral RNA synthesis. Nonetheless, NP53kD, NP47kD, and NP40kD all retain robust interferon antagonistic and 3′−5′ exonuclease activities. We suggest that the altered localization of these NP isoforms allows them to be more efficiently targeted by activated caspases for cleavage as decoy substrates, and to be better positioned to degrade viral double-stranded (ds)RNA species that accumulate in the cytoplasm during virus infection and/or interact with cytosolic RNA sensors, thereby limiting dsRNA-mediated innate immune responses. Taken together, this work provides insight into the mechanism by which JUNV leverages apoptosis during infection to generate biologically distinct pools of NP and contributes to our understanding of the expression and biological relevance of alternative protein isoforms during virus infection. IMPORTANCE A limited coding capacity means that RNA viruses need strategies to diversify their proteome. The nucleoprotein (NP) of the highly pathogenic arenavirus Junín virus (JUNV) produces three N-terminally truncated isoforms: two (NP47kD and NP40kD) are known to be produced by caspase cleavage, while, here, we show that NP53kD is produced by alternative translation initiation. Recombinant JUNVs lacking individual NP isoforms revealed that all three isoforms contribute to inhibiting caspase activation during infection, but cleavage to generate NP40kD makes the biggest contribution. Importantly, all three isoforms retain their ability to digest double-stranded (ds)RNA and inhibit interferon promoter activation but have a diffuse cytoplasmic distribution. Given the cytoplasmic localization of both aberrant viral dsRNAs, as well as dsRNA sensors and many other cellular components of innate immune activation pathways, we suggest that the generation of NP isoforms not only contributes to evasion of apoptosis but also robust control of the antiviral response. [ABSTRACT FROM AUTHOR]

The nucleoprotein (NP) is a vital target for the heterosubtypic immunity of CD8+ cytotoxic T lymphocytes (CTLs) due to its conservation among influenza virus subtypes. To further enhance the T cell immunity of NP, autophagy-inducing peptide C5 (AIP-C5) from the CFP10 protein of Mycobacterium tuberculosis was used. Mice were immunized intranasally (i.n.) with human adenoviral vectors, HAd-C5-NP(H7N9) or HAd-NP(H7N9), expressing NP of an H7N9 influenza virus with or without the AIP-C5, respectively. Both vaccines developed similar levels of NP-specific systemic and mucosal antibody titers; however, there was a significantly higher number of NP-specific CD8 T cells secreting interferon-gamma (IFN-γ) in the HAd-C5-NP(H7N9) group than in the HAd-NP(H7N9) group. The HAd-C5-NP(H7N9) vaccine provided better protection following the challenge with A/Puerto Rico/8/1934(H1N1), A/Hong Kong/1/68(H3N2), A/chukkar/MN/14951-7/1998(H5N2), A/goose/Nebraska/17097/2011(H7N9), or A/Hong Kong/1073/1999(H9N2) influenza viruses compared to the HAd-NP(H7N9) group. The autophagy transcriptomic gene analysis of the HAd-C5-NP(H7N9) group revealed the upregulation of some genes involved in the positive regulation of the autophagy process. The results support further exploring the use of NP and AIP-C5 for developing a universal influenza vaccine for pandemic preparedness. [ABSTRACT FROM AUTHOR]

Australian bat lyssavirus (ABLV) shows similar clinical symptoms as rabies, but there are currently no protein structures available for ABLV proteins. In lyssaviruses, the interaction between nucleoprotein (N) and phosphoprotein (N) in the absence of RNA generates a complex (N0P) that is crucial for viral assembly, and understanding the interface between these two proteins has the potential to provide insight into a key feature: the viral lifecycle. In this study, we used recombinant chimeric protein expression and X-ray crystallography to determine the structure of ABLV nucleoprotein bound to residues 1–40 of its phosphoprotein chaperone. Comparison of our results with the recently generated structure of RABV CVS-11 N0P demonstrated a highly conserved interface in this complex. Because the N0P interface is conserved in the lyssaviruses of phylogroup I, it is an attractive therapeutic target for multiple rabies-causing viral species. [ABSTRACT FROM AUTHOR]

Simple Summary: Parrot bornavirus (PaBV) causes Proventricular Dilatation Disease (PDD), a fatal neurological disorder in Psittaciformes. Its diversity in South America is poorly known. We detected the first case of a genotype 2 (PaBV-2) infection in Cacatua galerita in Brazil, which presented severe neuropathies and PDD. Evolutionary analysis estimated a potential Brazilian (or South American) ancestor as the origin of this genotype and possibly other genotypes of the Orthobornavirus alphapsittaciforme species. Additionally, a progressive decline in the size of the effective population was observed, which could be related to various factors, such as biodiversity loss and ecosystem disruption. The emergence or reemergence of PaBVs related to ancestral strains in Brazil and South America is a matter of concern, as the spread of these viruses poses a risk to bird biodiversity and endangered species. These results underscore the need for surveillance studies in this region. Parrot bornavirus (PaBV) is an RNA virus that causes Proventricular Dilatation Disease (PDD), neurological disorders, and death in Psittaciformes. Its diversity in South America is poorly known. We examined a Cacatua galerita presenting neuropathies, PDD, and oculopathies as the main signs. We detected PaBV through reverse transcription polymerase chain reaction (RT-PCR) and partial sequencing of the nucleoprotein (N) and matrix (M) genes. Maximum likelihood and Bayesian phylogenetic inferences classified it as PaBV-2. The nucleotide identity of the sequenced strain ranged from 88.3% to 90.3% against genotype PaBV-2 and from 80.2% to 84.4% against other genotypes. Selective pressure analysis detected signs of episodic diversifying selection in both the N and M genes. No recombination events were detected. Phylodynamic analysis estimated the time to the most recent common ancestor (TMRCA) as the year 1758 for genotype PaBV-2 and the year 1049 for the Orthobornavirus alphapsittaciforme species. Substitution rates were estimated at 2.73 × 10−4 and 4.08 × 10−4 substitutions per year per site for N and M, respectively. The analysis of population dynamics showed a progressive decline in the effective population size during the last century. Timescale phylogeographic analysis revealed a potential South American ancestor as the origin of genotypes 1, 2, and 8. These results contribute to our knowledge of the evolutionary origin, diversity, and dynamics of PaBVs in South America and the world. Additionally, it highlights the importance of further studies in captive Psittaciformes and the potential impact on endangered wild birds. [ABSTRACT FROM AUTHOR]

Human respiratory syncytial virus (hRSV) is one of the major contagious viruses and causes complicated respiratory issues, especially in young children. The sensitive and fast detection of hRSV is critical for taking the most effective actions. In the present study, rabbit antibodies against the hRSV nucleoprotein (NP) were developed using phage display technology. A female rabbit was immunized with an hRSV strain A2 recombinant NP. A Fab library was built and sorted during two successive panning rounds for strain B and the A2 NP (recombinant preparations), respectively. The choice of candidates was performed using ELISA on the two NP strains. The obtained library was 3 × 106 cfu/mL, with an insertion rate of >95%. The two panning rounds permitted an enrichment factor of 100. ELISA screening allowed us to obtain 28 NP-specific Fab candidates. Among them, 10 retained candidates were reformatted into rabbit full IgG; thereafter, pairing tests on the recombinant strains and native lysate samples were performed. After the pairing tests on the recombinant strains, 53 pairs were identified. Eleven pairs were identified as being able to detect RSVs from native lysates. This work presents new high-potential monoclonal antibodies mAbs (mAbs), which would benefit from lateral flow testing data with patient materials. [ABSTRACT FROM AUTHOR]

The recent outbreaks of Marburg virus disease (MVD) in Guinea, Ghana, Equatorial Guinea, and Tanzania, none of which had reported previous outbreaks, imply increasing risks of spillover of the causative viruses, Marburg virus (MARV) and Ravn virus (RAVV), from their natural host animals. These outbreaks have emphasized the need for the development of rapid diagnostic tests for this disease. Using monoclonal antibodies specific to the viral nucleoprotein, we developed an immunochromatography (IC) assay for the rapid diagnosis of MVD. The IC assay was found to be capable of detecting approximately 102−4 50% tissue culture infectious dose (TCID50)/test of MARV and RAVV in the infected culture supernatants. We further confirmed that the IC assay could detect the MARV and RAVV antigens in the serum samples from experimentally infected nonhuman primates. These results indicate that the IC assay to detect MARV can be a useful tool for the rapid point-of-care diagnosis of MVD. [ABSTRACT FROM AUTHOR]

Annual vaccination is considered as the main preventive strategy against seasonal influenza. Due to the highly variable nature of major viral antigens, such as hemagglutinin (HA) and neuraminidase (NA), influenza vaccine strains should be regularly updated to antigenically match the circulating viruses. The influenza virus nucleoprotein (NP) is much more conserved than HA and NA, and thus seems to be a promising target for the design of improved influenza vaccines with broad cross-reactivity against antigenically diverse influenza viruses. Traditional subunit or recombinant protein influenza vaccines do not contain the NP antigen, whereas live-attenuated influenza vaccines (LAIVs) express the viral NP within infected cells, thus inducing strong NP-specific antibodies and T-cell responses. Many strategies have been explored to design broadly protective NP-based vaccines, mostly targeted at the T-cell mode of immunity. Although the NP is highly conserved, it still undergoes slow evolutionary changes due to selective immune pressure, meaning that the particular NP antigen selected for vaccine design may have a significant impact on the overall immunogenicity and efficacy of the vaccine candidate. In this review, we summarize existing data on the conservation of the influenza A viral nucleoprotein and review the results of preclinical and clinical trials of NP-targeting influenza vaccine prototypes, focusing on the ability of NP-specific immune responses to protect against diverse influenza viruses. [ABSTRACT FROM AUTHOR]

Abstract Background Severe fever with thrombocytopenia syndrome (SFTS) is an infectious disease caused by the Dabie bandavirus, [or SFTS virus (SFTSV)] that has become increasingly widespread since it was first reported in 2009. The SFTSV comprises three essential single-stranded RNA gene segments, with the S segment encoding the nucleocapsid (N) protein. Since the N protein is the most abundant and stable viral protein, it is a useful diagnostic marker of infection. Various SFTSV N-protein-based detection methods have been developed. However, given the limited research on antibodies of an SFTSV N-protein, here we report the characterization of the antibodies against SFTSV N protein especially their mapping results which is essential for more efficient and optimized detection of SFTSV. Methods To generate SFTSV-N-protein-specific monoclonal antibodies, recombinant full-length SFTSV N protein was expressed in E. coli, and the purified N protein was immunized to mice. The binding epitope positions of the antibodies generated were identified through binding-domain mapping. An antibody pair test using a lateral flow immunoassay (LFIA) was performed to identify effective diagnostic combinations of paired antibodies. Results Nine monoclonal antibodies specific for the SFTSV N protein were generated. Antibodies #3(B4E2) and #5(B4D9) were specific for sequential epitopes, while the remainder were specific for conformational epitopes. Antibody #4(C2G1) showed the highest affinity for the SFTSV N protein. The binding domain mapping results indicated the binding regions of the antibodies were divided into three groups. The antibody pair test demonstrated that #3(B4E2)/#4(C2G1) and #4(C2G1)/#5(B4D9) were effective antibody pairs for SFTSV diagnosis. Conclusions Effective virus detection requires at least two strong antibodies recognizing separate epitope binding sites of the virus antigen. Here, we generated SFTSV-N-protein-specific monoclonal antibodies and subsequently performed epitope mapping and an antibody pair test to enhance the diagnostic efficiency and accuracy of SFTSV. Confirmation of epitope mappings and their combination immune response to the N protein provide valuable information for effective detection of SFTSV as well as can respond actively to detect a variant SFTSV.

The relationship between pangolin-CoV and SARS-CoV-2 has been a subject of debate. Further evidence of a special relationship between the two viruses can be found by the fact that all known COVID-19 viruses have an abnormally hard outer shell (low M disorder, i.e., low content of intrinsically disordered residues in the membrane (M) protein) that so far has been found in CoVs associated with burrowing animals, such as rabbits and pangolins, in which transmission involves virus remaining in buried feces for a long time. While a hard outer shell is necessary for viral survival, a harder inner shell could also help. For this reason, the N disorder range of pangolin-CoVs, not bat-CoVs, more closely matches that of SARS-CoV-2, especially when Omicron is included. The low N disorder (i.e., low content of intrinsically disordered residues in the nucleocapsid (N) protein), first observed in pangolin-CoV-2017 and later in Omicron, is associated with attenuation according to the Shell-Disorder Model. Our experimental study revealed that pangolin-CoV-2017 and SARS-CoV-2 Omicron (XBB.1.16 subvariant) show similar attenuations with respect to viral growth and plaque formation. Subtle differences have been observed that are consistent with disorder-centric computational analysis.

ABSTRACT Filoviruses depend on the nucleoprotein (NP) to accomplish multiple functions during the viral life cycle. NP is the most abundantly expressed viral protein in infected cells and the main component of the viral nucleocapsid. It can be structurally divided into amino- and carboxy- terminal domains (NTD and CTD). The NTD can homo-oligomerize to interact and protect the (−) ssRNA genome, forming long helical structures. The flexible CTD is responsible for the binding of other nucleocapsid proteins and is involved in the formation of inclusion bodies (IBs)—the cytoplasmic sites of nucleocapsid formation and genome replication. The CTD ends in a ~100-residue globular tail. Měnglà virus (MLAV) is the only member of the new Dianlovirus genus within the Filoviridae family. Their differential characteristics and the possibility of becoming a threat for human health justify the interest in better understanding of its structure and function. In this work, we present the structure of the globular tail of the MLAV NP CTD, showing an overall conformation closely related to that previously reported for the equivalent NP region in MARV. Moreover, analyses of the CTD-CTD interactions in the crystal asymmetric unit revealed a higher-order helicoidal structure. Mutational studies underscore the crucial role of a number of residues, located at the CTD-CTD contact interface, for IB formation. Site-directed mutagenesis of amino acids L653 and F687, involved in the formation of these helicoidal assemblies, abrogate the growth of IBs when the full-length MLAV NP was ectopically expressed in cells. Our findings confirm the role of NP CTD in IB formation, also for MLAV, and focus the attention on particular residues as starting point for further analysis. IMPORTANCE Filoviruses are the causative agents of severe and often fatal hemorrhagic disease in humans. Měnglà virus (MLAV) is a recently reported filovirus, isolated from fruit bats that is capable to replicate in human cells, representing a potential risk for human health. An in-depth structural and functional knowledge of MLAV proteins is an essential step for antiviral research on this virus that can also be extended to other emerging filoviruses. In this study, we determined the first crystal structures of the C-terminal domain (CTD) of the MLAV nucleoprotein (NP), showing important similarities to the equivalent domain in MARV. The structural data also show that the NP CTD has the ability to form large helical oligomers that may participate in the control of cytoplasmic inclusion body formation during viral replication.

Swine H1N1/2009 influenza is a highly infectious respiratory disease in pigs, which poses a great threat to pig production and human health. In this study, we investigated the global expression profiling of swine-encoded genes in response to swine H1N1/2009 influenza A virus (SIV-H1N1/2009) in newborn pig trachea (NPTr) cells. In total, 166 genes were found to be differentially expressed (DE) according to the gene microarray. After analyzing the DE genes which might affect the SIV-H1N1/2009 replication, we focused on polo-like kinase 3 (PLK3). PLK3 is a member of the PLK family, which is a highly conserved serine/threonine kinase in eukaryotes and well known for its role in the regulation of cell cycle and cell division. We validated that the expression of PLK3 was upregulated after SIV-H1N1/2009 infection. Additionally, PLK3 was found to interact with viral nucleoprotein (NP), significantly increased NP phosphorylation and oligomerization, and promoted viral ribonucleoprotein assembly and replication. Furthermore, we identified serine 482 (S482) as the phosphorylated residue on NP by PLK3. The phosphorylation of S482 regulated NP oligomerization, viral polymerase activity and growth. Our findings provide further insights for understanding the replication of influenza A virus.

Bunyavirus is widely distributed, highly contagious, and has a high fatality rate. It is a negative- strand RNA virus that has a major impact on public health around the world. The development of vaccines and the search for drugs are the key to prevent bunyavirus infection. The nucleoprotein (NP) of viruses is necessary for the synthesis of viral RNA, which combines with viral RNA to form the nucleocapsid, participates in viral assembly and RNA transcription, and plays an important role in viral proliferation. In addition, NP also has B cell and T cell epitopes, which can induce cellular and humoral immunity, so NP is an ideal target for vaccine design and drug development. Given its abundance and specificity, NP is also commonly used in the detection of viral diseases. More and more bunyavirus NP structures and structures of NP-RNA complexes have been resolved. Researchers have discovered two important antiviral targets through these structures, the terminal arm and the RNA binding cleft. This paper reviews the function and three-dimensional structure of the bunyavirus NP and the research progress of NP as an antiviral target, in order to provide a theoretical basis for the prevention and treatment of the bunyavirus disease. [ABSTRACT FROM AUTHOR]

Improved broad-spectrum influenza virus vaccines are desperately needed to provide protection against both drifted seasonal and emerging pandemic influenza A viruses (IAVs). Antibody-based protection from influenza A virus-induced morbidity and mortality is traditionally associated with neutralizing antibodies. As such, vaccine efforts have solely focused on the hemagglutinin (HA) as a vaccine target; however, the HA is mutation prone resulting in the need for annual vaccine reformulation. Broad-spectrum vaccines could be achieved through non-neutralizing antibodies that target conserved influenza virus antigens. Here, we describe six human monoclonal antibodies (mAbs) isolated from two H3N2-infected donors that showed robust binding against the conserved internal nucleoprotein (NP) or matrix protein 1 (M1) of IAV strains. Despite the capacity for potent antigen binding, substantial morbidity was observed in mice prophylactically treated with these mAbs and then challenged with A/Netherlands/602/2009 (H1N1) or A/Switzerland/9715293/2013 (H3N2) viruses. While our findings need to be confirmed with a larger number of mAbs and with polyclonal serum, these findings suggest that human NP and M1 antibodies that are elicited following IAV infection/vaccination do not protect from substantial weight loss in the mouse model and imply that protection afforded targeting these antigens following vaccination/infection is most likely the result of cellular-based immunity. [ABSTRACT FROM AUTHOR]

Background: Severe fever with thrombocytopenia syndrome (SFTS) is an infectious disease caused by the Dabie bandavirus, [or SFTS virus (SFTSV)] that has become increasingly widespread since it was first reported in 2009. The SFTSV comprises three essential single-stranded RNA gene segments, with the S segment encoding the nucleocapsid (N) protein. Since the N protein is the most abundant and stable viral protein, it is a useful diagnostic marker of infection. Various SFTSV N-protein-based detection methods have been developed. However, given the limited research on antibodies of an SFTSV N-protein, here we report the characterization of the antibodies against SFTSV N protein especially their mapping results which is essential for more efficient and optimized detection of SFTSV. Methods: To generate SFTSV-N-protein-specific monoclonal antibodies, recombinant full-length SFTSV N protein was expressed in E. coli, and the purified N protein was immunized to mice. The binding epitope positions of the antibodies generated were identified through binding-domain mapping. An antibody pair test using a lateral flow immunoassay (LFIA) was performed to identify effective diagnostic combinations of paired antibodies. Results: Nine monoclonal antibodies specific for the SFTSV N protein were generated. Antibodies #3(B4E2) and #5(B4D9) were specific for sequential epitopes, while the remainder were specific for conformational epitopes. Antibody #4(C2G1) showed the highest affinity for the SFTSV N protein. The binding domain mapping results indicated the binding regions of the antibodies were divided into three groups. The antibody pair test demonstrated that #3(B4E2)/#4(C2G1) and #4(C2G1)/#5(B4D9) were effective antibody pairs for SFTSV diagnosis. Conclusions: Effective virus detection requires at least two strong antibodies recognizing separate epitope binding sites of the virus antigen. Here, we generated SFTSV-N-protein-specific monoclonal antibodies and subsequently performed epitope mapping and an antibody pair test to enhance the diagnostic efficiency and accuracy of SFTSV. Confirmation of epitope mappings and their combination immune response to the N protein provide valuable information for effective detection of SFTSV as well as can respond actively to detect a variant SFTSV. [ABSTRACT FROM AUTHOR]

Crimean-Congo hemorrhagic fever virus (CCHFV; family Nairoviridae) is a tick-borne pathogen that frequently causes lethal disease in humans. CCHFV has a wide geographic distribution, and cases have been reported in Africa, Asia, the Middle East, and Europe. Availability of a safe and efficacious vaccine is critical for restricting outbreaks and preventing disease in endemic countries. We previously developed a virus-like replicon particle (VRP) vaccine that provides complete protection against homologous and heterologous lethal CCHFV challenge in mice after a single dose. However, the immune responses induced by this vaccine are not well characterized, and correlates of protection remain unknown. Here we comprehensively characterized the kinetics of cell-mediated and humoral immune responses in VRP-vaccinated mice, and demonstrate that they predominantly target the nucleoprotein (NP). NP antibodies are not associated with protection through neutralizing activity, but VRP vaccination results in NP antibodies possessing Fc-mediated antibody effector functions, such as complement activation (ADCD) and antibody-mediated cellular phagocytosis (ADCP). This suggests that Fc-mediated effector functions may contribute to this vaccine'sefficacy. [ABSTRACT FROM AUTHOR]

Genetic toxin–antitoxin element hok/sok from the natural Escherichia coli R1 plasmid ensures segregational stability of plasmids. Bacterial cells that have lost all copies of the plasmid encoding the short-lived antitoxin are killed by the stable toxin. When introduced into bacterial expression vectors, the hok/sok element can increase the productive time of recombinant protein biosynthesis by slowing down accumulation of non-producing cells lacking the expression plasmid. In this work, we studied the effects of position and orientation of the hok/sok element in the standard pET28a plasmid with the inducible T7lac promoter and kanamycin resistance gene. It was found that the hok/sok element retained its functional activity regardless of its location and orientation in the plasmid. Bacterial cells retained the hok/sok-containing plasmids after four days of cultivation without antibiotics, while the control plasmid without this element was lost. Using three target proteins – E. coli type II asparaginase (ASN), human growth hormone (HGH), and SARS-CoV-2 virus nucleoprotein (NP) – it was demonstrated that the maximum productivity of bacteria for the cytoplasmic proteins (HGH and NP) was observed only when the hok/sok element was placed upstream of the target gene promoter. In the case of periplasmic protein localization (ASN), the productivity of bacteria during cultivation with the antibiotic decreased for all variants of the hok/sok location. When the bacteria were cultivated without the antibiotic, the productivity was better preserved when the hok/sok element was located upstream of the target gene promoter. The use of the pEHU vector with the upstream location of the hok/sok element allowed to more than double the yield of HGH (produced as inclusion bodies) in the absence of antibiotic and to maintain ASN biosynthesis at the level of at least 10 mg/liter for four days during cultivation without antibiotics. The developed segregation-stabilized plasmid vectors can be used to obtain various recombinant proteins in E. coli cells without the use of antibiotics. [ABSTRACT FROM AUTHOR]

Nairobi sheep disease (NSD), caused by the viral agent NSD virus (NSDV), is a haemorrhagic fever disease affecting and inducing high mortality in sheep and goat populations. NSDV belongs to the genus Orthonairovirus of the Nairoviridae family from the order Bunyavirales. Other viruses circulating in livestock such as Crimean–Congo haemorrhagic fever virus (CCHFV) and Dugbe virus (DUGV) are members of the same genus and are reported to share antigenic features. There are very few available materials to study NSDV infection both in vitro and in vivo. In the present work, we characterised two monoclonal antibodies generated in mice that recognise NSDV specifically but not CCHFV or DUGV, along with a potential use to define virus-infected cells, using flow cytometry. We believe this tool can be useful for research, but also NSDV diagnostics, especially through immunological staining. [ABSTRACT FROM AUTHOR]

The mammarenavirus Junín (JUNV) is the causative agent of Argentine hemorrhagic fever, a severe disease of public health concern. The most abundant viral protein is the nucleoprotein (NP), a multifunctional, two-domain protein with the primary role as structural component of the viral nucleocapsids, used as template for viral polymerase RNA synthesis activities. Here, we report that the C-terminal domain (CTD) of the attenuated Candid#1 strain of the JUNV NP can be purified as a stable soluble form with a secondary structure in line with known NP structures from other mammarenaviruses. We show that the JUNV NP CTD interacts with the viral matrix protein Z in vitro, and that the full-length NP and Z interact with each other in cellulo, suggesting that the NP CTD is responsible for this interaction. This domain comprises an arrangement of four acidic residues and a histidine residue conserved in the active site of exoribonucleases belonging to the DEDDh family. We show that the JUNV NP CTD displays metal-ion-dependent nuclease activity against DNA and single- and double-stranded RNA, and that this activity is impaired by the mutation of a catalytic residue within the DEDDh motif. These results further support this activity, not previously observed in the JUNV NP, which could impact the mechanism of the cellular immune response modulation of this important pathogen. [ABSTRACT FROM AUTHOR]

Background: Mumps virus (MuV) can trigger severe infections, such as parotitis, epididymo-orchitis, and meningitis. The effectiveness of MuV vaccine administration has been proven, but current outbreaks warrant the development of antivirals against MuV. Zingiber officinale var. Roscoe or ginger is often used as an alternative remedy. Currently, there are no known in vitro or in vivo studies that investigate ginger as an MuV antiviral. Purpose: This study aims to evaluate the antiviral potency of the bioactive compounds in Zingiber officinale var. Roscoe against MuV. Methods: Antiviral activity screening was conducted by druglikeness analysis, antiviral probability, molecular docking, and molecular dynamic simulation. Results: As an antiviral, 6-shogaol from Zingiber officinale var. Roscoe has potency against MuV. It has a good binding affinity and can establish interactions with the binding domain of the target protein by forming hydrogen, Van der Waals, and alkyl bonds. Conclusion: The complex of 6-shogaol_NP was predicted to be volatile but stable for triggering inhibitory activity. However, these results must be proved by in vivo and in vitro approaches to strengthen the scientific evidence.

Influenza A viruses (IAVs) possess a segmented genome consisting of eight viral RNAs (vRNAs) associated with multiple copies of viral nucleoprotein (NP) and a viral polymerase complex. Despite the crucial role of RNA structure in IAV replication, the impact of NP binding on vRNA structure is not well understood. In this study, we employed SHAPE chemical probing to compare the structure of NS and M vRNAs of WSN IAV in various states: before the addition of NP, in complex with NP, and after the removal of NP. Comparison of the RNA structures before the addition of NP and after its removal reveals that NP, while introducing limited changes, remodels local structures in both vRNAs and long-range interactions in the NS vRNA, suggesting a potentially biologically relevant RNA chaperone activity. In contrast, NP significantly alters the structure of vRNAs in vRNA/NP complexes, though incorporating experimental data into RNA secondary structure prediction proved challenging. Finally, our results suggest that NP not only binds single-stranded RNA but also helices with interruptions, such as bulges or small internal loops, with a preference for G-poor and C/U-rich regions.

Replication of the RNA genome of influenza A virus occurs in the nucleus of infected cells. The influenza nucleoprotein (NP) associated with the viral RNA into ribonucleoprotein complexes (vRNPs) is involved in the nuclear import of the viral genome. NP has two nuclear localization sequences (NLSs), NLS1 and NLS2. Most studies have concentrated on the role of NP's NLSs using in vitro-assembled or purified vRNPs, which may differ from incoming vRNPs released in the cytoplasm during an infection. Here, we study the contribution of the NP's NLSs to the nuclear import of vRNPs in a cell culture model system for influenza infection: human lung carcinoma cells infected with viruses containing NP-carrying mutations in NLS1 or NLS2 (NLS2MT), generated by reverse genetics. We found that cells infected with these mutant viruses were defective in the nuclear import of incoming vRNPs and produced reduced amounts of newly synthesized NP, newly assembled vRNP, and progeny virus. In addition, NLS2MT-infected cells were also defective in the nucleolar accumulation of NP, confirming the nucleolar localization role of NLS2. Our findings indicate that both NLS1 and NLS2 have to be present for successful infection and demonstrate the crucial role of these two NLSs in the infection cycle of the influenza A virus. [ABSTRACT FROM AUTHOR]

• A live rabies vaccine strain, ERA-NG2, with multiple mutations was established. • ERA-NG2 is highly and stably attenuated. • ERA-NG2 induces a neutralizing antibody response in mice and dogs. Live rabies vaccines have advantageous features that can facilitate mass vaccination for dogs, the most important reservoirs/transmitters of rabies. However, some live vaccine strains have problems in their safety, namely, risks from the residual pathogenicity and the pathogenic reversion of live vaccine strains. The reverse genetics system of rabies virus provides a feasible option to improve the safety of a live vaccine strain by, for example, artificially introducing attenuating mutations into multiple viral proteins. It was previously demonstrated in separate studies that introduction of amino acid residues Leu at position 333 in the viral glycoprotein (G333), Ser at G194, and Leu/His at positions 273/394 in the nucleoprotein (N273/394) enhance the safety of a live vaccine strain. In this study, to test our hypothesis that combinational introduction of these residues would significantly increase the safety level of a vaccine strain, we generated a novel live vaccine candidate, ERA-NG2, that is attenuated by mutations at N273/394 and G194/333, and we examined its safety and immunogenicity in mice and dogs. ERA-NG2 did not cause any clinical signs in mice after intracerebral inoculation. After 10 passages in suckling mouse brains, ERA-NG2 retained all of the introduced mutations except the mutation at N394 and the highly attenuated phenotype. These findings indicate that the ERA-NG2 is highly and stably attenuated. After confirming that ERA-NG2 induced a virus-neutralizing antibody (VNA) response and protective immunity in mice, we immunized dogs intramuscularly with a single dose (105-7 focus-forming units) of ERA-NG2 and found that, at all of the tested doses, the strain induced a VNA response in dogs without inducing any clinical signs. These findings demonstrate that ERA-NG2 has a high level of safety and a substantial level of immunogenicity in dogs and thus is a promising live vaccine candidate that can facilitate vaccination in dogs. [ABSTRACT FROM AUTHOR]

Influenza viruses pose a significant threat to human health worldwide due to seasonal epidemics and occasional global pandemics. These viruses can cause severe upper respiratory tract infections that contribute to high morbidity and mortality rates. The emergence of drug‐resistant influenza viruses has created the need for the development of novel broad‐spectrum antivirals. Here, we present a novel anti‐influenza agent with new targets and mechanisms of action to address this problem. Our findings led to the discovery of a novel influenza virus inhibitor, a ligustrazine derivative known as A9. We have found that it exhibits broad‐spectrum antiviral properties against influenza A and B viruses (IAV and IBV, respectively), including oseltamivir‐resistant strain. Through multiple bioassays such as time‐of‐addition assay, indirect immunofluorescence assay, and nuclear‐cytoplasmic fractionation assay, we demonstrated that A9 inhibits the nuclear export of the viral ribonucleoprotein (vRNP). Furthermore, escape mutant analyses and affinity studies determined by surface plasmon resonance indicated that A9 specifically targets the nucleoprotein. In addition, four chalcone derivatives developed from A9 (B14, B29, B31, and B32), were found to effectively inhibit the replication of influenza virus through the same mechanism of action. In this manuscript we highlight A9 and its four derivatives as potential leads for the treatment of IAV and IBV infections, and their unique and novel mechanism of action probable benefit the field of anti‐influenza drug discovery. [ABSTRACT FROM AUTHOR]

Mitophagy is a form of autophagy that plays a key role in maintaining the homeostasis of functional mitochondria in the cell. Viruses have evolved various strategies to manipulate mitophagy to escape host immune responses and promote virus replication. In this study, the nucleoprotein (NP) of H1N1 virus (PR8 strain) was identified as a regulator of mitophagy. We revealed that NP-mediated mitophagy leads to the degradation of the mitochondria-anchored protein MAVS, thereby blocking MAVS-mediated antiviral signaling and promoting virus replication. The NP-mediated mitophagy is dependent on the interaction of NP with MAVS and the cargo receptor TOLLIP. Moreover, Y313 of NP is a key residue for the MAVS-NP interaction and NP-mediated mitophagy. The NPY313F mutation significantly attenuates the virus-induced mitophagy and the virus replication in vitro and in vivo. Taken together, our findings uncover a novel mechanism by which the NP of influenza virus induces mitophagy to attenuate innate immunity. Abbreviations: ACTB: actin beta; ATG7: autophagy related 7; ATG12: autophagy related 12; CCCP: carbonyl cyanide 3-chlorophenyl hydrazone; co-IP: co-immunoprecipitation; COX4/COXIV: cytochrome c oxidase subunit 4; DAPI: 4ʹ,6-diamidino-2-phenylindole, dihydrochloride; EID50: 50% egg infective dose; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HEK: human embryonic kidney; hpi: hours post-infection; IAV: influenza A virus; IFN: interferon; IP: immunoprecipitation; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; Mdivi-1: mitochondrial division inhibitor 1; MLD50: 50% mouse lethal dose; MOI: multiplicity of infection; NBR1: NBR1 autophagy cargo receptor; NP: nucleoprotein; PB1: basic polymerase 1; RFP: red fluorescent protein; RIGI: RNA sensor RIG-I; RIGI-N: RIGI-CARD; SeV: Sendai virus; SQSTM1/p62: sequestosome 1; TIMM23: translocase of inner mitochondrial membrane 23; TOLLIP: toll interacting protein; TOMM20: translocase of outer mitochondrial membrane 20; TUBA: tubulin alpha; Vec: empty vector; vRNP: viral ribonucleoprotein. [ABSTRACT FROM AUTHOR]

The spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for target recognition, cellular entry, and endosomal escape of the virus. At the same time, it is the part of the virus that exhibits the greatest sequence variation across the many variants which have emerged during its evolution. Recent studies have indicated that with progressive lineage emergence, the positive charge on the spike protein has been increasing, with certain positively charged amino acids (AAs) improving the binding of the spike protein to cell receptors. We have performed a detailed analysis of dissociable AAs of more than 1400 different SARS-CoV-2 lineages, which confirms these observations while suggesting that this progression has reached a plateau with Omicron and its subvariants and that the positive charge is not increasing further. Analysis of the nucleocapsid protein shows no similar increase in positive charge with novel variants, which further indicates that positive charge of the spike protein is being evolutionarily selected for. Furthermore, comparison with the spike proteins of known coronaviruses shows that already the wild-type SARS-CoV-2 spike protein carries an unusually large amount of positively charged AAs when compared to most other betacoronaviruses. Our study sheds light on the evolutionary changes in the number of dissociable AAs on the spike protein of SARS-CoV-2, complementing existing studies and providing a stepping stone towards a better understanding of the relationship between the spike protein charge and viral infectivity and transmissibility. [ABSTRACT FROM AUTHOR]

Influenza, a respiratory disease mainly caused by influenza A and B, viruses of the Orthomyxoviridae, is still a burden on our society's health and economic system. Influenza A viruses (IAV) circulate in mammalian and avian populations, causing seasonal outbreaks with high numbers of cases. Due to the high variability in seasonal IAV triggered by antigenic drift, annual vaccination is necessary, highlighting the need for a more broadly protective vaccine against IAV. The safety tested Modified Vaccinia virus Ankara (MVA) is licensed as a third-generation vaccine against smallpox and serves as a potent vector system for the development of new candidate vaccines against different pathogens. Here, we generated and characterized recombinant MVA candidate vaccines that deliver the highly conserved internal nucleoprotein (NP) of IAV under the transcriptional control of five newly designed chimeric poxviral promoters to further increase the immunogenic properties of the recombinant viruses (MVA-NP). Infections of avian cell cultures with the recombinant MVA-NPs demonstrated efficient synthesis of the IAV-NP which was expressed under the control of the five new promoters. Prime-boost or single shot immunizations in C57BL/6 mice readily induced circulating serum antibodies' binding to recombinant IAV-NP and the robust activation of IAV-NP-specific CD8+ T cell responses. Moreover, the MVA-NP candidate vaccines protected C57BL/6 mice against lethal respiratory infection with mouse-adapted IAV (A/Puerto Rico/8/1934/H1N1). Thus, further studies are warranted to evaluate the immunogenicity and efficacy of these recombinant MVA-NP vaccines in other IAV challenge models in more detail. [ABSTRACT FROM AUTHOR]

Background: Tight junctions act as a barrier that prevents invasion of pathogens through epithelial cells. This study aims to elucidate the correlation between tight junctions and nairoviruses using Hazara orthonairovirus (HAZV) as a surrogate model for Crimean-Congo hemorrhagic fever virus. Methods: mRNA, total protein, and cell surface protein levels of tight junction proteins were examined by quantitative real-time reverse transcription polymerase chain reaction, immunoblot and flow cytometry, respectively. HAZV growth was measured by plaque assay. Immunofluorescence assay was used to examine viral cell-to-cell spread. The interaction between HAZV nucleoprotein and claudin-1 was analyzed by immunoprecipitation. Results: HAZV infection induced mRNA of several tight junction proteins, especially claudin-1. HAZV infection also induced cell surface expression of claudin-1 protein. Claudin-1 overexpression inhibited the growth of HAZV by blocking its cell-to-cell spread. In contrast, HAZV nucleoprotein completely inhibited HAZV-induced cell surface expression of claudin-1, and this inhibition required interaction between HAZV nucleoprotein and claudin-1. Conclusion: HAZV nucleoprotein was shown to bind to claudin-1 to negatively regulate its cell surface expression, and so can promote cell-to-cell spread of HAZV. This is the first presentation of a possible mechanism behind how nairoviruses counteract tight junction barrier function. [ABSTRACT FROM AUTHOR]