Introduction: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a betacoronavirus that emerged in late 2019, is the causative agent of Coronavirus Disease 2019 (COVID19). The viral spike (S) and Nucleocapsid Protein (NP) are key structural components and principal targets for antigen detection. Developing reliable in-house immunoassays targeting these proteins is essential to enhance laboratory capacity and reduce dependence on commercial diagnostic kits. Aim: To analytically develop and optimise semi-in-house direct and sandwich Enzyme Linked Immuno Sorbent Assay (ELISA) for individual and simultaneous detection of SARS-CoV-2 spike (S) and NP antigens. Materials and Methods: This was an analytical laboratorybased study conducted between April 2025 and October 2025 at King Fahd Medical Research Centre, King Abdulaziz University, Jeddah, Saudi Arabia. For direct ELISAs, assay conditions were optimised using checkerboard titration. Recombinant SARSCoV-2 S and/or NP antigens were used as positive controls, while MERS-CoV S and/or NP antigens were included to evaluate potential cross-reactivity. Horseradish Peroxidase (HRP)-conjugated monoclonal antibodies specific to SARSCoV-2 S and/or NP served as detection antibodies. Optimal antibody concentrations were defined as those producing the highest positive-to-negative Optical Density (OD) ratio, including blank controls. For sandwich ELISAs, plates were coated with monoclonal anti-SARS-CoV-2 antibodies targeting S/RBD and/ or NP as capture antibodies. Subsequent steps followed the optimised protocol established for the direct ELISA format. Analytical performance was evaluated by determining the assay cut-off value, Limit Of Detection (LOD), linear dynamic range, and intra- and inter-assay Coefficients of Variation (CV%). Cross-reactivity was assessed using MERS-CoV recombinant proteins and heat-inactivated virus preparations. Results: Direct ELISAs detected S antigen at ≥6.25 ng and NP antigen at ≥3.125 ng per well whereas sandwich ELISAs improved analytical sensitivity four-fold (LOD 0.78 ng). Detection using heat-inactivated virus demonstrated analytical sensitivity down to 0.049×105 TCID50/mL. Intra-assay CV ranged from 3.1- 6.1% and inter-assay CV from 6.2-9.2%. No cross-reactivity with MERS-CoV was observed under tested conditions. Conclusion: The developed semi-in-house ELISAs demonstrated acceptable analytical sensitivity and reproducibility for research applications. Clinical validation using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)-confirmed patient samples is required before diagnostic implementation.

PEDV nucleocapsid (N) protein induces liquid-liquid phase separation (LLPS) to selectively aggregate the viral genome.

Mice are valuable small animal models for studying SARS-CoV-2 pathogenesis. Ancestral SARS-CoV-2 strains do not efficiently utilize murine Ace2, rendering wild-type mice resistant to infection. Although human ACE2 transgenic models such as K18-hACE2 have provided critical insights, they express multiple copies of both murine and human ACE2, and random transgene insertion can result in non-physiological receptor expression. To overcome these limitations, we employed a human ACE2 knock-in (hACE2-KI) model in which the murine Ace2 coding sequence is replaced with human ACE2 using CRISPR/Cas9 technology, generating an mAce2-null background. This design allows human ACE2 expression under endogenous regulatory control while eliminating murine Ace2 expression, thereby providing a more physiologically relevant platform to investigate SARS-CoV-2 pathogenesis and evaluate therapeutic and preventive strategies. In this study, SARS-CoV-2-associated disease was evaluated and compared among hACE2-KI, K18-hACE2 and C57BL/6J mice. Mice were intranasally inoculated with 105 plaque-forming units of SARS-CoV-2 lineages B.1 or B.1.351. Both hACE2-KI and K18-hACE2 mice developed severe disease after SARS-CoV-2 infection. Following infection with B.1, both K18-hACE2 mice and hACE2-KI mice exhibited significant weight loss and mortality, with high viral loads detected in the lungs and brain. hACE2-KI mice infected with SARS-CoV-2 B.1.351 also showed significant weight loss and viral loads, resulting in high mortality. The pathology and inflammatory response within the lungs and brain of infected hACE2-KI mice revealed robust expression of viral nucleocapsid protein, histopathological changes, and upregulated cytokine and chemokine responses. Together, these findings demonstrate that the hACE2-KI knock-in mouse model supports robust SARS-CoV-2 replication and mimics severe COVID-19 disease.

The neurotropic betacoronavirus porcine hemagglutinating encephalomyelitis virus (PHEV) subverts early innate defenses to establish persistent neuronal infection. We show that PHEV activates RIG-I-MAVS signaling but hijacks this pathway to induce a delayed IRF7-dependent interferon (IFN-I) response (>12 h post-infection), permitting unchecked replication prior to late-phase immunity. Mechanistically, the viral nucleocapsid (N) protein directly engages RIG-I's caspase activation and recruitment domain (CARD) via its C-terminal domain (CTD), competitively blocking TRIM25-mediated K63-linked ubiquitination and silencing RIG-I activation. Concurrently, N protein disrupts IRF3 activation by disrupting homodimerization, phosphorylation, and nuclear translocation, abrogating its function as the dominant early antiviral mediator. Consequently, inadequate IRF7-driven IFN induction (<3-fold at mRNA level) fails to compensate for IRF3 inactivation, creating an immune-permissive window. Pharmacological blockade of replication (Remdesivir or Lopinavir) abolished RIG-I-IRF7 activation and IFN induction, confirming replication-derived dsRNA as the essential immune trigger. Thus, PHEV deploys its N protein to simultaneously sabotage RIG-I sensing and IRF3 effector functions, enabling covert immune evasion critical for neurotropic pathogenesis.IMPORTANCEPorcine hemagglutinating encephalomyelitis virus (PHEV) causes lethal encephalomyelitis in piglets by exploiting neuronal immune vulnerabilities. We reveal that PHEV nucleocapsid (N) protein directly binds RIG-I to block its antiviral activation signal (K63-ubiquitination) and concurrently disabling IRF3-the master regulator of early interferon defense. This unique strategy, distinct from nonstructural protein-mediated evasion in other coronaviruses, allows unchecked viral replication during critical early infection. Our work identifies the N protein as a central immunosuppressor evolved for neurotropism and exposes the RIG-I-IRF3 interface as a druggable target. These findings provide a blueprint for countermeasures against PHEV and related neuroinvasive coronaviruses threatening human and animal health.

Autophagy is involved in various stages of the viral life cycle and modulates viral replication. Coronaviruses have developed several strategies to exploit autophagy for their benefit. Nevertheless, the exact mechanisms through which the infectious bronchitis virus (IBV) influences autophagy remain inadequately understood. Here, we demonstrate that IBV infection of chicken embryonic kidney (CEK) cells activates the AKT-mTOR signaling pathway to suppress autophagosome formation and mitophagy. Further investigation reveals that the viral spike protein (S) inhibits cellular autophagy by interacting with the mitophagy receptor FUNDC1. However, FUNDC1-mediated mitophagy promotes degradation of the viral nucleocapsid (N) protein and restricts IBV replication. To counteract this host defense mechanism, the S protein competitively binds to the LC3-interacting region (LIR) motif of FUNDC1, thereby disrupting its interaction with LC3 and ultimately suppressing mitophagy. Molecular docking analysis revealed that a conserved asparagine residue at position 240 (N240) in the S1 subunit of the IBV S protein is essential for binding to FUNDC1. Furthermore, reverse genetics demonstrated that an IBV mutant with an N240A substitution exhibited reduced pathogenicity in the kidneys, trachea, and lungs of specific-pathogen-free (SPF) chickens compared to the wild-type virus. Collectively, these findings unveil a novel mechanism by which IBV antagonizes host mitophagy and provide new insights into the host-virus interplay within the context of autophagic regulation.IMPORTANCEIBV has evolved a mechanism to counteract the host's antiviral defense. Specifically, the viral spike (S) protein blocks a form of autophagy called mitophagy by binding to the mitochondrial receptor FUNDC1. Normally, FUNDC1 helps cells eliminate damaged mitochondria and restricts IBV replication by promoting the degradation of the viral nucleocapsid protein. By interfering with this process, the S protein enhances viral survival. We further identified a single conserved amino acid in the S protein that is critical for this function, and mutation of this residue weakened IBV in chickens. These findings reveal how IBV manipulates host defenses and suggest new strategies for controlling coronavirus infections.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a persistent threat to the global swine industry, owing to its high genetic variability and immune evasion capabilities. However, the mechanisms by which PRRSV manipulates host cell apoptosis to promote its own replication and evade the host immune response remain inadequately understood. This study reveals, for the first time, a critical role for caspase-6-a cysteine-aspartic protease of the apoptotic cascade-in PRRSV infection. We demonstrated that caspase-6 specifically cleaves the viral nucleocapsid (N) protein at aspartate residue 94 (D94), generating N-terminal and C-terminal fragments that subsequently inhibit the activation and nuclear translocation of the key host transcription factor, namely, interferon (IFN) regulatory factor 3 (IRF3). This leads to a significant reduction in the expression of host IFN-β, thereby promoting viral replication. Further investigation confirmed that this cleavage site is highly conserved across different PRRSV strains, suggesting it as a potential target for the development of broad-spectrum antiviral therapeutics. Moreover, a D94A mutant virus (PRRSV-D94A), constructed using reverse genetics, exhibited significantly attenuated replication and pathogenicity. This mutant induced a more robust host antiviral immune response, characterized by markedly elevated levels of IFNs and inflammatory cytokines, indicating its potential as an ideal live attenuated vaccine candidate. This study elucidated, from the perspective of a host protease, a novel molecular mechanism by which PRRSV evades the host immune response and promotes viral replication, providing a vital theoretical basis and a new strategy for PRRSV vaccine design and antiviral drug development.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) remains one of the most economically devastating pathogens in the swine industry, largely due to its ability to evade innate immunity and persist within its host. This study identifies, for the first time, a host-virus mechanism in which PRRSV recruits caspase-6 to cleave its N protein at a conserved D94 site, thereby suppressing type I IFN signaling and enhancing viral replication. We further demonstrate that disrupting this cleavage through the D94A mutation attenuates viral replication, enhances innate immune activation, and reduces pathogenicity in pigs. These findings not only reveal a previously unrecognized immune-evasion strategy of PRRSV but also highlight caspase-6 and the N-protein cleavage site as promising targets for host-directed antiviral interventions and rational vaccine design.

Protective efficacy of a candidate attenuated live vaccine derived from an NADC30-like strain against homologous porcine reproductive and respiratory syndrome virus challenge.

ObjectiveThis study aimed to develop a sensitive and reliable method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in various environmental samples using nano–liquid chromatography–tandem mass spectrometry parallel reaction monitoring (nanoLC-MS/MS PRM), targeting sequences of the viral spike (S) and nucleocapsid (N) proteins.Materials and MethodsA synthetic peptide screening approach was employed to systematically evaluate candidate sequences as surrogates for protein quantification. The process began with the digestion of purified SARS-CoV-2 proteins. The candidate sequences were then refined to a subset of target peptides, and stable isotope-labeled heavy peptides were used as standards in a PRM workflow. The detection limits were determined using the nanoLC-MS/MS PRM method.ResultsThe developed method achieved limits of detection in the 1 fmol/µL (2–8 × 10-15 viral copies/L) range on-column. This demonstrates high sensitivity for detecting SARS-CoV-2 in environmental samples using the PRM method.ConclusionThe study successfully developed a sensitive and reliable peptide probe-based approach for detecting SARS-CoV-2 in various environmental samples. The use of the PRM method targeting the S and N proteins of SARS-CoV-2 provides a robust approach for virus detection, particularly given the potential persistence of the virus on surfaces.Graphic Graphic

During infection in vitro with the strain 438/06 of bovine coronavirus (BCoV), a β-coronavirus similar to severe acute respiratory syndrome (SARS) CoV-2, treatment with 6-pentyl-α-pyrone (6PP), a fungal metabolite obtained from Trichoderma atroviride, was recently shown to influence viral load by reducing viral entry. Herein, the ability of 6PP to counteract the BCoV infection was further investigated both in vitro and in silico. Following the BCoV (strain 282/23) infection in bovine (MDBK) cells, the 6PP in co-treatment increased cell viability, reduced morphological signs of cell death, and significantly inhibited viral yield, by lessening the expression of the viral spike (S) protein, as well as the gene transcription of the viral nucleocapsid (NP) protein. In addition, a noticeable down-regulation in the expression of aryl hydrocarbon receptor (AhR) signaling, a strategic modulator of CoVs infection, was found. Molecular docking studies were performed to evaluate the potential interaction between 6PP and AhR involved in the BCoV infection. The docking 3D structural model showed that 6PP fits into a binding pocket positioned between the PASB and TAD domains of bovine AhR (bAhR), where the ligand is stabilized through hydrophobic interactions. In addition, the obtained computational data strongly suggest that the bAhR binding mechanism of 6PP is principally mediated by a well-conserved hydrophobic cavity playing a key role in the modulation of the receptor functions. Overall, our findings showed an antiviral action of 6PP versus BCoV infection in vitro and in silico.

Background: Rift Valley fever virus (RVFV) is a significant mosquito-borne zoonotic virus with high public health and veterinary importance in Africa and the Middle East. Reliable diagnostic assays for detecting antibodies and assessing their functional neutralizing capacity are essential for surveillance programs, vaccine monitoring, and outbreak preparedness. Objective: This study evaluates and compares the analytical performance of a competitive enzyme-linked immunosorbent assay (cELISA) and a virus neutralization test (VNT) for detecting RVFV antibodies in vaccinated sheep sera, establishing an integrated laboratory workflow for virus titration, serological detection, and functional neutralization. Methods: Twenty serum samples were collected from sheep pre-vaccination and one month post-vaccination with Smithburn live attenuated RVFV vaccine. Sera were tested using a commercial multispecies RVFV competitive ELISA to detect antibodies specific to the viral nucleocapsid protein. Viral titration was conducted in Vero cells, and 50% tissue culture infective dose (TCID50/0.1 mL) was calculated using the Reed and Muench method. VNT was performed at 24, 48, 72, and 96 h after infection with different viral doses (102 to 105TCID50/0.1 mL), and the neutralizing ability of serial serum dilutions (1:2 to 1:1024) was tested. Compared with the control, protection was determined by cytopathic effect (CPE) inhibition. Results: ELISA revealed robust antibody signals up to a 1:32 dilution, with signal-to-noise (S/N) < 40%, whereas for higher dilutions, antibody detection became inconclusive or negative. Virus titration was performed to verify a stock concentration of 106.5TCID50/0.1 mL. The VNT exhibited time- and dose-dependent kinetics; high protection rates (≥97) were observed at 1:2-1:8 dilutions against 102-103TCID50/0.1 mL challenge doses; however, neutralizing efficacy decreased significantly at higher viral loads and higher serum dilutions. While cELISA and VNT results correlated strongly at low serum dilutions, the cELISA showed decreased sensitivity at dilutions ≥ 1:64, where the VNT remained capable of detecting functional neutralizing activity. Conclusions/Discussion: The results demonstrate that while both assays correlate well at high antibody concentrations, they diverge at lower concentrations. This discrepancy highlights the functional difference between binding antibodies (N-protein) and neutralizing antibodies (Gn/Gc glycoproteins). Consequently, the cELISA is ideal for rapid screening, whereas the VNT is indispensable for confirming functional immunity. Integrating both assays provides a more accurate immunological profile for RVFV surveillance and vaccine evaluation.

Objective Prokaryotic expression and purification of the Rift Valley fever virus (RVFV) nucleocapsid protein (NP) were performed to establish a basis for serum NP antibody detection and to produce specific polyclonal antibodies against NP. Methods The constructed pET28a-ZH501-NP prokaryotic expression plasmid was transformed into BL21(DE3) competent cells, and the expression of the recombinant protein NP was induced by isopropyl-β-D-thiogalactoside (IPTG). The recombinant protein NP was purified using HisTrapTM HP and mixed with Freund's complete and incomplete adjuvants to immunize New Zealand white rabbits. NP polyclonal antibodies were prepared, and their specificity was identified by indirect immunofluorescence (IFA) and Western Blotting. Results The prokaryotic expression plasmid was successfully constructed, and expression was successfully induced in BL21(DE3) competent cells. The recombinant protein NP was expressed in the form of inclusion bodies. Western Blotting and IFA assays indicated that the polyclonal antibody could specifically recognize RVFV NP. Conclusion The pure and effective expression of RVFV NP protein was achieved. The experimental conditions for the indirect ELISA using it as the coating antigen were explored. Simultaneously, specific polyclonal antibodies against RVFV NP were prepared, providing technical support for the establishment of a reliable rapid diagnostic technology for RVF and the research and evaluation of new RVFV vaccines.

Since the beginning of the COVID-19 pandemic, vulnerable populations such as pregnant persons have been at higher risk of severe symptoms and poor outcomes. Although reports of SARS-CoV-2 vertical transmission remain rare, several studies showed that maternal infection during pregnancy can induce histomorphological and inflammatory alterations in the placenta. However, the permissiveness of human trophoblasts to various variants of the virus remains poorly characterized. In this study, human primary villous cytotrophoblasts isolated from term placentas, along with trophoblastic cell lines BeWo, JEG-3, and HIPEC-65 were infected with the ancestral SARS-CoV-2 strain, which disseminated worldwide in early 2020. Permissiveness was assessed with quantitative RT-PCR, immunostaining of viral protein Nucleocapsid, and plaque assays. To investigate viral entry routes, cells were treated with Camostat mesylate (an inhibitor of the co-entry factor TMPRSS2) or chloroquine phosphate (an endosomal entry inhibitor) and viral fitness was assessed by plaque assays. Primary villous cytotrophoblasts and JEG-3 cells were also tested for infection with three pre-omicron SARS-CoV-2 variants of concern. Our results show that primary villous cytotrophoblasts are permissive to all tested SARS-CoV-2 strains in vitro. Infection with the ancestral SARS-CoV-2 strain relies mainly on a non-canonical endosomal entry pathway. Notably, JEG-3 cells represent an appropriate and convenient model for studying trophoblast infection by SARS-CoV-2, as they exhibit high permissiveness to the ancestral strain, and the SARS-CoV-2 entry pathway is similar to that in villous cytotrophoblasts. Overall, this study reveals that the cytotrophoblastic permissiveness to SARS-CoV-2 depends on the viral genetic nature and provides new insights into its entry route in human trophoblasts.

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic alphacoronavirus that causes severe diarrhea. It has a high fatality rate among newborn piglets, posing a considerable economic burden to the swine industry. Therefore, elucidating the host-pathogen interaction is warranted to advance precision antiviral therapies. Herein, for the first time, we noted a marked upregulation of aldehyde dehydrogenase 1 family member L1 (ALDH1L1) during PEDV infection. Furthermore, ALDH1L1 exerts its antiviral effects by specifically binding to the viral nucleocapsid (N) and envelope (E) proteins and mediating their degradation via the autophagosome-lysosomal degradation pathway. Additional experiments revealed that this degradation process is mediated via the interactions of ALDH1L1 with the E3 ubiquitin ligase STUB1 and the cargo receptor TOLLIP, eliminating the N and E structural glycoproteins via the autophagolysosomal pathway. Our study findings suggest the ALDH1L1-STUB1-TOLLIP axis as a novel antiviral target and propose a new strategy for viral clearance based on the degradation of host protein. Furthermore, our research provides valuable information on how host antiviral factors impede PEDV replication as a regulator of the protein degradation pathway.IMPORTANCEPorcine epidemic diarrhea virus (PEDV) is a highly pathogenic alphacoronavirus that causes fatal hemorrhagic gastroenteritis among neonatal piglets. This causes significant financial losses. During infection, certain host factors can activate the innate immune regulatory network to antagonize the viral replication cycle, interfere with the virus invasion, inhibit virus replication, prevent virus assembly and release, and enhance the host's immune response. Our study revealed that the host metabolic enzyme ALDH1L1 acts as a novel antiviral restriction factor that mediates the autophagy-lysosome-targeted degradation of viral structural proteins (N/E) via the STUB1 (E3 ubiquitin ligase)-TOLLIP (autophagy adaptor protein) axis. Our study findings offer new perspectives on the mechanism by which host antiviral factors inhibit PEDV by regulating the protein degradation pathway.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne orthonairovirus associated with severe hemorrhagic fever and high mortality in humans. The genome is comprised of three segments of single-stranded, negative-sense RNA. The nucleocapsid (NP) protein, which is the focus of our study, encoded by the smallest (S) segment, plays a central role in viral RNA encapsidation and is known to be highly immunogenic, eliciting innate, humoral and cellular immune responses. This study aimed to identify the immunodominant regions of the NP that trigger T cell-mediated immune responses. To this end, three truncated variants of the NP from the CCHFV Kelkit'06 strain [NPdelN(1-135 aa), NPdelM(136-295 aa) and NPdelC(296-482 aa)] as well as the full-length NP(1-482 aa) were tested. Following mice immunisations, local lymph node cells were used as the source of lymphocytes and these cells were assessed to gauge the T cell responses. Stimulated splenocytes were analysed for lymphoproliferative responses and cytokine mRNA expression to evaluate T cell polarisation. Based on the data obtained, it appears that the amino-terminal region (1-135 amino acids) of NP is significantly more immunogenic than the other regions. In contrast, the carboxy-terminal region (296-482 aa) appears to play a suppressive role in cellular immune activation. Additionally, the middle region (136-295 aa) of the NP is identified as being responsible for inducing Th17-type cellular immune responses. In conclusion, this study points out to the specific regions of the CCHFV NP that are involved in shaping the cellular immune responses, representing a crucial step toward refining the structural elements contributing anti-viral immunity and providing a sound ground for modulation efforts.

Multiple mosquito species serve as competent vectors to carry and transmit numerous flaviviruses1,2. Several long-standing scientific questions remain to be answered, including identification of the fundamental factors that facilitate flavivirus infectivity in mosquitoes and the genetic basis that contributes to the naturally occurring interspecies specificity of mosquitoes to flaviviruses3-8, such as Aedes aegypti mosquitoes to dengue virus (DENV). Here we report that circulating mature virions are inactivated by the acidity of mosquito haemolymph; thus, extracellular vesicles carrying replication-competent viral nucleocapsids serve as the predominant means of intercellular viral dissemination. Mechanistically, mosquito valosin-containing protein (VCP) binds to the viral capsid, thereby allowing the incorporation of nucleocapsids into extracellular vesicles. The capsid of a flavivirus (such as DENV) selectively binds to the VCP of its natural vector (Ae. aegypti), but not to that of an incompetent vector (for example, Culex quinquefasciatus). Replacing the DENV capsid with that of Japanese encephalitis virus (JEV) renders DENV infectious in the haemolymph of the natural JEV vector, Cx. quinquefasciatus. Furthermore, two amino residues in Aedes (D723/N728) and Culex (E723/E728) VCP determine its binding specificity for viral capsid, thus contributing to interspecies specificity of mosquitoes to flaviviruses. In vivo ectopic expression of the Cx. quinquefasciatus VCP mutant E723D/E728N renders Cx. quinquefasciatus susceptible to DENV2 via intrathoracic microinjection. Our study provides a major molecular mechanism contributing to the selectivity and compatibility between mosquito vectors and flavivirus species, enabling systemic virus dissemination after the virus reaches the haemocoel. Upstream mechanisms that determine specificity at the midgut level remain to be determined.

To analyze the pathomorphological and immunohistochemical characteristics of the hearts of patients who died from COVID-19. A comprehensive pathomorphologic study of the heart in 88 autopsies of patients who died from severe COVID-19 was performed. The presence of SARS-CoV-2 infection was confirmed by positive PCR tests in all cases. Immunohistochemical study was used to analyze the expression of viral nucleocapsid protein (NP), CD3, CD68, CD31, CD34, Willebrand factor (vWF) in cell populations and myocardial structures. In most cases, polymorphism of structural changes was observed, which was expressed in a combination of signs of chronic and acute myocardial damage. Numerous foci of interstitial and perivascular fibrosis and lipomatosis, irregular hypertrophy of cardiomyocytes, amyloid deposits were indicative of previous cardiovascular diseases. Acute and subacute pathological changes in myocardium included circulatory disorders (hyperemia, intraluminal megakaryocytes, microvascular thrombosis, interstitial edema), vascular damage, and alterative changes in cardiomyocytes. Lymphomacrophage infiltration was present in 45% of cases and was associated with immunohistochemical detection of NP in cardiomyocytes and vascular cells. Weak expression of CD31 and high expression of vWF in microvascular endothelium was found. NP expression in cardiomyocytes, macrophages and endothelial cells of cardiac vessels indicates their direct infection with SARS-CoV-2 virus and possible long-term persistence of viral infection. It was found that in severe COVID-19 CD3- and CD68-positive cells are detected in the heart; endothelial dysfunction is observed, which is indicated by decreased expression of CD31 and high expression of vWF. The identified myocardial changes may influence the development of post-COVID cardiovascular complications.

The continuous emergence of variants of concern (VoC) represent a significant challenge to effectively control SARS-CoV-2. Although FDA-approved vaccines and antivirals have been successfully developed and implemented for the prophylactic and therapeutic intervention of SARS-CoV-2 infection, recent VoC could escape protection garnered by previous vaccine and antiviral approaches. Determining the efficacy of prophylactics and/or therapeutics against recent VoC will assist in efficiently controlling currently circulating SARS-CoV-2 strains. We used our previously described bacterial artificial chromosome (BAC)-based reverse genetics approach for Omicron BA.5 to generate a recombinant SARS-CoV-2 BA.5 encoding a fusion of ZsGreen to Nanoluciferase (rBA.5 ZsG-Nluc) from the locus of the viral nucleocapsid (N) protein separated by the porcine teschovirus-1 (PTV-1) 2A proteolytic cleavage site. The rBA5 ZsG-Nluc replicates to levels comparable to recombinant BA.5 wild-type (rBA.5 WT) and expresses high levels of ZsG and Nluc in cultured cells. This facilitates tracking viral infection and the identification of antivirals and neutralizing antibodies (NAbs) with EC50 and NT50 values, respectively, similar to those obtained with rBA.5 WT. Importantly, in K18 hACE2 mice, rBA.5 ZsG-Nluc retains the same pathogenicity and ability to replicate in the lungs of infected mice as rBA.5 WT. Using rBA.5 ZsG-Nluc, we detected Nluc activity systemically and Nluc and ZsG expression in the lungs of infected mice using an in vivo imaging system (IVIS). Our results demonstrate the feasibility of using rBA.5 ZsG-Nluc to track viral infections and identify prophylactics and therapeutics against recent SARS-CoV-2 VoC in vitro, ex vivo, and in vivo.

Vesicles are a class of naturally released, cell-derived, lipid bilayer enclosed compartments. Different kinds of vesicles can be produced within cells. Cellular processes are heavily dependent on the synergistic action of some vesicles within cells. Studies have showed diverse roles of vesicles including intracellular trafficking, cell-to-cell communication and intracellular digestion. Vesicles can deliver not only some proteins and lipids to target sites but also viral components between nucleus and cytoplasm. Transmembrane transport of some viral nucleocapsids through intranuclear microvesicle is necessary for some viral propagation in host. Viral infection and viral spread are directly involved with vesicle-mediated endocytosis and exocytosis. So, the production of some progeny virion and viral release can be regulated by vesicle-mediated cellular transport and exocytosis. In the study, the classification, biogenesis and function of vesicles were summarized to better understand their role in viral propagation. In conclusion, vesicular trafficking can play important roles in regulation of viral entry, transport of viral substance and viral spread.

Development and validation of a recombinant N protein-based indirect ELISA for serological detection of feline infectious peritonitis virus.

Introduction. Transboundary and emerging infections of cattle and small ruminants, such as peste des petits ruminants, Schmallenberg virus infection, etc., pose a serious animal health and economic threat in the context of developing globalization. Given the current geopolitical situation, the need for modern domestically produced diagnostic systems is particularly acute. Such systems can be developed using genetic engineering methods. Objective. Analysis of domestic and foreign publications on the production of recombinant proteins of pathogens of transboundary and emerging infections of cattle and small ruminants. Creation of genetic constructs based on the processed data for further development of diagnostic tools, in particular ELISA test systems. Materials and methods. Using bioinformatics tools, codon composition of the sequences encoding the nucleocapsid proteins of peste des petits ruminants virus (PPRV) and Schmallenberg virus (SBV) was analyzed and optimized. The optimized gene fragments were synthesized de novo and cloned into the pET-32b(+) expression vector. Successful insertion of the target sequence into the vector was confirmed by polymerase chain reaction and restriction analysis. Results . Information on ELISA test systems developed on the basis of recombinant antigens for the diagnosis of peste des petits ruminants and Schmallenberg virus infection is presented. The main technological aspects of obtaining recombinant antigens for their further use in a diagnostic system factored in the biological features of a particular pathogen are highlighted. Our proprietary methodology for creating protein expression vectors for the pathogens of the diseases under review is additionally described. Conclusion. The most promising recombinant antigens for use in ELISA test systems designed to detect antibodies against PPRV and SBV are full-length and truncated virion nucleocapsid proteins. Furthermore, the biophysical properties and antigenic structure of these proteins enable their production in Escherichia coli. It should be noted that production of significant amounts of functional proteins in soluble form may require their expression as part of fusion proteins with tags enhancing solubility and facilitating correct folding.