Large-Scale Serological Survey of Influenza A Virus in South Korean Wild Boar (Sus scrofa).

In this comprehensive large-scale study, conducted from 2015 to 2019, 7,209 wild boars across South Korea were sampled to assess their exposure to influenza A viruses (IAVs). Of these, 250 (3.5%) were found to be IAV-positive by ELISA, and 150 (2.1%) by the hemagglutination inhibition test. Detected subtypes included 23 cases of pandemic 2009 H1N1, six of human seasonal H3N2, three of classical swine H1N1, 13 of triple-reassortant swine H1N2, seven of triple-reassortant swine H3N2, and seven of swine-origin H3N2 variant. Notably, none of the serum samples tested positive for avian IAV subtypes H3N8, H5N3, H7N7, and H9N2 or canine IAV subtype H3N2. This serologic analysis confirmed the exposure of Korean wild boars to various subtypes of swine and human influenza viruses, with some serum samples cross-reacting between swine and human strains, indicating potential infections with multiple IAVs. The results highlight the potential of wild boar as a novel mixing vessel, facilitating the adaptation of IAVs and their spillover to other hosts, including humans. In light of these findings, we recommend regular and frequent surveillance of circulating influenza viruses in the wild boar population as a proactive measure to prevent potential human influenza pandemics and wild boar influenza epizootics.

H1N1 nanobody development and therapeutic efficacy verification in H1N1-challenged mice.

Influenza A virus causes numerous deaths and infections worldwide annually. Therefore, we have considered nanobodies as a potential treatment for patients with severe cases of influenza. We developed a nanobody that was expected to have protective efficacy against the A/California/04/2009 (CA/04; pandemic 2009 flu strain) and evaluated its therapeutic efficacy against CA/04 in mice experiments. This nanobody was derived from the immunization of the alpaca, and the inactivated CA/04 virus was used as an immunogen. We successfully generated a nanobody library through bio-panning, phage ELISA, and Bio-layer interferometry. Moreover, we confirmed that administering nanobodies after lethal doses of CA/04 reduced viral replication in the lungs and influenza-induced clinical signs in mice. These research findings will help to develop nanobodies as viral therapeutics for CA/04 and other infectious viruses.

Genetic characterization and pathogenicity in a mouse model of newly isolated bat-originated mammalian orthoreovirus in South Korea.

Mammalian orthoreoviruses (MRVs) infect a wide range of hosts, including humans, livestock, and wildlife. In the present study, we isolated a novel Mammalian orthoreovirus from the intestine of a microbat (Myotis aurascens) and investigated its biological and pathological characteristics. Phylogenetic analysis indicated that the new isolate was serotype 2, sharing the segments with those from different hosts. Our results showed that it can infect a wide range of cell lines from different mammalian species, including human, swine, and non-human primate cell lines. Additionally, media containing trypsin, yeast extract, and tryptose phosphate broth promoted virus propagation in primate cell lines and most human cell lines, but not in A549 and porcine cell lines. Mice infected with this strain via the intranasal route, but not via the oral route, exhibited weight loss and respiratory distress. The virus is distributed in a broad range of organs and causes lung damage. In vitro and in vivo experiments also suggested that the new virus could be a neurotropic infectious strain that can infect a neuroblastoma cell line and replicate in the brains of infected mice. Additionally, it caused a delayed immune response, as indicated by the high expression levels of cytokines and chemokines only at 14 days post-infection (dpi). These data provide an important understanding of the genetics and pathogenicity of mammalian orthoreoviruses in bats at risk of spillover infections.IMPORTANCEMammalian orthoreoviruses (MRVs) have a broad range of hosts and can cause serious respiratory and gastroenteritis diseases in humans and livestock. Some strains infect the central nervous system, causing severe encephalitis. In this study, we identified BatMRV2/SNU1/Korea/2021, a reassortment of MRV serotype 2, isolated from bats with broad tissue tropism, including the neurological system. In addition, it has been shown to cause respiratory syndrome in mouse models. The given data will provide more evidence of the risk of mammalian orthoreovirus transmission from wildlife to various animal species and the sources of spillover infections.

Self-Assembled Nanostructures Presenting Repetitive Arrays of Subunit Antigens for Enhanced Immune Response.

Infectious diseases pose persistent threats to public health, demanding advanced vaccine technologies. Nanomaterial-based delivery systems offer promising solutions to enhance immunogenicity while minimizing reactogenicity. We introduce a self-assembled vaccine (SAV) platform employing antigen-polymer conjugates designed to facilitate robust immune responses. The SAVs exhibit efficient cellular uptake by dendritic cells (DCs) and macrophages, which are crucial players in the innate immune system. The high-density antigen presentation of this SAV platform enhances the affinity for DCs through multivalent recognition, significantly augmenting humoral immunity. SAV induced high levels of immunoglobulin G (IgG), IgG1, and IgG2a, suggesting that mature DCs efficiently induced B cell activation through multivalent antigen recognition. Universality was confirmed by applying it to respiratory viruses, showcasing its potential as a versatile vaccine platform. Furthermore, we have also demonstrated strong protection against influenza A virus infection with SAV containing hemagglutinin, which is used in influenza A virus subunit vaccines. The efficacy and adaptability of this nanostructured vaccine present potential utility in combating infectious diseases.

Label-Free and Colorimetric Detection of Influenza A Virus via Receptor-Mediated Viral Fusion with Plasmonic Vesicles.

The rapid transmission and numerous re-emerging human influenza virus variants that spread via the respiratory system have led to severe global damage, emphasizing the need for detection tools that can recognize active and intact virions with infectivity. Here, this work presents a plasmonic vesicle-mediated fusogenic immunoassay (PVFIA) comprising gold nanoparticle (GNP) encapsulating fusogenic polymeric vesicles (plasmonic vesicles; PVs) for the label-free and colorimetric detection of influenza A virus (IAV). The PVFIA combines two sequential assays: a biochip-based immunoassay for target-specific capture and a PV-induced fusion assay for color change upon the IAV-PV fusion complex formation. The PVFIA demonstrates excellent specificity in capturing the target IAV, while the fusion conditions and GNP induce a significant color change, enabling visual detection. The integration of two consecutive assays results in a low detection limit (100.7919 EID50 mL-1 ) and good reliability (0.9901), indicating sensitivity that is 104.208 times higher than conventional immunoassay. Leveraging the PV viral membrane fusion activity renders the PVFIA promising for point-of-care diagnostics through colorimetric detection. The innovative approach addresses the critical need for detecting active and intact virions with infectivity, providing a valuable tool with which to combat the spread of the virus.

Development of a neutralization monoclonal antibody with a broad neutralizing effect against SARS-CoV-2 variants.

BACKGROUND: The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has challenged the effectiveness of current therapeutic regimens. Here, we aimed to develop a potent SARS-CoV-2 antibody with broad neutralizing effect by screening a scFv library with the spike protein receptor-binding domain (RBD) via phage display. METHODS: SKAI-DS84 was identified through phage display, and we performed pseudovirus neutralization assays, authentic virus neutralization assays, and in vivo neutralization efficacy evaluations. Furthermore, surface plasmon resonance (SPR) analysis was conducted to assess the physical characteristics of the antibody, including binding kinetics and measure its affinity for variant RBDs. RESULTS: The selected clones were converted to human IgG, and among them, SKAI-DS84 was selected for further analyses based on its binding affinity with the variant RBDs. Using pseudoviruses, we confirmed that SKAI-DS84 was strongly neutralizing against wild-type, B.1.617.2, B.1.1.529, and subvariants of SARS-CoV-2. We also tested the neutralizing effect of SKAI-DS84 on authentic viruses, in vivo and observed a reduction in viral replication and improved lung pathology. We performed binding and epitope mapping experiments to understand the mechanisms underlying neutralization and identified quaternary epitopes formed by the interaction between RBDs as the target of SKAI-DS84. CONCLUSIONS: We identified, produced, and tested the neutralizing effect of SKAI-DS84 antibody. Our results highlight that SKAI-DS84 could be a potential neutralizing antibody against SARS-CoV-2 and its variants.

Advanced Strategies for Developing Vaccines and Diagnostic Tools for African Swine Fever.

African swine fever (ASF) is one of the most lethal infectious diseases affecting domestic pigs and wild boars of all ages. Over a span of 100 years, ASF has continued to spread over continents and adversely affects the global pig industry. To date, no vaccine or treatment has been approved. The complex genome structure and diverse variants facilitate the immune evasion of the ASF virus (ASFV). Recently, advanced technologies have been used to design various potential vaccine candidates and effective diagnostic tools. This review updates vaccine platforms that are currently being used worldwide, with a focus on genetically modified live attenuated vaccines, including an understanding of their potential efficacy and limitations of safety and stability. Furthermore, advanced ASFV detection technologies are presented that discuss and incorporate the challenges that remain to be addressed for conventional detection methods. We also highlight a nano-bio-based system that enhances sensitivity and specificity. A combination of prophylactic vaccines and point-of-care diagnostics can help effectively control the spread of ASFV.

A Robust Quadruple Protein-Based Indirect ELISA for Detection of Antibodies to African Swine Fever Virus in Pigs.

African swine fever (ASF) emerged in domestic pigs and wild boars in China in 2018 and rapidly spread to neighboring Asian countries. Currently, no effective vaccine or diagnostic tests are available to prevent its spread. We developed a robust quadruple recombinant-protein-based indirect enzyme-linked immunosorbent assay (QrP-iELISA) using four antigenic proteins (CD2v, CAP80, p54, and p22) to detect ASF virus (ASFV) antibodies and compared it with a commercial kit (IDvet) using ASFV-positive and -negative serum samples. The maximum positive/negative value was 24.033 at a single antigen concentration of 0.25 µg/mL and quadruple ASFV antigen combination of 1 µg/mL at a 1:100 serum dilution. Among 70 ASFV-positive samples, 65, 67, 65, 70, 70, and 14 were positive above the cut-offs of 0.121, 0.121, 0.183, 0.065, 0.201, and 0.122, for CD2v, CAP80, p54, p22-iELISA, QrP-iELISA, and IDvet, respectively, with sensitivities of 92.9%, 95.7%, 92.9%, 100%, 100%, and 20%, respectively, all with 100% specificity. The antibody responses in QrP-iELISA and IDvet were similar in pigs infected with ASFV I. QrP-iELISA was more sensitive than IDvet for early antibody detection in pigs infected with ASFV II. These data provide a foundation for developing advanced ASF antibody detection kits critical for ASF surveillance and control.

Mucosal and cellular immune responses elicited by nasal and intramuscular inoculation with ASFV candidate immunogens.

African swine fever (ASF) is an infectious disease caused by African swine fever virus (ASFV) that is highly contagious and has an extremely high mortality rate (infected by virulent strains) among domestic and wild pigs, causing huge economic losses to the pig industry globally. In this study, SDS-PAGE gel bands hybridized with ASFV whole virus protein combined with ASFV-convalescent and ASFV-positive pig serum were identified by mass spectrometry. Six antigens were detected by positive serum reaction bands, and eight antigens were detected in ASFV-convalescent serum. In combination with previous literature reports and proteins corresponding to MHC-II presenting peptides screened from ASFV-positive pig urine conducted in our lab, seven candidate antigens, including KP177R (p22), K78R (p10), CP204L (p30), E183L (p54), B602L (B602L), EP402R-N (CD2V-N) and F317L (F317L), were selected. Subunit-Group 1 was prepared by mixing above-mentioned seven ASFV recombinant proteins with MONTANIDETM1313 VG N mucosal adjuvant and immunizing pigs intranasally and intramuscularly. Subunit-Group 2 was prepared by mixing four ASFV recombinant proteins (p22, p54, CD2V-N1, B602L) with Montanide ISA 51 VG adjuvant and immunizing pigs by intramuscular injection. Anticoagulated whole blood, serum, and oral fluid were collected during immunization for flow cytometry, serum IgG as well as secretory sIgA antibody secretion, and cytokine expression testing to conduct a comprehensive immunogenicity assessment. Both immunogen groups can effectively stimulate the host to produce ideal humoral, mucosal, and cellular immune responses, providing a theoretical basis for subsequent functional studies, such as immunogens challenge protection and elucidation of the pathogenic mechanism of ASFV.

A Whole-Genome Analysis of the African Swine Fever Virus That Circulated during the First Outbreak in Vietnam in 2019 and Subsequently in 2022.

Since its initial report in Vietnam in early 2019, the African swine fever (ASF), a highly lethal and severe viral swine disease worldwide, continues to cause outbreaks in other Southeast Asian countries. This study analyzed and compared the genomic sequences of ASF viruses (ASFVs) during the first outbreak in Hung Yen (VN/HY/2019-ASFV1) and Quynh Phu provinces (VN/QP/2019-ASFV1) in Vietnam in 2019, and the subsequent outbreak in Hung Yen (VN/HY/2022-ASFV2) in 2022, to those of other ASFV strains. VN/HY/2019-ASFV1, VN/QP/2019-ASFV1, and VN/HY/2022-ASFV2 genomes were 189,113, 189,081, and 189,607 bp in length, encoding 196, 196, and 203 open reading frames (ORFs), respectively. VN/HY/2019-ASFV1 and VN/QP/2019-ASFV1 shared a 99.91-99.99% average nucleotide identity with genotype II strains. Variations were identified in 28 ORFs in VN/HY/2019-ASFV1 and VN/QP/2019-ASFV1 compared to 20 ASFV strains, and 16 ORFs in VN/HY/2022-ASFV2 compared to VN/HY/2019-ASFV1 and VN/QP/2019-ASFV1. Vietnamese ASFV genomes were classified as IGR II variants between the I73R and I329L genes, with two copy tandem repeats between the A179L and A137R genes. A phylogenetic analysis based on the whole genomes of 27 ASFV strains indicated that the Vietnamese ASFV strains are genetically related to Estonia 2014, ASFV-SY18, and Russia/Odintsovo_02/14. These results reveal the complete genome sequences of ASFV circulating during the first outbreak in 2019, providing important insights into understanding the evolution, transmission, and genetic variation of ASFV in Vietnam.

Establishment of a p30-based lateral flow assay for African swine fever virus detection.

African swine fever virus (ASFV) has continuously devastated the global pig industry. Viral persistence causes problems in large pig farms and kills small farms. Timely diagnostic tools play an important role in controlling outbreaks and minimizing losses. In this study, we developed a lateral flow assay to detect ASFV on-site. The VDRG® ASFV Ag Rapid Kit was established using two monoclonal antibodies (mAbs) against the p30 protein. The conjunction pad of the kit was coated with a mixture of the mAb and colloidal gold. This rapid kit was capable of detecting 11.5 ng of antigen and 0.16 HAD50 of virus from samples, in 20 min for the entire procedure. It passed cross-specific tests using common viruses that cause infectious diseases in pigs. ASFV was detected after 4 days in experimental infection in pigs by the kit. The specificity and sensitivity of the kit for clinical samples were 99.88% and 84.52% (93.8% for samples with a Ct value below 30), respectively. Finally, the kit can detect 100% positive herd outbreaks. The VDRG® ASFV Ag Rapid Kit presents a useful point-of-care tool for ASFV detection.

Rapid Visible Detection of African Swine Fever Virus Using Hybridization Chain Reaction-Sensitized Magnetic Nanoclusters and Affinity Chromatography.

African swine fever virus (ASFV) is a severe and persistent threat to the global swine industry. As there are no vaccines against ASFV, there is an immense need to develop easy-to-use, cost-effective, and rapid point-of-care (POC) diagnostic platforms to detect and prevent ASFV outbreaks. Here, a novel POC diagnostic system based on affinity column chromatography for the optical detection of ASFV is presented. This system employs an on-particle hairpin chain reaction to sensitize magnetic nanoclusters with long DNA strands in a target-selective manner, which is subsequently fed into a column chromatography device to produce quantitatively readable and colorimetric signals. The detection approach does not require expensive analytical apparatus or immobile instrumentation. The system can detect five genes constituting the ASFV whole genome with a detection limit of ≈19.8 pm in swine serum within 30 min at laboratory room temperature. With an additional pre-amplification step using polymerase chain reaction (PCR), the assay is successfully applied to detect the presence of ASFV in 30 suspected swine samples with 100% sensitivity and specificity, similar to quantitative PCR. Thus, this simple, inexpensive, portable, robust, and customizable platform for the early detection of ASFV can facilitate the timely surveillance and implementation of control measures.

Susceptibility to SARS-CoV-2 and MERS-CoV in Beagle Dogs.

The coronavirus disease 19 (COVID-19) pandemic, caused by the severe acute respiratory syndrome, coronavirus 2 (SARS-CoV-2), has resulted in unprecedented challenges to healthcare worldwide. In particular, the anthroponotic transmission of human coronaviruses has become a common concern among pet owners. Here, we experimentally inoculated beagle dogs with SARS-CoV-2 or Middle East respiratory syndrome (MERS-CoV) to compare their susceptibility to and the pathogenicity of these viruses. The dogs in this study exhibited weight loss and increased body temperatures and shed the viruses in their nasal secretions, feces, and urine. Pathologic changes were observed in the lungs of the dogs inoculated with SARS-CoV-2 or MERS-CoV. Additionally, clinical characteristics of SARS-CoV-2, such as increased lactate dehydrogenase levels, were identified in the current study.

Experimental Infection and Transmission of SARS-CoV-2 Delta and Omicron Variants among Beagle Dogs.

We assessed susceptibility of dogs to SARS-COV-2 Delta and Omicron variants by experimentally inoculating beagle dogs. Moreover, we investigated transmissibility of the variants from infected to naive dogs. The dogs were susceptible to infection without clinical signs and transmitted both strains to other dogs through direct contact.

Establishment of multicenter COVID-19 therapeutics preclinical test system in Republic of Korea.

Throughout the recent COVID-19 pandemic, South Korea led national efforts to develop vaccines and therapeutics for SARS-CoV-2. The project proceeded as follows: 1) evaluation system setup (including Animal Biosafety Level 3 (ABSL3) facility alliance, standardized nonclinical evaluation protocol, and laboratory information management system), 2) application (including committee review and selection), and 3) evaluation (including expert judgment and reporting). After receiving 101 applications, the selection committee reviewed pharmacokinetics, toxicity, and efficacy data and selected 32 final candidates. In the nonclinical efficacy test, we used golden Syrian hamsters and human angiotensin-converting enzyme 2 transgenic mice under a cytokeratin 18 promoter to evaluate mortality, clinical signs, body weight, viral titer, neutralizing antibody presence, and histopathology. These data indicated eight new drugs and one repositioned drug having significant efficacy for COVID-19. Three vaccine and four antiviral drugs exerted significant protective activities against SARS-CoV-2 pathogenesis. Additionally, two anti-inflammatory drugs showed therapeutic effects on lung lesions and weight loss through their mechanism of action but did not affect viral replication. Along with systematic verification of COVID-19 animal models through large-scale studies, our findings suggest that ABSL3 multicenter alliance and nonclinical evaluation protocol standardization can promote reliable efficacy testing against COVID-19, thus expediting medical product development.

Rapid discovery and classification of inhibitors of coronavirus infection by pseudovirus screen and amplified luminescence proximity homogeneous assay.

To identify potent antiviral compounds, we introduced a high-throughput screen platform that can rapidly classify hit compounds according to their target. In our platform, we performed a compound screen using a lentivirus-based pseudovirus presenting a spike protein of coronavirus, and we evaluated the hit compounds using an amplified luminescence proximity homogeneous assay (alpha) test with purified host receptor protein and the receptor binding domain of the viral spike. With our screen platform, we were able to identify both spike-specific compounds (class I) and broad-spectrum antiviral compounds (class II). Among the hit compounds, thiosemicarbazide was identified to be selective to the interaction between the viral spike and its host cell receptor, and we further optimized the binding potency of thiosemicarbazide through modification of the pyridine group. Among the class II compounds, we found raloxifene and amiodarone to be highly potent against human coronaviruses including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2. In particular, using analogs of the benzothiophene moiety, which is also present in raloxifene, we have identified benzothiophene as a novel structural scaffold for broad-spectrum antivirals. This work highlights the strong utility of our screen platform using a pseudovirus assay and an alpha test for rapid identification of potential antiviral compounds and their mechanism of action, which can lead to the accelerated development of therapeutics against newly emerging viral infections.

A flap endonuclease 1-assisted universal viral nucleic acid sensing system using surface-enhanced Raman scattering.

The continued uncertainty of emerging infectious viral diseases has led to an extraordinary urgency to develop advanced molecular diagnostic tests that are faster, more reliable, simpler to use, and readily available than traditional methods. This study presents a system that can accurately and rapidly trace viral nucleic acids by employing flap endonuclease 1 (FEN1)-assisted specific DNA cleavage reactions and surface-enhanced Raman scattering (SERS)-based analysis. The designed Raman tag-labeled 5'- and 3'-flap provider DNA yielded structurally defined DNA substrates on magnetic nanoparticle surfaces when a target was present. The FEN1 enzyme subsequently processes the substrates formed via an invasive cleavage reaction, producing 5'-flap DNA products. Magnetic separation allows efficient purification of flap products from reaction mixtures. The isolated solution was directly applied onto high aspect-ratio plasmonic silver nanopillars serving as SERS-active substrates to induce amplified SERS signals. We verified the developed SERS-based sensing system using a synthetic target complementary to an avian influenza A (H9N2) virus gene and examined the detection performance of the system using complementary DNA (cDNA) derived from H9N2 viral RNA. As a result, we could detect a synthetic target with a detection limit of 41.1 fM with a single base-pair discrimination ability and achieved multiplexed detection capability for two targets. Using cDNA samples from H9N2 viruses, we observed a high concordance of R2 = 0.917 between the data obtained from SERS and the quantitative polymerase chain reaction. We anticipate that this enzyme-assisted SERS sensor may provide insights into the development of high-performance molecular diagnostic tools that can respond rapidly to viral pathogens.

Laboratory information management system for COVID-19 non-clinical efficacy trial data.

BACKGROUND: As the number of large-scale studies involving multiple organizations producing data has steadily increased, an integrated system for a common interoperable format is needed. In response to the coronavirus disease 2019 (COVID-19) pandemic, a number of global efforts are underway to develop vaccines and therapeutics. We are therefore observing an explosion in the proliferation of COVID-19 data, and interoperability is highly requested in multiple institutions participating simultaneously in COVID-19 pandemic research. RESULTS: In this study, a laboratory information management system (LIMS) approach has been adopted to systemically manage various COVID-19 non-clinical trial data, including mortality, clinical signs, body weight, body temperature, organ weights, viral titer (viral replication and viral RNA), and multiorgan histopathology, from multiple institutions based on a web interface. The main aim of the implemented system is to integrate, standardize, and organize data collected from laboratories in multiple institutes for COVID-19 non-clinical efficacy testings. Six animal biosafety level 3 institutions proved the feasibility of our system. Substantial benefits were shown by maximizing collaborative high-quality non-clinical research. CONCLUSIONS: This LIMS platform can be used for future outbreaks, leading to accelerated medical product development through the systematic management of extensive data from non-clinical animal studies.

Cell-mimetic biosensors to detect avian influenza virus via viral fusion.

Avian influenza virus (AIV) causes acute infectious diseases in poultry, critically impacting food supply. Highly pathogenic avian influenza viruses (HPAIVs), in particular, cause morbidity and mortality, resulting in significant economic losses in the poultry industry. To prevent the spread of HPAIVs, detection at early stages is critical to implement effective countermeasures such as quarantine and isolation. Through a viral fusion mechanism, cell-mimetic nanoparticles (CMPs), developed in the current study, can rapidly detect HPAIV and low pathogenic AIV (LPAIV). The CMPs comprise polymeric nanoparticles, which are constructed using sialic acid and fluorescence resonance energy transfer (FRET) dye pairs that expose the FRET off signal in response to LPAIV and HPAIV, after activation by enzymatic cleavage in the endosomal environment. The CMPs detect a wide variety of LPAIVs and HPAIVs in biological environments. Additionally, the cross-reactivity of CMPs is determined by testing their function with different viral species. Therefore, these findings demonstrate the significant potential of the proposed strategy for mimicking viral infection in vitro and using them as a highly effective diagnostic assay to rapidly detect LPAIV and HPAIV, preventing economic losses associated with viral outbreaks.