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.

Assessment of the immune interference effects of multivalent vaccine for influenza epidemic strain in 2022-2023 and evaluation of its efficacy.

The various strains of influenza virus cause respiratory symptoms in humans every year and annual vaccinations are recommended. Due to its RNA-type genes and segmented state, it belongs to a virus that mutates frequently with antigenic drift and shift, giving rise to various strains. Each year, the World Health Organization identifies the epidemic strains and operates a global surveillance system to suggest the viral composition for the influenza vaccine. Influenza viruses, which have multiple viral strains, are produced in the format of multivalent vaccine. However, the multivalent vaccine has a possibility of causing immune interference by introducing multiple strain-specific antigens in a single injection. Therefore, evaluating immune interference phenomena is essential when assessing multivalent vaccines. In this study, the protective ability and immunogenicity of multivalent and monovalent vaccines were evaluated in mice to assess immune interference in the multivalent vaccine. Monovalent and multivalent vaccines were manufactured using the latest strain of the 2022-2023 seasonal influenza virus selected by the World Health Organization. The protective abilities of both types of vaccines were tested through hemagglutination inhibition test. The immunogenicity of multivalent and monovalent vaccines were tested through enzyme-linked immunosorbent assay to measure the cellular and humoral immunity expression rates. As a result of the protective ability and immunogenicity test, higher level of virus neutralizing ability and greater amount of antibodies in both IgG1 and IgG2 were confirmed in the multivalent vaccine. No immune interference was found to affect the protective capacity and immune responses of the multivalent vaccines.

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.

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.

Intranasal immunization with the recombinant measles virus encoding the spike protein of SARS-CoV-2 confers protective immunity against COVID-19 in hamsters.

BACKGROUND: As the nasal mucosa is the initial site of infection for COVID-19, intranasal vaccines are more favorable than conventional vaccines. In recent clinical studies, intranasal immunization has been shown to generate higher neutralizing antibodies; however, there is a lack of evidence on sterilizing immunity in the upper airway. Previously, we developed a recombinant measles virus encoding the spike protein of SARS-CoV-2 (rMeV-S), eliciting humoral and cellular immune responses against SARS-CoV-2. OBJECTIVES: In this study, we aim to provide an experiment on nasal vaccines focusing on a measles virus platform as well as injection routes. STUDY DESIGN: Recombinant measles viruses expressing rMeV-S were prepared, and 5 × 105 PFUs of rMeV-S were administered to Syrian golden hamsters via intramuscular or intranasal injection. Subsequently, the hamsters were challenged with inoculations of 1 × 105 PFUs of SARS-CoV-2 and euthanized 4 days post-infection. Neutralizing antibodies and RBD-specific IgG in the serum and RBD-specific IgA in the bronchoalveolar lavage fluid (BALF) were measured, and SARS-CoV-2 clearance capacity was determined via quantitative reverse-transcription PCR (qRT-PCR) analysis and viral titer measurement in the upper respiratory tract and lungs. Immunohistochemistry and histopathological examinations of lung samples from experimental hamsters were conducted. RESULTS: The intranasal immunization of rMeV-S elicits protective immune responses and alleviates virus-induced pathophysiology, such as body weight reduction and lung weight increase in hamsters. Furthermore, lung immunohistochemistry demonstrated that intranasal rMeV-S immunization induces effective SARS-CoV-2 clearance that correlates with viral RNA content, as determined by qRT-PCR, in the lung and nasal wash samples, SARS-CoV-2 viral titers in lung, nasal wash, BALF samples, serum RBD-specific IgG concentration, and RBD-specific IgA concentration in the BALF. CONCLUSION: An intranasal vaccine based on the measles virus platform is a promising strategy owing to the typical route of infection of the virus, the ease of administration of the vaccine, and the strong immune response it elicits.

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.

Potential for transmission of naturally mutated H10N1 avian influenza virus to mammalian hosts and causing severe pulmonary disease.

Subtype H10 avian influenza viruses (AIV) are distributed worldwide in wild aquatic birds, and can infect humans and several other mammalian species. In the present study, we investigated the naturally mutated PB2 gene in A/aquatic bird/South Korea/SW1/2018 (A/SW1/18, H10N1), isolated from wild birds during the 2018-2019 winter season. This virus was originally found in South Korea, and is similar to isolates from mainland China and Mongolia. It had low pathogenicity, lacked a multi-basic cleavage site, and showed a binding preference for α2,3-linked sialic acids. However, it can infect mice, causing severe disease and lung pathology. SW1 was also transmitted by direct contact in ferrets, and replicated in the respiratory tract tissue, with no evidence of extrapulmonary spread. The pathogenicity and transmissibility of SW1 in mouse and ferret models were similar to those of the pandemic strain A/California/04/2009 (A/CA/04, H1N1). These factors suggest that subtype H10 AIVs have zoonotic potential and may transmit from human to human, thereby posing a potential threat to public health. Therefore, the study highlights the urgent need for closer monitoring of subtype H10 AIVs through continued surveillance of wild aquatic birds.

Evidence for the circulation of genotype 4 Eurasian avian-like lineage swine H1N1 influenza A viruses on Korean swine farms, obtained using a newly developed one-step multiplex RT-qPCR assay.

Genotype 4 (G4) Eurasian avian-like lineage swine H1N1 influenza A viruses, which are reassortants containing sequences from the pandemic 2009 H1N1 virus lineage, triple-reassortant-lineage internal genes, and EA-lineage external genes, have been reported in China since 2013. These have been predominant in pig populations since 2016 and have exhibited pandemic potential. In this study, we developed a one-step multiplex RT-qPCR assay targeting the M, HA1, and PB2 genes to detect G4 and related EA H1N1 viruses, with detection limits of 1.5 × 101 copies/µL and 1.15 × 10-2 ng/µL for the purified PCR products and RNA templates, respectively. The specificity of the detection method was confirmed using various influenza virus subtypes. When the one-step multiplex RT-qPCR assay was applied to swine respiratory samples collected between 2020 and 2022 in Korea, a virus related to G4 EA H1N1 strains was detected. Phylogenetic analysis based on portions of all eight genome segments showed that the positive sample contained HA, NA, PB2, NS, and NP genes closely related to those of G4 EA H1N1 viruses, confirming the ability of our assay to accurately detect G4 EA H1N1 viruses in the field.

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.

Human-to-Animal Transmission of SARS-CoV-2, South Korea, 2021.

To investigate SARS-CoV-2 transmission from humans to animals in Seoul, South Korea, we submitted samples from companion animals owned by persons with confirmed COVID-19. Real-time PCR indicated higher SARS-CoV-2 viral infection rates for dogs and cats than previously reported from the United States and Europe. Host-specific adaptations could introduce mutant SARS-CoV-2 to humans.

Corrigendum: Development of a microneedle swab for acquisition of genomic DNA from buccal cells.

[This corrects the article DOI: 10.3389/fbioe.2022.829648.].

Characterization of replication and variations in genome segments of a bat reovirus, BatMRV/B19-02, by RNA-seq in infected Vero-E6 cells.

Mammalian orthoreoviruses (MEVs) that can cause enteric, respiratory, and encephalitic infections have been identified in a wide variety of mammalian species. Here, we report a novel MRV type 1 strain detected in Miniopterus schreibersii that may have resulted from reassortment events. Using next-generation RNA sequencing (RNA-seq), we found that the ratios of the RNA levels of the 10 reovirus segments in infected cells were constant during the late stages of infection. We also discovered that the relative abundance of each segment differed. Notably, the relative abundance of M2 (encoding the µ1 protein) and S4 (encoding the σ3 protein) RNAs was higher than that of the others throughout the infection. Additionally, massive junctions were identified. These results support the hypothesis that defective genome segments are generated and that cross-family recombination occurs. These data may further the study of gene function, viral replication, and virus evolution.

Severe acute respiratory syndrome coronavirus 2 and influenza A virus co-infection alters viral tropism and haematological composition in Syrian hamsters.

The ongoing coronavirus disease 2019 pandemic and its overlap with the influenza season lead to concerns over severe disease caused by the influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) co-infections. Using a Syrian hamster co-infection model with SARS-CoV-2 and the pandemic influenza virus A/California/04/2009 (H1N1), we found (a) more severe disease in co-infected animals, compared to those infected with influenza virus alone but not SARS-CoV-2 infection alone; (b) altered haematological changes in only co-infected animals and (c) altered influenza virus tropism in the respiratory tracts of co-infected animals. Overall, our study revealed that co-infection with SARS-CoV-2 and influenza virus is associated with altered disease severity and tissue tropism, as well as haematological changes, compared to infection with either virus alone.

A COVID-19 Vaccine for Dogs Prevents Reverse Zoonosis.

COVID-19 is caused by severe acute respiratory syndrome virus type 2 (SARS-CoV-2), which can infect both humans and animals. SARS-CoV-2 originated from bats and can affect various species capable of crossing the species barrier due to active mutation. Although reports on reverse zoonosis (human-to-animal transmission) of SARS-CoV-2 remain limited, reverse zoonosis has been reported in many species such as cats, tigers, minks, etc. Therefore, transmission to more animals cannot be ruled out. Moreover, the wide distribution of SARS-CoV-2 in the human population could result in an increased risk of reverse zoonosis. To counteract reverse zoonosis, we developed the first COVID-19 subunit vaccines for dogs, which are representative companion animals, and the vaccine includes the SARS-CoV-2 recombinant protein of whole S1 protein and the receptor-binding domain (RBD). A subunit vaccine is a vaccine developed by purifying only the protein region that induces an immune response instead of the whole pathogen. This type of vaccine is safer than the whole virus vaccine because there is no risk of infection and proliferation through back-mutation of the virus. Vaccines were administered to beagles twice at an interval of 3 weeks subcutaneously and antibody formation rates were assessed in serum. We identified a titer, comparable to that of vaccinated people, shown to be sufficient to protect against SARS-CoV-2. Therefore, the vaccination of companion animals, such as dogs, may prevent reverse zoonosis by protecting animals from SARS-CoV-2; thus, reverse zoonosis of COVID-19 is preventable.

Impeding the Medical Protective Clothing Contamination by a Spray Coating of Trifunctional Polymers.

The risk of fomite-mediated transmission in the clinic is substantially increasing amid the recent COVID-19 pandemic as personal protective equipment (PPE) of hospital workers is easily contaminated by direct contact with infected patients. In this context, it is crucial to devise a means to reduce such transmission. Herein, we report an antimicrobial, antiviral, and antibiofouling trifunctional polymer that can be easily coated onto the surface of medical protective clothing to effectively prevent pathogen contamination on the PPE. The coating layer is formed on the surfaces of PPE by the simple spray coating of an aqueous solution of the trifunctional polymer, poly(dodecyl methacrylate (DMA)-poly(ethylene glycol) methacrylate (PEGMA)-quaternary ammonium (QA)). To establish an optimal ratio of antifouling and antimicrobial functional groups, we performed antifouling, antibacterial, and antiviral tests using four different ratios of the polymers. Antifouling and bactericidal results were assessed using Staphylococcus aureus, a typical pathogenic bacterium that induces an upper respiratory infection. Regardless of the molar ratio, polymer-coated PPE surfaces showed considerable antiadhesion (∼65-75%) and antibacterial (∼75-87%) efficacies soon after being in contact with pathogens and maintained their capability for at least 24 h, which is sufficient for disposable PPEs. Further antiviral tests using coronaviruses showed favorable results with PPE coated at two specific ratios (3.5:6:0.5 and 3.5:5.5:1) of poly(DMA-PEGMA-QA). Moreover, biocompatibility assessments using the two most effective polymer ratios showed no recognizable local or systemic inflammatory responses in mice, suggesting the potential of this polymer for immediate use in the field.

Development of a Microneedle Swab for Acquisition of Genomic DNA From Buccal Cells.

A swab is a tool for obtaining buccal DNA from buccal mucus for biological analysis. The acquisition of a sufficient amount and high quality of DNA is an important factor in determining the accuracy of a diagnosis. A microneedle swab (MN swab) was developed to obtain more oral mucosal tissues non-invasively. Eight types of MN swabs were prepared with varying combinations of patterns (zigzag or straight), number of MNs, intervals of MNs, and sharpness of tips. When MN swab was applied up to 10 times, the tissue amount and DNA yield increased compared to commercial swabs. A zigzag pattern of microneedles was found to be more efficient than a straight pattern and increasing the number of microneedles in an array increased the DNA yield. The MN swab collected about twice the DNA compared to the commercial swab. In an in vivo test using mini pigs, the lower cycle threshold values of mucosal samples collected with MN swabs compared to samples collected with commercial swabs indicated that a greater amount of DNA was collected for SNP genotyping. A polymer MN swab is easy to manufacture by a single molding process, and it has a greater sampling capacity than existing commercial swabs.

Direct neuronal infection of SARS-CoV-2 reveals cellular and molecular pathology of chemosensory impairment of COVID-19 patients.

Patients with recent pandemic coronavirus disease 19 (COVID-19) complain of neurological abnormalities in sensory functions such as smell and taste in the early stages of infection. Determining the cellular and molecular mechanism of sensory impairment is critical to understand the pathogenesis of clinical manifestations, as well as in setting therapeutic targets for sequelae and recurrence. The absence of studies utilizing proper models of human peripheral nerve hampers an understanding of COVID-19 pathogenesis. Here, we report that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly infects human peripheral sensory neurons, leading to molecular pathogenesis for chemosensory impairments. An in vitro system utilizing human embryonic stem cell (hESC)-derived peripheral neurons was used to model the cellular and molecular pathologies responsible for symptoms that most COVID-19 patients experience early in infection or may develop as sequelae. Peripheral neurons differentiated from hESCs expressed viral entry factor ACE2, and were directly infected with SARS-CoV-2 via ACE2. Human peripheral neurons infected with SARS-CoV-2 exhibited impaired molecular features of chemosensory function associated with abnormalities in sensory neurons of the olfactory or gustatory organs. Our results provide new insights into the pathogenesis of chemosensory dysfunction in patients with COVID-19.