Detection of Recombinant African Swine Fever Virus Strains of p72 Genotypes I and II in Domestic Pigs, Vietnam, 2023.

African swine fever virus (ASFV) genotype II is endemic to Vietnam. We detected recombinant ASFV genotypes I and II (rASFV I/II) strains in domestic pigs from 6 northern provinces in Vietnam. The introduction of rASFV I/II strains could complicate ongoing ASFV control measures in the region.

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.

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.

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.

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.

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.

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.

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.

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.

Evaluation of a biotin-based surrogate virus neutralization test for detecting postvaccination antibodies against SARS-CoV-2 variants in sera.

A severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) surrogate virus neutralization test (sVNT) was used to determine the degree of inhibition of binding between human angiotensin converting enzyme 2 (hACE2) and the receptor binding domain (RBD) of spike protein by neutralizing antibodies in a biosafety level 2 facility. Here, to improve the sensitivity and specificity of the commercial sVNT, we developed a new biotin based sVNT using biotinylated RBD and HRP conjugated streptavidin instead of HRP conjugated RBD for direct detection in an ELISA assay that strongly correlated to the FDA approved cPass sVNT commercial kit (R2 = 0.8521) and pseudo virus neutralization test (R2 = 0.9006) (pVNT). The biotin based sVNT was evaluated in 535 postvaccination serum samples corresponding to second and third boosts of AZD1222 and BNT162b2 vaccines of the wild type strain. We confirmed that the neutralizing antibodies against SARS-CoV-2 variants in second vaccination sera decreased after a median of 141.5 days. Furthermore, vaccination sera from BNT162b2-BNT162b2 vaccines maintained neutralizing antibodies for longer than those of AZD1222 only vaccination. In addition, both vaccines maintained high neutralizing antibodies in third vaccination sera against Omicron BA.2 after a median of 27 days, but neutralizing antibodies significantly decreased after a median of 141.5 days. Along with the cPass sVNT commercial kit, biotin based sVNTs may also be suitable for specifically detecting neutralizing antibodies against multiple SARS-CoV-2 variants; however, to initially monitor the neutralizing antibodies in vaccinated sera using high throughput screening, conventional PRNT could be replaced by sVNT to circumvent the inconvenience of a long test time.

Magnetic-bead-based DNA-capture-assisted real-time polymerase chain reaction and recombinase polymerase amplification for the detection of African swine fever virus.

African swine fever (ASF) is a deadly disease in swine caused by African swine fever virus (ASFV). The global spread of ASFV has resulted in significant economic losses worldwide. Improved early detection has been the most important first line of defense for preventing ASF outbreaks and for activating control measures. Despite the availability of rapid amplification methods, nucleic acid extraction from specimens still needs to be performed in a laboratory. To facilitate this step, we exploited the strong affinity of biotin-streptavidin binding by functionalizing streptavidin-coated magnetic beads with biotinylated oligonucleotide capture probes to efficiently capture genotype II ASFV DNA directly from crude clinical samples. The captured DNA is suitable for detection using real-time quantitative PCR (qPCR) and recombinase polymerase amplification (RPA). In this study, ASFV DNA was efficiently captured from swine feces, serum, and tissue samples. Both DNA-capture-assisted qPCR and RPA-based detection methods have a limit of detection (LOD) of 102 copies/µl, which is comparable to those of commercially available kits. In addition, an RPA-SYBR Green I method was developed for the immediate visual detection of ASFV DNA, which is time-saving and efficient for resource-limited field settings. In summary, a rapid, versatile, sequence-specific DNA capture method was developed to efficiently capture ASFV DNA from swine clinical samples and subsequent detection by qPCR and RPA, which has the potential to be used for robust screening and surveillance of ASFV and in point-of-care (POC) diagnostics.