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.

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.

Pipetting-based immunoassay for point-of-care testing: Application for detection of the influenza A virus.

Point-of-care tests (POCT) for pathogens are considered important for low-resource countries and facilities. Although lateral flow immunoassays (LFIA) have many advantages including speed and ease of use, their sensitivity is limited without specific equipment. Furthermore, their response cannot be enhanced through enzymatic reactions. Owing to these limitations, LFIAs have not yet been generally adopted as the standard protocol for in vitro analysis of infectious pathogens. We aimed to develop a novel pipetting-based immunoassay using a removable magnetic ring-coupled pipette tip. The "magnetic bead-capture antibody-targeted protein complex" was simply purified by pipetting and quantified by enzymatic colour development or using a lateral flow system. This pipetting-based immunoassay was applied to detect the nucleoprotein (NP) of the influenza A virus. Using an HRP-conjugated monoclonal antibody as a probe, the assay allowed for specific and sensitive detection. Furthermore, when this assay was applied exclusively for antigen capture in the lateral flow system, the limit of detection improved 100-fold and displayed greater sensitivity than the lateral flow system alone. Therefore, the pipetting-based immunoassay may be potentially used as a sensitive POCT to clinically detect a target antigen.

Identification of canine norovirus in dogs in South Korea.

BACKGROUND: Canine noroviruses (CaNoVs) are classified into genogroups GIV, GVI, and GVII and have been detected in fecal samples from dogs since their first appearance in a dog with enteritis in Italy in 2007. CaNoVs may be a public health concern because pet animals are an integral part of the family and could be a potential reservoir of zoonotic agents. Nonetheless, there was no previous information concerning the epidemiology of CaNoV in South Korea. In the present study, we aimed to detect CaNoV antigens and to investigate serological response against CaNoV in dogs. RESULTS: In total, 459 fecal samples and 427 sera were collected from small animal clinics and animal shelters housing free-roaming dogs in geographically distinct areas in South Korea. For the detection of CaNoV, RT-PCR was performed using target specific primers, and nucleotide sequences of CaNoV isolates were phylogenetically analyzed. Seroprevalence was performed by ELISA based on P domain protein. CaNoVs were detected in dog fecal samples (14/459, 3.1%) and were phylogenetically classified into the same cluster as previously reported genogroup GIV CaNoVs. Seroprevalence was performed, and 68 (15.9%) of 427 total dog serum samples tested positive for CaNoV IgG antibodies. CONCLUSION: This is the first study identifying CaNoV in the South Korean dog population.

Reduction of 4(5)-Methylimidazole Using Cookie Model Systems.

The objective of this study was to determine the reduction of 4(5)-methylimidazole (4-MI) under various baking conditions. For 4-MI analysis, an analytical method using gas chromatography-mass spectrometry was developed. The developed method was validated with linearity (r2 > 0.999), recovery (101% to 103%, 3 levels), and precision (1.5% to 4.3%, 3 levels). Limits of detection and quantification were 18.5 and 56.0 µg/kg, respectively. This method was used to monitor the level of 4-MI in 11 commercial cookies, which ranged from 71.5 to 1254.8 µg/kg. Time and temperature were modified in the cookie model system to reduce 4-MI. The largest reduction in 4-MI (56%) was achieved by baking at 140 °C for 8 min; however the cookies baked at this condition were not well accepted by consumers. With combination of consumer liking test result, baking cookies at 140 °C for 16 min is optimal for 4-MI reduction (28% reduction), while it has minimal impact on consumer acceptance. A strong correlation (r2 = 0.9981) was found between caramel colorant and 4-MI in the cookie model system. PRACTICAL APPLICATION: A consumer awareness toward toxicity of 4-MI has been arising, and method to reduce the levels of 4-MI in food products are being developed in many studies. Yet, these reduction studies in food model systems only focused on use of food additives for 4-MI reduction. Current study investigated the use of process modification on 4-MI reduction in cookie, and suggested that baking cookies longer at lower temperature, in turn, reduces the levels of 4-MI in cookies without compromising consumer acceptance. Finding from current study can practically aid bakery industry to ensure safety of bakery products without affecting consumer likings.