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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 10

Coronavirus disease 2019 (COVID-19) is an acute respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Other coronaviruses (CoVs) can also infect humans, although the majority cause only mild respiratory symptoms. Because early diagnosis of SARS-CoV-2 is critical for preventing further transmission events and improving clinical outcomes, it is important to be able to distinguish SARS-CoV-2 from other SARS-related CoVs in respiratory samples. Therefore, we developed and evaluated a novel reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay targeting the genes encoding the spike (S) and membrane (M) proteins to enable the rapid identification of SARS-CoV-2, including several new circulating variants and other emerging SARS-like CoVs. By analysis of in vitro-transcribed mRNA, we established multiplex RT-qPCR assays capable of detecting 5 × 10° copies/reaction. Using RNA extracted from cell culture supernatants, our multiple simultaneous SARS-CoV-2 assays had a limit of detection of 1 × 10° TCID50/mL and showed no cross-reaction with human CoVs or other respiratory viruses. We also validated our method using human clinical samples from patients with COVID-19 and healthy individuals, including nasal swab and sputum samples. This novel one-step multiplex RT-qPCR assay can be used to improve the laboratory diagnosis of human-pathogenic CoVs, including SARS-CoV-2, and may be useful for the identification of other SARS-like CoVs of zoonotic origin.

Sites of live poultry trade and marketing are hot spots for avian influenza virus (AIV) transmission. We conducted active surveillance at a local live poultry market (LPM) in northern Vietnamese provinces in December 2016. Feces samples from the market were collected and tested for AIV. A new reassorted AIV strain was isolated from female chickens, named A/chicken/Vietnam/AI-1606/2016 (H5N6), and was found to belong to group C of clade 2.3.4.4 H5N6 highly pathogenic (HP) AIVs. The neuraminidase gene belongs to the reassortant B type. The viral genome also contained polymerase basic 2 and polymerase acidic, which were most closely related to domestic-duck-origin low pathogenic AIVs in Japan (H3N8) and Mongolia (H4N6). The other six genes were most closely related to poultry-origin H5N6 HP AIVs in Vietnam and had over 97% sequence identity with human AIV isolate A/Guangzhou/39715/2014 (H5N6). The new reassorted AIV isolate A/chicken/Vietnam/AI-1606/2016 (H5N6) identified in this study exemplifies AIVs reassortment and evolution through contact among wild birds, poultry farms, and LPMs. Therefore, active surveillance of AIVs is necessary to prevent potential threats to human and animal health. Supplementary Information The online version contains supplementary material available at 10.1007/s00284-021-02661-z.

In recent years, several novel circular single-stranded DNA viruses have been detected in various mammals, birds, insects, and environmental samples using metagenomic and high-throughput sequencing approaches. In this study, we tested for the presence of circoviruses in 243 bat fecal samples collected between 2018 and 2019 from 48 sampling sites across Korea. To detect circoviruses, nested PCR was performed with degenerate primers targeting a conserved replication-associated protein (rep) gene of circovirus/cyclovirus. Among 243 samples tested, a total of 37 fecal samples from 14 sampling sites were PCR-positive for circoviruses at a frequency rate of 15.23%. We obtained 36 partial rep gene sequences of circoviruses and one complete genome sequence of bat-associated circovirus 12, encompassing a genome size of 2097 nt containing two inversely arranged open reading frames and a conserved nonamer sequence in the apex of a stem-loop structure. In addition, we found four bat species that were harboring circoviruses in Korea based on species identification PCR of circovirus-positive bat fecal samples. Detailed sequence analysis indicated that the bat-associated circovirus sequences identified in this study were related to those of known bat and avian groups of circoviruses. Herein, we report evidence for the presence of bat-associated circoviruses in Korean bats.

African swine fever (ASF) is a highly infectious disease of pigs caused by African swine fever virus (ASFV). In order to identify potential genetic variations among ASFV strains circulating in Vietnam, 26 ASFV isolates from organs and blood samples collected from domestic pigs from 23 different provinces of northern, central and southern Vietnam during 2019-2020 ASF outbreaks were genetically characterized. Nucleotide sequences were determined for a portion of the B646L (p72) gene, the complete E183L (p54) gene, the variable region of EP402R (CD2v), the central variable region (CVR) of pB602L, and a tandem repeat sequence (TRS) between the I73R and I329L genes. Analysis of the partial B646L (p72) and EP402R (CD2v) gene sequences and the full-length E183L (p54) gene sequence showed that all 26 ASFV isolates belonged to genotype II and serotype VIII and that they were identical to the strain Georgia/2007/1 and all ASFV strains sequenced in China. The TRS between the I73R and I329L genes contained a 10-nucleotide insertion that was observed in the Chinese ASFV strain CN201801 isolated from domestic pigs in 2018, but not in the Georgia/2007/1 and China/Jilin/2018/boar strains isolated from wild boar in China. This is the first intra-epidemic genome analysis reported for the ASFV strains circulating in Vietnam.

Aquatic birds are known to be a reservoir for the most common influenza A viruses (IAVs). In the annual surveillance program, we collected the feces of migratory birds for the detection of IAVs in South Korea in November 2016. A novel reassorted H3N3 avian influenza virus (AIV) containing genes from viruses of wild and domestic birds was identified and named A/aquatic bird/South Korea/sw006/2016(H3N3). The polymerase basic 2 (PB2) and non-structural (NS) genes of this isolate are most closely related to those of wild-bird-origin AIV, while the polymerase basic 1 (PB1), polymerase acidic (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), and matrix (M) genes are most closely related to those of domestic-bird-origin AIV. A/aquatic bird/South Korea/sw006/2016 contains PA, NP, M, and NS genes were most closely related to those of AIV subtype H4 and PB2, PB1, and HA genes that are most closely related to those of AIV subtype H3N8, while the NA gene was most closely related to those of subtype H10, which was recently detected in humans in China. These results suggest that novel reassortment of AIV strains occurred due to interaction between wild and domestic birds. Hence, we emphasize the need for continued surveillance of avian influenza virus in bird populations.

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.

Fraser’s dolphin (Lagenodelphis hosei Fraser, 1956) is one of the least known and most recently described species in the family Delphinidae, and its complete mitogenome has yet to be determined. Herein, we report the first complete mitogenome of L. hosei. The newly obtained 16,390 bp L. hosei mitogenome showed the highest average nucleotide identity value with the Burrunan dolphin (Tursiops australis, 97.3%), and its overall structure was similar to those from other delphinid species. Multigene phylogeny using the 13 protein coding genes in the mitogenome revealed that L. hosei was well clustered with other species within the subfamily Delphininae, and the overall tree topology was congruent with recent phylogenetic studies of this species. These results provide important insights for conservation genetics and further evolutionary studies of the oceanic dolphins.

In Korea, H5-subtype highly pathogenic avian influenza (HPAI) has caused huge economic losses in poultry farms through outbreaks of H5N1 since 2003, H5N8 since 2013 and H5N6 since 2016. Although it was reported that long-distance migratory birds may play a major role in the global spread of avian influenza viruses (AIVs), transmission from such birds to poultry has not been confirmed. Intermediate hosts in the wild also may be a potential factor in viral transmission. Therefore, a total of 367 serum samples from wild animals were collected near major migratory bird habitats from 2011 to 2016 and tested by AIV-specific blocking ELISA and hemagglutination inhibition (HI) test. Two mammalian and eight avian species were seropositive according to the ELISA test. Among these, two mammalian (Hydropotes inermis and Prionailurus bengalensis) and three avian (Aegypius monachus, Cygnus cygnus, and Bubo bubo) species showed high HI titres (> 1,280) against one or two H5-subtype AIVs. As H. inermis (water deer), P. bengalensis (leopard cat), and B. bubo (Eurasian eagle owl) are indigenous animals in Korea, evidence of H5-subtype AIV in these animals implies that continuous monitoring of indigenous animals should be followed to understand interspecies transmission ecology of H5-subtype influenza viruses.

Stejneger’s beaked whale (Mesoplodon stejnegeri, True in Proc US Natl Mus 8:584–585, 1885) is one of the least known cold temperate and subarctic species of the beaked whales (Zhiphiidae) found in the North Pacific Ocean, and its complete mitogenome has yet to be investigated. Herein, we report the complete mitogenome of M. stejnegeri stranded in South Korea. Although its overall structure was similar to those from the other cetacean species, the obtained 16,349 bp mitogenome showed only 92.5% average nucleotide identity value with its closest relative, Blainville’s beaked whale, M. densirostris, and less with other species in the Zhiphiidae family. Multigene phylogeny using the whole mitogenome revealed that M. stejnegeri was well separated from other Mesoplodon species, and the overall tree topology was in accordance with recent phylogenetic analyses. These results provide information fundamental for the genetic conservation and further evolutionary studies of the beaked whales.

Although several strandings of the leatherback turtle (Dermochelys coriacea Vandelli, 1761) have been reported in Korea, its haplotype or mitogenome have yet to be investigated. Herein, we report the first complete mitogenome of D. coriacea. The 16,501-bp sequenced mitogenome is similar to those of other marine turtles, and the particular genetic features reported in birds and turtles were also found in ND3 and ATP8. The comparison of the control region verified that D. coriacea stranded in Korea belonged to the haplotype JD1 (identical to haplotypes I and Dc9.1). Multigene phylogeny revealed that D. coriacea was well separated from other Cheloniidae species, and the overall tree topology was congruent with the recent phylogenetic analysis of marine turtles. These results provide information fundamental for genetic and conservation studies on leatherbacks, especially the critically endangered West Pacific Ocean subpopulation.

The nutria or coypu (Myocastor coypus, Molina, 1782) is one of the world’s worst invasive alien species. Nutria have significant harmful effects on natural ecosystems and agricultural industries on a global scale, including in South Korea. Herein, we report the complete mitochondrial genome of M. coypus. The 16,874 bp sequenced genome exhibited a typical rodential mitochondrial gene arrangement, and consisted of the typical set of 37 genes, one replication origin, and a D-loop. The mitogenome of nutria displayed the highest similarity with that of Cuvier’s spiny rat, Proechimys cuvieri, and was distinct from that of the house mouse, Mus musculus. Multigene phylogenetic analysis also revealed that M. coypus was well clustered with other species in Myocastorini, and the overall tree topology accorded well with recent molecular phylogenetic analysis of South American spiny rats. The results will provide information fundamental for the scientific management of the nutria populations in South Korea.

The Pacific white-sided dolphin (Lagenorhynchus obliquidens Gill, 1865) is one of the most abundant, widely distributed delphinids in the North Pacific Ocean, whose existence is threatened by fisheries and environmental contamination. Herein, we report the first complete mitochondrial genome of L. obliquidens. The 16,392-bp sequenced genome exhibited typical cetacean mitochondrial gene arrangement, consisted of the typical set of 37 genes, one replication origin, and a D-loop. As expected, the genome displayed the highest similarity with that of Cephalorhynchus heavisidii and was distinct from that of L. albirostris. Multigene phylogeny also revealed that L. obliquidens was closely related to C. heavisidii, thus suggesting that the genus Lagenorhynchus is polyphyletic, in accordance with the results of recent molecular phylogenetic studies. The results provide information fundamental for genetic and conservation studies for L. obliquidens.

The beluga whale Delphinapterus leucas (Mammalia: Cetacea: Monodontidae) is a small, toothed, Arctic and sub-Arctic cetacean threatened by human activities and environmental contamination. Herein, we report the first complete mitochondrial genome sequence of D. leucas, comprising 16,386 bp encoding the typical set of 37 mitochondrial genes, one replication origin, and a putative control region. It exhibits the classic vertebrate mitochondrial gene arrangement and demonstrated the highest similarity with the mitochondrial genome of the narwhal Monodon monoceros, a member of the only other extant genus in the Monodontidae family. Phylogenetic analysis using the 35 available Odontoceti (toothed whale) mitochondrial genomes clustered D. leucas with M. monoceros, with a tree topology that accorded well with traditional and recent molecular taxonomies. These data will provide information fundamental to conservation genetic studies of this endangered Arctic whale.

An outbreak of a canine influenza virus (CIV) H3N2 reassortant derived from pandemic (pdm) H1N1 and CIV H3N2 in companion animals has underscored the urgent need to monitor CIV infections for potential zoonotic transmission of influenza viruses to humans. In this study, we assessed the virulence of a novel CIV H3N2 reassortant, VC378, which was obtained from a dog that was coinfected with pdm H1N1 and CIV H3N2, in ferrets, dogs, and mice. Significantly enhanced virulence of VC378 was demonstrated in mice, although the transmissibility and pathogenicity of VC378 were similar to those of classical H3N2 in ferrets and dogs. This is notable because mice inoculated with an equivalent dose of classical CIV H3N2 showed no clinical signs and no lethality. We found that the PA and NS gene segments of VC378 were introduced from pdmH1N1, and these genes included the amino acid substitutions PA-P224S and NS-I123V, which were previously found to be associated with increased virulence in mice. Thus, we speculate that the natural reassortment between pdm H1N1 and CIV H3N2 can confer virulence and that continuous surveillance is needed to monitor the evolution of CIV in companion animals.

The bat paramyxovirus B16-40 was first isolated in Korea in this study. Using the isolated virus, we could obtain not only genomic information, but also several biological characteristics of the virus. In the phylogenetic analysis, the virus was found to belong to the recently proposed genus Shaanvirus. Through sequence analyses and in vitro testing, the isolated virus was also found to have haemagglutinin-neuraminidase (HN) protein as one of the structural proteins. When mouse antiserum was generated against the isolated virus and tested, it was cross-reactive to human parainfluenza virus 1 in an indirect immunofluorescence assay but could not cross-neutralize human parainfluenza virus 1. In addition, the bat paramyxovirus B16-40 was not infectious in the mouse model. Collectively, this study provided basic information on further classification of the bat paramyxovirus B16-40 and related viruses in the proposed genus Shaanvirus.

Dual-specificity phosphatase 26 (DUSP26) has recently proved to be a promising therapeutical target for the treatment of human cancers. Here, we report the first example for a successful application of the structure-based virtual screening approach to identify nine novel inhibitors of DUSP26. These inhibitors are also screened for having desirable physicochemical properties as drug candidates and reveal a high potency with IC50 values ranging from 8 to 42 μM. Therefore, they deserve consideration for further development by structure–activity relationship (SAR) studies to optimize the anticancer activities. Structural features relevant to the stabilization of the newly identified inhibitors in the active site of DUSP26 are addressed in detail.

Glutathione peroxidases (Gpxs) are the key anti-oxidant enzymes found in Saccharomyces cerevisiae. Among the three Gpx isoforms, glutathione peroxidase 3 (Gpx3) is ubiquitously expressed and modulates the activities of redox-sensitive thiol proteins involved in various biological reactions. By using a proteomic approach, glyceralde-hyde-3-phosphate dehydrogenase 2 (GAPDH2; EC 1.2.1.12) was found as a candidate protein for interaction with Gpx3. GAPDH, a key enzyme in glycolysis, is a multi-functional protein with multiple intracellular localizations and diverse activities. To validate the interaction between Gpx3 and GAPDH2, immunoprecipitation and a pull-down assay were carried out. The results clearly showed that GAPDH2 interacts with Gpx3 through its carboxyl-terminal domain both in vitro and in vivo. Additionally, Gpx3 helps to reduce the S-nitrosylation of GAPDH upon nitric oxide (NO) stress; this subsequently increases cellular viability. On the basis of our findings, we suggest that Gpx3 protects GAPDH from NO stress and thereby contributes to the maintenance of homeostasis during exposure to NO stress.

Heat shock protein 33 (Hsp33) inhibits aggregation of partially denatured proteins during oxidative stress. The chaperone activity of Hsp33 is unique among heat shock proteins because the activity is reversibly regulated by cellular redox status. We report here the crystal structure of the N-terminal region of Hsp33 fragments with constitutive chaperone activity. The structure reveals that the N-terminal portion of Hsp33 forms a tightly associated dimer formed by a domain crossover. A concave groove on the dimeric surface contains an elongated hydrophobic patch that could potentially bind denatured protein substrates. The termini of the subunits are located near the hydrophobic patch, indicating that the cleaved C-terminal domain may shield the hydrophobic patch in an inactive state. Two of the four conserved zinc-coordinating cysteines are in the end of the N-terminal domain, and the other two are in the cleaved C-terminal domain. The structural information and subsequent biochemical characterizations suggest that the redox switch of Hsp33 occurrs by a reversible dissociation of the C-terminal regulatory domain through oxidation of zinc-coordinating cysteines and zinc release.