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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.

Since severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) share similar characteristics with respect to clinical signs, etiology, and transmission, methods for a rapid and accurate differential diagnosis are important. Therefore, the aim of this study was to develop a duplex real-time reverse transcription (RT)-PCR method for the simultaneous detection of these viruses. Primers and probes that target the conserved spike S2 region of human SARS-CoV, MERS-CoV, and their related bat CoVs were designed. The results of real-time RT-PCR showed specific reactions for each virus with adequate detection limits of 50-100 copies/mL and 5-100 copies/mL using pUC57-SARS-pS2 (a template for SARS-CoV) and pGEM-MERS-S2 (a template for MERS-CoV), respectively. In addition, this real-time RT-PCR system was able to detect the target viruses SARS-like bat CoV and MERS-CoV in bat fecal samples and sputum of MERS patients, respectively. Therefore, this newly developed real-time RT-PCR method is expected to detect not only SARS-CoV and MERS-CoV in humans but also several bat CoVs that are closely related to these viruses in bats.

Influenza is an acute respiratory disease and a major health problem worldwide. Since mucosal immunity plays a critical role in protection against influenza virus infection, mucosal immunization is considered a promising vaccination route. However, except for live-attenuated vaccines, there are no effective killed or recombinant mucosal influenza vaccines to date. Outer membrane vesicles (OMVs) are nano-sized vesicles produced by gram-negative bacteria, and contain various bacterial components capable of stimulating the immune system of the host. We generated an OMV with low endotoxicity (fmOMV) by modifying the structure of the lipid A moiety of lipopolysaccharide and investigated its effect as an intranasal vaccine adjuvant in an influenza vaccine model. In this model, fmOMV exhibited reduced toll-like receptor 4-stimulating activity and attenuated endotoxicity compared to that of native OMV. Intranasal injection of the vaccine antigen with fmOMV significantly increased systemic antibody and T cell responses, mucosal IgA levels, and the frequency of lung-resident influenza-specific T cells. In addition, the number of antigen-bearing CD103+ dendritic cells in the mediastinal lymph nodes was significantly increased after fmOMV co-administration. Notably, the mice co-immunized with fmOMV showed a significantly higher protection rate against challenge with a lethal dose of homologous or heterologous influenza viruses without adverse effects. These results show the potential of fmOMV as an effective mucosal adjuvant for intranasal vaccines.

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.

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.

Objectives Staphylococcus cohnii has become increasingly recognized as a potential pathogen of clinically significant nosocomial and farm animal infections. This study was designed to determine the genome of a multidrug-resistant S. cohnii subsp. urealyticus strain SNUDS-2 isolated from a farmed duck in Korea. Methods Genomic DNA was sequenced using the PacBio RS II system. The complete genome was annotated and the presence of antimicrobial resistance and virulence genes were identified. Results The annotated 2,625,703 bp genome contained various antimicrobial resistance genes conferring resistance to β-lactam, aminoglycosides, fluoroquinolones, phenicols and trimethoprim. The virulence-associated three synergistic hemolysins have been identified in the strain. Conclusions To the best of our knowledge, this is the first complete genome of S. cohnii , and will provide important insights into the biodiversity of CoNS and valuable information for the control of this emerging pathogen.

Bats are considered reservoirs of severe emerging human pathogens. Notably, bats host major mammalian paramyxoviruses from the family Paramyxoviridae, order Mononegavirales. In this study, paramyxoviruses were investigated by reverse transcription semi-nested polymerase chain reaction (RT-semi-nested PCR) and reverse transcription polymerase chain reaction (RT-PCR), based on the RT-semi-nested PCR using the consensus paramyxovirus primers targeting the RNA dependent-RNA-polymerase (RdRp) region. In addition, RT-PCR was performed using newly designed primers targeting regions of the fusion protein (F) and hemagglutinin-neuraminidase (HN). The dominant bat species in the collection site of paramyxoviruses were Miniopterus schreibersii, Myotis macrodactylus, Myotis petax, and Rhinolophus ferrumequinum. Paramyxoviruses were detected in four samples in 2016 and six in 2019. Meanwhile, in samples collected in 2017 and 2018, no paramyxoviruses were detected. Phylogenetic analysis based on the partial nucleotide sequences of RdRp, F, and HN proteins suggested that the viruses belonged to the proposed genus Shaanvirus. In conclusion, this study revealed that bat paramyxoviruses in Korea belonged to a single genus and circulated sporadically in several provinces, including Chungbuk, Gangwon, Jeju, and Jeonnam.

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.

We report here the complete genome sequence of Aeromonas rivipollensis KN-Mc-11N1, which was isolated from a wild nutria (Myocastor coypus) in South Korea. Genomic analysis indicated that A. rivipollensis may have zoonotic potential similar to that of other aeromonads, and nutria could be one of the sources of transmission of zoonotic pathogens to humans.

Lacinutrix venerupis has recently been considered a potential fish pathogen. Here, we report the complete genome sequence of L. venerupis DOK2-8, which possesses several virulence-related genes. This strain may be potentially virulent to other marine organisms, and its genomic information will provide important insights into the biodiversity of the genus Lacinutrix .

Summary Bovine coronavirus (BCoV) is a causative agent of respiratory and enteric diseases in cattle and calves. BCoV infection was also evident in captive wild ruminants. Recently, water deer are recognized as the most common wildlife to approach farmhouses and livestock barns in Korea. Therefore, we investigated 77 nasal swab samples from non‐captive wild water deer (Hydropotes inermis) between November 2016 and September 2017 and identified three samples positive for coronavirus, indicating potential for respiratory shedding. The full genomic sequences of the water deer coronavirus were closely related to BCoV (>98%). Therefore, effective biosecurity system in bovine farms would be necessary to prevent contact between farm ruminants and free‐ranging wild water deer.

Gene segments from avian H1N1 influenza A viruses have reassorted with other influenza viruses to generate pandemic strains over the past century. Nevertheless, little effort has been invested in understanding the characteristics of avian H1N1 influenza viruses. Here, we present the genome sequence and a molecular and virological characterization of an avian influenza A virus, A/wild bird/Korea/SK14/2014 (A/SK14, H1N1), isolated from migratory birds in South Korea during the winter season of 2014-2015. Full-genome sequencing and phylogenetic analysis revealed that the virus belongs to the Eurasian avian lineage. Although it retained avian-receptor binding preference, A/SK14 virus also exhibited detectable human-like receptor binding and was able to replicate in differentiated primary normal human bronchial epithelial cells. In animal models, A/SK14 virus was moderately pathogenic in mice, and virus was detected in nasal washes from inoculated guinea pigs, but not in direct-contact guinea pigs. Although A/SK14 showed moderate pathogenicity and no evidence of transmission in a mammalian model, our results suggest that the dual receptor specificity of A/SK14-like virus might allow for a more rapid adaptation to mammals, emphasizing the importance of further continuous surveillance and risk-assessment activities.

Photobacterium damselae subsp. damselae (PDD) is a marine bacterium that can infect a variety of marine animals and humans. Although this bacterium has been isolated from several stranded dolphins and whales, its pathogenic role in cetaceans is still unclear. In this study, we report the complete genome of PDD strain KC-Na-1 isolated from a finless porpoise (Neophocaena asiaeorientalis) rescued from the South Sea (Republic of Korea). The sequenced genome comprised two chromosomes and four plasmids. Among the recently identified major virulence factors in PDD, only phospholipase (plpV) was found in strain KC-Na-1. Interestingly, two genes homologous to Vibrio thermostable direct hemolysin (tdh) and its transcriptional regulator toxR, which are known virulence factors associated with Vibrio parahaemolyticus, were encoded on the plasmid pPDD-Na-1-3. Based on these results, strain KC-Na-1 may have potential pathogenicity in humans and other marine animals and also could act as a potential virulent strain. To the best of our knowledge, this is the first report of the complete genome sequence of P. damselae.

We report here the complete genome of Vibrio coralliilyticus strain 58, which was originally isolated from inactive Pacific oyster (Crassostrea gigas) larvae in Japan. The assembled genome consisted of two chromosomes and one plasmid. These data will provide valuable information and important insights into the biodiversity of this organism.

We report here the complete genome of Vibrio coralliilyticus strain 58, which was originally isolated from inactive Pacific oyster ( Crassostrea gigas ) larvae in Japan. The assembled genome consisted of two chromosomes and one plasmid. These data will provide valuable information and important insights into the biodiversity of this organism.

Here, we report the complete genome sequence of an H1N1 avian influenza virus (AIV), which was isolated from the feces of migratory birds in the Republic of Korea during the winters of 2014 and 2015. Full-genome sequencing and phylogenetic analysis revealed that all genome segments belonged to the Eurasian lineage.

Influenza A viruses must undergo adaptation to acquire virulence in new host species. In mouse models, host adaptation for virulence is generally performed through 5 to 20 lung-to-lung passages. However, highly pathogenic avian influenza viruses (e.g., H5N1 and H7N7 subtypes) have been observed to acquire virulence in mice after only a few in vivo passages. In this study, a low-pathogenic avian influenza H5N2 virus, A/Aquatic Bird/Korea/CN2/2009, which was a prevalent subtype in South Korea in 2009, was serially passaged in mice to evaluate its potential to become highly pathogenic. Unexpectedly, the virus became highly pathogenic in mice after a single lung-to-lung passage, resulting in 100% lethality with a mean death time (MDT) of 6.1 days postinfection (DPI). Moreover, the pathogenicity gradually increased after subsequent in vivo passages with an MDT of 5.2 and 4.2 DPI after the second and third passage, respectively. Our molecular analysis revealed that two amino acid changes in the polymerase complex (a glutamate-to-lysine substitution at position 627 of PB2 and a threonine-to-isoleucine substitution at position 97 of PA) were associated with the increased pathogenicity; the PB2 E627K mutation was responsible for the initial virulence conversion (0 to 100% lethality), while the PA T97I mutation acted as an accessory for the increased virulence.

Since the emergence of highly pathogenic avian influenza (HPAI) H5N1 in Asia, the haemagglutinin (HA) gene of this virus lineage has continued to evolve in avian populations, and H5N1 lineage viruses now circulate concurrently worldwide. Dogs may act as an intermediate host, increasing the potential for zoonotic transmission of influenza viruses. Virus transmission and pathologic changes in HPAI clade 1.1.2 (H5N1)-, 2.3.2.1c (H5N1)- and 2.3.4.4 (H5N6)-infected dogs were investigated. Mild respiratory signs and antibody response were shown in dogs intranasally infected with the viruses. Lung histopathology showed lesions that were associated with moderate interstitial pneumonia in the infected dogs. In this study, HPAI H5N6 virus replication in dogs was demonstrated for the first time. Dogs have been suspected as a "mixing vessel" for reassortments between avian and human influenza viruses to occur. The replication of these three subtypes of the H5 lineage of HPAI viruses in dogs suggests that dogs could serve as intermediate hosts for avian-human influenza virus reassortment if they are also co-infected with human influenza viruses.

In this study, we developed a further-modified outer membrane vesicle (fmOMV) from the ΔmsbB/ΔpagP mutant of Escherichia coli transformed with the plasmid, pLpxF, in order to use it as an adjuvant for pandemic H1N1 (pH1N1) influenza vaccine. We evaluated the efficacy of the pH1N1 influenza vaccine containing the fmOMV in animal models as compared to the commercial adjuvants, alum or AddaVaxTM. The fmOMV-adjuvanted pH1N1 influenza vaccine induced a significant increase in the humoral immunity; however, this effect was less than that of the AddaVaxTM. The fmOMV-adjuvanted vaccine displayed pronounced an enhanced protective efficacy with increased T cell immune response and reduced the viral load in the lungs of the infected mice after challenging them with a lethal dose of the homologous virus. Moreover, it resulted in a significantly higher cross-protection against heterologous virus challenge than that of the pH1N1 vaccine with alum or with no adjuvants. In ferrets, the fmOMV-adjuvanted vaccine elicited a superior antibody response based on the HI titer and efficiently protected the animals from the lethal viral challenges. Taken together, the nontoxic fmOMV could be a promising adjuvant for inducing robust T cell priming into the pH1N1 vaccine and might be broadly applicable to the development of preventive measures against influenza virus infection.

PRL-3, phosphatase of regenerating liver-3, plays a role in cancer progression through its involvement in invasion, migration, metastasis, and angiogenesis. We synthesized rhodanine derivatives, CG-707 and BR-1, which inhibited PRL-3 enzymatic activity with IC50 values of 0.8 μM and 1.1 μM, respectively. CG-707 and BR-1 strongly inhibited the migration and invasion of PRL-3 overexpressing colon cancer cells without exhibiting cytotoxicity. The specificity of the inhibitors on PRL-3 phosphatase activity was confirmed by the phosphorylation recovery of known PRL-3 substrates such as ezrin and cytokeratin 8. The compounds selectively inhibited PRL-3 in comparison with other phosphatases, and CG-707 regulated epithelial-to-mesenchymal transition (EMT) marker proteins. The results of the present study reveal that rhodanine is a specific PRL-3 inhibitor and a good lead molecule for obtaining a selective PRL-3 inhibitor.

Cyclophilins belong to a family of proteins that bind to the immunosuppressive drug cyclosporin A (CsA). Several members of this protein family catalyze the cis-trans isomerization of peptide bonds preceding prolyl residues. The present study describes the biochemical and structural characteristics of a cytosolic cyclophilin (TaCypA-1) cloned from wheat (Triticum aestivum L.). Purified TaCypA-1 expressed in Escherichia coli showed peptidyl-prolyl cis-trans isomerase activity, which was inhibited by CsA with an inhibition constant of 78.3 nM. The specific activity and catalytic efficiency (kcat/Km) of the purified TaCypA-1 were 99.06 ± 0.13 nmol s(-1) mg(-1) and 2.32 × 10(5) M(-1) s(-1), respectively. The structures of apo TaCypA-1 and the TaCypA-1-CsA complex were determined at 1.25 and 1.20 Å resolution, respectively, using X-ray diffraction. Binding of CsA to the active site of TaCypA-1 did not result in any significant conformational change in the apo TaCypA-1 structure. This is consistent with the crystal structure of the human cyclophilin D-CsA complex reported at 0.96 Å resolution. The TaCypA-1 structure revealed the presence of a divergent loop of seven amino acids (48)KSGKPLH(54) which is a characteristic feature of plant cyclophilins. This study is the first to elucidate the structure of an enzymatically active plant cyclophilin which shows peptidyl-prolyl cis-trans isomerase activity and the presence of a divergent loop.

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.

International audience; AbstractEquine influenza virus (EIV) causes a highly contagious disease in horses and other equids. Recently, we isolated an H3N8 EIV (A/equine/Kyonggi/SA1/2011) from a domestic horse in South Korea that exhibited symptoms of respiratory disease, and found that the EIV strain contained a naturally mutated NS gene segment encoding a truncated NS1 protein. In order to determine whether there was an association between the NS gene truncation and viral virulence, a reverse genetics system was applied to generate various NS gene recombinant viruses using the backbone of the H1N1 A/Puerto Rico/8/1934 (PR/8) virus. In a mouse model, the recombinant PR/8 virus containing the mutated NS gene of the Korean H3N8 EIV strain showed a dramatically reduced virulence: it induced no weight loss, no clinical signs and no histopathological lesions. However, the mice infected with the recombinant viruses with NS genes of PR/8 and H3N8 A/equine/2/Miami/1963 showed severe clinical signs including significant weight loss and 100% mortality. In addition, the levels of the pro-inflammatory cytokines; IL-6, CCL5, and IFN-γ, in the lungs of mice infected with the recombinant viruses expressing a full-length NS1 were significantly higher than those of mice infected with the virus with the NS gene from the Korean H3N8 EIV strain. In this study, our results suggest that the C-terminal moiety of NS1 contains a number of virulence determinants and might be a suitable target for the development of a vaccine candidate against equine influenza.

H3N2 canine influenza virus emerged in South Korea in 2007 and subsequently spread to China and Thailand, causing epidemic or endemic respiratory diseases in dogs. Through intermammalian species transmission, the virus has also infected cats. However, no direct evidence of significant genetic evolution has been reported since its first emergence. Here, we describe in depth the genetic and molecular characteristics of the ancestral strain (i.e., the first virus isolate from South Korea) of the H3N2 canine influenza virus currently circulating in East Asia.

The prognosis of pancreatic cancer has not improved despite considerable and continuous effort. Dual-specificity phosphatase 28 (DUSP28) is highly expressed in human pancreatic cancers and exerts critical effects. However, knowledge of its function in pancreatic cancers is extremely limited. Here, we demonstrate the peculiar role of DUSP28 in pancreatic cancers. Analysis using the Gene Expression Omnibus public microarray database indicated higher DUSP28, MUC1, MUC4, MUC5B, MUC16 and MUC20 messenger RNA (mRNA) levels in pancreatic cancers compared with normal pancreas tissues. DUSP28 expression in human pancreatic cancer correlated positively with those of MUC1, MUC4, MUC5B, MUC16 and MUC20. In contrast, there were no significant correlations between DUSP28 and mucins in normal pancreas tissues. Decreased DUSP28 expression resulted in down-regulation of MUC5B and MUC16 at both the mRNA and protein levels; furthermore, transfection with small interfering RNA (siRNA) for MUC5B and MUC16 inhibited the migration and survival of AsPC-1 cells. In addition, transfection of siRNA for MUC5B and MUC16 resulted in a significant decrease in phosphorylation of FAK and ERK1/2 compared with transfection with scrambled-siRNA. These results collectively indicate unique links between DUSP28 and MUC5B/MUC16 and their roles in pancreatic cancer; moreover, they strongly support a rationale for targeting DUSP28 to inhibit development of malignant pancreatic cancer.

Dual-specificity protein phosphatases (DUSPs) constitute a family of protein phosphatase characterized by the ability to dephosphorylate phospho-tyrosyl and phospho-seryl/threonyl residues. Most DUSPs are involved in regulation of cell survival and differentiation. In this study, a human dual-specificity protein phosphatase, DUSP28, was isolated from a human kidney cDNA. The recombinant protein was successfully produed in E.coli and showed sufficient phosphatase activity toward DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate). Various phosphatase inhibitors and divalent metals were tested for their effects on the DUSP28 phosphatase activity. As a result, Zn 2+ was found to strongly inhibit DUSP28 phosphatase activity, suggesting DUSP28 is involved in Zn-related signal transduction pathway. Furthermore, the DUSP28 protein preferred phospho-tyrosyl residues to phospho-threonyl residues, implying its physiological roles in the cellular process.

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.

Map kinase phosphatase 4 (MKP-4), which has been implicated in signalling pathways that negatively regulate glucose uptake, belongs to the dual-specificity phosphatase (DUSP) family. An inherent property of MKPs is an ability to undergo structural rearrangement, transitioning from a partially active to a fully active conformation. Here, a 2.7 Å resolution crystal structure of the catalytic domain of MKP-4 (MKP-4C) is presented. It was determined that the MKP-4C structure seriously deviates from canonical conformations of DUSPs and this characteristic feature results in significant gaps between the catalytic core and several surrounding loops which are unique compared with other MKP counterparts that adopt an active conformation. Using virtual library screening, it was found that inhibitors bind to MKP-4C with high affinity near these gaps. Inhibitors that target other binding sites instead of the active site can be utilized to prevent transition to a fully active conformation. Compounds that are able to make contacts with these sites in MKP-4 would not only provide a beneficial increase in affinity but may also permit greater specificity relative to other protein tyrosine phosphatases.

Highly pathogenic avian influenza virus (HPAIV) infections have occurred continuously and crossed the species barrier to humans, leading to fatalities. A polymerase chain reaction based molecular test is currently the most sensitive diagnostic tool for HPAIV; however, the results must be analyzed in centralized diagnosis systems by a trained individual. This requirement leads to delays in quarantine and isolation. To control the spread of HPAIV, rapid and accurate diagnostics suitable for field testing are needed, and the tests must facilitate a differential diagnosis between HPAIV and low pathogenic avian influenza virus (LPAIV), which undergo cleavage specifically by trypsin‐ or furin‐like proteases, respectively. In this study, a differential avian influenza virus rapid test kit is developed and evaluated in vitro and using clinical specimens from HPAIV H5N1‐infected animals. It is demonstrated that this rapid test kit provides highly sensitive and specific detection of HPAIV and LPAIV and is thus a useful field diagnostic tool for H5N1 HPAIV outbreaks and for rapid quarantine control of the disease., Since highly pathogenic avian influenza virus (HPAIV) has crossed the species barrier to human resulting in fatalities, early and accurate diagnosis between HPAIV and low pathogenic avian influenza virus is considered the key issue. FluSome is a novel surveillance tool with high specificity, which can carry out early preventative procedures for controlling the HPAIV outbreaks.

Cdc25 phosphatases have been considered as attractive drug targets for anticancer therapy due to the correlation of their overexpression with a wide variety of cancers. We have been able to identify five novel Cdc25 phosphatase inhibitors with micromolar activity by means of a structure-based de novo design method with a known inhibitor scaffold. Because the newly discovered inhibitors are structurally diverse and have desirable physicochemical properties as a drug candidate, they deserve further investigation as anticancer drugs. The differences in binding modes of the identified inhibitors in the active sites of Cdc25A and B are addressed in detail.

In mammalian cells, the DNA damage-related histone H2A variant H2A.X is characterized by a C-terminal tyrosyl residue, Tyr-142, which is phosphorylated by an atypical kinase, WSTF. The phosphorylation status of Tyr-142 in H2A.X has been shown to be an important regulator of the DNA damage response by controlling the formation of gammaH2A.X foci, which are platforms for recruiting molecules involved in DNA damage repair and signaling. In this work, we present evidence to support the identification of the Eyes Absent (EYA) phosphatases, protein-tyrosine phosphatases of the haloacid dehalogenase superfamily, as being responsible for dephosphorylating the C-terminal tyrosyl residue of histone H2A.X. We demonstrate that EYA2 and EYA3 displayed specificity for Tyr-142 of H2A.X in assays in vitro. Suppression of eya3 by RNA interference resulted in elevated basal phosphorylation and inhibited DNA damage-induced dephosphorylation of Tyr-142 of H2A.X in vivo. This study provides the first indication of a physiological substrate for the EYA phosphatases and suggests a novel role for these enzymes in regulation of the DNA damage response.

Cdc25 phosphatases have been considered as attractive drug targets for anticancer therapy because of the correlation of their overexpression with a wide variety of cancers. We have been able to identify five novel Cdc25 phosphatase inhibitors with micromolar activity by means of a computer-aided drug design protocol involving the homology modeling of Cdc25A and the virtual screening with the automated AutoDock program implementing the effects of ligand solvation in the scoring function. Because the newly discovered inhibitors are structurally diverse and reveal a significant potency with IC 50 values lower than 10 microM, they can be considered for further development by structure-activity relationship studies or de novo design methods. The differences in binding modes of the identified inhibitors in the active sites of Cdc25A and B are discussed in detail.

The M37 lipase from Photobacterium lipolyticum shows an extremely low activation energy and strong activity at low temperatures, with optimum activity seen at 298 K and more than 75% of the optimum activity retained down to 278 K. Though the M37 lipase is most closely related to the filamentous fungal lipase, Rhizomucor miehei lipase (RML) at the primary structure level, their activity characteristics are completely different. In an effort to identify structural components of cold adaptation in lipases, we determined the crystal structure of the M37 lipase at 2.2 A resolution and compared it to that of nonadapted RML. Structural analysis revealed that M37 lipase adopted a folding pattern similar to that observed for other lipase structures. However, comparison with RML revealed that the region beneath the lid of the M37 lipase included a significant and unique cavity that would be occupied by a lid helix upon substrate binding. In addition, the oxyanion hole was much wider in M37 lipase than RML. We propose that these distinct structural characteristics of M37 lipase may facilitate the lateral movement of the helical lid and subsequent substrate hydrolysis, which might explain its low activation energy and high activity at low temperatures. Proteins 2008. © 2008 Wiley-Liss, Inc.

Cdc25C (cell division cycle 25C) phosphatase triggers entry into mitosis in the cell cycle by dephosphorylating cyclin B-Cdk1. Cdc25C exhibits basal phosphatase activity during interphase and then becomes activated at the G2/M transition after hyperphosphorylation on multiple sites and dissociation from 14-3-3. Although the role of Cdc25C in mitosis has been extensively studied, its function in interphase remains elusive. Here, we show that during interphase Cdc25C suppresses apoptosis signal-regulating kinase 1 (ASK1), a member of mitogen-activated protein (MAP) kinase kinase kinase family that mediates apoptosis. Cdc25C phosphatase dephosphorylates phospho-Thr-838 in the activation loop of ASK1 in vitro and in interphase cells. In addition, knockdown of Cdc25C increases the activity of ASK1 and ASK1 downstream targets in interphase cells, and overexpression of Cdc25C inhibits ASK1-mediated apoptosis, suggesting that Cdc25C binds to and negatively regulates ASK1. Furthermore, we showed that ASK1 kinase activity correlated with Cdc25C activation during mitotic arrest and enhanced ASK1 activity in the presence of activated Cdc25C resulted from the weak association between ASK1 and Cdc25C. In cells synchronized in mitosis following nocodazole treatment, phosphorylation of Thr-838 in the activation loop of ASK1 increased. Compared with hypophosphorylated Cdc25C, which exhibited basal phosphatase activity in interphase, hyperphosphorylated Cdc25C exhibited enhanced phosphatase activity during mitotic arrest, but had significantly reduced affinity to ASK1, suggesting that enhanced ASK1 activity in mitosis was due to reduced binding of hyperphosphorylated Cdc25C to ASK1. These findings suggest that Cdc25C negatively regulates proapoptotic ASK1 in a cell cycle-dependent manner and may play a role in G2/M checkpoint-mediated apoptosis.

The testis- and skeletal-muscle-specific dual-specificity phosphatase (TMDP) is a member of the dual-specificity phosphatase (DSP) subgroup of protein tyrosine phosphatases. TMDP has similar activities toward both tyrosine and threonine phosphorylated substrates, and is supposed to be involved in spermatogenesis. Here, we report the crystal structure of human TMDP at a resolution of 2.4 A. In spite of high sequence similarity with other DSPs, the crystal structure of TMDP shows distinct structural motifs and surface properties. In TMDP, the alpha1-beta1 loop, a substrate recognition motif is located further away from the active site loop in comparison to prototype DSP Vaccinia H1 related phophatase (VHR), which preferentially dephosphorylates tyrosine phosphorylated substrates and down-regulates MAP kinase signaling. Residues in the active site residues of TMDP are smaller in size and more hydrophobic than those of VHR. In addition, TMDP cannot be aligned with VHR in loop beta3-alpha4. These differences in the active site of TMDP result in a flat and wide pocket structure, allowing equal binding of phosphotyrosine and phosphothreonine substrates.

MAP kinase phosphatase 5 (MKP5) is a member of the mitogen-activated protein kinase phosphatase (MKP) family and selectively dephosphorylates JNK and p38. We have determined the crystal structure of the catalytic domain of human MKP5 (MKP5-C) to 1.6 A. In previously reported MKP-C structures, the residues that constitute the active site are seriously deviated from the active conformation of protein tyrosine phosphatases (PTPs), which are accompanied by low catalytic activity. High activities of MKPs are achieved by binding their cognate substrates, representing substrate-induced activation. However, the MKP5-C structure adopts an active conformation of PTP even in the absence of its substrate binding, which is consistent with the previous results that MKP5 solely possesses the intrinsic activity. Further, we identify a sequence motif common to the members of MKPs having low catalytic activity by comparing structures and sequences of other MKPs. Our structural information provides an explanation of constitutive activity of MKP5 as well as the structural insight into substrate-induced activation occurred in other MKPs.

A series of rhodanine derivatives was synthesized and evaluated for their ability to inhibit PRL-3. Benzylidene rhodanine derivative showed good biological activity, while compound 5e was the most active in this series exhibiting an IC50 value of 0.9 microM in vitro and showed a reduced invasion in cell-based assay.

Two biflavonoids, ginkgetin (1) and sciadopitysin (2), were isolated from the MeOH extract of the young branches of Taxus cuspidata, which inhibited phosphatase of regenerating liver-3 (PRL-3) with IC50 values of 25.8 and 46.2 microM, respectively. This is the first report on PRL-3 inhibitors, isolated from natural sources.

In Vietnam, highly pathogenic avian influenza (HPAI), such as that caused by H5N1 viruses, is the most highly contagious infectious disease that has been affecting domestic poultry in recent years. Vietnam might be an evolutionary hotspot and a potential source of globally pandemic strains. However, few studies have reported viruses circulating in the south-central region of Vietnam. In the present study, 47 H5N1-positive samples were collected from both vaccinated and unvaccinated poultry farms in the South Central Coast region of Vietnam during 2013-2014, and their genetic diversity was analyzed. A common sequence motif for HPAI virus was identified at HA-cleavage sites in all samples: either RERRRKR/G (clades 2.3.2.1c and 2.3.2.1a) or REGRRKKR/G (clade 1.1.2). Phylogenetic analysis of HA genes identified three clades of HPAI H5N1: 1.1.2 (n=1), 2.3.2.1a (n=1), and 2.3.2.1c (n=45). The phylogenetic analysis indicated that these Vietnamese clades may have evolved from Chinese and Cambodian virus clades isolated in 2012-2013 but are less closely related to the clades detected from the Tyva Republic, Bulgaria, Mongolia, Japan, and Korea in 2009-2011. Detection of the coexistence of virus clades 2.3.2.1 and the very virulent 1.1.2 in the south-central regions suggests their local importance and highlights concerns regarding their spread, both northwards and southwards, as well as the potential for reassortment. The obtained data highlight the importance of regular identification of viral evolution and the development and use of region-specific vaccines.