A novel defective recombinant porcine enterovirus G virus carrying a porcine torovirus papain-like cysteine protease gene and a putative anti-apoptosis gene in place of viral structural protein genes.
ABSTRACT: Enterovirus G (EV-G) belongs to the family of Picornaviridae. Two types of recombinant porcine EV-Gs carrying papain-like cysteine protease (PLCP) gene of porcine torovirus, a virus in Coronaviridae, are reported. Type 1 recombinant EV-Gs are detected in pig feces in Japan, USA, and Belgium and carry the PLPC gene at the junction site of 2C/3A genes, while PLPC gene replaces the viral structural genes in type 2 recombinant EV-G detected in pig feces in a Chinese farm. We identified a novel type 2 recombinant EV-G carrying the PLCP gene with flanking sequences in place of the viral structural genes in pig feces in Japan. The ~0.3?kb-long upstream flanking sequence had no sequence homology with any proteins deposited in GenBank, while the downstream ~0.9?kb-long flanking sequence included a domain having high amino acid sequence homology with a baculoviral inhibitor of apoptosis repeat superfamily. The pig feces, where the novel type 2 recombinant EV-G was detected, also carried type 1 recombinant EV-G. The amount of type 1 and type 2 recombinant EV-G genomes was almost same in the pig feces. Although the phylogenetic analysis suggested that these two recombinant EV-Gs have independently evolved, type 1 recombinant EV-G might have served as a helper virus by providing viral structural proteins for dissemination of the type 2 recombinant EV-G.
Project description:Enterovirus species G (EV-G) comprises a highly diversity of 20 genotypes that is prevalent in pig populations, with or without diarrhoea. In the present study, a novel EV-G strain (KOR/KNU-1811/2018) that resulted from cross-order recombination was discovered in diagnostic faecal samples from neonatal pigs with diarrhoea that were negative for swine enteric coronaviruses and rotavirus. The recombinant EV-G genome possessed an exogenous 594-nucleotide (198-amino acid) sequence, flanked by two viral 3Cpro cleavage sites at the 5' and 3' ends in its 2C/3A junction region. This insertion encoded a predicted protease similar to the porcine torovirus papain-like cysteine protease (PLCP), which was recently found in the EV-G1, -G2, and -G17 genomes. The complete KNU-1811 genome shared 73.7% nucleotide identity with a prototype EV-G1 strain, but had 83.9%-86.7% sequence homology with the global EV-G1-PLCP strains. Genetic and phylogenetic analyses demonstrated that the Korean recombinant EV-G's own VP1 and inserted foreign PLCP genes are most closely related independently to contemporary chimeric G1-PLCP and G17-PLCP strains respectively. These results implied that the torovirus-derived PLCP gene might have undergone continuous nucleotide mutations in the respective EV-G genome following its independent acquisition through naturally occurring recombination. Our results advance the understanding of the genetic evolution of EV-G driven by infrequent viral recombination events, by which EV-G populations laterally gain an exotic gene encoding a virulence factor from heterogeneous virus families, thereby causing clinical disease in swine.
Project description:Plant proteases are key regulators of plant cell processes such as seed development, immune responses, senescence and programmed cell death (PCD). Apoplastic papain-like cysteine proteases (PL) are hubs in plant-microbe interactions and play an important role during abiotic stresses. The apoplast is a crucial interface for the interaction between plant and microbes. So far, apoplastic maize PL and their function have been mostly described for aerial parts. In this study, we focused on apoplastic PLCPs in the roots of maize plants. We have analyzed the phylogeny of maize PLCPs and investigated their protein abundance after salicylic acid (SA) treatment. Using activity-based protein profiling (ABPP) we have identified a novel root-specific PLCP belonging to the RD21-like subfamily, as well as three SA activated PLCPs. The root specific PLCP CP1C shares sequence and structural similarities to known CP1-like proteases. Biochemical analysis of recombinant CP1C revealed different substrate specificities and inhibitor affinities compared to the related proteases. This study characterized a root-specific PLCP and identifies differences between the SA-dependent activation of PLCPs in roots and leaves.
Project description:The identification and characterization of viruses of the genus Enterovirus in healthy and infected livestock, including cattle and goats, have been increasing. Enterovirus E (EV-E) and Enterovirus F (EV-F) are commonly found in cattle, whereas Enterovirus G (EV-G) is found in goats. In this study, molecular and phylogenetic analyses were performed to determine the prevalence of EVs in cattle and goat feces from Kanchanaburi Province, Thailand. The presence of EVs in water samples and the feces of other animals collected from the areas surrounding cattle and goat farms was also investigated. By use of 5'-untranslated region (5' UTR) real-time reverse transcription-PCR (RT-PCR), EVs were detected in 39.5% of cattle samples, 47% of goat samples, 35.3% of water samples, and one pool of chicken feces. Phylogenetic analysis revealed the presence of EV-E and EV-F in cattle, EV-E and EV-G in goats, and EV-F in water samples and chicken feces. Analysis of enteroviral VP1 sequences from cattle revealed that the EV-E genotypes circulating in the study region were EV-E1, with a possible new genotype that is closely related to EV-E2. Analysis of enteroviral VP1 sequences from goats suggested the circulation of EV-G5 and a possible new genotype that is closely related to EV-G20. Sequence analyses also suggested that although the VP1 sequences from goats were closely related to those of EV-G, which were considered porcine enterovirus sequences, their 5' UTRs form a separated cluster with sequences of sheep and goat origin, suggesting a new classification of the ovine/caprine-specific enterovirus group.IMPORTANCE Possible new EV-E and EV-G genotypes were identified for EVs detected in this study. The EV-E viruses were also successfully isolated from MDBK cells. The goat EV sequence analysis suggested the presence of an ovine/caprine-specific EV group that is different from EV-G of porcine origin. The significance of our research is that it identifies and characterizes possible novel EVs, thereby indicating that enteroviruses in animals are continually evolving. The facts that enteroviruses can persist in the environment, contaminate it for long periods, and be transmitted between animals raise serious concerns regarding this group of viruses as emerging livestock pathogens.
Project description:<h4>Background</h4>The large livestock operations and dense human population of Southeast Asia are considered a hot-spot for emerging viruses.<h4>Objectives</h4>To determine if the pathogens adenovirus (ADV), coronavirus (CoV), encephalomyocarditis virus (EMCV), enterovirus (EV), influenza A-D (IAV, IBV, ICV, and IDV), porcine circovirus 2 (PCV2), and porcine rotaviruses A and C (RVA and RVC), are aerosolized at the animal-interface, and if humans working in these environments are carrying these viruses in their nasal airways.<h4>Study</h4>This cross-sectional study took place in Sarawak, Malaysia among 11 pig farms, 2 abattoirs, and 3 animal markets in June and July of 2017. Pig feces, pig oral secretions, bioaerosols, and worker nasal wash samples were collected and analyzed via rPCR and rRT-PCR for respiratory and diarrheal viruses.<h4>Results</h4>In all, 55 pig fecal, 49 pig oral or water, 45 bioaerosol, and 78 worker nasal wash samples were collected across 16 sites. PCV2 was detected in 21 pig fecal, 43 pig oral or water, 3 bioaerosol, and 4 worker nasal wash samples. In addition, one or more bioaerosol or pig samples were positive for EV, IAV, and RVC, and one or more worker samples were positive for ADV, CoV, IBV, and IDV.<h4>Conclusions</h4>This study demonstrates that nucleic acids from a number of targeted viruses were present in pig oral secretions and pig fecal samples, and that several viruses were detected in bioaerosol samples or in the nasal passages of humans with occupational exposure to pigs. These results demonstrate the need for future research in strengthening viral surveillance at the human-animal interface, specifically through expanded bioaerosol sampling efforts and a seroepidemiological study of individuals with exposure to pigs in this region for PCV2 infection.
Project description:Arcobacter species are prevalent in pigs, and strains have been isolated from pig feces and pork meat; some Arcobacter strains may be porcine abortifacients. Arcobacter suis was recovered from pork meat in Spain. This study describes the whole-genome sequence of the A. suis type strain LMG 26152 (=F41T =CECT 7833T).
Project description:The goal of this study was to evaluate methanogen diversity in animal hosts to develop a swine-specific archaeal molecular marker for fecal source tracking in surface waters. Phylogenetic analysis of swine mcrA sequences compared to mcrA sequences from the feces of five animals (cow, deer, sheep, horse, and chicken) and sewage showed four distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. Only one mcrA sequence (P23-2) showed specificity for swine and therefore was used for environmental testing. PCR primers for the P23-2 clone mcrA sequence were developed and evaluated for swine specificity. The P23-2 primers amplified products in P23-2 plasmid DNA (100%), pig feces (84%), and swine waste lagoon surface water samples (100%) but did not amplify a product in 47 bacterial and archaeal stock cultures and 477 environmental bacterial isolates and sewage and water samples from a bovine waste lagoon and a polluted creek. Amplification was observed in only one sheep sample out of 260 human and nonswine animal fecal samples. Sequencing of PCR products from pig feces demonstrated 100% similarity to pig mcrA sequence from clone P23-2. The minimal amount of DNA required for the detection was 1 pg for P23-2 plasmid, 1 ng for pig feces, 50 ng for swine waste lagoon surface water, 1 ng for sow waste influent, and 10 ng for lagoon sludge samples. Lower detection limits of 10(-6) g of wet pig feces in 500 ml of phosphate-buffered saline and 10(-4) g of lagoon waste in estuarine water were established for the P23-2 marker. This study was the first to utilize methanogens for the development of a swine-specific fecal contamination marker.
Project description:The glnA gene from the human pathogen Streptococcus agalactiae was cloned from a genomic library prepared with the lambda phage vector lambdaDASHII. A 4.6-kb DNA fragment of one of the recombinant phages was subcloned in pUC18. This Escherichia coli clone expressed a 52-kDa protein encoded by a 1,341-bp open reading frame. The nucleotide sequence of the open reading frame and the deduced amino acid sequence shared a significant degree of homology with the sequences of other glutamine synthetases (GS). The highest homology was between our deduced protein and GS of gram-positive bacteria such as Bacillus subtilis, Bacillus cereus, and Staphylococcus aureus. Plasmids with the cloned streptococcal glnA were able to complement E. coli glnA mutants grown on minimal media. Rabbit antisera to streptococcal GS recombinant protein recognized not only the recombinant protein but also a similar-sized band in mutanolysin extracts of all group B streptococcal strains tested, regardless of polysaccharide type or surface protein profile. The amino acid sequence of the deduced protein had similarities to other streptococcal cell-surface-bound proteins. The possible functional role of the immunological features of streptococcal GS is discussed.
Project description:Co-expression of protease inhibitors like the tomato cystatin SlCYS8 is useful to increase recombinant protein production in plants, but key proteases involved in protein proteolysis are still unknown. Here, we performed activity-based protein profiling to identify proteases that are inhibited by SlCYS8 in agroinfiltrated Nicotiana benthamiana. We discovered that SlCYS8 selectively suppresses papain-like cysteine protease (PLCP) activity in both apoplastic fluids and total leaf extracts, while not affecting vacuolar-processing enzyme and serine hydrolase activity. A robust concentration-dependent inhibition of PLCPs occurred in vitro when purified SlCYS8 was added to leaf extracts, indicating direct cystatin-PLCP interactions. Activity-based proteomics revealed that nine different Cathepsin-L/-F-like PLCPs are strongly inhibited by SlCYS8 in leaves. By contrast, the activity of five other Cathepsin-B/-H-like PLCPs, as well as 87 Ser hydrolases, was unaffected by SlCYS8. SlCYS8 expression prevented protein degradation by inhibiting intermediate and mature isoforms of granulin-containing proteases from the Resistant-to-Desiccation-21 (RD21) PLCP subfamily. Our data underline the key role of endogenous PLCPs on recombinant protein degradation and reveal candidate proteases for depletion strategies.
Project description:In vast areas of the ocean, microbes must adapt to the availability of scarce nutrients, and a key strategy for reducing the cellular phosphorus (P) quota is to remodel membranes by replacing phospholipids with non-P surrogate lipids. A metallophosphoesterase, PlcP, is essential for lipid remodeling in cosmopolitan marine bacteria of the Roseobacter (e.g., Phaeobacter sp. strain MED193) and SAR11 (e.g., Pelagibacter sp. strain HTCC7211) clades, and transcription of plcP is known to be induced by P limitation. In order to better understand PlcP-mediated lipid remodeling, we sought to characterize PlcP for its metal ion requirement and to determine its selectivity for native bacterial phospholipids. Here, we report the occurrence of a highly conserved binuclear ion center in PlcPs from MED193 and HTCC7211 and show that manganese is the preferred metal for metallophosphoesterase activity. PlcP displayed high activity towards the major bacterial phospholipids, e.g., phosphatidylglycerol but also phosphatidic acid, a key intermediate in phospholipid biosynthesis. In contrast, phosphatidylserine and phosphatidylinositol, both of which are rare lipids in bacteria, are not preferred substrates. These data suggest that PlcP undertakes a generic lipid remodeling role during the cellular response of marine bacteria to P deficiency and that manganese availability may play a key role in regulating the lipid remodeling process.IMPORTANCE Membrane lipids form the structural basis of all cells. In the marine environment, it is well established that phosphorus availability significantly affects lipid composition in cosmopolitan marine bacteria, whereby non-phosphorus-containing lipids are used to replace phospholipids in response to phosphorus stress. Central to this lipid remodeling pathway is a newly identified phospholipase C-type metallophosphoesterase (PlcP). However, little is known about how PlcP activity is regulated. Here, we determined the role of metal ions in regulating PlcP activity and compared PlcP substrate specificities in PlcP enzymes from two model marine bacteria from the marine Roseobacter clade and the SAR11 clade. Our data provide new insights into the regulation of lipid remodeling in these marine bacteria.
Project description:Mammalian orthoreoviruses (MRVs), which cause gastrointestinal and respiratory illness, have been isolated from a wide variety of mammalian species including bats, minks, pigs and humans. Here we report the isolation and genetic and pathogenic characterization of a novel MRV type 3 (MRV3), named MRV-ZJ2013, from the diarrheic feces of piglets in Zhejiang province, China. Genomic and phylogenetic analysis shows that MRV-ZJ2013 may have originated from reassortments among mink, bat, and pig MRVs, suggesting the hypothesis that interspecies transmission has occurred in pig herds. Neonatal piglets infected with MRV-ZJ2013 displayed mild clinical signs such as poor appetite and soft feces, but vomiting and diarrhea were not observed. Fecal virus shedding was detected only in three out of six piglets, each for one- or two-day post-infection. In contrast, piglets inoculated with a virulent porcine epidemic diarrhea virus (PEDV) strain as the control group had severe signs characterized by acute vomiting and watery diarrhea. These findings suggest that the virulence of MRV-ZJ2013, if any, was likely not significant compared to that of PEDV. A seroepidemiological survey of MRV by means of an indirect enzyme-linked immune-sorbent assay (ELISA) based on a recombinant MRV3 capsid protein sigma1 as antigen revealed a high seroprevalence (77%) in 1037 samples from diarrheic pigs of different ages from 24 herds in seven provinces of east China between 2015 and 2016, indicating that MRV3 is endemic in pig herds in China, and may contribute collectively to enteric disease along with other porcine pathogens.