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Since November 2016, severe infectious diseases characterized by gout and kidney swelling and caused by goose astrovirus (GoAstV) have affected goslings in major goose-producing areas in China. In 2021, a similar serious infectious disease broke out in commercial goose farms in Heilongjiang Province, China. In this study, strain HLJ2021 was successfully isolated from goose embryos. Electron microscopy showed that the viral particles are spherical, with a diameter of about 28 nm. The complete genomic length of strain HLJ2021 is 7210 nt, and it encodes three viral proteins. A phylogenetic analysis showed that strain HLJ2021 belongs to GoAstV-2 (G2). Compared with the two original GoAstV strains, amino acid site 540

In 2020, to trace the prevalence and evolution of bovine coronavirus (BCoV) in China, a total of 1383 samples (1016 fecal samples and 367 nasal swab samples) were collected from 1016 cattle exhibiting diarrhea symptoms on dairy farms and beef cattle farms in Heilongjiang Province, Northeast China. All samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) detection of the BCoV N gene, followed by an analysis of its epidemiology and genetic evolution. The results indicated that of the 1016 diarrhea-affected cattle, 15.45% (157/1016) were positive for BCoV, in which positive rates of the fecal and nasal swab samples were 12.20% (124/1016) and 21.53% (79/367), respectively. Of the 367 cattle whose nasal swab samples were collected, the BCoV positive rate of the corresponding fecal samples was 15.26% (56/367). BCoV infection was significantly associated with age, farming pattern, cattle type, farm latitude, sample type, and clinical symptom (p 0.05). Of the 203 BCoV-positive samples, 20 spike (S) genes were successfully sequenced. The 20 identified BCoV strains shared nucleotide homologies of 97.7-100.0%, and their N-terminal domain of S1 subunit (S1-NTD: residues 15-298) differed genetically from the reference strains of South Korea and Europe. The 20 identified BCoV strains were clustered in the Asia-North America group (GII group) in the global strain-based phylogenetic tree and formed three clades in the Chinese strain-based phylogenetic tree. The HLJ/HH-10/2020 strain was clustered into the Europe group (GI group) in the S1-NTD-based phylogenetic tree, exhibiting N

Subversion of the host cell cycle to facilitate viral replication is a common feature of coronavirus infections. Coronavirus nucleocapsid (N) protein can modulate the host cell cycle, but the mechanistic details remain largely unknown. Here, we investigated the effects of manipulation of porcine epidemic diarrhea virus (PEDV) N protein on the cell cycle and the influence on viral replication. Results indicated that PEDV N induced Vero E6 cell cycle arrest at S-phase, which promoted viral replication (P

Organic amendments are usually carried out at field-scale for efficient remediation of organic pollutants; however, their effects on pollutant distribution and the corresponding microbial mechanisms were rarely discussed. The main aim of this study was to compare the fate of benz[a]anthracene in soil amended by several bioremediation materials and underlying microbial mechanisms. In this study, the potential for biotransformation of polycyclic aromatic hydrocarbon in a farmland soil was investigated in microcosms spiked with 14C-benz[a]anthracene as the tracer. A series of organic amendments including lignin, straw, mushroom culture waste, and cow manure, as well as a fungal inoculum of Pleurotus ostreatus, were compared. Illumina sequencing was introduced to reveal the bacterial community in different amendments. The metagenomic function was predicted with the bioinformatics tool of phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt). From the results, the lignin-contained substrates (lignin, straw, mushroom culture waste) showed increase trend in the dissipation of benz[a]anthracene, while Pleurotus ostreatus and cow manure resulted in opposite trends. Specifically, mushroom culture waste mainly increased 14C to the formation of humin-bound residue (39.5 ± 6.8%); lignin amendment significantly (P

Per- and polyfluoroalkyl substances (PFAS) are complex emerging pollutants that are widely distributed in soils. The compositions of PFAS vary according to the emission sources. However, the soil distributions of PFAS from different sources are still poorly understood. In this study, the concentrations and compositions of 18 PFAS in soils close to potential sources (industrial areas, airports, landfills, fire stations and agricultural areas) were investigated in Shanghai. The total PFAS concentrations varied from 0.64 to 294 μg kg

Laccases are capable of rapidly oxidizing benzo[a]pyrene. It is thought that the metabolites with an increase in water solubility caused by the oxidation of benzo[a]pyrene may stimulate the subsequent mineralization. However, to date, there has been no experimental evidence to support this. In this study, the fate of benzo[a]pyrene in soil affected by laccase amendment and the resulting soil bacterial responses were investigated. Laccase amendment promoted benzo[a]pyrene dissipation (15.6%) from soil, accompanied by trace mineralization (0.58 ± 0.02%) and substantial bound residue formation (∼80%). An increase of ∼15% in the bound residue fraction was observed by laccase amendment, which mainly resulted from covalent binding of the residues to humin fraction. During the incubation, the abundance of bacterial 16S rRNA and polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase genes did not change markedly. In contrast, benzo[a]pyrene treated with laccase resulted in a smaller shift in the bacterial community composition, indicating a reduced disturbance to the soil microbial communities. These results here suggest that benzo[a]pyrene contaminated soil can be detoxified by laccase amendment mainly due to the enhanced bound residue formation to soil organic matter via covalent binding.

Bioremediation of polycyclic aromatic hydrocarbon (PAH)-contaminated soil is often limited by inadequate microbial activity and/or low PAH bioavailability. A pot experiment was performed with an aged contaminated arable soil to explore the remediation potential of ryegrass and lignin, which are believed to improve microbial degradation, as well as the biosurfactant rhamnolipid. On average, the concentration of 15 priority PAHs was reduced by 41.7% in the combined ryegrass, lignin and rhamnolipid treatment after 90 days. In contrast, there was no reduction in PAH concentration when each treatment was used alone. The rhamnolipid was beneficial for successful remediation, as shown by the lack of PAH transformation in all non-rhamnolipid treatments. The total amount of PAHs that accumulated in ryegrass biomass was less than 0.1% of the initial amount in the pot. When the theoretical estimate of plant uptake was considered, it suggested that rhizoremediation rather than direct uptake contributed to PAH dissipation. High-throughput sequencing analysis demonstrated that lignin addition substantially changed the fungal and bacterial communities; however, there was no indication that lignin selected for known bacterial PAH degraders. Nevertheless, a [14C]benz(a)anthracene-spiked microcosm experiment showed that lignin amendment led to enhanced PAH mineralization and nonextractable residue formation. Taken together, these findings highlight the importance of selecting bioremediation treatments that can simultaneously stimulate microbial activity and increase PAH bioavailability to achieve remediation effectively. Treatments incorporating rhizoremediation, biostimulation and biosurfactant addition hold promise for detoxifying aged PAH-contaminated agricultural soil.

Thaumarchaeota is an ecologically relevant archaeal phylum which may significantly contribute to global nitrogen cycling. Thaumarchaeotal abundance, composition, and activity can be changed by soil pH and pollutants such as toxic metals. This study aims to examine the responses of thaumarchaeotal community to soil pH variation and polycyclic aromatic hydrocarbon (PAH) pollution which may co-occur in agricultural soils. Field soil samples were collected from agricultural land impacted by both acidification and PAH contamination. Thaumarchaeotal abundance and composition were assessed using molecular approaches targeting 16S rRNA or amoA genes and were linked to environmental factors by correlation and canonical correspondence analysis (CCA). To evaluate the short-term responses of Thaumarchaeota to PAHs, additional soil microcosms amended with either three selected PAHs were established. Changes in thaumarchaeotal communities during the incubation were monitored. A significant correlation between thaumarchaeotal gene abundance and soil pH was observed within field samples, with the I.1a-associated group enriched when pH

Bacteria able to degrade pyrene play a critical role in the biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs). However, the traditional isolation procedure only obtains strains related to the genus Mycobacterium. The aim of the present study was to develop a modified method to isolate taxonomically distinct pyrene-degrading strains. The results indicated that replacing pyrene with phenanthrene in the isolation step improved the isolation efficiency. Using the modified method, six PAH degraders belonging to the genera Bosea, Arthrobacter, Paenibacillus, Bacillus, and Rhodococcus were obtained. They were capable of effectively utilizing pyrene (∼100%) as their sole carbon source, and could co-metabolize the degradation of benzo [a]pyrene (26.9-71.5%). In contrast, a small amount of pyrene (5.6%) and benzo [a]pyrene (8.6%) were degraded by a phenanthrene-degrading Sphingobium sp. NS7 under the same conditions. The difference in PAHs degradation between agar plate culture and liquid culture may lead to the low isolation efficiency in the traditional procedure. Hereditary stability analysis showed that PAH degradation capability of the Bosea, Paenibacillus, and Rhodococcus strains were easily lost without PAH pressure, which may partly explain why those strains were difficult to obtain using the traditional method.

The fate of polycyclic aromatic hydrocarbons (PAHs) determines their potential risk in soil, which may be directly affected by abiotic conditions and indirectly through the changes in decomposer communities. In comparison, the indirect effects on the fate remain largely elusive. In this study, the fate of phenanthrene and benzo[a]pyrene and the corresponding bacterial changes were investigated in three contaminated farmland soils using a 14C tracer method and Miseq sequencing. The results showed that most benzo[a]pyrene was consistently extractable with dichloromethane (DCM) after the 60-day incubation (60.4%–78.2%), while phenanthrene was mainly mineralized to CO2 during the 30-day incubation (40.4%–58.7%). Soils from Guangzhou (GZ) showed a different distribution pattern of 14C-PAHs exemplified by low mineralization and disparate bound residue formation. The PAH fate in the Shenyang (SY) and Nanjing (NJ) soils were similar to each other than to that in the GZ soil. The fate in the GZ soil seemed to be linked to the distinct edaphic properties, such as organic matter content, however soil microbial community could have influenced the distribution pattern of PAHs. This potential role of microorganisms was reflected by the unique changes in the copy numbers of Gram positive RHDα gene, and by the distinct shifts in bacterial community composition during the incubation. A quite different shift in bacterial communities was found in the GZ microcosms which may influence PAH mineralization and non-extractable residue (NER) formation.

Acidification and pollution are two major threats to agricultural ecosystems; however, microbial community responses to co-existed soil acidification and pollution remain less explored. In this study, arable soils of broad pH (4.26–8.43) and polycyclic aromatic hydrocarbon (PAH) gradients (0.18–20.68 mg kg−1) were collected from vegetable farmlands. Bacterial community characteristics including abundance, diversity and composition were revealed by quantitative PCR and high-throughput sequencing. The bacterial 16S rRNA gene copies significantly correlated with soil carbon and nitrogen contents, suggesting the control of nutrients accessibility on bacterial abundance. The bacterial diversity was strongly related to soil pH, with higher diversity in neutral samples and lower in acidic samples. Soil pH was also identified by an ordination analysis as important factor shaping bacterial community composition. The relative abundances of some dominant phyla varied along the pH gradient, and the enrichment of a few phylotypes suggested their adaptation to low pH condition. In contrast, at the current pollution level, PAH showed marginal effects on soil bacterial community. Overall, these findings suggest pH was the primary determinant of bacterial community in these arable soils, indicative of a more substantial influence of acidification than PAH pollution on bacteria driven ecological processes.

The purified C subunit of the recombinant porcine aminopeptidase N (rpAPN-C) protein was used as an immobilized target to screen potential ligands against rpAPN-C from a 12-mer phage display random peptide library. After five rounds of biopanning, five phage clones showed specific binding affinities to rpAPN-C. In 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assays, the phage clone PM1, which contained the HDAISWTHYHPW peptide sequence, had a protective effect against TGEV infection in swine testis cells. Therefore, the HDAISWTHYHPW peptide sequence has a potential use as a small molecular therapeutic agent against TGEV infection.

The variable regions of the heavy chain (VH) and light chain (VL) were amplified by RT-PCR from the hybridoma 6E6, which secretes the monoclonal antibody against PEDV S protein. The VL and VH amplicons were combined using SOE-PCR by a 12 amino acid flexible linker (SSGGGGSGGGGS), which produced the scFv gene (named scFv/6E6). After sequence analysis, the scFv/6E6 gene was cloned into the prokaryotic expression vector pGEX-6p-1 with a GST-tag. The recombinant scFv/6E6 protein was successfully expressed in recombinant Escherichia coli by IPTG induction. Moreover, the recombinant scFv/6E6 protein was purified from the inclusion body form by the gel-cutting measure followed by electroelution and dialysis. The recombinant scFv/6E6 protein reported here will provide some basis for further antiviral drug research based on the scFv molecule.

Ring-hydroxylating dioxygenases (RHDs) play a critical role in the biodegradation of polycyclic aromatic hydrocarbons (PAHs). In this study, genes pdoAB encoding a dioxygenase capable of oxidizing various PAHs with up to five-ring benzo[a]pyrene were cloned from Mycobacterium sp. NJS-P. The α-subunit of the PdoAB showed 99% and 93% identity to that from Mycobacterium sp. S65 and Mycobacterium sp. py136, respectively. An Escherichia coli expression experiment revealed that the enzyme is able to oxidize anthracene, phenanthrene, pyrene and benzo[a]pyrene, but not to fluoranthene and benzo[a]anthracene. Furthermore, the results of in silico analysis showed that PdoAB has a large substrate-binding pocket satisfying for accommodation of HMW PAHs, and suggested that the binding energy of intermolecular interaction may predict the substrate conversion of RHDs towards HMW PAHs, especially those may have steric constraints on the substrate-binding pocket, such as benzo[a]pyrene and benzo[a]anthracene.

Bacterial laccase CueO from Escherichia coli can oxidize polycyclic aromatic hydrocarbons (PAHs); however, its application in the remediation of PAH-contaminated soil mainly suffers from a low oxidation rate and copper dependence. It was reported that a laccase with a higher redox potential tended to have a higher oxidation rate; thus, the present study investigated the oxidation of PAHs using another bacterial laccase CotA from Bacillus subtilis with a higher redox potential (525 mV) than CueO (440 mV). Recombinant CotA was overexpressed in E. coli and partially purified, exhibiting a higher laccase-specific activity than CueO over a broad pH and temperature range. CotA exhibited moderate thermostability at high temperatures. CotA oxidized PAHs in the absence of exogenous copper. Thereby, secondary heavy metal pollution can be avoided, another advantage of CotA over CueO. Moreover, this study also evaluated some unexplained phenomena in our previous study. It was observed that the oxidation of PAHs with bacterial laccases can be promoted by copper. The partially purified bacterial laccase oxidized only two of the 15 tested PAHs, i.e., anthracene and benzo[a]pyrene, indicating the presence of natural redox mediators in crude cell extracts. Overall, the recombinant CotA oxidizes PAHs with high laccase activity and copper independence, indicating that CotA is a better candidate for the remediation of PAHs than CueO. Besides, the findings here provide a better understanding of the oxidation of PAHs using bacterial laccases.

Graphical abstract, Highlights • Protein profile of PEDV-infected Vero cells was analyzed by quantitative proteomics. • 126 differentially expressed proteins were identified in PEDV-infected Vero cells. • The identified proteins provide valuable insight into PEDV-Vero cell interactions., Recent outbreaks of porcine epidemic diarrhea virus (PEDV) have caused widespread concern. The identification of proteins associated with PEDV infection might provide insight into PEDV pathogenesis and facilitate the development of novel antiviral strategies. We analyzed the differential protein profile of PEDV-infected Vero E6 cells using mass spectrometry and an isobaric tag for relative and absolute quantification. A total of 126 proteins were identified that were differentially expressed between the PEDV-infected and mock-infected groups (P

Vero cells are highly susceptible to many viruses in humans and animals, and its membrane proteins (MPs) are responsible for virus entry. In our study, the MP proteome of the Vero cells was investigated using a shotgun LC-MS/MS approach. Six hundred twenty-seven proteins, including a total of 1839 peptides, were identified in MP samples of the Vero cells. In 627 proteins, 307 proteins (48.96%) were annotated in terms of biological process of gene ontology (GO) categories; 356 proteins (56.78%) were annotated in terms of molecular function of GO categories; 414 proteins (66.03%) were annotated in terms of cellular components of GO categories. Of 627 identified proteins, seventeen proteins had been revealed to be virus receptor proteins. The resulting protein lists and highlighted proteins may provide valuable information to increase understanding of virus infection of Vero cells.

Full-length cDNA for the pig metallothionein 1A (pMT1A) gene was synthesized based on the pig MT1A gene sequence in Genbank and cloned into the pMD18-T vector. After sequence analysis and structure prediction, the pMT1A gene was cloned into vector pET-32a (+) containing a His-tag. The recombinant pMT1A (rpMT1A) was expressed in a soluble form using Escherichia coli Rosetta™ (DE3) plysS cells. Western blotting showed that the purified rpMT1A protein bound an anti-His-tag monoclonal antibody. Further investigation revealed that the rpMT1A protein showed high metal-binding activity with the divalent metal ions copper (Cu2+), zinc (Zn2+), and cadmium (Cd2+).

We characterized alcohol dehydrogenase from Rhodococcus erytropolis to catalyze ketoesters or ketones.We cloned alcohol dehydrogenase gene (adh) of 1047 bp from Rhodococcus erythropolis ATCC 4277, inserted the open reading frame of adh into vector pET-22b(+) and expressed in auto-inducing media for 24 h at 15 degrees C. The enzyme activity was determined at 30 degrees C using acetophenone as substrate.Under the above conditions, the specific enzyme activity of crude extract was 2.6 U/mg. The optimal pH was between 6.0 and 6.5 and the enzyme can survived up to 60 degrees C. After incubation at 60 degrees C for 5 h, 80% enzyme activity remained. The optimal substrate among beta-ketoesters examined was ethyl acetoaetate. Ethyl 4-chloroacetoacetate was catalyzed by whole cell in aqueous phase. After chiral liquid chromatography, the product showed (R)-enantioselective.The study shows that the enzyme might have potential in beta-ketoesters transformation on industrial scale.

The entire pig aminopeptidase N (pAPN) gene was amplified by RT-PCR using total RNA extracted from intestinal brush border membrane of a newborn piglet. The amplified products of the pAPN gene were cloned into the vector pMD18-T, generating a recombinant plasmid pMD18-T-pAPN. The C subunit of pAPN (pAPN-C) produced by PCR from the plasmid pMD18-T-pAPN was expressed in Escherichia coli using vector pET-32a with His tag. After confirming reactivity of the recombinant protein pAPN-C to antibody against native pAPN, polyclonal antibody against the recombinant protein pAPN-C was prepared in rabbit using purified protein as immunogen. In Western blot analysis, the antibody elicited by the recombinant protein pAPN-C could recognize the native pAPN. These data demonstrate that the pAPN-C recombinant protein and its polyclonal antibody can provide some basis for further receptor antagonist.

Highlights ► A simple and rapid measure for generation of a scFv library against pAPN using T7Select phage display. ► The scFv library possesses a sufficiently large capacity. ► The scFv library has a sufficient diversity., Porcine aminopeptidase N (pAPN) is a common cellular receptor for swine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV). To investigate single-chain fragment variable (scFv) repertoire against pAPN, the genes encoding the immunoglobulin light chain variable region (VL) and heavy chain variable region (VH) were amplified by reverse transcript polymerase chain reaction (RT-PCR) using a series of degenerate primers from the spleen of BABL/c mice immunized with native pAPN. The VL and VH amplicons were combined randomly by a 12 amino acid flexible linker by splicing by overlap extension PCR (SOE-PCR), which produced the scFv gene repertoire. After ligation of the scFv gene repertoire into the T7Select10-3b vector, a mouse scFv phage library specific for pAPN was produced through in vitro packaging. The primary scFv library against pAPN contained 2.0 × 107 recombinant phage clones, and the titer of the amplified library was 3.6 × 109 pfu/mL. BstNI restriction analysis and DNA sequencing revealed that 28 phage clones from the primary pAPN scFv library showed excellent diversity. The effectiveness of the scFv library against pAPN was verified further by phage ELISA using the recombinant protein of the pAPN C subunit as coating antigen. The construction and evaluation of a murine scFv library against the common receptor pAPN of porcine coronaviruses TGEV and PEDV using the T7 phage display system are described.

Excess application with the poor efficiency of fertilizers is one of the major problems confronting the sustainable agricultural development. Whereas, loss of N and P nutrients from farmlands to surface waters is considered as one of the major sources inducing eutrophication. In order to work out a minimum fertilizer application rate for both keeping conventional yield and reducing nutrient loading of non point source pollution, an experiment of formulated fertilization scheme on rice at different rates and nutrient ratios by incorporating the soil supply potential was conducted in a paddy soil Wunitu in Kunshan City, Suzhou. The soil nutrient can supply a yield at 6.75 t/hm2 at least for one cropping season. Among the fertilization treatments, a maximum yield of 9.79 t/hm2 was obtained at fertilization (N + P) rate of 300 kg/hm2kg N and P at a ratio of N to P being 3:2 or 1:1. However, with much lower fertilization (N + P) rate at 240 kg/hm2 with the same ratio a high yield of 9.69 t/hm2 was reached. The fertilizer efficiency of the later was 3 times as that of the former rate. Thus, a economical and environmentally sound mode of fertilization in such paddy soils as total N and P 240 kg/hm2, with N:P:Kratio being 3:2:0 or 1:1:0 was developed. Further study would be directed to a innovation of combination of reducing fertilization rate with rotated fertilization for alternative crops.

Soil samples of different soil types under different land use patterns and environmental impacts were collected in Wuxian City in the Taihu Lake region. The contents of SAS were determined by Azure A colorimeter. Degradation of LAS was studied in the water and soil mediums by using laboratory incubation. The SAS concentration in the studied soils ranged from 2 to 10 mg.kg-1, varied with soil conditions. Profile distribution of SAS showed decreasing trend down to the soil bottom, where 0.7 mg.kg-1 of SAS was still detected. The degradation of LAS in three dominant types of paddy soils obeyed the primary reaction equation like that of soil organic matter with the half residence time in range of 9-16 days. The data indicates the relative accumulation of SAS in the soils in the Taihu Lake region and longer residence of such synthetic organic compounds in the soil environments.