Epidemiological and molecular investigations of Salmonella isolated from duck farms in southwest and around area of Shandong, China.

Salmonella is a zoonotic pathogen posing a serious risk to the farming industry and public health due to food animals serving as reservoirs for future contamination and spread of Salmonella. The present study is designed to monitor the contamination status of Salmonella in duck farms and the main control points during breeding. 160 strains of duck-derived Salmonella were isolated from the 736 samples (cloacal swabs, feces, water, feed, soil, air and dead duck embryos) collected in southwest Shandong Province and the province's surrounding area. The percentage of Salmonella-positive samples collected was 21.74 % (160/736), and the greatest prevalence from duck embryo samples (40.00 %, 36/90). These Salmonella were classified into 23 serotypes depending on their O and H antigens, in which S. Typhimurium (30.15 %), S. Kottbus (13.97 %) and S. Enteritidis (10.29 %) were the prevailing serotypes. Subsequently, the molecular subtyping was done. Clustered regularly interspaced short palindromic repeats (CRISPR) analysis showed that 41 strains of S. Typhimurium and 14 strains of S. Enteritidis were classified into 13 and 3 genotypes, respectively. 19 S. Kottbus isolates from different sources featured ST1546, ST198, ST321, and ST1690 by multilocus sequence typing (MLST) analysis, among which ST1546 belongs to S. Kottbus was a new ST. The minimum spanning tree analysis based on the two CRISPR loci and seven MLST loci from all S. Typhimurium, S. Enteritidis and S. Kottbus isolates revealed that duck embryos, feed and water were key control points to the spread of Salmonella along the breeding chain. Meanwhile, the emergence of S. Kottbus in duck flocks was considered a potential public health hazard.

A single-nucleus transcriptomic atlas characterizes cell types and their molecular features in the ovary of adult Nile tilapia.

In fish species, there is limited analysis of signature transcriptome profiles at the single-cell level in gonadal cells. Here, the molecular signatures of distinct ovarian cell categories in adult Nile tilapia (Oreochromis niloticus) were analysed using single-nucleus RNA sequencing (snRNA-seq). We identified four cell types (oogonia, oocytes, granulosa cell, and thecal cell) based on their specifically expressed genes and biological functions. Similarly, we found some key pathways involved in ovarian development that may affect germline-somatic interactions. A cell-to-cell communication network between the distinct cell types was constructed. We found that the bidirectional communication is mandatory for the development of germ cells and somatic cells in fish ovaries, and the granulosa cells and thecal cells play a central regulating role in the cell network in fish ovary. Additionally, we identified some novel candidate marker genes for various types of ovarian cells and also validated them using in situ hybridization. Our work reveals an ovarian atlas at the cellular and molecular levels and contributes to providing insights into oogenesis and gonad development in fish.

Identification and Characterization of a Novel Prophage Lysin against Streptococcus dysgalactiae.

Streptococcus dysgalactiae infection can cause bovine mastitis and lead to huge economic losses for the dairy industry. The abuse of antibiotics has resulted in growing drug resistance of S. dysgalactiae, which causes hard-to-treat infections. Bacteriophage lysin, as a novel antibacterial agent, has great potential for application against drug-resistant gram-positive bacteria. However, few studies have been conducted on the prophage lysin of S. dysgalactiae. In this study, we mined a novel prophage lysin, named Lys1644, from a clinical S. dysgalactiae isolate by genome sequencing and bioinformatic analysis. Lys1644 was expressed and purified, and the lytic activity, antibacterial spectrum, optimal pH and temperature, lytic activity in milk in vitro, and synergistic bacteriostasis with antibiotics were assessed. The Lys1644 prophage lysin showed high bacteriolysis activity specifically on S. dysgalactiae, which resulted in CFU 100-fold reduction in milk. Moreover, Lys1644 maintained high activity over a wide pH range (pH 5-10) and a wide temperature range (4-42 °C). Synergistic bacteriostatic experiments showed that the combination of low-dose Lys1644 (50 µg/mL) with a subinhibitory concentration of aminoglycoside antibiotics (kanamycin or spectinomycin) can completely inhibit bacterial growth, suggesting that the combination of Lys1644 and antibiotics could be an effective therapeutic strategy against S. dysgalactiae infection.

Stimulation of lipopolysaccharide from Pseudomonas aeruginosa following H9N2 IAV infection exacerbates inflammatory responses of alveolar macrophages and decreases virus replication.

H9N2 IAV infection contributed to P. aeruginosa coinfection, causing severe hemorrhagic pneumonia in mink. In this study, the in vitro alveolar macrophage models were developed to investigate the innate immune responses to P. aeruginosa LPS stimulation following H9N2 IAV infection, using MH-S cells. The cytokine levels, apoptosis levels and the viral nucleic acid levels were detected and analyzed. As a result, the levels of IFN-α, IL-1ß, TNF-α, and IL-10 in MH-S cells with P. aeruginosa LPS stimulation following H9N2 IAV infection were significantly higher than those in MH-S cells with single H9N2 IAV infection and single LPS stimulation (P < 0.05), exacerbating inflammatory responses. LPS stimulation aggravated the apoptosis of MH-S cells with H9N2 IAV infection. Interestingly, LPS stimulation influences H9N2 IAV replication and indirectly reduced H9N2 IAV replications in in vitro AMs. It implied that LPS should play an important role in the pathogenesis of H9N2 IAV and P. aeruginosa coinfection.

Establishment of indirect ELISA method for Salmonella antibody detection from ducks based on PagN protein.

BACKGROUND: Salmonella as an important food-borne zoonotic bacterial pathogen, infection in ducks is a recessive infection, however, it can also cause high mortality and threat to food safety. Preventing and controlling the infection and transmission of Salmonella in ducks critically require rapid and sensitive detection method. Full-length Salmonella-specific protein PagN was induced and expressed in E.coil BL21 and was purified as an antigen to establish an indirect enzyme-linked immunosorbent assays (iELSA) detection kit. RESULTS: The recombinant PagN protein has a molecular weight of 43 kDa containing a His-tag, was recognized by an anti-Salmonella positive serum by Western blot assay. The optimal concentration of PagN as a coating antigen in the iELISA was 1 µg/mL, and the optimal dilution of enzyme-labeled secondary antibody was 1:4000 (0.025 µg/mL). The cutoff OD450 value was established at 0.268. The iELISA kit showed high selectivity since no cross-reaction with E. coli, Staphylococcus aureus and Streptococcus was observed. iELISA method and Dot-blot test were performed on 100 clinical sera samples collected from duck farms, and the actual coincidence rate was 89% (89/100). 613 duck serum samples from 3 different farms were tested using established method and commercial ELISA kit. The concordance between the two methods was 94.1%. CONCLUSION: Anti-PagN based iELISA can serve as a useful tool for diagnosis of Salmonella infection.

Substrate access tunnel engineering for improving the catalytic activity of a thermophilic nitrile hydratase toward pyridine and pyrazine nitriles.

Nitrile hydratase (NHase) is able to bio-transform nitriles into amides. As nitrile hydration being an exothermic reaction, a NHase with high activity and stability is needed for amide production. However, the widespread use of NHase for amide bio-production is limited by an activity-stability trade-off. In this study, through the combination of substrate access tunnel calculation, residue conservative analysis and site-saturation mutagenesis, a residue located at the substrate access tunnel entrance of the thermophilic NHase from extremophile Caldalkalibacillus thermarum TA2. A1, ßLeu48, was semi-rationally identified as a potential gating residue that directs the enzymatic activity toward various pyridine and pyrazine nitriles. The specific activity of the corresponding mutant ßL48H towards 3-cyanopyridine, 2-cyanopyridine and cyanopyrazine were 2.4-fold, 2.8-fold and 3.1-fold higher than that of its parent enzyme, showing a great potential in the industrial production of high-value pyridine and pyrazine carboxamides. Further structural analysis demonstrated that the ßHis48 could form a long-lasting hydrogen bond with αGlu166, which contributes to the expansion of the entrance of substrate access tunnel and accelerate substrate migration.

Identification of a common conserved neutralizing linear B-cell epitope in the VP3 protein of waterfowl parvoviruses.

Waterfowl parvoviruses (WPVs) including goose parvovirus (GPV), novel GPV-related virus (NGPV) and Muscovy duck parvovirus (MDPV) cause significant economic losses and epizootic threat to the waterfowl industries, and little is known about the B-cell epitopes of WPVs. In this study, a monoclonal antibody (mAb) 5B5 against the VP3 protein of NGPV was used to identify the possible epitope in the three kinds of WPVs. The mAb 5B5 had neutralizing activities to the three viruses, and reacted with the conserved linear B-cell epitopes of 438LHNPPP443 in VP3 protein of GPV, NGPV and MDPV. To the authors' best knowledge, this is the first report on identification of the common conserved neutralizing linear B-cell epitope on VP3 protein of three different WPVs, which would facilitate the development of a novel immunodiagnostic assay for rapid detection of WPV infection.

Computational Design of Nitrile Hydratase from Pseudonocardia thermophila JCM3095 for Improved Thermostability.

High thermostability and catalytic activity are key properties for nitrile hydratase (NHase, EC 4.2.1.84) as a well-industrialized catalyst. In this study, rational design was applied to tailor the thermostability of NHase from Pseudonocardia thermophila JCM3095 (PtNHase) by combining FireProt server prediction and molecular dynamics (MD) simulation. Site-directed mutagenesis of non-catalytic residues provided by the rational design was subsequentially performed. The positive multiple-point mutant, namely, M10 (αI5P/αT18Y/αQ31L/αD92H/ßA20P/ßP38L/ßF118W/ßS130Y/ßC189N/ßC218V), was obtained and further analyzed. The Melting temperature (Tm) of the M10 mutant showed an increase by 3.2 °C and a substantial increase in residual activity of the enzyme at elevated temperatures was also observed. Moreover, the M10 mutant also showed a 2.1-fold increase in catalytic activity compared with the wild-type PtNHase. Molecular docking and MD simulations demonstrated better substrate affinity and improved thermostability for the mutant.

A novel method to rescue and culture duck Astrovirus type 1 in vitro.

BACKGROUND: Reverse genetics systems enable the manipulation of viral genomes and therefore serve as robust reverse genetic tools to study RNA viruses. A DNA-launched rescue system initiates the transcription of viral genomic cDNA from eukaryotic promoter in transfected cells, generating homogenous RNA transcripts in vitro and thus enhancing virus rescue efficiency. As one of the hazardous pathogens to ducklings, the current knowledge of the pathogenesis of duck astrovirus type 1 (DAstV-1) is limited. The construction of a DNA-launched rescue system can help to accelerate the study of the virus pathogenesis. However, there is no report of such a system for DAstV-1. METHODS: In this study, a DNA-launched infectious clone of DAstV-1 was constructed from a cDNA plasmid, which contains a viral cDNA sequence flanked by hammerhead ribozyme (HamRz) and a hepatitis delta virus ribozyme (HdvRz) sequence at both terminals of the viral genome. A silent nucleotide mutation creating a Bgl II site in the ORF2 gene was made to distinguish the rescued virus (rDAstV-1) from the parental virus (pDAstV-1). Immunofluorescence assay (IFA) and western blot were conducted for rescued virus identification in duck embryo fibroblast (DEF) cells pre-treated with trypsin. The growth characteristics of rDAstV-1 and pDAstV-1 in DEF cells and the tissue tropism in 2-day-old ducklings of rDAstV-1 and pDAstV-1 were determined. RESULTS: The infectious DAstV-1 was successfully rescued from baby hamster kidney (BHK-21) cells and could propagate in DEF cells pre-treated with 1 µg/ml trypsin. Upon infection of DEF cells pre-treated with trypsin, DAstV-1 mRNA copies were identified after serial passaging, and the result showed that rDAstV-1 and pDAstV-1 shared similar replication kinetics. Animal experiment showed that the rDAstV-1 had an extensive tissue tropism, and the virus was capable of invading both the central and the peripheral immune organs in infected ducklings. CONCLUSIONS: An improved DNA-launched reverse genetics system for DAstV-1 was firstly constructed. Infectious virus recovered from BHK-21 cells could propagate in DEF cells pre-treated with trypsin. This is the first report of the successful in vitro cultivation of DAstV-1. We believe this valuable experimental system will contribute to the further study of DAstV-1 genome function and pathogenesis.

Suppression of lymphocyte apoptosis in spleen by CXCL13 after porcine circovirus type 2 infection and regulatory mechanism of CXCL13 expression in pigs.

Porcine circovirus-associated disease (PCVAD) is one of the most serious infectious diseases in pigs worldwide. The primary causative agent of PCVAD is porcine circovirus type 2 (PCV2), which can cause lymphoid depletion and immunosuppression in pigs. Our previous study demonstrated that Laiwu (LW) pigs, a Chinese indigenous pig breed, have stronger resistance to PCV2 infection than Yorkshire × Landrace (YL) pigs. In this study, we found that the YL pigs showed more severe lymphocyte apoptosis and higher viral load in the spleen tissue than LW pigs. To illustrate the differential gene expression between healthy and infected spleens, transcriptome profiling of spleen tissues from PCV2-infected and control YL pigs was compared by RNA sequencing. A total of 90 differentially expressed genes (DEGs) was identified, including CD207, RSAD2, OAS1, OAS2, MX2, ADRB3, CXCL13, CCR1, and ADRA2C, which were significantly enriched in gene ontology (GO) terms related to the defense response to virus and cell-cell signaling, and another nine DEGs, KLF11, HGF, PTGES3, MAP3K11, XDH, CYCS, ACTC1, HSPH1, and RYR2, which were enriched in GO terms related to regulation of cell proliferation or apoptosis. Among these DEGs, the CXCL13 gene, which can suppress lymphocyte apoptosis during PCV2 infection, was significantly down-regulated in response to PCV2 infection in YL but not in LW pigs. By analysis of the regulatory elements in the promoter and 3'-untranslated region (3'-UTR) of porcine CXCL13, we found that the single nucleotide polymorphism (SNP) -1014 G (LW) > A (YL) and the Sus scrofa microRNA-296-5p (ssc-miR-296-5p) participated in regulating CXCL13 expression during the response to PCV2 infection.

Quantitative Proteomic Analysis Uncovers the Mediation of Endoplasmic Reticulum Stress-Induced Autophagy in DHAV-1-Infected DEF Cells.

Autophagy is a tightly regulated catabolic process and is activated in cells in response to stress signals. Despite extensive study, the interplay between duck hepatitis A virus type 1 (DHAV-1) and the autophagy of host cells is not clear. In this study, we applied proteomics analysis to investigate the interaction mechanism between DHAV-1 and duck embryo fibroblast (DEF) cells. In total, 507 differentially expressed proteins (DEPs) were identified, with 171 upregulated proteins and 336 downregulated proteins. The protein expression level of heat shock proteins (Hsps) and their response to stimulus proteins and zinc finger proteins (ZFPs) were significantly increased while the same aspects of ribosome proteins declined. Bioinformatics analysis indicated that DEPs were mainly involved in the "response to stimulus", the "defense response to virus", and the "phagosome pathway". Furthermore, Western blot results showed that the conversion of microtubule-associated protein 1 light chain 3-I (LC3-I) to the lipidation form of LC3-II increased, and the conversion rate decreased when DEF cells were processed with 4-phenylbutyrate (4-PBA). These findings indicated that DHAV-1 infection could cause endoplasmic reticulum (ER) stress-induced autophagy in DEF cells, and that ER stress was an important regulatory factor in the activation of autophagy. Our data provide a new clue regarding the host cell response to DHAV-1 and identify proteins involved in the DHAV-1 infection process or the ER stress-induced autophagy process.

Construction and characterization of an improved DNA-launched infectious clone of duck hepatitis a virus type 1.

BACKGROUND: DNA-launched infectious system is a useful tool with high rescue efficiency that allows the introduction of mutations in specific positions to investigate the function of an individual viral element. Rescued virus particles could be harvested by directly transfecting the DNA-launched recombinant plasmid to the host cells, which will reduce labor and experimental cost by skipping the in vitro transcription assay. METHODS: A total of four overlapping fragments covering the entire viral genome were amplified and then were assembled into a transformation vector based on pIRES2-EGFP to establish the DNA-launched infectious system of duck hepatitis A virus type 1 (DHAV-1), named pIR-DHAV-1. Reverse transcription polymerase chain reaction (RT-PCR) detection, quantitative real-time polymerase chain reaction (qRT-PCR), western blotting assay and indirect immunofluorescence (IFA) were conducted for rescued virus identification. A total of 4.0 µg of recombinant plasmid of pIR-DHAV-1 and in vitro transcribed product of 4.0 µg of RNA-launched infectious clone named pR-DHAV-1 were transfected into BHK-21 cells to analyze the rescue efficiency. Following that, tissue tropism of rescued virus (rDHAV-1) and parental virus (pDHAV-1) were assayed for virulence testing in 1-day-old ducklings. RESULTS: Rescued virus particles carry the designed genetic marker which could be harvested by directly transfecting pIR-DHAV-1 to BHK-21 cells. The qRT-PCR and western blotting results indicated that rDHAV-1 shared similar growth characteristics with pDHAV-1. Furthermore, DNA-launched infectious system possessed much higher rescue efficiency assay compared to RNA-launched infectious system. The mutation at position 3042 from T to C has no impact on viral replication and tissue tropism. From 1 h post infection (hpi) to 48 hpi, the viral RNA copies of rDHAV-1 in liver were the highest among the six tested tissues (with an exception of thymus at 6 hpi), while the viral RNA copy numbers in heart and kidney were alternately the lowest. CONCLUSION: We have constructed a genetically stable and highly pathogenic DNA-launched infectious clone, from which the rescued virus could be harvested by direct transfection with recombinant plasmids. rDHAV-1 shared similar growth characteristics and tissue tropism with pDHAV-1. The DNA-launched infectious system of DHAV-1 possessed higher rescue efficiency compared to the traditional RNA-launched infectious system.

Development of an Indirect Dot-PPA-ELISA using glutamate dehydrogenase as a diagnostic antigen for the rapid and specific detection of Streptococcus suis and its application to clinical specimens.

Streptococcus suis is an important zoonotic pathogen causing infections in pigs and humans. Bacterial surface-related proteins are often explored as potential vaccine candidates and diagnostic antigens. In the present study, glutamate dehydrogenase, a highly conserved immunogenic extracellular protein, was used to establish a dot horseradish peroxidase enzyme-linked staphylococcal protein A immunosorbent assay (Dot-PPA-ELISA) for diagnosis of S. suis infection. The antigen-antibody reaction was optimised through checkerboard titration involving serial dilutions, followed by selective blocking tests and evaluations of cross-reaction, repeatability, and stability. Comparative analysis by using a conventional plate ELISA kit showed that the specificity and sensitivity of the Dot-PPA-ELISA were 97.5 and 96.6%, respectively. Furthermore, dynamic changes in the levels of antibody in rabbits immunised with a propolis inactivated vaccine were monitored by Dot-PPA-ELISA. A total seroprevalence of 73.1% in 305 pig serum samples indicated the method's applicability to detect S. suis infection. Cumulatively, the results suggested that Dot-PAA-ELISA is a convenient, rapid, sensitive, and specific diagnostic method suitable for studying large numbers of samples obtained from clinical and epidemiological studies, thereby helping reduce important economic losses.

Circulation and in vivo distribution of duck hepatitis A virus types 1 and 3 in infected ducklings.

The circulation of duck hepatitis A virus types 1 (DHAV-1) and 3 (DHAV-3) in Southeast Asia has resulted in a continuously changing epidemiological scenario. In this study, a duplex real-time PCR assay for simultaneous quantitative detection of DHAV-1 and DHAV-3 was established, and 200 liver samples from dead ducklings collected from 31 different flocks in Shandong province, China, were tested. Fifty-eight (29.0 %) samples from 13 flocks were positive for DHAV-1 single infection, 113 (56.5 %) samples from 13 other flocks were positive for DHAV-3 single infection, and 24 samples (12.0 %) from four flocks were positive for both viruses. DHAV-1 and DHAV-3 were detected with high viral loads in all of the organs tested (liver, spleen, pancreas, kidney, heart, thymus, bursa of Fabricius and brain). No significant difference in DHAV-1 and DHAV-3 viral loads was found between singly infected and coinfected samples, and there was no correlation between the viral loads of the two viruses and the age of dead ducklings. To the best of our knowledge, this is the first report about the in vivo distribution of DHAV-1 and DHAV-3 in clinically infected ducklings.

Duck hepatitis A virus type 1 infection induces hepatic metabolite and gut microbiota changes in ducklings.

Duck hepatitis A virus type 1 (DHAV-1) can cause severe liver damage in infected ducklings and is a fatal and contagious pathogen that endangers the Chinese duck industry. The objective of this study was to explore the correlation mechanism of liver metabolism-gut microbiota in DHAV-1 infection. Briefly, liquid chromatography-mass spectrometry and 16S rDNA sequencing combined with multivariate statistical analysis were used to evaluate the effects of DHAV-1 infection on liver metabolism, gut microbiota regulation, and other potential mechanisms in ducklings. In DHAV-1-infected ducklings at 72 h postinfection, changes were found in metabolites associated with key metabolic pathways such as lipid metabolism, sugar metabolism, and nucleotide metabolism, which participated in signaling networks and ultimately affecting the function of the liver. The abundance and composition of gut microbiota were also changed, and gut microbiota is significantly involved in lipid metabolism in the liver. The evident correlation between gut microbiota and liver metabolites indicates that DHAV-host gut microbiome interactions play important roles in the development of duck viral hepatitis (DVH).

2A2 protein of DHAV-1 induces duck embryo fibroblasts gasdermin E-mediated pyroptosis.

The duck hepatitis A virus type 1 (DHAV-1) causes rapid death in ducklings by triggering a severe cytokine storm. Pyroptosis is an inflammatory form of programmed cell death that is directly related to an increase in pro-inflammatory cytokine levels. Only a few studies have explored the mechanisms underlying pyroptosis in virus-infected avian cells. In this study, we established an avian infection model in vitro by infecting duck embryo fibroblasts (DEFs) with the virulent DHAV-1 LY0801 strain. DHAV-1 infection induced pyroptosis in the DEFs by activating gasdermin E (GSDME) protein via caspase-3-mediated cleavage. The genes encoding the different structural and non-structural DHAV-1 proteins were cloned into eukaryotic expression plasmids, and the 2A2 protein was identified as the key protein involved in pyroptosis. The HPLC-tandem mass spectrometry (HPLC-MS/MS) and co-immunoprecipitation (Co-IP) analysis established that DHAV-1 2A2 directly interacted with the mitochondrial anti-viral signaling protein (MAVS) both intracellularly and in vitro. Furthermore, we got the results that N-terminal 1-130 aa of 2A2 was involved in the interaction with MAVS and the C-terminal TM domain of MAVS is necessary for the interaction with 2A2 by Co-IP analysis. To our knowledge, this is the first study to reveal that DHAV-1 protein interacts with host proteins to induce pyroptosis. Our findings provide new insights into the molecular pathogenesis of DHAV-1 infection, and a scientific basis for the prevention and treatment of duck viral hepatitis.

Coagulase-negative staphylococci are the main causes of bacterial meningitis in duck.

Since September 2018, serious meningitis has been found on some breeding-duck farms in Shandong Province, China. A large number of ducks exhibit severe neurological symptoms. The ducks were randomly selected for laboratory testing. Duck brain samples were collected using standard sterile techniques, and the staphylococci isolates were detected in 404 (70.14%) out of 576 brain samples. A total of 525 coagulase-negative staphylococci (CoNS) strains were isolated, including 6 species: Staphylococcus sciuri (S. sciuri) (67.24%, 353/525), Staphylococcus epidermidis (S. epidermidis) (9.71%, 51/525), Staphylococcus saprophyticus (S. saprophyticus) (8.38%, 44/525), Staphylococcus lentus (S. lentus) (7.62%, 40/525), Staphylococcus haemolyticus (S. haemolyticus) (2.48%, 13/525), and Staphylococcus xylosus (S. xylosus) (4.57%, 24/525). Mixed strain infections were detected in 121 (29.95%) infected presentations. The antimicrobial susceptibility testing indicated that 40.38% of the isolates exhibited multi-drug resistance, and 53.90% of the strains were methicillin-resistant strains by amplification of the methicillin resistance gene (mecA) gene. Through experimental reproduction of the disease, we determined that the CoNS strains were the leading pathogens causing bacterial meningitis in ducks. Although these CoNS strains does not directly cause the death of sick ducks, they still cause large economic losses due to the retarded growth and development of the sick ducks, lower feed returns, and lower grades of processed duck products. The results of this study will contribute to our understanding of the epidemiology and pathogenesis of CoNS and be helpful in the prevention and treatment of the infection.

Evidence for cross-species transmission of human coronavirus OC43 through bioinformatics and modeling infections in porcine intestinal organoids.

Cross-species transmission of coronaviruses has been continuously posing a major challenge to public health. Pigs, as the major animal reservoirs for many zoonotic viruses, frequently mediate viral transmission to humans. This study comprehensively mapped the relationship between human and porcine coronaviruses through in-depth bioinformatics analysis. We found that human coronavirus OC43 and porcine coronavirus PHEV share a close phylogenetic relationship, evidenced by high genomic homology, similar codon usage patterns and comparable tertiary structure in spike proteins. Inoculation of infectious OC43 viruses in organoids derived from porcine small and large intestine demonstrated that porcine intestinal organoids (pIOs) are highly susceptible to human coronavirus OC43 infection and support infectious virus production. Using transmission electron microscopy, we visualized OC43 viral particles in both intracellular and extracellular compartments, and observed abnormalities of multiple organelles in infected organoid cells. Robust OC43 infections in pIOs result in a significant reduction of organoids viability and widespread cell death. This study bears essential implications for better understanding the evolutionary origin of human coronavirus OC43, and provides a proof-of-concept for using pIOs as a model to investigate cross-species transmission of human coronavirus.

KP177R-based visual assay integrating RPA and CRISPR/Cas12a for the detection of African swine fever virus.

Introduction: Early detection of the virus in the environment or in infected pigs is a critical step to stop African swine fever virus (ASFV) transmission. The p22 protein encoded by ASFV KP177R gene has been shown to have no effect on viral replication and virulence and can serve as a molecular marker for distinguishing field virus strains from future candidate KP177R deletion vaccine strains. Methods: This study established an ASFV detection assay specific for the highly conserved ASFV KP177R gene based on recombinase polymerase amplification (RPA) and the CRISPR/Cas12 reaction system. The KP177R gene served as the initial template for the RPA reaction to generate amplicons, which were recognized by guide RNA to activate the trans-cleavage activity of Cas12a protein, thereby leading to non-specific cleavage of single-stranded DNA as well as corresponding color reaction. The viral detection in this assay could be determined by visualizing the results of fluorescence or lateral flow dipstick (LFD) biotin blotting for color development, and was respectively referred to as fluorescein-labeled RPA-CRISPR/Cas12a and biotin-labeled LFD RPA-CRISPR/Cas12a. The clinical samples were simultaneously subjected to the aforementioned assay, while real-time quantitative PCR (RT-qPCR) was employed as a control for determining the diagnostic concordance rate between both assays. Results: The results showed that fluorescein- and biotin-labeled LFD KP177R RPA-CRISPR/Cas12a assays specifically detected ASFV, did not cross-react with other swine pathogens including PCV2, PEDV, PDCoV, and PRV. The detection assay established in this study had a limit of detection (LOD) of 6.8 copies/µL, and both assays were completed in 30 min. The KP177R RPA-CRISPR/Cas12a assay demonstrated a diagnostic coincidence rate of 100% and a kappa value of 1.000 (p < 0.001), with six out of ten clinical samples testing positive for ASFV using both KP177R RPA-CRISPR/Cas12a and RT-qPCR, while four samples tested negative in both assays. Discussion: The rapid, sensitive and visual detection assay for ASFV developed in this study is suitable for field application in swine farms, particularly for future differentiation of field virus strains from candidate KP177R gene-deleted ASFV vaccines, which may be a valuable screening tool for ASF eradication.

Isolation and characterization of duck sewage source Salmonella phage P6 and antibacterial activity for recombinant endolysin LysP6.

Salmonella is a globally prevalent foodborne pathogen, and adverse events caused by S. Enteritidis and S. Typhimurium are extremely common. With the emergence of drug resistance, there is an urgent need for efficient and specific lytic bacteriophages as alternative to antibiotics in clinical practice. In this study, phage P6 was isolated and screened from effluent and fecal samples from duck farm environments to specifically lyse the duck sources S. Typhimurium and S. Enteritidis. Phage P6 belongs to the genus Lederbergvirus, unclassified Lederbergvirus species. The phage P6 genome did not contained non-coding RNA, virulence genes and drug resistance genes, indicating that phage P6 was biologically safe for clinical applications. Phage P6 lysed 77.78% (28/36) of multidrug-resistant Salmonella and reduced biofilms formed by S. Enteritidis CVCC 3377, 4, and 24, and S. Typhimurium 44 by 44% to 75% within 3 h, and decreased Salmonella in duckling feces by up to 1.64 orders of magnitude. Prokaryotic expression of endolysin LysP6 lysed the chloroform-treated bacterial outer membrane from different serotypes of duck-derived Salmonella and E. coli standard strain ATCC 25922. The host range was expanded compared to phage P6, and the growth of Salmonella was effectively inhibited by LysP6 in conjunction with the membrane permeabilizer EDTA within 24 h. Therefore, phage P6 and phage-derived endolysins LysP6 are suitable for application as potent biocontrol agents to improve poultry health and food safety.