In vitro suppression of porcine epidemic diarrhea virus by Panax notoginseng saponins: assessing antiviral potential.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high mortality in neonatal suckling piglets, leading to significant economic losses to the swine industry. Panax notoginseng saponins (PNS) are bioactive extracts derived from the P. notoginseng plant. In this study, we investigated the anti-PEDV effect of PNS by employing various methodologies to assess their impact on PEDV in Vero cells. Using a CCK-8 (Cell Counting Kit-8) assay, we found that PNS had no significant cytotoxicity below the concentration of 128 µg/mL in Vero cells. Using immunofluorescence assays (IFAs), an enzyme-linked immunosorbent assay (ELISA), and plaque formation assays, we observed a dose-dependent inhibition of PEDV infection by PNS within 24-48 hours postinfection. PNS exerts its anti-PEDV activity specifically at the genome replication stage, and mRNA-seq analysis demonstrated that treatment with PNS resulted in increased expression of various genes, including IFIT1 (interferon-induced protein with tetratricopeptide repeats 1), IFIT3 (interferon-induced protein with tetratricopeptide repeats 3), CFH (complement factor H), IGSF10 (immunoglobulin superfamily member 10), ID2 (inhibitor of DNA binding 2), SPP1 (secreted phosphoprotein 1), PLCB4 (phospholipase C beta 4), and FABP4 (fatty acid binding protein 4), but it resulted in decreased expression of IL1A (interleukin 1 alpha), TNFRSF19 (TNF receptor superfamily member 19), CDH8 (cadherin 8), DDIT3 (DNA damage inducible transcript 3), GADD45A (growth arrest and DNA damage inducible alpha), PTPRG (protein tyrosine phosphatase receptor type G), PCK2 (phosphoenolpyruvate carboxykinase 2), and ADGRA2 (adhesion G protein-coupled receptor A2). This study provides insights into the potential mechanisms underlying the antiviral effects of PNS. Taken together, the results suggest that the PNS might effectively regulate the defense response to the virus and have potential to be used in antiviral therapies.

Development of a Competitive Enzyme-Linked Immunosorbent Assay Targeting the-p30 Protein for Detection of Antibodies against African Swine Fever Virus.

African swine fever (ASF) is a highly contagious hemorrhagic viral disease of domestic and wild pigs of all breeds and ages, caused by African swine fever virus (ASFV). Due to the absence of a safe and efficacious vaccine, accurate laboratory diagnosis is critical for the control of ASF prevention. The p30 protein is immunogenic and stimulates a high level of antibody response to ASFV infection. We developed a panel of 4 monoclonal antibodies (mAbs) against p30 protein, and mAb-2B4 showed the highest percent of inhibition (PI) of 70% in the solid phase blocking ELISA (bELISA). Epitope mapping revealed the mAb-2B4 recognized the epitope of aa 12-18 of p30, which is conserved among various ASFV genotypes. Subsequently, a competitive enzyme-linked immunosorbent assay (cELISA) was established using HRP-labeled mAb-2B4. The cutoff for discrimination between 98 negative sera and 40 positive sera against ASFV was determined by plotting a receiver operating characteristic (ROC) curve. It yielded the area under the curve (AUC) of 0.998, and a diagnostic specificity of 97.96% and a sensitivity of 97.5% were achieved when the cutoff value was determined at 37.1%. Furthermore, the results showed an excellent repeatability of the established cELISA and no cross-reaction to antisera against six other pig pathogens. Additionally, the cELISA detected a titer of 1:256 in the positive standard serum. Overall, mAb-2B4 showed a conserved epitope and high ability to be inhibited by positive sera in ASFV antibody detection. The cELISA based on HRP-labeled mAb-2B4 offers an alternative to other assays for a broader diagnostic coverage of ASFV infection.

Comparison of pathogenicity of porcine deltacoronavirus CZ2020 from cell culture and intestinal contents in 27-day-old piglets.

Porcine deltacoronavirus (PDCoV) is an emenging swine enteropathogenic coronavirus that can cause high mortality rate. It affects pigs of all ages, but most several in neonatal piglets. Little is known regarding the pathogenicity of PDCoV against 27-day-old piglets. In this study, 27-day-old piglets were experimentally infected with PDCoV CZ2020 from cell culture, the challenged piglets do not have obvious symptoms from 1 to 7 days post-challenge (DPC), while viral shedding was detected in rectal swab at 1 DPC. Tissues of small intestines displayed slight macroscopic and microscopic lesions with no viral antigen detection. On the other hand, 27-day-old piglets were infected with PDCoV from intestinal contents, the piglets developed mild to severe diarrhea, shedding increasing from 2 to 7 DPC, and developed macroscopic and microscopic lesions in small intestines with clear viral antigen confirmed by immunohistochemistry staining. Indicating the small intestine was still the major target organ in PDCoV-challenged pigs at the age of 27-day-old. Diarrhea caused by PDCoV from intestinal contents in 27-day-old piglets is less reported. Thus, our results might provide new insights into the pathogenesis of PDCoV.

Comprehensive analysis of codon usage patterns of porcine deltacoronavirus and its host adaptability.

The porcine deltacoronavirus (PDCoV) is a newly discovered pig enteric coronavirus that can infect cells from various species. In Haiti, PDCoV infections in children with acute undifferentiated febrile fever were recently reported. Considering the great potential of inter-species transmission of PDCoV, we performed a comprehensive analysis of codon usage patterns and host adaptation profiles of 54 representative PDCoV strains with the spike (S) gene. Phylogenetic analysis of the PDCoV S gene indicates that the PDCoV strains can be divided into five genogroups. We found a certain codon usage bias existed in the S gene, in which the synonymous codons are often ended with U or A. Heat map analysis revealed that all the PDCoV strains shared a similar codon usage trend. The PDCoV S gene with a dN/dS ratio lower than 1 reveals a negative selection on the PDCoV S gene. Neutrality analysis showed that natural selection is the dominant force in shaping the codon usage bias of the PDCoV S gene. Unexpectedly, host adaptation analysis reveals a higher adaptation level of PDCoV to Homo sapiens and Gallus gallus than to Sus scrofa. Compared to the USA lineage, the PDCoV strains in the Early China lineage and Thailand lineage were less adapted to their hosts, which indicates that the evolutionary process plays an important role in the adaptation ability of PDCoV. These findings of this study add to our understanding of PDCoV's evolution, adaptability, and inter-species transmission.

Evaluation of the transcriptional regulatory efficacy of transcription regulatory sequences of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteropathogenic coronavirus causing severe watery diarrhea and high mortality in piglets. In order to investigate the role of the transcription regulatory sequences (TRSs) in regulation of gene expression and replication of PEDV, the enhanced green fluorescent protein (EGFP) gene, under control of different TRSs of PEDV, were inserted between the N gene and 3' UTR of the PEDV genome using a reverse genetic system. The EGFP expression from different chimeric PEDVs was analyzed for each TRS. TRSs of all the structural and accessory protein genes of PEDV positively regulate EGFP expression at different levels, and the TRS of M protein gene produced the highest level of EGFP. Moreover, this is the first study to show that exogenous gene could be inserted between N gene and 3' UTR of PEDV, and the EGFP insertion had no effect on PEDV replication. Taken together, our study enriched the information of PEDV TRSs on gene expression and replication of PEDV.

Pathogenicity, infective dose and altered gut microbiota in piglets infected with porcine deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that cause severe diarrhea, resulting in high mortality in neonatal piglets. Little is known regarding the pathogenicity of PDCoV in different infective dose and the dynamic changes in the composition of the gut microbiota in PDCoV-induced diarrhea piglets. In this study, 5-day-old piglets were experimentally infected with different dose of PDCoV. The challenged piglets developed typical symptoms, characterized by acute and severe watery diarrhea from 1 to 8 days post-inoculation (DPI), and viral shedding was detected in rectal swab until 11 DPI. Tissues of small intestines displayed significant macroscopic and microscopic lesions with clear viral antigen expression. However, no significant differences among groups were found in challenged piglets. Then alteration in gut microbiota in the jejunum and colon of PDCoV infected-piglets were analyzed using 16S rRNA sequencing. PDCoV infection reduced bacterial diversity and richness, and significantly altered the structure and abundance of the microbiota from the phylum to genus. Fusobacterium, and Proteobacteria was significantly increased (P < 0.05), while the abundance of Bacteroidota was markedly decreased in the infected-piglets. Furthermore, microbial function prediction indicated that the changes in intestinal bacterial also affected the immune system, excretory system, circulatory system, neurodegenerative disease, cardiovascular disease, xenobiotics biodegradation and metabolism, etc. These findings suggest that regulating gut microbiota community may be an effective approach for preventing PDCoV infection.

Quantitative proteomic analysis reveals that serine/threonine kinase is involved in Streptococcus suis virulence and adaption to stress conditions.

The eukaryotic-type serine/threonine kinase of Streptococcus suis serotype 2 (SS2) performs critical roles in bacterial pathogenesis. In this study, isobaric tags for relative and absolute quantification (iTRAQ) MS/MS were used to analyze the protein profiles of wild type strain SS2-1 and its isogenic STK deletion mutant (Δstk). A total of 281 significant differential proteins, including 147 up-regulated and 134 down-regulated proteins, were found in Δstk. Moreover, 69 virulence factors (VFs) among these 281 proteins were predicted by the Virulence Factor Database (VFDB), including 38 downregulated and 31 up-regulated proteins in Δstk, among which 15 down regulated VFs were known VFs of SS2. Among the down-regulated proteins, high temperature requirement A (HtrA), glutamine synthase (GlnA), ferrichrome ABC transporter substrate-binding protein FepB, and Zinc-binding protein AdcA are known to be involved in bacterial survival and/or nutrient and energy acquisition under adverse host conditions. Overall, our results indicate that STK regulates the expression of proteins involved in virulence of SS2 and its adaption to stress environments.

Co-infection analysis of bacterial and viral respiratory pathogens from clinically healthy swine in Eastern China.

Porcine respiratory disease complex (PRDC) is one of the most challenging health concerns for pig production worldwide. The aim of the present study was to determine the prevalence of pathogens associated with PRDC, including porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) and bacterial agents, such as Streptococcus suis, Haemophilus parasuis and Actinobacillus pleuropneumoniae, in clinically healthy pigs in Eastern China. Molecular detection revealed positive single-pathogen detection rates of 59.9%, 27.2%, 52.3%, 33.2% and 0.4% for PCV2, PRRSV, S. suis, H. parasuis and A. pleuropneumoniae, respectively. Co-infection with more than one pathogen was frequently detected in these samples, with PCV2/S. suis, H. parasuis and PCV2/H. parasuis mixed infection rates of 35.4%, 33.2% and 21.6%, respectively, and PCV2/S. suis/H. parasuis and PRRSV/PCV2/S. suis co-infection rates of 21.6% and 6.2%, respectively. These results suggest that mixed infections are prevalent among PRDC cases in swine, which may pose a greater threat to the health of herds compared with single-pathogen infections.

In vitro and In vivo Antibacterial Effects of Nisin Against Streptococcus suis.

Nisin is a promising therapeutic candidate because of its potent activity against Gram-positive bacteria. The present study aimed to describe the in vitro and in vivo antibacterial effects of nisin against Streptococcus suis, an important zoonotic pathogen. The minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of nisin against different S. suis strains ranged from 0.12 to 4.0 µg/mL and from 0.25 to 8.0 µg/mL, respectively. Time-killing curve assays illustrated that nisin killed 100% of tested virulent S. suis strains within 4 h when used at 2× MIC, which indicates the rapid bactericidal activity of nisin against the bacteria. Transmission and scanning electron microscopy revealed that nisin destroyed S. suis cell membrane integrity and affected its cellular ultrastructure, including a significantly wrinkled surface, intracellular content leakage, and cell lysis. In addition, nisin inhibited biofilm formation by S. suis in a concentration-dependent manner and exhibited strong degrading activities against preformed biofilms. More importantly, nisin displayed antimicrobial activity against S. suis infection in vivo. Upon treatment with 5.0-10 mg/kg nisin solution, the survival rates of mice challenged with a lethal dose of virulent S. suis virulent ranged 87.5-100%. Nisin significantly decreased bacterial proliferation and translocation in the mouse spleen, brain, and blood. These results indicate that nisin has potential as a novel antimicrobial agent for the clinical treatment and prevention of infection caused by S. suis in animals.

The Redox-Sensing Regulator Rex Contributes to the Virulence and Oxidative Stress Response of Streptococcus suis Serotype 2.

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen responsible for septicemia and meningitis. The redox-sensing regulator Rex has been reported to play critical roles in the metabolism regulation, oxidative stress response, and virulence of various pathogens. In this study, we identified and characterized a Rex ortholog in the SS2 virulent strain SS2-1 that is involved in bacterial pathogenicity and stress environment susceptibility. Our data show that the Rex-knockout mutant strain Δrex exhibited impaired growth in medium with hydrogen peroxide or a low pH compared with the wildtype strain SS2-1 and the complementary strain CΔrex. In addition, Δrex showed a decreased level of survival in whole blood and in RAW264.7 macrophages. Further analyses revealed that Rex deficiency significantly attenuated bacterial virulence in an animal model. A comparative proteome analysis found that the expression levels of several proteins involved in virulence and oxidative stress were significantly different in Δrex compared with SS2-1. Electrophoretic mobility shift assays revealed that recombinant Rex specifically bound to the promoters of target genes in a manner that was modulated by NADH and NAD+. Taken together, our data suggest that Rex plays critical roles in the virulence and oxidative stress response of SS2.

Complete Genome Sequence of Streptococcus suis Serotype 2 Virulent Strain SS2-1.

Streptococcus suis is an important swine pathogen that can also cause severe diseases in humans. Herein, we describe the genome sequence of Streptococcus suis serotype 2 virulent strain SS2-1, which was isolated from a diseased dead pig amid the 1998 Streptococcus suis outbreak in Jiangsu Province in China.

Exudative epidermitis of piglets caused by non-toxigenic Staphylococcus sciuri.

Infections with strains of Staphylococcus sciuri are a potential threat to animal and public health, and a cause for considerable concern. We isolated and identified S. sciuri as a pathogen from an acute outbreak of exudative epidermitis in piglets for further genetic identification using experimental infections. The results of this study showed that S. sciuri strain NJ1306 reproduced exudative epidermitis in experimentally infected 5-day-old piglets. The isolated bacteria also caused sudden death in BALB/c mice following intraperitoneal injection with 5×108 CFU of the isolate. The data indicated that strain NJ1306 of S. sciuri was pathogenic to piglets and mice, and the study provided the first known report of clinical lung lesions and endocarditis in piglets due to S. sciuri.

Novel L-lactic acid biosensors based on conducting polypyrrole-block copolymer nanoparticles.

The development of advanced nanomaterials for the highly efficient electrical detection of biological species has attracted great attention. Here, novel polypyrrole-Pluronic F127 nanoparticles (PPy-F127 NPs) with conducting and biocompatibility properties were synthesized and used to construct a L-lactic acid biosensor that could be applied in biochemical assays. The PPy-F127 NPs were characterized by transmission electron microscopy (TEM), elemental analysis and UV-vis spectroscopy. Lactate oxidase (LOx) structure variation on the PPy-F127 NPs was investigated by circular dichroism (CD). The cyclic voltammetric results indicated that LOx immobilized on the PPy-F127 NPs exhibited direct electron transfer reaction with a formal potential value (E(0)') of 0.154 V vs. SCE. Moreover, the biosensor had good electrocatalytic activity toward L-lactic acid with a wide linear range (0.015-37.5 mM) and a low detection limit of 0.0088 mM. The regression equation was I (µA) = 0.02353c (mM) + 1.4135 (R(2) = 0.9939). The L-lactic acid biosensor had a good anti-interference property towards uric acid (UA), ascorbic acid (AA), glucose and cysteine. The idea and method provide a promising platform for the rapid development of biosensors that can be used in the detection of biological species.

Contribution of eukaryotic-type serine/threonine kinase to stress response and virulence of Streptococcus suis.

Streptococcus suis serotype 2 (SS2) is an important swine and human pathogen responsible for septicemia and meningitis. The bacterial homologues of eukaryotic-type serine/threonine kinases (ESTKs) have been reported to play critical roles in various cellular processes. To investigate the role of STK in SS2, an isogenic stk mutant strain (Δstk) and a complemented strain (CΔstk) were constructed. The Δstk showed a significant decrease in adherence to HEp-2 cells, compared with the wild-type strain, and a reduced survival ratio in whole blood. In addition, the Δstk exhibited a notable reduced tolerance of environmental stresses including high temperature, acidic pH, oxidative stress, and high osmolarity. More importantly, the Δstk was attenuated in both the CD1 mouse and piglet models of infection. The results of quantitative reverse transcription-PCR (qRT-PCR) analysis indicated that the expressions of a few genes involving in adherence, stress response and virulence were clearly decreased in the Δstk mutant strain. Our data suggest that SsSTK is required for virulence and stress response in SS2.

Characterization and proteome analysis of inosine 5-monophosphate dehydrogenase in epidemic Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes severe disease symptoms in pigs and humans. In the present study, we found one isogenic mutant lacking inosine 5-monophosphate dehydrogenase (IMPDH) ΔZY05719 was attenuated in pigs compared with the wild-type SS2 strain ZY05719. Comparative proteome analysis of the secreted proteins expression profiles between ZY05719 and ΔZY05719 allowed us to identify Triosephosphate isomerase (TPI) and glyceraldehyde phosphate dehydrogenase (GAPDH), which were down expressed in the absence of the IMPDH. Both of them are glycolytic enzymes participating in the glycolytic pathway. Compared with ZY05719, ΔZY05719 lost the ability of utilize mannose, which might relate to down expression of TPI and GAPDH. In addition, GAPDH is a well-known factor that involved in adhesion to host cells, and we demonstrated ability of adhesion to HEp-2 and PK15 by ΔZY05719 was significantly weakened, in contrast to ZY05719. The adhesion to host cells is the crucial step to cause infection for pathogen, and the reduction adhesion of ΔZY05719, to some extent illustrates the attenuated virulence of ΔZY05719.

Monoclonal antibodies against Nsp2 protein of the highly pathogenic porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) continues to be a serious threat, causing an economically significant impact on the swine industry worldwide. In this study, non-structural protein Nsp2 of porcine reproductive and respiratory syndrome virus (PRRSV) was expressed in Escherichia coli and purified by dialysis. An important monoclonal antibody (MAb 2H6) against Nsp2 protein was generated by fusing mouse myeloma cell line SP2/0 with spleen lymphocytes from Nsp2 protein immunized mice. Then activity of the MAb was characterized by enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and indirect immunofluorescence assays. The results demonstrated that the MAb has a positive reaction to HP-PRRSV in IFA at 1:100 dilution and in Western blot analysis at 1:500 dilution, and no reaction with classic PRRSV. These indicated that this MAb against Nsp2 protein of PRRSV might be a good candidate for a specific diagnostic method and functional exploration of the Nsp2 protein.

Development of a loop-mediated isothermal amplification assay for the detection of porcine hokovirus.

Hokoviruses have recently been detected as pathogens belonging to the family Parvoviridae, which comprises porcine hokovirus (PHoV) and bovine hokovirus (BHoV). In this study, we developed a loop-mediated isothermal amplification (LAMP) assay for the rapid, specific and sensitive detection of PHoV. A set of four primers specific for six regions within the PHoV VP1/2 genes was designed using online software. The reaction temperature and time were optimized at 65°C and 60 min, respectively. LAMP products were detected by agarose gel electrophoresis or by visual inspection of a color change caused by a fluorescent dye. The method was highly specific for PHoV, and no cross-reaction was observed with porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine bocavirus (PBoV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), classic swine fever virus (CSFV), or Japanese encephalitis virus (JEV). The detection limit was approximately 10 copies per reaction, which was 10 times more sensitive than conventional PCR. Furthermore, the efficiency of detection of PHoV in clinical samples was comparable to that of PCR and sequencing. These results show that the LAMP assay is a simple, rapid, sensitive and specific method for detecting PHoV. It does not require specialized equipment and can be used to detect PHoV both in the laboratory and in the field.

Complete genome sequence of a recombinant porcine epidemic diarrhea virus strain from eastern china.

A field porcine epidemic diarrhea virus (PEDV) strain, JS2008, was isolated from stool samples of a piglet with acute diarrhea on a vaccinated farm in eastern China. We sequenced and analyzed the complete genome of strain JS2008, which will help increase our understanding of the molecular characteristics of the epidemic PEDV in China.

In vitro and in vivo isolation of a novel rearranged porcine circovirus type 2.

Here, we present the first report of a novel rearranged porcine circovirus type 2 (PCV2) strain named BIV, isolated from both in vitro and in vivo sources. The complete circular genome of BIV is 896 nucleotides in length. The data will help us to update current knowledge of the replication of PCV2 viruses in cell culture and of their molecular evolution, as well as their diagnosis.

A novel porcine circovirus-like agent P1 is associated with wasting syndromes in pigs.

A novel porcine pathogen tentatively named P1, which was obtained from the sera of the pigs exhibiting clinical signs of postweaning multisystemic wasting syndrome (PMWS) experimentally caused the classical clinic signs and pathologic lesions of the disease in pigs by direct in vivo injection with P1 DNA plasmids. Twenty colostrum-fed (CF) pigs that were free of PCV2 and P1 at 1 month of age were randomly designated equally to two groups. Group 1 pigs were each injected with 400 µg of the cloned P1 plasmid DNA into the superficial inguinal lymph nodes and Group 2 were injected with same amount of the empty pSK vector DNA and served as controls. Viremias were positively detected in 8 of 10 P1 infected pigs from 14-21 days post-inoculation (dpi). The 8 infected animals showed pallor of skin and diarrhea. Gross lesions in the pigs euthanized on 35 dpi were similarly characterized by encephalemia, haemorrhage of the bladder mucosa, haemorrhage of the superficial inguinal lymph nodes, lung atrophy and haemorrhage. Histopathological lesions were arteriectasis and telangiectasia of the cavitas subarachnoidealis, interstitial pneumonia, mild atrophy of the cardiac muscle cells, histiocytic hyperplasia of the follicles in the tonsils, and haemorrhage of the inguinal lymph nodes. P1 DNA and antigens were confirmed by PCR and immunohistochemistry in the tissues and organs of the infected pigs, including the pancreas, bladders, testicles/ovaries, brains, lungs and liver. There were no obvious clinical signs and pathological lesions in the control pigs. This study demonstrated that P1 infection is one of the important pathologic agents on pig farms.