Establishment of a duplex fluorescent quantitative PCR assay for detection of Porcine circovirus type 2 and type 3

To establish a method for simultaneous detection of porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3), specific primers and TaqMan probes were designed after sequence alignment according to the specific sequences of PCV2 Cap gene and PCV3 Cap gene on GenBank. By optimizing the reaction conditions, a duplex fluorescence quantitative PCR detection method for simultaneous detection of porcine circovirus type 2 and 3 was established, and the specificity, sensitivity, and reproducibility were tested. Specificity test results showed that in addition to the positive test results for PCV2 and PCV3, tests for PRRSV, CSFV, PPV, PRV, PEDV, and TGEV were all negative with no cross-reaction, indicating its good specificity. Sensitivity test results showed that the minimum detection limit for detection of PCV2 and PCV3 can both reach 10 copies.L-1, indicating its high sensitivity. The coefficient of variation within and between groups of this method was less than 2%, indicating its good stability. A total of 181 pork and whole blood samples collected from Zhejiang Province were tested using the detection method established in this article and the standard common fluorescent PCR detection method. The results showed that the positive rate of PCV2 was 50.83% (92/181), the positive rate of PCV3 was 37.57% (68/181), and the co-infection rate of PCV2 and PCV3 was 12.15% (22/181). The above detection results of ordinary fluorescent PCR were 50.28% (91/181), 36.46% (66/181), and the co-infection rate was 11.60% (21/181). The coincidence rates of the two methods for PCV2 and PCV3 can reach 98.91% and 97.06%, and the coincidence rate for PCV2 and PCV3 mixed infection were 95.45%. In summary, the duplex fluorescence quantitative PCR detection method established in this experiment can distinguish PCV2 and PCV3 rapidly, which can be used for pathogen detection and epidemiological investigation.

Effect of Drebrin protein on the proliferation of PEDV in Vero cells

Previous studies have revealed that developmental regulated brain protein (Drebrin) is involved in cell- to-cell communication, nerve transmission, tumor metastasis, spermatogenesis and other life activities, but there are few studies on viruses. The aim of the current research was therefore, to study the function of Drebrin and its effect on the proliferation of porcine epidemic diarrhea virus (PEDV). The Drebrin gene was cloned according to the Drebrin gene sequence (XM_008015438.2) of Chlorocebus sabaeus registered by GenBank, and the phylogenetic tree was constructed to analyze its homology. The results showed that the CDS region of Vero cells Drebrin gene was 2088 bp long, encoding 695 amino acids, and was relatively conserved and had high homology with all species. To investigate the effect of Drebrin on the proliferation of PEDV in Vero cells, the eukaryotic expression vector pcDNA3.1-Drebrin-Flag was constructed. After transfection of Vero cells with different concentrations of pcDNA3.1-Drebrin-Flag, cells were infected with PEDV. Our results showed that overexpression of Drebrin in Vero cells could significantly inhibit the intracellular PEDV mRNA level and N protein expression, reduce the extracellular virus titer and inhibit the proliferation of PEDV. Further study on the interaction between Drebrin and PEDV S proteins by laser confocal technique was also performed. The results showed that Drebrin and S protein were co-located in the cytoplasm, suggesting that the two proteins may interact with each other. This study demonstrated for the first time that Drebrin can inhibit PEDV proliferation in Vero cells, laying a foundation for further research in to Drebrin function and provides a valuable information for anti-PEDV research.

Development and application of a multiplex fluorescent quantitative RT-PCR for detection of Porcine epidemic diarrhea virus, porcine coronavirus and porcine acute diarrhea syndrome coronavirus

To develop a multiplex fluorescent quantitative RT-PCR for the detection of porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and swine acute diarrhea syndrome coronavirus (SADS-CoV), in this study, specific primers/probes were designed based on the conserved regions of M, M and N gene sequences of PEDV, PDCoV and SADS-CoV, respectively. After optimization of the reaction conditions, a multiplex fluorescent quantitative RT-PCR for PEDV, PDCoV and SADS-CoV was established. The results of specificity assay showed that the method was positive for detection of PEDV, PDCoV and SADS-CoV, and negative for detection of porcine transmissible gastroenteritis virus, porcine rotavirus, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, porcine circovirus type 2, porcine parvovirus, classical swine fever virus and foot-and-mouth disease virus. The results of sensitivity assay showed that the detection limit of this method for PEDV, PDCoV, and SADS-CoV plasmids standard was 1.0x101 copies/L, and had a good linear relationship with their Ct values in the range of 101 copies/L to 106 copies/L. The results of repeatability assay showed that the coefficients of variation (CVs) of intra- and inter-assay reproducibility ranged from 0.33% to 2.53%, indicating good repeatability and stability. To evaluate the effects of the developed method, 100 clinical samples collected from different parts of Henan province were used for detection of these three viruses and compared with those of single RT-PCR and standard methods. The results of multiplex fluorescent quantitative RT-PCR showed that the positive rates of PEDV, PDCoV and SADS-CoV were 38% (38/100), 14% (14/100) and 5% (5/100), respectively. There was no mixed infection. The coincidence rate with the standard detection methods of PEDV and PDCoV was 100%, and the sensitivity was higher than that of single RT-PCR. In this study, a specific, sensitive and rapid multiplex fluorescent quantitative RTPCR method was established for the first time, which could be used for the differential detection of PEDV, PDCoV and SADS-CoV, and laid a foundation for the differential diagnosis and control of porcine diarrheal diseases.

Porcine epidemic diarrhoea virus (PEDV) infection activates AMPK and JNK through TAK1 to induce autophagy and enhance virus replication.

Autophagy plays an important role in defending against invading microbes. However, numerous viruses can subvert autophagy to benefit their replication. Porcine epidemic diarrhoea virus (PEDV) is an aetiological agent that causes severe porcine epidemic diarrhoea. How PEDV infection regulates autophagy and its role in PEDV replication are inadequately understood. Herein, we report that PEDV induced complete autophagy in Vero and IPEC-DQ cells, as evidenced by increased LC3 lipidation, p62 degradation, and the formation of autolysosomes. The lysosomal protease inhibitors chloroquine (CQ) or bafilomycin A and Beclin-1 or ATG5 knockdown blocked autophagic flux and inhibited PEDV replication. PEDV infection activated AMP-activated protein kinase (AMPK) and c-Jun terminal kinase (JNK) by activating TGF-beta-activated kinase 1 (TAK1). Compound C (CC), an AMPK inhibitor, and SP600125, a JNK inhibitor, inhibited PEDV-induced autophagy and virus replication. AMPK activation led to increased ULK1^S777 phosphorylation and activation. Inhibition of ULK1 activity by SBI-0206965 (SBI) and TAK1 activity by 5Z-7-Oxozeaenol (5Z) or by TAK1 siRNA led to the suppression of autophagy and virus replication. Our study provides mechanistic insights into PEDV-induced autophagy and how PEDV infection leads to JNK and AMPK activation.

Insights and progress on epidemic characteristics, genotyping, and preventive measures of PEDV in China: A review.

Porcine Epidemic Diarrhoea (PED) is an acute, extremely infectious intestinal disease of pigs caused by the Porcine Epidemic Diarrhoea Virus (PEDV). The virus can affect pigs of all breeds and age groups and shows varying degrees of symptoms, with piglets, in particular, being infected with mortality rates of up to 100%. PEDV was first identified in China in the 1980s and in October 2010 a large-scale PED outbreak caused by a variant of PEDV occurred in China, resulting in huge economic losses. Initially, vaccination can effectively prevent the classical strain, but since December 2010, the PEDV variant has caused "persistent diarrhoea" with severe vomiting, watery diarrhoea, and high morbidity and mortality in newborn piglets as the dominant clinical features, with a significant increase in morbidity and mortality. This indicates that PEDV strains have mutated during evolution and that traditional vaccines no longer provide effective cross-immune protection, so it is necessary to optimize immunization programs and find effective treatments through epidemiological surveys of PEDV to reduce the economic losses caused by infections with mutated strains. This article reviews the progress of research on the aetiology, epidemiological characteristics, genotyping, pathogenesis, transmission routes, and comprehensive control of PEDV infection in China.

Interaction between target peptide and complementary peptide by bimolecular fluorescence complementary technology

[Objective] Using the bimolecular fluorescence complementation (BiFC) technology, the present experiment aimed to study the interaction relationship and localization of the target peptide and the complementary peptide based on the porcine epidemic diarrhea virus (PEDV) S protein receptor binding site peptide in living cells, so as to provide the foundation and theoretical support for the further use of the peptide in the detection of porcine epidemic diarrhea virus. [Method] The target peptide was designed according to the physical and chemical characteristics of the target protein, such as the amino acid composition, the type of charge, the ability to form intennolecular hydrogen bonds, the strength of polarity, and hydrophobicity;According to the amino acid composition of the target protein, a complementary peptide that interacted with it in theory was designed, and the target peptide and complementary peptide were predicted and analyzed by using bioinfonnatics tools;The target peptide and complementary peptide were inserted into the pBiFC-VC155 and pBiFC-VN173 vector, which was double digested by the EcoRI/XhoI and NotI/SalI, respectively, verified by enzyme digestion and sequencing, and then transfected into Vero cells to study the interaction between the target peptide and the complementary peptide, and the precise localization of BiFC complex in cells. [Result] Bioinfonnatics analysis showed that the target peptide and complementary peptide had hydrophilic and hydrophobic domains, respectively, and the hydrophilic domains were both positively and negatively charged, which could generate electrostatic attraction. The results of enzyme digestion and sequencing showed that the pBiFC-VC155-target peptide and pBiFC-VNI73-complementary peptide plasmids were successfully constructed;Cell transfection experiments showed that the target peptide and complementary peptide could form BiFC complexes in Vcro cells after co-transfection of recombinant plasmids, indicating that they could interact with each other;Indirect immuttolluorescence assay confirmed that the BiFC complex was mainly distributed in the nucleus. [Conclusion] It was confirmed that the peptide designed based on the PEW/ S protein receptor binding site can interact with each other in living cells, demonstrating the feasibility of the peptide for detection.

Improved virus inhibition by microencapsulated monoclonal antibody against porcine epidemic diarrhea virus

Porcine epidemic diarrhea virus (PEDV) has been affecting the swine industry, especially in suckling pigs in with a high mortality rate. Among all the strategies to overcome PEDV, boosting mucosal immunity in pig intestine via oral administration appears to be more efficient than other routes. However, there are biological obstacles such as acidic environment that could damage biologics, a product from organisms often used for PEDV treatment. The plant-derived 2C10 monoclonal antibody (mAb) from Nicotiana benthamiana produced by transient expression was revealed as one of the potential candidates against PEDV through oral delivery. Herein, we demonstrated the calcium-alginate microencapsulation system to protect the 2C10 mAb from the harsh condition in the stomach and to be released the 2C10 mAb when arriving in the intestine. The pH-responsive encapsulated 2C10 mAb microbeads were constructed from the calcium-alginate system. The microbeads were well-tolerated under the acidic environment of simulated gastric fluid (SGF) and were digested under the alkaline condition of simulated intestinal fluid (SIF). The encapsulated 2C10 mAb in the SPF-treated microbeads exhibited high virus neutralization efficiency in Vero cells when compared to the unencapsulated 2C10 mAb treated by SPF that cannot neutralize the virus. For these reasons, calcium-alginate microencapsulation system is an attractive platform to be considered as a candidate for the next generation of oral vaccine development.

Duplex Taq man qPCR for detecting porcine epidemic diarrhea and transmissible gastroenteritis viruses and epidemic study in Fujian

Objective: A Taq Man probe-based duplex real-time PCR for rapid detection of porcine epidemic diarrhea virus(PEDV) and transmissible gastroenteritis virus(TGEV) was developed. A study was conducted using the methodology to analyze the related 2019-2021 epidemic occurred in Fujian. Method: Specific primers and probes labeled with FAM and VIC were designed to amplify the N gene of PEDV and the S gene of TGEV, respectively. A reaction system for the assay was established, optimized, and tested for sensitivity, specificity, and repeatability. The assay was used for the viral detection on297 suspected clinic specimens collected from 2019 to 2021 for an epidemiology study. Result: The newly developed duplex qPCR methodology showed a sensitivity of 10 copies.L-1 on PEDV and TGEV, which was 100 times higher than that of regular PCR. There were no cross reactions with other common viruses. The inter-and intra-assays had variations on Ct values below 1%. On the 297 specimens, the infection rate of PEDV was 88.89%, that of TGEV 1.01%, and that of both PEDV and TGEV 3.37%. Conclusion: The established duplex qPCR had high sensitivity, specificity, repeatability, and reproducibility for detecting PEDV and TGEV. The 2019-2021 epidemic involving the viruses appeared to be mostly PEDV with low incidents of mixed TGEV and PEDV/TGEV infection.

Isolation, identification and genetic evolution analysis of porcine epidemic diarrhea virus SNJ-P strain in Sichuan Province

Purpose: To investigate the epidemic variation of porcine epidemic diarrhea virus (PEDV) strains in Sichuan Province, and to analyze the causes of poor vaccination effect. Methods: Piglet intestinal samples were collected from a pig farm in Sichuan Province for PCR detection, virus purification, determination of virus titer and virus infection experiments. Whole genome sequencing of isolated strains was determined. The S gene sequence of the isolated strain was compared with the strains from other regions and vaccine strains, and the phylogenetic tree was established. The amino acid site variation of S protein between the isolated strain and the classical vaccine strain CV777 was compared. Results: A PEDV strain was successfully isolated and named as PEDV SNJ-P. The determination of virus titer was 1..107.5/100 L. Animal infection experiments showed that the isolated strain could cause diarrhea, dehydration and other symptoms and lead to death in piglets. Genome sequencing and phylogenetic tree analysis showed that the whole gene of PEDV SNJ-P strain was 28003 bp, and the genotype of the strain was S non-INDEL type. The strains were closely related to the strains of PEDV-WS, CH/JLDH/2016 and CH/HNLH/2015 isolated from China, and were relatively distant with the same type vaccine strain, and were far from the classical vaccine strain. Compared with the classical vaccine strain CV777, the S protein of SNJ-P strain had multiple amino acid mutations, deletions and insertions. Conclusion: Due to the continuous variation of strains, SNJ-P strain is far from the vaccine strain, and the current vaccines cannot provide effective protection. The results of this study are expected to provide reference for the study of PEDV strains and vaccine development in China.

Current understanding of the airborne transmission of important viral animal pathogens in spreading disease

Current research on airborne transmission of African swine fever virus (ASFV), porcine epidemic diarrhoea virus (PEDV), avian influenza (AIV), porcine reproductive and respiratory syndrome virus (PRRSV), and foot and mouth disease virus (FMDV) was reviewed to evaluate commonalities, knowledge gaps, and methodologies of studying airborne transmission of animal diseases. The reviewed studies were categorised as short-range transmission (within a single facility) and long-range transmission (beyond a single site). Short-range airborne transmission was demonstrated for at least one strain of the above-mentioned pathogens in experimental settings. Most studies reported in the literature concern FMDV, with limited information for ASFV and PEDV, particularly for short-range airborne transmission. Air sampling upwind, downwind, and within infected facilities has been commonly used to demonstrate long-range airborne transmission. The amount of evidence from air sampling for each of the reviewed viruses varies from no evidence on ASFV to evidence from multiple settings for AIV. Computer modelling has been used to study past outbreaks of infectious diseases to assess the contribution of airborne transmission with a multitude of computer models reported in the literature for simulating long-range airborne transmission of FMDV based on past outbreaks. This has resulted in predictive tools for assessing future risk of airborne transmission. Some important computer models are based on epidemiology analysis, weather analysis, and air dispersion. Few models are reported for ASFV, PEDV, and PRRSV. Studies in the literature indicate that airborne transmission is generally affected by virus strain, aerosol type, shedding duration and concentration, environmental conditions, and infectious dose.

PEDV orf3 independently regulates IB kinase beta-mediated NF-B and IFN-beta promoter activities

The Open Reading Frame 3 (ORF3), an accessory protein of porcine epidemic diarrhea virus (PEDV), has been shown to interact with a myriad of cellular proteins, among which include the IB kinase beta (IKBKB). Here, specific IKBKB domains responsible for ORF3-IKBKB interaction were identified. Dysregulation of NF-B and Type I interferon (IFN) in the presence of ORF3 was also demonstrated. We showed that while ORF3 was capable of up-regulating IKBKB-meditated NF-B promoter activity, it surprisingly down-regulated the activation of IKBKB-meditated IFN-beta promoter and expression of IFN-beta mRNA. When overexpressed, ORF3 could suppress Poly I:C mediated type I IFN production and induction. Finally, we demonstrated that IKBKB- and RIG-I-mediated type I IFN induction by ORF3 resulted in different outcomes. Our study is the first to demonstrate the potential and complex roles of ORF3 in the involvement of aberrant immune signaling as well as in the virus-host interaction.

One-step multiple TaqMan real-time RT-PCR for simultaneous detection of swine diarrhea viruses

Objective: The aim of this study was to establish a one-step multiplex real-time RT-PCR method to simultaneously detect and quantify five swine diarrhea related viruses, PEDV, GARV, PDCoV, SADS-CoV and PTV, so as to provide an efficient and sensitive tool for rapid diagnosis and epidemiological investigation of porcine diarrhea. Method: The ORF3 gene sequences of several genotypes of PEDV were analyzed, and then the primers and probes were designed for detection of PEDV field strains by referring to the ORF3 genes, which contained deletion mutations in attenuated strains. The 5'-end conserved region of NSP5 genes of GARV G3, G4, G5 and G9 strains were analyzed for design of probes and primers. The specific primers and probes targeting to the conserved regions of PDCoV M, PTV 5'UTR and SADS-CoV N genes were designed for detection of the pathogens. The ROC curves were completed by referring to parameters that were set in RStudio. The specificity value, sensitivity value, and areas under the curves (AUC) and Youden value were calculated according to ROC curves to determine the cut-off CT value. The amplified fragments were cloned into pEASY-T1 vector. The standards prepared through in vitro transcription were named as cRNA-PEDV, cRNA-GARV, cRNA-PDCoV, cRNA-PTV and cRNA-SADS-CoV. The sensitivity, specificity and repeatability of one-step multiplex real-time RT-PCR were evaluated. Coincidence rate between this and another similar method were compared in the detection of clinical samples. Result: Both the annealing temperature and optimal concentrations of primers and probes were obtained for detection of the five pathogens. According to the ROC curve, the CT cut off values for detection of PEDV, GARV, PDCoV, PTV, and SADS-CoV were set as 35.78, 34.25, 34.98, 34.60, and 35.70, respectively. The detection sensitivity of this method for the five pathogens could reach 1..102 copies/L. The standard curves had a good linear relationship and the amplification efficiency was between 96.3% and 104%. The established method could not detect the PEDV vaccine strains and other swine infecting viruses and bacteria including TGEV, CSFV, PRV, PRRSV, S.choleraesuis, P.multocida, E.coli, S.suis and S.aureus. The repeatability test showed the range of intra-assay and inter-assay coefficients of variability: 0.22% to 3.08% and 0.89% to 4.0%, respectively. The detection coincidence rates of the established detection method and another similar method for the five pathogens in 242 clinical samples were 97.9%, 98.8%, 100%, 98.3% and 100% for PEDV, GARV, PDCoV, PTV and SADS-CoV, respectively. The Kappa values were all higher than 0.9. The method had advantage over a commercial diagnostic kit for detection of PEDV wild strains in accuracy. Detection results with clinical samples showed that positive rates of PEDV, GARV, PDCoV and PTV was 10.7% (26/242), 13.6% (33/242), 18.2% (44/242) and 14.5% (35/242), respectively, demonstrating the prevalence state of the four pathogens in Sichuan province in the years. SADS-CoV was not detectable in any areas, but the phenomenon of coinfection with different diarrhea causing viruses was common. Therefore, it was necessary to strengthen the surveillance of several porcine diarrhea viruses in Sichuan province for preventive control. Conclusion: In this study, a one-step multiplex real-time RT-PCR was established for simultaneous detection of PEDV wild strains, PDCoV, SADS-COV and GARV, PTV multiple genotypes, which provided an efficient and sensitive tool for the differential diagnosis and epidemiological investigation of swine diarrhea disease.

Epidemiological investigation of bovine viral diarrhea virus infection in swinery

From 2017 to 2020, 1 078 piglet diarrhea samples were collected from 6 pig farms in different districts of Shanghai. Multiple RT-PCR method was used for detection and analysis to study the infection status of bovine viral diarrhea virus (BVDV) in swinery in Shanghai. The results showed that the overall detection rate of BVDV in swinery in Shanghai was 7.14% (77/1 078), and showed an increasing trend year by year. The mixed infection rate of BVDV and other diarrhea pathogens was high, with the highest dual infection rate (65%, 26/40), mainly BVDV/PASTV (61.54%, 16/26). On this basis, the triple infection rate was 25% (10/40), mainly BVDV/PAStV/PKoV (40%, 4/10) infection mode;The quadruple infection rate was 10% (4/40), which was dominated by BVDV/PAStV/PEDV/PSV (50%, 2/4) infection. The BVDV prevalence in swinery was seasonal, and the prevalence in spring (10.36%) and autumn (13.59%) was higher than that in summer (6.8%) and winter (2.66%). The positive rate of BVDV in different pig farms was significantly different by 0-24.07%. In view of the detection rate of diarrhea virus dominated by PEDV in pig farm 2 had been high in recent years, this study further monitored the infection of BVDV in this pig farm, and found that the detection rate of BVDV in this pig farm was increasing year by year from 2017 to 2019, with the highest detection rate in 2019 (8.61%, 42/488);The mixed infection of BVDV and other diarrhea pathogens was also serious, with the dual infection rate of 57.58% (19/32), triple infection rate of 21.21% (7/32), quadruple infection rate of 21.21% (7/32), respectively. This study enriched the epidemic data of BVDV in swinery in Shanghai, and could provide reference for the prevention and control of pig epidemics.

Complete genome sequencing and molecular genetic evolution analysis of Guangxi mutant of porcine epidemic diarrhea virus

In order to understand the genomic characteristics and molecular genetic diversity of porcine epidemic diarrhea virus(PEDV) in Guangxi in recent years, 11 pairs of specific primers were designed to detect the whole genome of PEDV GXNN isolated from porcine diarrhea in Nanning, Guangxi, China, and similarity comparison, genetic evolution, gene variation and S gene recombination were also analyzed. The results showed that full length of the GXNN strain was 28 035 bp, had similar genomic characteristics with other PEDV isolates, about 96.4%-98.7% nucleotide similarity with different reference strains, and the nucleotide similarity of S, ORF3, M and N genes was 93.7%-98.9%, 90.9%-99.4%, 97.4%-99.7% and 95.6%-99.2%;the amino acid similarity of them was 92.9%-99.5%, 91.3%-99.1%, 97.4%-99.1% and 96.4%-99.5%. GXNN is closely related to most domestic isolates in recent years. Phylogenetic tree showed that GXNN closely related to most strains isolated in China recent years, belonged to GII-b subtype. However, it was low relatedness to classic vaccine strains, domestic early epidemic strains, foreign epidemic strains and Guangxi CH-GX-2015-750 A, they belong to different subtypes. Compared with the 5 vaccine strains, the S gene of GXNN stain has a large variation, by inserting amino acid Q at positions 118 844 and 905 sizes, four unique amino acid mutations in the core neutralizing epitope(COE)region and the main epitope region, and 14 mutations in other regions. 126 T/A, 199 A/V and 103 T/A site mutations of ORF3, M and N genes were happened at position 126, 4 D4 region and PN-D4 region, respectively. Recombination analysis revealed that there was a potential recombination region in the hypervariable region of S gene at 826-3 142 nt. This study successfully obtained the complete genome sequence of a PEDV strain, and analyzed its genetic variation and provided a reference for PEDV molecular epidemiology research and new vaccine development.

STING gene knockout pig alveolar macrophage cell model constructed by CRISPR/Cas9 technology and its effect on type I interferon transcription. (Special Issue: Swine industry science.)

Objective: To elucidate the mechanism of interferon gene stimulating factor (STING) in the anti-pathogenic microbial infection of pigs, so as to further provide a reference for the scientific prevention and control of viral diseases such as porcine transmissible gastroenteritis, epidemic diarrhea and porcine pseudorabies. Method: High-scored targets were found in exons 4 and 8 of STING gene and corresponding sgRNA sequences were designed based on CRISPR/ Cas9 technology. The annealed sgRNAs were linked with the enzyme digested LentiCRISPRV2 carrier with T4 DNA ligase to obtain LentiCRISPRV2-STING-sgRNA lentivirus carrier(STING-sgRNA);Different combinations of STING sgRNA lentivirus carriers, packaging plasmid psPAX2 and envelope plasmid pMD2.G were transfected into 293T cells to obtain lentivirus containing sgRNA and then transduced into 3D4/21 cells. Monoclonal cell lines were obtained by puromycin screening and limited dilution method. The knockout efficiencies of the STING gene were identified by PCR amplification, Sequencing and Western blotting;The effect of STING gene knockout on the expression of type I interferon was verified by real-time fluorescent quantitative PCR. Result: When 293T cells were transfected with different combinations of STING-sgRNA lentivirus carrier and HA-STING over expression vector, the editing effect of STING eukaryotic expression carrier could be detected in cells, and the combination of STING-sgRNA(1+5)lentivirus carrier showed the supreme editing efficiency. Thus, the STING-sgRNA(1+5)lentivirus carrier combined with the packaging plasmid psPAX2 and the envelope plasmid pMD2.G were transfected 293T cells to package lentivirus, and then infected 3D4/21 cells with lentivirus. The results showed that a 3D4/21 cell line with a large deletion of the STING gene(4989 bp)was obtained. The STING protein was not observed by Western blotting, indicating that the STING gene knockout 3D4/21 cells(3D4/ 21-STING-/-)were successfully constructed. The transcription level of IFN-beta in 3D4/21-STING-/- cells decreased significantly (P<0.05) compared with parental cells when stimulated by transfection of Haemophilusparasuis DNA. Conclusion : By applying CRISPR/Cas9 technology, STING gene is successfully knock out in 3D4/21 cells, resulting in loss of function of STING gene;STING knockout leads to the transcription disorder of type I interferon when cells are stimulated by DNA, which also suggests that STING gene may be a key factor in the anti-pathogenic microbial infection of pigs.

The S protein of a novel recombinant PEDV strain promotes the infectivity and pathogenicity of PEDV in mid-west China.

Porcine epidemic diarrhoea virus (PEDV) is an emerging and re-emerging swine enterovirus that causes highly contagious diarrhoea and mortality in piglets. To better understand the current prevalence of PEDV in mid-west China, and to find out the reason for the re-emergence of PEDV from the viral genomic characteristics. Herein, we firstly investigated epidemiology of PEDV in mid-west China from 2019 to 2020. A total of 62.23% (257/413) of diarrhoea samples were positive for PEDV, and the PEDV-positive cases were mainly detected in winter. Then, we selected the SXSL strain as a representative strain to study the genetic and pathogenic characterization of PEDV pandemic strains in mid-west China. The recombination analysis showed that SXSL strain was a recombinant strain, and the major and minor parent strains of the recombination are CH/SCZJ/2018 strain and GDS48 strain, respectively. Complete genome sequencing and homology analysis showed that the S protein of SXSL strain contained multiple amino acid indels and mutations compared to the PEDV representative strains. Furthermore, we evaluated the effect of S protein on the infectivity and pathogenicity of PEDV by the PEDV reverse genetics system, and results showed that SXSL S protein increased the infectivity and pathogenicity of chimeric virus. Overall, our findings provided important information for understanding the roles of S protein in the prevalence of PEDV in mid-west China and developing vaccines based on PEDV pandemic strains.

Characterisation of ORF3, M, N and E Gene Sequences of Porcine Epidemic Diarrhoea Virus from Domestic Pigs in Poland.

Introduction: Porcine epidemic diarrhoea virus (PEDV) is an enteric pathogen causing porcine epidemic diarrhoea and acute gastroenteritis in pigs of all ages. Previous analysis of the viral genome of PEDV in Poland was only based on the spike protein (S) gene sequences and no analysis of other genes has been performed. The aim of this study was to analyse the envelope (E), membrane (M) and nucleocapsid (N) protein and open reading frame 3 (ORF3) gene sequences. Material and Methods: Viral RNA from 18 Polish pig faecal samples that were quantitative reverse transcription PCR-positive for PEDV was analysed in four genomic regions (E, M, N and ORF3). Results: Phylogenetic analysis based on these regions' sequences revealed that Polish PEDV isolates were highly related and were clustered into group G2a across the four genes compared. Moreover, the Polish strains were located in distinct subclusters on the phylogenetic trees, which suggests the presence of at least three independently evolving PEDV genetic lines circulating in Poland. The occurrence of unique mutations in the sequences of Polish PEDV strains suggests that PEDV continues to undergo evolutionary processes, accumulating the mutations necessary for viral fitness in its natural hosts. The Polish PEDV strains differed genetically from the CV777 vaccine strain, suggesting the risk of relatively low vaccine efficacy if this strain is used. Conclusion: Our results promote a better understanding of the genetic diversity of PEDV field isolates in Poland and highlight the importance of molecular characterisation of PEDV field strains for the development of an effective vaccine against PEDV.

Establishment of ELISA for detecting porcine epidemic diarrhea virus specific SIgA in mucus

The aim of this study is to establish an indirect ELISA technique for detecting the SIgA antibody against porcine epidemic diarrhea virus (PEDV) to evaluate its mucosal immunity. Firstly, the S1D gene (534-789 aa) of PEDV was cloned into the pET-28a(+) vector, and induced in Escherichia coli BL21 (DE3) by IPTG, the product of which was in the form of inclusion bodies. According to Western-blot, the target protein S1D with antigenic activity was 32 ku in molecular weight and could be well detected. Then, the S1D protein was denatured by 8 mol/L urea, purified and gradient as the coating antigen to establish an indirect ELISA for detecting the PEDV specific SIgA antibody in nasal or oral mucus by optimizing conditions. And the optimal antigen coating concentration of ELISA was 2 micro g mL, the working concentrations of nasal mucus was 1:1 and the optimal blocking solution was 50 g/L skimmed milk, while the working concentrations and optimal blocking solution were 1:2 and 30 g/L BSA in oral mucus, the working concentrations of the enzyme-labeled antibody was 1:2 000 in nasal and oral mucus. Finally, 84 samples of oral and nasal mucus from immunized pigs were detected by S1D of ELISA, and the coincidence rate could reach 95.2% compared with purified PEDV of ELISA. In conclusion, the indirect ELISA established in this study provided a quick, simple, sensitive, and specific method to detect PEDV specific SigA for evaluating the level of PEDV mucosal immunity.

Co-infection of porcine deltacoronavirus and porcine epidemic diarrhoea virus alters gut microbiota diversity and composition in the colon of piglets.

Porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhoea virus (PEDV) are the main porcine enteric coronaviruses that cause severe diarrhoea in piglets, posing huge threat to the swine industry. Our previous study verified that the co-infection of PDCoV and PEDV is common in natural swine infections and obviously enhances the disease severity in piglets. However, the effects of co-infection of PDCoV and PEDV on intestinal microbial community are unknown. In current study, the microbial composition and diversity in the colon of piglets were analyzed. Our results showed that both of PDCoV and PEDV were mainly distributed in the small intestines and caused severe damage of ileum but not colon in the co-inoculated piglets. Furthermore, we observed that PDCoV and PEDV co-infection alters the gut microbiota composition at the phylum, family and genus levels. The abundance of Mitsuokella and Collinsella at genus level were significantly increased in PDCoV-PEDV co-infection piglets. Spearman's correlation analysis further suggested that there existed strong positive correlation between Mitsuokella and TNF-α, IL-6 and IL-8 secretion, these two factors may together aggravating the small intestine pathological lesions. These results proved there existed obvious correlation between the disease severity caused by PDCoV-PEDV co-infection and intestinal microbial community.

Establishment and application of multiplex RT-PCR procedure for detection of three virus from swine

Based on the M gene sequence of TGEV and PEDV and VP2 gene sequence of PoRV, the optimal reaction system and amplification procedure were established by optimizing primer, probe concentration and annealing temperature, and the Quantitative PCR method of TaqMan probes for three viruses is successfully established. On this basis, after further optimization of conditions, a triple real-time fluorescent quantitative PCR method for detecting TGEV, PEDV, and PoRV was established. The detection sensitivity of this method for TGEV, PEDV, and PoRV were 2.49 copies/ L, 4.36 copies/ L, and 4.96 copies/ L respectively. The maximum value of CV in repeated trials detected by TGEV, PEDV and PoRV were 2.5%, 3.8%, 4.3%, and the maximum value of CV in repeated trials between groups were 3.7%, 3.4%, 3.2%, which are no more than 5%.indicating that the established method has good reproducibility. Using this method to detect PRV, PCV1, and PRRSV virus samples, there is no cross-reaction, indicating that the method is specific. Using the established method to detect 40 clinical diseases, the samples were tested, and the positive rates of TGEV, PEDV, and PoRV were 5%, 30%, and 12.5%respectively. The mixed infection rate of TGEV and PEDV was 2.5%, the mixed infection rate of PEDV and PoRV was 5%. The results of the multiple fluorescence quantitative PCR method are consistent with those of the detection of a single fluorescent RT-PCR method, indicating that the established method has good clinical application value.