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.

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.

Production of singlet oxygen from photosensitizer erythrosine for facile inactivation of coronavirus on mask.

The global health crisis caused by the COVID-19 pandemic has led to a surge in demand and use of personal protective equipment (PPE) such as masks, putting great pressure on social production and the environment.It is urgent to find an efficient and non-destructive disinfection method for the safe reuse of PPE. This study proposes a PPE disinfection method that uses erythrosine, a U.S. Food and Drug Administration-approved food dye, as photosensitizer to produce singlet oxygen for virus inactivation, and indicates the completion of disinfection by its photobleaching color change.After spraying 100 µL of 10 µM erythrosine on the surface of the mask for 3 times and light exposure for 25 min, the titer of coronavirus decreased by more than 99.999%, and the color of erythrosine on the mask surface disappeared. In addition, the structure of the mask was intact and the filtration efficiency was maintained at > 95% after 10 cycles of erythrosine treatment.Therefore, this disinfection method can provide at least 10 cycles of reuse with the advantages of high safety and convenient, and the completion of disinfection can be indicated by its photobleaching, which is suitable for hospitals and daily life to reduce the consumption of PPE.

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.

Genetic comparison of transmissible gastroenteritis coronaviruses.

Transmissible gastroenteritis virus (TGEV) is a porcine coronavirus that threatens animal health and remains elusive despite years of research efforts. The systematical analysis of all available full-length genomes of TGEVs (a total of 43) and porcine respiratory coronaviruses PRCVs (a total of 7) showed that TGEVs fell into two independent evolutionary phylogenetic clades, GI and GII. Viruses circulating in China (until 2021) clustered with the traditional or attenuated vaccine strains within the same evolutionary clades (GI). In contrast, viruses latterly isolated in the USA fell into GII clade. The viruses circulating in China have a lower similarity with that isolated latterly in the USA all through the viral genome. In addition, at least four potential genomic recombination events were identified, three of which occurred in GI clade and one in GII clade. TGEVs circulating in China are distinct from the viruses latterly isolated in the USA at either genomic nucleotide or antigenic levels. Genomic recombination serves as a factor driving the expansion of TGEV genomic diversity.

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.

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.

Development of a multiplex qRT-PCR assay for the detection of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine Deltacoronavirus.

Currently, porcine coronaviruses are prevalent in pigs, and due to the outbreak of COVID-19, porcine coronaviruses have become a research hotspot. porcine epidemic diarrhea virus (PEDV), Transmissible Gastroenteritis Virus (TGEV), and Porcine Deltacoronavirus (PDCoV) mentioned in this study mainly cause diarrhea in pigs. These viruses cause significant economic losses and pose a potential public health threat. In this study, specific primers and probes were designed according to the M gene of PEDV, the S gene of TGEV, and the M gene of PDCoV, respectively, and TaqMan probe-based multiplex real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was developed for the simultaneous detection of PEDV, TGEV, and PDCoV. This method has high sensitivity and specificity, and the detection limit of each virus can reach 2.95 × 100 copies/µl. An assay of 160 clinical samples from pigs with diarrhea showed that the positive rates of PEDV, TGEV, and PDCoV were 38.13, 1.88, and 5.00%; the coinfection rates of PEDV+TGEV, PEDV+PDCoV, TGEV+PDCoV, PEDV+TGEV+PDCoV were 1.25, 1.25, 0, 0.63%, respectively. The positive coincidence rates of the multiplex qRT-PCR and single-reaction qRT-PCR were 100%. This method is of great significance for clinical monitoring of the porcine enteric diarrhea virus and helps reduce the loss of the breeding industry and control the spread of the disease.

The TGEV Membrane Protein Interacts with HSC70 To Direct Virus Internalization through Clathrin-Mediated Endocytosis.

Coronavirus membrane protein is a major component of the viral envelope and plays a central role in the viral life cycle. Studies of the coronavirus membrane protein (M) have mainly focused on its role in viral assembly and budding, but whether M protein is involved in the initial stage of viral replication remains unclear. In this study, eight proteins in transmissible gastroenteritis virus (TGEV)-infected cells coimmunoprecipitated with monoclonal antibodies (MAb) against M protein in PK-15 cells, heat shock cognate protein 70 (HSC70), and clathrin were identified by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF MS). Further studies demonstrated that HSC70 and TGEV M colocalized on the cell surface in early stages of TGEV infection; specifically, HSC70 bound M protein through its substrate-binding domain (SBD) and preincubation of TGEV with anti-M serum to block the interaction of M and HSC70 reduced the internalization of TGEV, thus demonstrating that the M-HSC70 interaction mediates the internalization of TGEV. Remarkably, the process of internalization was dependent on clathrin-mediated endocytosis (CME) in PK-15 cells. Furthermore, inhibition of the ATPase activity of HSC70 reduced the efficiency of CME. Collectively, our results indicated that HSC70 is a newly identified host factor involved in TGEV infection. Taken together, our findings clearly illustrate a novel role for TGEV M protein in the viral life cycle and present a unique strategy used by HSC70 to promote TGEV infection in which the interaction with M protein directs viral internalization. These studies provide new insights into the life cycle of coronaviruses. IMPORTANCE TGEV is the causative agent of porcine diarrhea, a viral disease that economically affects the pig industry in many countries. However, the molecular mechanisms underlying viral replication remain incompletely understood. Here, we provide evidence of a previously undescribed role of M protein in viral replication during early stages. We also identified HSC70 as a new host factor affecting TGEV infection. We demonstrate that the interaction between M and HSC70 directs TGEV internalization in a manner dependent on CME, thus revealing a novel mechanism for TGEV replication. We believe that this study may change our understanding of the first steps of infection of cells with coronavirus. This study should facilitate the development of anti-TGEV therapeutic agents by targeting the host factors and may provide a new strategy for the control of porcine diarrhea.

Generation of APN-chimeric gene-edited pigs by CRISPR/Cas9-mediated knock-in strategy

The prevalence of porcine enteric coronaviruses (PECs), including transmissible gastroenteritis virus (TGEV), swine acute diarrhea syndrome coronavirus (SADS-CoV), porcine delta coronavirus (PDCoV), and porcine epidemic diarrhea virus (PEDV), poses a serious threat to animal and public health. Here, we aimed to further optimize the porcine aminopeptidase N (pAPN) gene editing strategy to explore the balance between individual antiviral properties and the biological functions of pAPN in pigs. Finally, APN-chimeric gene-edited pigs were produced through a CRISPR/Cas9-mediated knock-in strategy. Further reproductive tests indicated that these gene-edited pigs exhibited normal pregnancy rates and viability. Notably, in vitro viral challenge assays further demonstrated that porcine kidney epithelial cells isolated from F1-generation gene-edited pigs could effectively inhibit TGEV infection. This study is the first to report the generation of APN-chimeric pigs, which may provide a natural host animal for characterizing PEC infection with APN and help in the development of better antiviral solutions. © 2022 Elsevier B.V.

Eugenol Alleviates TGEV-Induced Intestinal Injury via Suppressing ROS/NLRP3/GSDMD-Dependent Pyroptosis.

Transmissible gastroenteritis virus (TGEV), a coronavirus, is one of the main causative agents of diarrhea in piglets and significantly impacts the global swine industry. Pyroptosis is involved in the pathogenesis of coronavirus, but its role in TGEV-induced intestinal injury has yet to be fully elucidated. Eugenol, an essential plant oil, plays a vital role in antiviral innate immune responses. We demonstrate the preventive effect of eugenol on TGEV infection. Eugenol alleviates TGEV-induced intestinal epithelial cell pyroptosis and reduces intestinal injury in TGEV-infected piglets. Mechanistically, eugenol reduces the activation of NLRP3 inflammasome, thereby inhibiting TGEV-induced intestinal epithelial cell pyroptosis. In addition, eugenol scavenges TGEV-induced reactive oxygen species (ROS) increase, which in turn prevents TGEV-induced NLRP3 inflammasome activation and pyroptosis. Overall, eugenol protects the intestine by reducing TGEV-induced pyroptosis through inhibition of NLRP3 inflammasome activation, which may be mediated through intracellular ROS levels. These findings propose that eugenol may be an effective strategy to prevent TGEV infection.

A Serological Investigation of Porcine Reproductive and Respiratory Syndrome and Three Coronaviruses in the Campania Region, Southern Italy.

Porcine coronaviruses and reproductive and respiratory syndrome (PRRS) are responsible for severe outbreaks that cause huge economic losses worldwide. In Italy, three coronaviruses have been reported historically: porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV). Although repeated outbreaks have been described, especially in northern Italy, where intensive pig farming is common, there is a worrying lack of information on the spread of these pathogens in Europe. In this work, we determined the seroprevalence of three porcine coronaviruses and PRRSV in the Campania region, southern Italy. A total of 443 samples were tested for the presence of antibodies against porcine coronaviruses and PRRSV using four different commercial ELISAs. Our results indicated that PEDV is the most prevalent among porcine coronaviruses, followed by TGEV, and finally PRCV. PRRSV appeared to be the most prevalent virus (16.7%). For coronaviruses, seroprevalence was higher in pigs raised in intensive farming systems. In terms of distribution, TGEV is more widespread in the province of Avellino, while PEDV and PRRSV are more prevalent in the province of Naples, emphasizing the epidemic nature of both infections. Interestingly, TGEV-positive animals are more common among growers, while seropositivity for PEDV and PRRSV was higher in adults. Our research provides new insights into the spread of swine coronaviruses and PRRSV in southern Italy, as well as a warning about the need for viral surveillance.

Different Mechanisms Are Utilized by Coronavirus Transmissible Gastroenteritis Virus To Regulate Interferon Lambda 1 and Interferon Lambda 3 Production.

Type III interferons (IFN-λ) are shown to be preferentially produced by epithelial cells, which provide front-line protection at barrier surfaces. Transmissible gastroenteritis virus (TGEV), belonging to the genus Alphacoronavirus of the family Coronaviridae, can cause severe intestinal injuries in porcine, resulting in enormous economic losses for the swine industry, worldwide. Here, we demonstrated that although IFN-λ1 had a higher basal expression, TGEV infection induced more intense IFN-λ3 production in vitro and in vivo than did IFN-λ1. We explored the underlying mechanism of IFN-λ induction by TGEV and found a distinct regulation mechanism of IFN-λ1 and IFN-λ3. The classical RIG-I-like receptor (RLR) pathway is involved in IFN-λ3 but not IFN-λ1 production. Except for the signaling pathways mediated by RIG-I and MDA5, TGEV nsp1 induces IFN-λ1 and IFN-λ3 by activating NF-κB via the unfolded protein responses (UPR) PERK-eIF2α pathway. Furthermore, functional domain analysis indicated that the induction of IFN-λ by the TGEV nsp1 protein was located at amino acids 85 to 102 and was dependent on the phosphorylation of eIF2α and the nuclear translocation of NF-κB. Moreover, the recombinant TGEV with the altered amino acid motif of nsp1 85-102 was constructed, and the nsp1 (85-102sg) mutant virus significantly reduced the production of IFN-λ, compared with the wild strain. Compared to the antiviral activities of IFN-λ1, the administration of IFN-λ3 showed greater antiviral activity against TGEV infections in IPEC-J2 cells. In summary, our data point to the significant role of IFN-λ in the host innate antiviral responses to coronavirus infections within mucosal organs and in the distinct mechanisms of IFN-λ1 and IFN-λ3 regulation. IMPORTANCE Coronaviruses cause infectious diseases in various mammals and birds and exhibit an epithelial cell tropism in enteric and respiratory tracts. It is critical to explore how coronavirus infections modulate IFN-λ, a key innate cytokine against mucosal viral infection. Our results uncovered the different processes of IFN-λ1 and IFN-λ3 production that are involved in the classical RLR pathway and determined that TGEV nsp1 induces IFN-λ1 and IFN-λ3 production by activating NF-κB via the PERK-eIF2α pathway in UPR. These studies highlight the unique regulation of antiviral defense in the intestine during TGEV infection. We also demonstrated that IFN-λ3 induced greater antiviral activity against TGEV replication than did IFN-λ1 in IPEC-J2 cells, which is helpful in finding a novel strategy for the treatment of coronavirus infections.

Development of a multiplex RT-PCR method for the detection of four porcine enteric coronaviruses.

Porcine enteric coronaviruses are pathogens that cause viral diarrhea in pigs and are widely prevalent worldwide. Moreover, studies have shown that some porcine enteric coronaviruses can infect humans and poultry. In order to effectively monitor these viruses, it is necessary to establish a multiple detection method to understand their prevalence and conduct in-depth research. Common porcine enteric coronaviruses include Porcine epidemic diarrhea virus (PEDV), Porcine transmissible gastroenteritis virus (TGEV), Porcine delta coronavirus (PDCoV), and Swine acute diarrhea syndrome coronavirus (SADS-CoV). Pigs infected with these viruses have the common clinical symptoms that are difficult to distinguish. A quadruplex RT-PCR (reverse transcription-polymerase chain reaction) method for the simultaneous detection of PEDV, PDCoV, TGEV and SADS-CoV was developed. Four pairs of specific primers were designed for the PEDV M gene, PDCoV N gene, TGEV S gene and SADS-CoV RdRp gene. Multiplex RT-PCR results showed that the target fragments of PDCoV, SADS-CoV, PEDV and TGEV could be amplified by this method. and the specific fragments with sizes of 250 bp, 368 bp, 616 bp and 801 bp were amplified, respectively. This method cannot amplify any fragment of nucleic acids of Seneca Valley virus (SVV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Atypical Porcine Pestivirus (APPV), and has good specificity. The lowest detection limits of PDCoV, PEDV, TGEV and SADS-CoV were 5.66 × 105 copies/µL, 6.48 × 105 copies/µL, 8.54 × 105 copies/µL and 7.79 × 106 copies/µL, respectively. A total of 94 samples were collected from pig farms were analyzed using this method. There were 15 positive samples for PEDV, 3 positive samples for mixed infection of PEDV and PDCoV, 2 positive samples for mixed infection of PEDV and TGEV, and 1 positive sample for mixed infection of PEDV, TGEV, and PDCoV. Multiplex RT-PCR method could detect four intestinal coronaviruses (PEDV, PDCoV, TGEV, and SADS-CoV) in pigs efficiently, cheaply and accurately, which can be used for clinical large-scale epidemiological investigation and diagnosis.

A Quadruplex qRT-PCR for Differential Detection of Four Porcine Enteric Coronaviruses.

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) are four identified porcine enteric coronaviruses. Pigs infected with these viruses show similar manifestations of diarrhea, vomiting, and dehydration. Here, a quadruplex real-time quantitative PCR (qRT-PCR) assay was established for the differential detection of PEDV, TGEV, PDCoV, and SADS-CoV from swine fecal samples. The assay showed extreme specificity, high sensitivity, and excellent reproducibility, with the limit of detection (LOD) of 121 copies/µL (final reaction concentration of 12.1 copies/µL) for each virus. The 3236 clinical fecal samples from Guangxi province in China collected between October 2020 and October 2022 were evaluated by the quadruplex qRT-PCR, and the positive rates of PEDV, TGEV, PDCoV, and SADS-CoV were 18.26% (591/3236), 0.46% (15/3236), 13.16% (426/3236), and 0.15% (5/3236), respectively. The samples were also evaluated by the multiplex qRT-PCR reported previously by other scientists, and the compliance rate between the two methods was more than 99%. This illustrated that the developed quadruplex qRT-PCR assay can provide an accurate method for the differential detection of four porcine enteric coronaviruses.

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.

Establishment and preliminary application of a TaqMan real-time quantitative PCR method for PastV3 detection

In order to build a specific, sensitive and rapid detection method for PAstV3 detection, the PAstVB gene sequences in Genbank were used and the conserved region in ORFlb was selected to design specific primers and TaqMan probe. Clinical stool samples were collected and preliminary detected by this newly established real-time RT-PCR method after reaction systems and conditions optimization. This detection method established in this study has a good linear relationship with the standard curve, with R2 value up to 0.9971. The sensitivity is 100 times higher than conventional PCR method, The variation co-efficient of in-batch and inter-batch repeatability test is less than 2.0%, indicating good repeatability. The detection results of Clinical samples showed that the positive rate of this method is higher than conventional PCR method. The establishment of this method provides a rapid detection means for PAstV3 laboratory diagnosis and epidemiological investigation.

Eugenol Attenuates Transmissible Gastroenteritis Virus-Induced Oxidative Stress and Apoptosis Via ROS-NRF2-ARE Signaling.

Transmissible gastroenteritis virus (TGEV), a coronavirus that causes severe diarrhea due to oxidative stress in the piglet intestine, is a major cause of economic loss in the livestock industry. However, limited interventions have been shown to be effective in the treatment of TGEV. Here, we demonstrate the therapeutic activity of eugenol in TGEV-induced intestinal oxidative stress and apoptosis. Our data show that eugenol supplementation protects intestine and IPEC-J2 cells from TGEV-induced damage. Mechanistically, eugenol reduces TGEV-induced oxidative stress in intestinal epithelial cells by reducing reactive oxygen species levels. Interestingly, eugenol also inhibits TGEV-induced intestinal cell apoptosis in vitro and in vivo. In conclusion, our data suggest that eugenol prevents TGEV-induced intestinal oxidative stress by reducing ROS-mediated damage to antioxidant signaling pathways. Therefore, eugenol may be a promising therapeutic strategy for TGEV infection.

Eugenol alleviates transmissible gastroenteritis virus-induced intestinal epithelial injury by regulating NF-κB signaling pathway.

Increasing evidence supports the ability of eugenol to maintain intestinal barrier integrity and anti-inflammatory in vitro and in vivo; however, whether eugenol alleviates virus-mediated intestinal barrier damage and inflammation remains a mystery. Transmissible gastroenteritis virus (TGEV), a coronavirus, is one of the main causative agents of diarrhea in piglets and significantly impacts the global swine industry. Here, we found that eugenol could alleviate TGEV-induced intestinal functional impairment and inflammatory responses in piglets. Our results indicated that eugenol improved feed efficiency in TGEV-infected piglets. Eugenol not only increased serum immunoglobulin concentration (IgG) but also significantly decreased serum inflammatory cytokine concentration (TNF-α) in TGEV-infected piglets. In addition, eugenol also significantly decreased the expression of NF-κB mRNA and the phosphorylation level of NF-κB P65 protein in the jejunum mucosa of TGEV-infected piglets. Eugenol increased villus height and the ratio of villus height to crypt depth in the jejunum and ileum, and decreased serum D-lactic acid levels. Importantly, eugenol increased tight junction protein (ZO-1) and mRNA expression levels of nutrient transporter-related genes (GluT-2 and CaT-1) in the jejunum mucosa of TGEV-infected piglets. Meanwhile, compared with TGEV-infected IPEC-J2 cells, treatment with eugenol reduced the cell cytopathic effect, attenuated the inflammatory response. Interestingly, eugenol did not increase the expression of ZO-1 and Occludin in IPEC-J2 cells. However, western blot and immunofluorescence results showed that eugenol restored TGEV-induced down-regulation of ZO-1 and Occludin, while BAY11-7082 (The NF-κB specific inhibitor) enhanced the regulatory ability of eugenol. Our findings demonstrated that eugenol attenuated TGEV-induced intestinal injury by increasing the expression of ZO-1 and Occludin, which may be related to the inhibition of NF-κB signaling pathway. Eugenol may offer some therapeutic opportunities for coronavirus-related diseases.

Establishment of interferon membrane protein IFITML knockdown cells and investigation of its effect on the replication of PRV, PCV2 and TGEV

To establish a J2-KD (knockdown) cell line stably expressing interfered IFITM1 and study the effect of interference with IFITMI gene on the infection of PCV2, PRV and TGEV. Gene cloning tech- niques were used to constructed pLKO. l-EGFP-Puro-IFITMI recombinant vector, which was co-transfected into 293 FT cells with lentiviral packaging plasmids psPAXZ and pMDZ. G to produce green fluorescent protein labeled lentiviruses expression IFITMlshRNA, the viral supernatant was collected at 48 hours after post transfection. J2 cells were infected with the harvested lentiviruses, screened by puromycin and cloned via cell limited dilution. Real-time PCR identify that the cell lines with stable interference with IFITMl gene were obtained, and via MTT method verify that interference with IFITMI expression had no effect on the growth of J2 cells, the successfully constructed J2 stable cell line interfere with IFITMl expression was named as JZ-KD. PRV, PCV2 and TGEV infected J2-KD cells, respectively. Using real-time fluorescence quantitative PCR detect virus replication. The results showed that J2-KD cell line was successfully generated with interfered IFITMl expression;the copy number of PCV2 and TGEV were in- creased, while PRV was decreased in J'Z-KD cell. Indicating that the interference of IFITMI gene expression markedly inhibited the replication of PRV while promoted that. of TGEV and PCV2, providing a basis for further study on the function of porcine IFITMI protein and elucidates its antiviral mechanism.