Transmissible Gastroenteritis Research Articles

Radiation Inactivation of Coronavirus Infection Pathogen by the Example of Transmissible Gastroenteritis Virus.

In recent years, members of the Coronaviridae family have caused outbreaks of respiratory diseases (MERS, SARS, and COVID-19). At the same time, the potential of radiation-induced inactivation of this group of viruses have been little studied, although radiation technologies can be widely used both in the processing of personal protective equipment and in the sterilization of vaccines. In the present work, the effect of 10 MeV electron beams and 7.6 MeV bremsstrahlung on the coronavirus infection pathogen (transmissible gastroenteritis virus) has been studied in vitro. In the given experimental conditions, irradiation with photons turned out to be more effective. The virus-containing suspension frozen at –86°C was the most resistant to radiation: the dose required for complete inactivation of the virus in this case was from 15 kGy, while for the liquid suspension and lyophilized form the sterilizing dose was from 10 kGy. At lower radiation doses for all samples during passaging in cell culture, residual infectious activity of the virus was observed. These differences in the efficiency of inactivation of liquid and frozen virus-containing samples indicate a significant contribution of the direct effect of radiation.

Melatonin and other indoles show antiviral activities against swine coronaviruses in vitro at pharmacological concentrations.

The current coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights major gaps in our knowledge on the prevention control and cross-species transmission mechanisms of animal coronaviruses. Transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), and porcine delta coronavirus (PDCoV) are three common swine coronaviruses and have similar clinical features. In the absence of effective treatments, they have led to significant economic losses in the swine industry worldwide. We reported that indoles exerted potent activity against swine coronaviruses, the molecules used included melatonin, indole, tryptamine, and L-tryptophan. Herein, we did further systematic studies with melatonin, a ubiquitous and versatile molecule, and found it inhibited TGEV, PEDV, and PDCoV infection in PK-15, Vero, or LLC-PK1 cells by reducing viral entry and replication, respectively. Collectively, we provide the molecular basis for the development of new treatments based on the ability of indoles to control TGEV, PEDV, and PDCoV infection and spread.

Genetic characterization and phylogenetic analysis of porcine epidemic diarrhea virus in Guangdong, China, between 2018 and 2019.

Porcine epidemic diarrhea virus (PEDV), a leading cause of piglet diarrhea outbreaks, poses a significant danger to the swine industry. The aim of this study was to investigate the epidemic characteristics of PEDV that was circulating in Guangdong province, one of China’s major pig producing provinces. Clinical samples were collected from eight pig farms in Guangdong province between 2018 and 2019 and tested for the major porcine enteric pathogens, including PEDV, transmissible gastroenteritis virus (TGEV), Swine enteric coronavirus (SeCoV), Swine acute diarrhea syndrome coronavirus (SADS-CoV), porcine deltacoronavirus (PDCoV), and porcine rotavirus (RV). As a result, only PEDV and RV were detected at a rate of 47.0% (16/34) and 18.6% (8/34), respectively. Coinfectoin with PEDV and RV occurred at a rate of PEDV 12.5% (2/16). Subsequently, the full-length S gene sequences of 13 PEDV strains were obtained, and phylogenetic analysis suggested the presence of GII-c group PEDV strains in this region (non-S-INDEL). Two novel common amino acid insertions (55T/IG56 and 551L) and one novel glycosylation site (1199G+) were detected when the CV777 and ZJ08 vaccine strains were compared. Furthermore, intragroup recombination events in the S gene regions 51–548 and 2478–4208 were observed in the PEDV strains studied. In summary, the observations provide current information on the incidence of viral agents causing swine diarrhea in southern China and detailed the genetic characteristics and evolutionary history of the dominant PEDV field strains. Our findings will aid in the development of an updated vaccine for the prevention and control of PEDV variant strains.

The Murine Reg3a Stimulated by Lactobacillus casei Promotes Intestinal Cell Proliferation and Inhibits the Multiplication of Porcine Diarrhea Causative Agent in vitro.

Lactobacillus casei (L. casei), a normal resident of the gastrointestinal tract of mammals, has been extensively studied over the past few decades for its probiotic properties in clinical and animal models. Some studies have shown that some bacterium of Lactobacillus stimulate the production of antimicrobial peptides in intestinal cells to clear enteric pathogens, however, which antimicrobial peptides are produced by L. casei stimulation and its function are still not completely understood. In this study, we investigated the changes of antimicrobial peptides’ expression after intragastric administration of L. casei to mice. The bioinformatics analysis revealed there were nine genes strongly associated with up-regulated DEGs. But, of these, only the antimicrobial peptide mReg3a gene was continuously up-regulated, which was also confirmed by qRT-PCR. We found out the mReg3a expressed in engineering E.coli promoted cell proliferation and wound healing proved by CCK-8 assay and wound healing assay. Moreover, the tight junction proteins ZO-1 and E-cadherin in mReg3a treatment group were significantly higher than that in the control group under the final concentration of 0.2 mg/ml both in Porcine intestinal epithelial cells (IPEC-J2) and Mouse intestinal epithelial cells (IEC-6) (p < 0.05). Surprisingly, the recombinant mReg3a not only inhibited Enterotoxigenic Escherichia coli (ETEC), but also reduced the copy number of the piglet diarrheal viruses, porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine rotavirus (PoRV), indicating the antimicrobial peptides mReg3a may be feed additives to resist the potential of the intestinal bacterial and viral diarrhea disease.

Novel universal primer-pentaplex PCR assay based on chimeric primers for simultaneous detection of five common pig viruses associated with diarrhea

Viral pathogens associated with diarrhea in pigs include porcine circovirus 2 (PCV2), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus A (RVA) and C (RVC) among others. In this study, a novel universal primer-based pentaplex PCR (UP-M-PCR) assay was developed for simultaneous detection and differentiation of these five viruses. The assay uses a short-cycle multiplex amplification by chimeric primers (CP), which are virus specific, with a tail added at the 5′ end of the universal primer (UP), followed by universal amplification using UPs and a regular cycle amplification. Five universal primers with CPs (UP1-5) were designed and evaluated in an UP-based single PCR (UP–S-PCR). All five UPs were found to work efficiently and UP2 exhibited the best performance. After system optimizations, the analytical sensitivity of the UP-M-PCR, using plasmids containing the specific viral target fragments, was 5 copies/reaction for each of the five viruses irrespective of presence of a single or multiple viruses in the reaction. No cross-reaction was observed with other non-target viruses. When 273 fecal samples from clinically healthy pigs were tested, the assay sensitivity was 90.9–100%, the specificity was 98.0–100%, and the agreement rate with the UP-S-PCR was 98.5–99.6% with a Kappa value being 0.95–0.98. In summary, the UP-M-PCR developed here is a rapid and highly sensitive and specific detection method that can be used to demonstrate mixed infections in pigs with diarrhea.

APB-13 improves the adverse outcomes caused by TGEV infection by correcting the intestinal microbial disorders in piglets.

Porcine transmissible gastroenteritis virus (TGEV) is an enteric coronavirus that has caused high morbidity and mortality of piglets worldwide. Previous studies have shown that the TGEV can lead to severe diarrhoea, vomiting and dehydration in 2-week-old piglets and weaned piglets, resulting in a large number of piglet deaths. Antimicrobial peptides have broad-spectrum antimicrobial activity and a strong killing effect on bacteria, especially on the drug-resistant pathogenic bacteria, and it has attracted broad concern. However, there are very few reports on the effect of APB-13 (an antimicrobial peptide) on the intestinal microbes of piglets infected with TGEV. In this study, 16S rRNA gene sequencing was used to compare the microbial phylum and the genus of piglet's enteric microorganism in different experimental groups, and to predict the metabolic function of the microbial flora. At the same time, the apparent digestibility of nutrients, digestive enzyme activity, daily weight gain and survival rate were also measured. TGEV infection could cause the imbalance of intestinal microbes in piglets, and increase of the relative abundance of Proteobacteria, and decrease of the relative abundance of Firmicutes, Bacteroidetes and Actinobacteri. With the addition of APB-13, this problem can be alleviated, which can reduce the relative abundance of Proteobacteria and improve the balance of intestinal microorganisms. At the microbial genus level, after adding APB-13, the relative abundance of Catenibacterium, Enterobacter and Streptococcus in the intestinal tract of piglets infected with TGEV showed significant decrease, while the relative abundance of Lactobacillus and Ruminococcus increased. Finally, we found that APB-13 can significantly increase the activity of digestive enzyme in the intestinal tract of piglet, thereby improving the apparent digestibility of nutrients and the growth performance of piglets. This study demonstrates that APB-13 can alleviate the adverse outcomes caused by TGEV infection by correcting the intestinal microbial disorders.

The effect of vitamin and mineral supplements on the replication of coronaviruses in the SPEV cell culture

The article describes studies on the effect of vitamin and mineral supplements (VMS) on the replication of coronavirus in tissue culture. The cytopathic effect of the porcine transmissible gastroenteritis virus (strain TO36SD192 (Japan) and the vaccine strain "RIMS" was studied on the SPEV cell line in the presence of mineral and vitamin supplements (VMS) with an increased content of vitamins A and D. The results of the study showed that the introduction of vitamin and mineral supplements into the growth medium provides an increase in the yield of the RIMS virus by 16.7 - 23%. Interestingly, at the minimum infectious dose, the presence or absence of VMS does not in any way affect the titer of the virus of the reference strain of the coronavirus TO36SD192. However, an increase in the infectious dose by only one order of magnitude (1000 TDC50 / ml) stimulates an increase in the virus titer by 22.5% in the presence of VMS. With the addition of VMS, the RIMS strain reproduced more actively and the virus titer in the experiment with the addition of VMS was 30% higher than in the control (without VMS). A further increase in the infectious dose showed a significant increase in the virus titer from 13.4% to 6.25%. It should be noted that the TO36SD192 virus, well adapted to the SPEV culture, can be compared with viruses that cause seasonal ARVI in humans, which are also well adapted in the human body, and the use of VMS during the peak of the epidemic can stimulate viral replication. Since the RIMS strain virus is poorly adapted to the SPEV culture, its action can be compared with a virus that has entered the human population, for example, SARS-CoV-2. This means that taking vitamins at any infectious dose (during an epidemic) will stimulate the disease.

Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium.

Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response without any interference such as low protein expression, enzyme inactivity, or structural changes. This makes APN an attractive candidate in the development of vaccines that selectively target the mucosal epithelium. Previous studies have shown that APN is a receptor protein for both enterotoxigenic Escherichia coli (E. coli) F4 and transmissible gastroenteritis virus. Thus, APN shows promise in the development of antibody-drug conjugates or novel vaccines based on APN-specific antibodies. In this study, we compared production of APN-specific monoclonal antibodies (mAbs) using traditional hybridoma technology and recombinant antibody expression method. We also established a stably transfected Chinese hamster ovary (CHO) cell line using pIRES2-ZSGreen1-rAbs-APN and an E. coli expression BL21(DE3) strain harboring the pET28a (+)-rAbs-APN vector. The results show that antibodies expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and mAbs produced using hybridomas could recognize and bind to the APN protein. This provides the basis for further elucidation of the APN receptor function for the development of therapeutics targeting different APN-specific epitopes.

Activation of Interleukin-1β Release and Pyroptosis by Transmissible Gastroenteritis Virus Is Dependent on the NOD-Like Receptor Protein 3 Inflammasome in Porcine Intestinal Epithelial Cell Line.

Transmissible gastroenteritis virus (TGEV) is a coronavirus, which causes fatal severe diarrhea and leads to high mortality in newborn piglets. Inflammasomes are hub molecules that induce proinflammatory cytokine production and maturation to initiate innate immune defenses upon cellular infection. To date, the potential role of inflammasome in TGEV infection in porcine intestinal epithelial cells has not been elucidated. The present study aims to investigate the function of the inflammasome in response to TGEV infection in porcine intestinal epithelial cells. Our results revealed that TGEV infection induced the production of pro-interleukin-1β (pro-IL-1β) and enhanced its processing and maturation in porcine intestinal epithelial cells through caspase-1 activation. In addition, TGEV infection in porcine intestinal epithelial cells induced pyroptosis, indicated by cell death and the production and cleavage of gasdermin D (GSDMD). Meanwhile, TGEV infection sufficiently activated the expression and assembly of the NOD-like receptor protein 3 (NLRP3) inflammasome in porcine intestinal epithelial cells, and inhibition of NLRP3 blocked TGEV-induced IL-1β release. We also found that inhibition of NLRP3 enhanced the replication of TGEV without inducing cell death. In conclusion, these data demonstrated that activation of IL-1β release and pyroptosis is dependent on NLRP3 inflammasome, thus NLRP3 inflammasome may play a central role in the innate immune response to TGEV infection.

Evaluation of mobile real-time polymerase chain reaction tests for the detection of severe acute respiratory syndrome coronavirus 2

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), calls for prompt and accurate diagnosis and rapid turnaround time for test results to limit transmission. Here, we evaluated two independent molecular assays, the Biomeme SARS-CoV-2 test, and the Precision Biomonitoring TripleLock SARS-CoV-2 test on a field-deployable point-of-care real-time PCR instrument, Franklin three9, in combination with Biomeme M1 Sample Prep Cartridge Kit for RNA 2.0 (M1) manual extraction system for rapid, specific, and sensitive detection of SARS-COV-2 in cell culture, human, and animal clinical samples. The Biomeme SARS-CoV-2 assay, which simultaneously detects two viral targets, the orf1ab and S genes, and the Precision Biomonitoring TripleLock SARS-CoV-2 assay that targets the 5′ untranslated region (5′ UTR) and the envelope (E) gene of SARS-CoV-2 were highly sensitive and detected as low as 15 SARS-CoV-2 genome copies per reaction. In addition, the two assays were specific and showed no cross-reactivity with Middle Eastern respiratory syndrome coronavirus (MERS-CoV), infectious bronchitis virus (IBV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis (TGE) virus, and other common human respiratory viruses and bacterial pathogens. Also, both assays were highly reproducible across different operators and instruments. When used to test animal samples, both assays equally detected SARS-CoV-2 genetic materials in the swabs from SARS-CoV-2-infected hamsters. The M1 lysis buffer completely inactivated SARS-CoV-2 within 10 min at room temperature enabling safe handling of clinical samples. Collectively, these results show that the Biomeme and Precision Biomonitoring TripleLock SARS-CoV-2 mobile testing platforms could reliably and promptly detect SARS-CoV-2 in both human and animal clinical samples in approximately an hour and can be used in remote areas or health care settings not traditionally serviced by a microbiology laboratory.

SENSITIVITY OF SWINE CORONAVIRUS TO ACTION OF DIFFERENT TEMPERATURES

Coronavirus infections are currently receiving a lot of attention due to the emergence of the covid-19 pandemic in the world. The search for models with which it is possible to obtain an adequate result to identify the properties of coronaviruses is intensive. For comparison with the results of our experiments, the article also presents data on the duration of preservation of the infectious properties of the SARS-Cov-2 virus by contamination of surfaces made of different materials and at different temperatures.&#x0D; We studied the long-term effect on coronavirus, the causative agent of transmissible swine gastroenteritis (TGE), a number of temperatures: + 4ºC, + 25ºC, minus 13ºC, minus 20ºC and thirteen-fold change in temperature, which was in the range of 31-33ºC. It was found that both vaccine and epizootic strains of TGE coronavirus after long-term storage reduce the infectious properties, but when in contact with a sensitive biological system (in vitro) quickly enough (in the case of successive passages in this system) restore them. We proved that the TGE coronavirus during storage for more than two years reduced, but did not lose infectious properties at temperatures minus 13ºC, minus 20ºC, which were restored during subsequent passage in sensitive biological systems in vitro. The same trend was observed for storage of the virus for 8 years at a temperature of + 4ºC. The fastest decrease in coronavirus titer occurred at a temperature of + 25ºC, but more stable under these conditions was the epizootic strain of the virus, which requires attention when working with field isolates of coronavirus.&#x0D; The general tendency of animal and human coronaviruses to decrease the survival time of the pathogen with increasing exposure temperature has been established. Resistance of the virus to repeated sharp changes in temperature (from minus 13 ± 0.5°C to room temperature) without loss of infectious properties was revealed.

Data standardization implementation and applications within and among diagnostic laboratories: integrating and monitoring enteric coronaviruses.

Every day, thousands of samples from diverse populations of animals are submitted to veterinary diagnostic laboratories (VDLs) for testing. Each VDL has its own laboratory information management system (LIMS), with processes and procedures to capture submission information, perform laboratory tests, define the boundaries of test results (i.e., positive or negative), and report results, in addition to internal business and accounting applications. Enormous quantities of data are accumulated and stored within VDL LIMSs. There is a need for platforms that allow VDLs to exchange and share portions of laboratory data using standardized, reliable, and sustainable information technology processes. Here we report concepts and applications for standardization and aggregation of data from swine submissions to multiple VDLs to detect and monitor porcine enteric coronaviruses by RT-PCR. Oral fluids, feces, and fecal swabs were the specimens submitted most frequently for enteric coronavirus testing. Statistical algorithms were used successfully to scan and monitor the overall and state-specific percentage of positive submissions. Major findings revealed a consistently recurrent seasonal pattern, with the highest percentage of positive submissions detected during December-February for porcine epidemic diarrhea virus, porcine deltacoronavirus, and transmissible gastroenteritis virus (TGEV). After 2014, very few submissions tested positive for TGEV. Monitoring VDL data proactively has the potential to signal and alert stakeholders early of significant changes from expected detection. We demonstrate the importance of, and applications for, data organized and aggregated by using LOINC and SNOMED CTs, as well as the use of customized messaging to allow inter-VDL exchange of information.

Porcine Coronaviruses: Overview of the State of the Art

Like RNA viruses in general, coronaviruses (CoV) exhibit high mutation rates which, in combination with their strong tendency to recombine, enable them to overcome the host species barrier and adapt to new hosts. It is currently known that six CoV are able to infect pigs. Four of them belong to the genus Alphacoronavirus [transmissible gastroenteritis coronavirus (TEGV), porcine respiratory coronavirus (PRCV), porcine epidemic diarrhea virus (PEDV), swine acute diarrhea syndrome coronavirus (SADS-CoV)], one of them to the genus Betacoronavirus [porcine hemagglutinating encephalomyelitis virus (PHEV)] and the last one to the genus Deltacoronavirus (PDCoV). PHEV was one of the first identified swine CoV and is still widespread, causing subclinical infections in pigs in several countries. PRCV, a spike deletion mutant of TGEV associated with respiratory tract infection, appeared in the 1980s. PRCV is considered non-pathogenic since its infection course is mild or subclinical. Since its appearance, pig populations have become immune to both PRCV and TGEV, leading to a significant reduction in the clinical and economic importance of TGEV. TGEV, PEDV and PDCoV are enteropathogenic CoV and cause clinically indistinguishable acute gastroenteritis in all age groups of pigs. PDCoV and SADS-CoV have emerged in 2014 (US) and in 2017 (China), respectively. Rapid diagnosis is crucial for controlling CoV infections and preventing them from spreading. Since vaccines are available only for some porcine CoV, prevention should focus mainly on a high level of biosecurity. In view of the diversity of CoV and the potential risk factors associated with zoonotic emergence, updating the knowledge concerning this area is essential.

Acoustical Slot Mode Sensor for the Rapid Coronaviruses Detection.

A method for the rapid detection of coronaviruses is presented on the example of the transmissible gastroenteritis virus (TGEV) directly in aqueous solutions with different conductivity. An acoustic sensor based on a slot wave in an acoustic delay line was used for the research. The addition of anti-TGEV antibodies (Abs) diluted in an aqueous solution led to a change in the depth and frequency of resonant peaks on the frequency dependence of the insertion loss of the sensor. The difference in the output parameters of the sensor before and after the biological interaction of the TGE virus in solutions with the specific antibodies allows drawing a conclusion about the presence/absence of the studied viruses in the analyzed solution. The possibility for virus detection in aqueous solutions with the conductivity of 1.9–900 μs/cm, as well as in the presence of the foreign viral particles, has been demonstrated. The analysis time did not exceed 10 min.

Isolation and Characterization of a Porcine Transmissible Gastroenteritis Coronavirus in Northeast China.

Transmissible gastroenteritis virus (TGEV) is a coronavirus (CoV) that is a major pathogenity of viral enteritis and diarrhea in suckling piglets, causing high morbidity and mortality. In this study, a TGEV strain HQ2016 was isolated from northeast China and characterized its genome sequence and pathogenicity. The phylogenetic analysis indicated that the TGEV HQ2016 strain was more similar to the TGEV Purdue cluster than to the Miller cluster. Both recombination and phylogenetic analysis based on each structural and non-structural gene revealed no recombination event in the HQ2016 strain. Experimental infection study using colostrum-deprived newborn piglets successfully showed that the HQ2016 can cause clinical symptoms including anorexia and yellow-to-whitish watery diarrhea, which are characteristics of TGE, in the inoculated piglets 48 h post-inoculation. These results provide valuable information about the evolution of the porcine CoVs.

DEVELOPMENT OF A UPL PROBE-BASED REAL-TIME PCR ASSAY OF PORCINE DELTACORONAVIRUS IN TAIWAN

Porcine deltacoronavirus (PDCoV) causes clinical symptoms characterized by severe diarrhea and vomiting in neonatal piglets and pregnant sows, which is similar to those resulted from transmissible gastroenteritis coronavirus (TGEV) and porcine epidemic diarrhea virus (PEDV). Since PEDV was considered as the dominant enteric virus all round the world, PDCoV has been unwittingly overlooked due to its indistinguishable clinical signs with other porcine coronaviruses and relatively low death rates in the pig farm. Specimens which have been previously performed for the detection of PEDV in Animal Disease and Diagnostic Center, National Pingtung University of Science and Technology (NPUST) from January 5, 2015 to January 11, 2016 were examined by a novel universal probe library (UPL) probe-based real-time polymerase chain reaction (PCR). A total of 527 clinical specimens from pigs with diarrhea suspected were examined for PDCoV. Positive rates of PDCoV in small intestine and rectal swab were 4.3% (13/305) and 1.8% (4/222), respectively. Collectively, as to the total specimens, the detection rate is 3.2% (17/527). Our results provide development of a UPL probe-based real-time PCR assay and retrospective investigation of potentially circulating PDCoVs in the field in the whole 2015 and early 2016.

Detection and Genetic Diversity of Porcine Coronavirus Involved in Diarrhea Outbreaks in Spain.

Porcine enteric coronaviruses include some of the most relevant viral pathogens to the swine industry such as porcine epidemic diarrhea virus (PEDV) or porcine transmissible gastroenteritis virus (TGEV) as well as several recently identified virus such as swine enteric coronavirus (SeCoV), porcine deltacoronavirus (PDCoV) or swine enteric alphacoronavirus (SeACoV). The aim of this study is the identification and characterization of enteric coronaviruses on Spanish pig farms between 2017 and 2019. The study was carried out on 106 swine farms with diarrhea outbreaks where a viral etiology was suspected by using two duplex RT-PCRs developed for the detection of porcine enteric coronaviruses. PEDV was the only coronavirus detected in our research (38.7% positive outbreaks, 41 out of 106) and neither TGEV, SeCoV, PDCoV nor SeACoV were detected in any of the samples. The complete S-gene of all the PEDV isolates recovered were obtained and compared to PEDV and SeCoV sequences available in GenBank. The phylogenetic tree showed that only PEDV of the INDEL 2 or G1b genogroup has circulated in Spain between 2017 and 2019. Three different variants were detected, the recombinant PEDV-SeCoV being the most widespread. These results show that PEDV is a relevant cause of enteric disorders in pigs in Spain while new emerging coronavirus have not been detected so far. However, the monitoring of these virus is advisable to curtail their emergence and spread.

Replicative capacity of four porcine enteric coronaviruses in LLC-PK1 cells.

Enteric coronaviruses (CoVs) are major pathogens that cause diarrhea in piglets. To date, four porcine enteric CoVs have been identified: transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and HKU2-like porcine enteric alphacoronavirus (PEAV). In this study, we investigated the replicative capacity of these four enteric CoVs in LLC-PK1 cells, a porcine kidney cell line. The results showed that LLC-PK1 cells are susceptible to all four enteric CoVs, particularly to TGEV and PDCoV infections, indicating that LLC-PK1 cells can be applied to porcine enteric CoV research in vitro, particularly for coinfection studies.

A portable, 3D printed, microfluidic device for multiplexed, real time, molecular detection of the porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine deltacoronavirus at the point of need.

The porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV) are emerging/reemerging coronaviruses (CoVs) of neonatal pigs that cause great economic losses to pig farms and pork processors. Specific, rapid, and simple multiplex detection of these viruses is critical to enable prompt implementation of appropriate control measures. Conventional methods for molecular diagnosis require skilled personnel and relatively sophisticated equipment, restricting their use in centralized laboratories. We developed a low-cost, rapid, semi-quantitative, field deployable, 3D-printed microfluidic device for auto-distribution of samples and self-sealing and real-time and reverse transcription-loop-mediated isothermal amplification (RT-LAMP), enabling the co-detection of PEDV, TGEV and PDCoV within 30 minutes. Our assay's analytical performance is comparable with a benchtop, real-time RT-LAMP assay and the gold standard quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay with limits of detection of 10 genomic copies per reaction for PEDV and PDCoV, and 100 genomic copies per reaction for TGEV. Evaluation of clinical specimens from diseased pigs with our microfluidic device revealed excellent concordance with both benchtop RT-LAMP and qRT-PCR. Our portable RT-LAMP microfluidic chip will potentially facilitate simple, specific, rapid multiplexed detection of harmful infections in minimally equipped veterinary diagnostic laboratories and on-site in pigs' farms.

Downregulation of miR-218 by porcine reproductive and respiratory syndrome virus facilitates viral replication via inhibition of type I interferon responses

Porcine reproductive and respiratory syndrome virus (PRRSV) is a devastating pathogen in the swine industry worldwide. miRNAs are reported to be involved in virus–host interaction. Here, we used high-throughput sequencing and miRNA inhibitors to screen possible miRNAs that can inhibit PRRSV infection on its target cell, porcine alveolar macrophages. We observed that miR-218 was downregulated upon virus infection, and knockdown of miR-218 significantly enhanced PRRSV replication. Overexpression of miR-218 resulted in a decrease in PRRSV replication, and this overexpression did not alter viral genomic RNA levels, but rather increased antiviral interferon signaling. Further analysis revealed that miR-218 regulated PRRSV replication by directly targeting porcine suppressor of cytokine signaling 3 (SOCS3), a JAK2 kinase inhibitor. Knockdown of the endogenous SOCS3 expression led to augmentation of type I interferon genes and resulted in decreased PRRSV replication, and vice versa. During PRRSV infection in vivo and in vitro, cellular miR-218 expression was downregulated and SOCS3 expression was upregulated, further supporting the inverse correlation between miR-218 and SOCS3 expression. The data on SOCS3 depletion in combination with miR-218 inhibition suggested that the antiviral activity of miR-218 required the SOCS3-mediated signaling pathway. Similarly, miR-218 negatively regulated PRRSV replication in Marc-145 cells, as well as the replication of porcine epidemic diarrhea virus and transmissible gastroenteritis virus in Vero and ST cells respectively. Taken together, these results demonstrate that PRRSV-induced miR-218 downregulation serves to inhibit the type I interferon response and may provide a novel therapeutic target for treatment of PRRSV and other viral infections.