Porcine Transmissible Gastroenteritis Virus Research Articles

Molecular and phylogenetic analysis of transmissible gastroenteritis virus strain VET-16, isolated from piglets in Vietnam.

Porcine transmissible gastroenteritis virus (TGEV) is a major pathogen that causes viral enteritis and severe diarrhea in newborn piglets. TGEV strains have been isolated in the USA, Europe, and China, and their molecular characteristics are well known. However, there have been few reports of molecular analysis of TGEV strains isolated in Southeast Asia. In 2016, we isolated TGEV strain VET-16 from fecal samples collected from piglets in Vietnam and determined its complete genome sequence by Sanger sequencing. We found that, while the full genome of the VET-16 strain was 92.4-99.9% identical to those of other TGEV strains, the ORF3 gene showed very little sequence similarity. Phylogenetic analysis suggested that the VET-16 strain belongs to the Purdue subgroup. Comparison of the predicted amino acid (aa) sequence of the spike protein of strain VET-16 with those of other TGEV strains revealed three aa substitutions (V378L, S379T, and D380N) and a 3-aa insertion (F383_F387insWEK) in antigenic site D of the VET-16 strain. Also, a single aa deletion (∆F1413) was found in the transmembrane domain of the spike gene of VET-16. Like the ORF3 gene from the TGEV Miller M60 vaccine strain, the VET-16 strain has a large deletion (∆725 nt) in the ORF3 gene. Previous studies have suggested that these mutations in the spike and ORF3 genes might be associated with a reduction in pathogenicity. The data from this study will facilitate further genetic analysis and research into the evolution of TGEV in pigs in Vietnam.

Serial cell culture passaging in vitro led to complete attenuation and changes in the characteristic features of a virulent porcine deltacoronavirus strain.

Porcine deltacoronavirus (PDCoV) is an important enteric coronavirus that has caused enormous economic losses in the pig industry worldwide. However, no commercial vaccine is currently available. Therefore, developing a safe and efficacious live-attenuated vaccine candidate is urgently needed. In this study, the PDCoV strain CH/XJYN/2016 was continuously passaged in LLC-PK cells until passage 240, and the virus growth kinetics in cell culture, pathogenicity in neonatal piglets, transcriptome differences after LLC-PK infection, changes in the functional characteristics of the spike (S) protein in the high- and low-passage strains, genetic variation of the virus genome, resistance to pepsin and acid, and protective effects of this strain when used as a live-attenuated vaccine were examined. The results of animal experiments demonstrated that the virulent PDCoV strain CH/XJYN/2016 was completely attenuated and not pathogenic in piglets following serial cell passage. Genome sequence analysis showed that amino acid mutations in nonstructural proteins were mainly concentrated in Nsp3, structural protein mutations were mainly concentrated in the S protein, and the N, M, and E genes were conserved. Transcriptome comparison revealed that compared with negative control cells, P10-infected LLC-PK cells had the most differentially expressed genes (DEGs), while P0 and P240 had the least number of DEGs. Analysis of trypsin dependence and related structural differences revealed that the P10 S protein interacted more strongly with trypsin and that the P120 S protein interacted more strongly with the APN receptor. Moreover, the infectivity of P240 was not affected by pepsin but was significantly decreased after exposure to low pH. Furthermore, the P240-based live-attenuated vaccine provided complete protection to piglets against the challenge of virulent PDCoV. In conclusion, we showed that a PDCoV strain was completely attenuated through serial passaging in vitro. These results provide insights into the potential molecular mechanisms of PDCoV attenuation and the development of a promising live-attenuated PDCoV vaccine.IMPORTANCEPorcine deltacoronavirus (PDCoV) is one of the most important enteropathogenic pathogens that cause diarrhea in pigs of various ages, especially in suckling piglets, and causes enormous economic losses in the global commercial pork industry. There are currently no effective measures to prevent and control PDCoV. As reported in previous porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus studies, inactivated vaccines usually elicit less robust protective immune responses than live-attenuated vaccines in native sows. Therefore, identifying potential attenuation mechanisms, gene evolution, pathogenicity differences during PDCoV passaging, and immunogenicity as live-attenuated vaccines is important for elucidating the mechanism of attenuation and developing safe and effective vaccines for virulent PDCoV strains. In this study, we demonstrated that the virulence of the PDCoV strain CH/XJYN/2016 was completely attenuated following serial cell passaging in vitro, and changes in the biological characteristics and protection efficacy of the strain were evaluated. Our results help elucidate the mechanism of PDCoV attenuation and support the development of appropriate designs for the study of live PDCoV vaccines.

Development and Clinical Application of a Molecular Assay for Four Common Porcine Enteroviruses.

Porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA) are the four main pathogens that cause viral diarrhea in pigs, and they often occur in mixed infections, which are difficult to distinguish only according to clinical symptoms. Here, we developed a multiplex TaqMan-probe-based real-time RT-PCR method for the simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA for the first time. The specific primers and probes were designed for the M protein gene of PEDV, N protein gene of TGEV, N protein gene of PDCoV, and VP7 protein gene of PoRVA, and corresponding recombinant plasmids were constructed. The method showed extreme specificity, high sensitivity, and excellent repeatability; the limit of detection (LOD) can reach as low as 2.18 × 102 copies/μL in multiplex real-time RT-PCR assay. A total of 97 clinical samples were used to compare the results of the conventional reverse transcription PCR (RT-PCR) and this multiplex real-time RT-PCR for PEDV, TGEV, PDCoV, and PoRVA detection, and the results were 100% consistent. Subsequently, five randomly selected clinical samples that tested positive were sent for DNA sequencing verification, and the sequencing results showed consistency with the detection results of the conventional RT-PCR and our developed method in this study. In summary, this study developed a multiplex real-time RT-PCR method for simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA, and the results of this study can provide technical means for the differential diagnosis and epidemiological investigation of these four porcine viral diarrheic diseases.

Development of a colloidal gold immunochromatographic strip for the simultaneous detection of porcine epidemic diarrhea virus and transmissible gastroenteritis virus.

In recent years, porcine diarrhea-associated viruses have caused significant economic losses globally. These viruses present similar clinical symptoms, such as watery diarrhea, dehydration, and vomiting. Co-infections with porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are common. For the rapid and on-site preliminary diagnosis on the pig farms, this study aimed to develop a colloidal gold immunochromatography assay (GICA) strip for the detection of PEDV and TGEV simultaneously. The GICA kit showed that there was no cross-reactivity with the other five common porcine viruses. With visual observation, the lower limits were approximately 104 TCID50/mL and 104 TCID50/mL for PEDV and TGEV, respectively. The GICA strip could be stored at 4°C or 25°C for 12 months without affecting its efficacy. To validate the GICA strip, 121 clinical samples were tested. The positive rates of PEDV and TGEV were 42.9 and 9.9%, respectively, and the co-infection rate of the two viruses was 5.8% based on the duplex GICA strip. Thus, the established GICA strip is a rapid, specific, and stable tool for on-site preliminary diagnosis of PEDV- and TGEV-associated diarrhea.

THE CO-ADMINISTRATION OF POIL-2 WITH TGEV INACTIVATED VACCINE ENHANCES IMMUNE RESPONSE OF PIGLETS TO TGEV

Porcine interleukin-2 (poIL-2) has not yet been demonstrated to be immune-enhancing against porcine transmissible gastroenteritis virus (TGEV) inactivated vaccine (IV), despite IL-2 having been proven to have immunological adjuvant effects for a variety of vaccinations. In this work, the impact of poIL-2 on TGEV IV in terms of immunological enhancement was investigated. Twenty four (24) SPF piglets were utilized and divided into six groups: PBS group, poIL-2 group, IV group, 10μg poIL-2+IV group, 50μg poIL-2 +IV group, and 250μg poIL-2+IV group. They received a second vaccine at 28 days point following the initial immunization. Serum and blood samples were obtained at various periods throughout the experiment. By using ELISA assay, neutralization assay, MTT assay, and flow cytometry assay, the TGEV-specific antibody expressions, neutralizing antibodies generations, interleukin-4 (IL-4), and interferon-gamma (IFN-γ) productions, peripheral blood mononuclear cells (PBMCs) proliferation response and lymphocyte phenotype subpopulations (CD3+, CD4+, and CD8+ immune cells) reflections were determined. The results showed that piglets inoculated with IV supplemented with poIL-2 significantly not only increased more piglet cellular immunity against TGEV by raising the degrees of IL-4, IFN-γ, Stimulation Index (SI), and the ratio of CD4+ /CD8+ cell subgroups, but also promoted more humoral immunity against TGEV by increasing levels of anti-TGEV specific antibodies and neutralizing antibodies (NAs) than those piglets inoculated with the TGEV IV alone. Additionally, the results suggested that porcine interleukin-2 (poIL-2) may improve pigs' immune responses in a dosage-dependent way.Our study revealed that poIL-2 had an immune-enhancing effect on the immunization of TGEV IV, and it possessed the potential to be applied as an immune-stimulating agent. Keywords: Inactivated vaccine (IV); adjuvant; porcine transmissible gastroenteritis virus (TGEV); porcine interleukin-2 (poIL-2); immune response.

Development of a triplex quantitative reverse transcription-polymerase chain reaction for the detection of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, and porcine rotavirus A.

Porcine viral diarrhea is caused by many pathogens and can result in watery diarrhea, dehydration and death. Various detection methods, such as polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR), have been widely used for molecular diagnosis. We developed a triplex real-time quantitative reverse transcription PCR (qRT-PCR) for the simultaneous detection of three RNA viruses potentially associated with porcine viral diarrhea: porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine rotavirus A (PoRVA). The triplex qRT-PCR had R2 values of 0.999 for the standard curves of PEDV, TGEV and PoRVA. Importantly, the limits of detection for PEDV, TGEV and PoRVA were 10 copies/μL. The specificity test showed that the triplex qRT-PCR detected these three pathogens specifically, without cross-reaction with other pathogens. In addition, the approach had good repeatability and reproducibility, with intra-and inter-assay coefficients of variation <1%. Finally, this approach was evaluated for its practicality in the field using 256 anal swab samples. The positive rates of PEDV, TGEV and PoRVA were 2.73% (7/256), 3.91% (10/256) and 19.14% (49/256), respectively. The co-infection rate of two or more pathogens was 2.73% (7/256). The new triplex qRT-PCR was compared with the triplex RT-PCR recommended by the Chinese national standard (GB/T 36871-2018) and showed 100% agreement for PEDV and TGEV and 95.70% for PoRVA. Therefore, the triplex qRT-PCR provided an accurate and sensitive method for identifying three potential RNA viruses for porcine viral diarrhea that could be applied to diagnosis, surveillance and epidemiological investigation.

Pyrococcus furiosus Argonaute Based Detection Assays for Porcine Deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is a major cause of diarrhea and diarrhea-related deaths among piglets and results in massive losses to the overall porcine industry. The clinical manifestations of porcine diarrhea brought on by the porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and PDCoV are oddly similar to each other. Hence, the identification of different pathogens through molecular diagnosis and serological techniques is crucial. Three novel detection methods for identifying PDCoV have been developed utilizing recombinase-aided amplification (RAA) or reverse transcription recombinase-aided amplification (RT-RAA) in conjunction with Pyrococcus furiosus Argonaute (PfAgo): RAA-PfAgo, one-pot RT-RAA-PfAgo, and one-pot RT-RAA-PfAgo-LFD. The indicated approaches have a detection limit of around 60 copies/μL of PDCoV and do not cross-react with other viruses including PEDV, TGEV, RVA, PRV, PCV2, or PCV3. The applicability of one-pot RT-RAA-PfAgo and one-pot RT-RAA-PfAgo-LFD were examined using clinical samples and showed a positive rate comparable to the qPCR method. These techniques offer cutting-edge technical assistance for identifying, stopping, and managing PDCoV.

Pathological and immunohistochemical assessment of the impact of three different strains of swine enteric coronaviruses in the intestinal barrier

Swine enteric coronaviruses, such as porcine epidemic diarrhea virus (PEDV) or transmissible gastroenteritis virus (TGEV), have risen concern for the porcine industry and research community due to the increase in their virulence, their potential recombination capacity and the emergence of new variants. This in vivo study aims to compare the impact of three different strains of swine enteric coronaviruses [(two G1b (S-INDEL) PEDV strains and a recombinant TGEV-PEDV or Swine enteric coronavirus (SeCoV)] in the intestine of 3-weeks-old infected piglets, focusing on the pathology and main components of the intestinal barrier, including the number of goblet cells, and the expression of IgA as well as FoxP3, a regulatory T cell marker. Severity of lesions was evidenced in the three infected groups and was highly correlated with the viral load in feces and the frequency of viral antigen-positive cells. Furthermore, higher cellular death together with an increase in the expression of the FoxP3 marker was detected in the duodenum and jejunum of infected animals at 3 days post-infection. Our results highlight a recruitment of FoxP3+ cells in the small intestine of infected animals which may represent a response to the tissue damage caused by viral replication and cell death. Further studies should be addressed to determine the potential role of these cells during swine enteric coronavirus infections.

Virus-like particle vaccines with epitopes from porcine epidemic virus and transmissible gastroenteritis virus incorporated into self-assembling ADDomer platform provide clinical immune responses in piglets.

Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are major intestinal coronaviruses that cause vomiting, diarrhea, dehydration, and mortality in piglets. These viruses coexist and lead to significant economic losses in the swine industry. Virus-like particles (VLPs) have emerged as promising alternatives to conventional inactivated vaccines due to their exceptional safety, efficacy, and ability to provide multi-disease protection with a single dose. Our study focused on specific antigenic epitopes from the PEDV S protein (SS2 and 2C10 regions) and the TGEV S protein (A and D sites) as target candidates. These epitopes were integrated into the ADDomer framework, and we successfully generated recombinant proteins AD, AD-P, AD-T, and AD-PT using the baculovirus expression vector system (BEVS). By meticulously optimizing conditions in High Five cells, we successfully expressed and purified the recombinant proteins. Subsequently, we developed the recombinant ADDomer-VLP vaccine and conducted a comprehensive evaluation of its efficacy in piglets. Following ultrafiltration concentration and sucrose gradient centrifugation purification, the recombinant proteins self-assembled into VLPs as observed by transmission electron microscopy (TEM). Administration of the vaccine did not result in any adverse reactions in the immunized piglets. Additionally, no significant instances of fever were detected in any of the experimental groups, and there were no notable changes in average daily weight gain compared to the control group that received PBS. The recombinant ADDomer-VLP vaccines demonstrated strong immunogenicity, effectively stimulating the production of neutralizing antibodies against both PEDV and TGEV. Moreover, the recombinant ADDomer-VLP vaccine induced elevated levels of IFN-γ, IL-2, and IL-4, and enhanced cytotoxic T lymphocyte (CTL) activity in the peripheral blood of piglets. These recombinant VLPs have demonstrated the ability to induce strong cellular and humoral immune responses in piglets, making them an incredibly promising platform for the rapid and simplified development of epitope vaccines.

MAb Targeting a Link Between ExoN and MTase of TGEV NSP14.

Porcine transmissible gastroenteritis virus (TGEV) infection results in severe gastrointestinal disease manifesting vomiting, diarrhea in neonatal porcine, with extremely high mortality. Monoclonal antibody (MAb) specific to TGEV nonstructural protein (NSP)14 that contains two functional domains, exonuclease (ExoN) and methyltransferase (MTase) domains, may help elucidate the role of NSP14 in the viral life-cycle. In this study, we developed a murine MAb, designated 12F1, against TGEV NSP14 using traditional cell-fusion technique. It was shown the MAb can exclusively bind to viral NSP14, as evidenced by the results of indirect fluorescent assay and western blotting. Intriguingly, epitope screening assay shown that 12F1 targets a hinge region connecting ExoN and N7-MTase of NSP14.

Ultrasensitive detection porcine transmissible gastroenteritis virus based on the dual-photoelectrode photofuel cell with in-situ isothermal amplification

Loop-mediated isothermal amplification (LAMP) as a nucleic acid detection technology has many advantages than conventional polymerase chain reaction in terms of sensitivity, specificity, and equipment requirements. And combining photocatalytic fuel cell (PFC) power density readout with high sensitivity, we developed a LAMP-based dual-photoelectrode photocatalytic fuel cell based self-powered biosensors (LAMP PFC-SPBs) for the ultra-sensitive detection of transmissible gastroenteritis virus (TGEV). In this sensor, the specially designed primers were immobilized on the photosensitive material CdS-modified sensing substrate with a distinct electrochemical signal and amplified TGEV within 1 h. A wide detection range of the LAMP PFC-SPBs was demonstrated, ranging from 0.075 fg μL−1 to 7.5 ng μL−1, with a lower detection limit of 0.025 fg μL−1. In-situ LAMP PFC-SPBS had great merits in simplicity (no complex thermal cycles, no need for expensive instruments), sensitivity, specificity, and provides a new perspective on the combination of nucleic acid amplification and electrochemistry.

Determination of optimum nanofiltration conditions for the manufacturing process of human normal immunoglobulin G for intravenous administration

Scientific relevance. Medicinal products based on immunoglobulin class G (IgG) from human plasma are widely used in clinical practice to treat bacterial and viral infections, primary and secondary immunodeficiencies, and autoimmune diseases. Nanofiltration is a way to mitigate the risk of in-process contamination of raw materials with various pathogens, including viruses. Therefore, it is relevant to investigate the development and implementation of additional viral inactivation and/or elimination steps.Aim. This study aimed to develop and validate optimum nanofiltration conditions and to scale up the nanofiltration step for the manufacturing of human IgG for intravenous administration.Materials and methods. The study used a solution of IgG from plasma fractions II and III. The authors paired nanofilters manufactured by Planova 20N and BioEx (Asahi Kasei, Japan), Viresolve Pro (Merck Millipore, USA), Virosart HC and HF (Sartorius, Germany), and Pegasus SV4 and Prime (Pall, USA) with Sartopore polyethersulphone prefilters by Sartorius (Germany), Virosart MAX polyamide prefilters by Sartorius (Germany), and EKX-P regenerated cellulose prefilters by Pall (Germany). Virus reduction validation studies were performed with model viruses (human immunodeficiency virus type 1, porcine transmissible gastroenteritis virus, porcine parvovirus, murine encephalomyocarditis virus, and bovine viral diarrhoea virus) in the laboratories of the N.F. Gamaleya centre. The sample data analysis involved calculating mean values with 95% confidence intervals.Results. For all the selected combinations of prefilters and filters, the maximum nanofiltration throughput depended on the IgG concentration in the test solution. With the combination of an EKX-P filter with a Pegasus SV4 nanofilter, the maximum throughput and the IgG yield reached 6300 g/m2 and 95%, respectively. When combined with a Planova 20N nanofilter, EKX-P and Sartopore (polyethersulphone) filters provided a maximum throughput of up to 2980 g/m2 and an IgG yield of almost 100%, provided that the test solution had an IgG concentration of 10 g/L. With different filter combinations, virus reduction levels ranged from 4.00±0.05 to 4.75±0.04 log10 for human immunodeficiency virus type 1, from 4.30±0.04 to 4.55±0.06 log10 for porcine transmissible gastroenteritis virus, from 5.38±0.08 log10 to 5.57±0.04 log10 for murine encephalomyocarditis virus, 5.12±0.10 log10 to 5.25±0.08 log10 for porcine parvovirus, and exceeded 5.00 log10 for bovine viral diarrhoea virus. The virus reduction levels achieved were not statistically associated with prefilter brands.Conclusions. The study demonstrated that nanofiltration was effective at removing viruses with various virion sizes and physicochemical characteristics, including viruses as small as parvovirus B19. The levels of virus reduction exceeded 4 log10 and met the acceptance criteria.The laboratory-scale nanofiltration parameters and the corresponding filtration times, as well as IgG yields, did not change when the process was scaled up. Therefore, nanofiltration is an effective and productive technique that helps eliminate various types of viruses and considerably improve viral safety without affecting the quality of biological medicinal products.

Antiviral efficacy of nanomaterial-treated textiles in real-life like exposure conditions

Due to the growing interest towards reducing the number of potentially infectious agents on critical high-touch surfaces, the popularity of antimicrobially and antivirally active surfaces, including textiles, has increased. The goal of this study was to create antiviral textiles by spray-depositing three different nanomaterials, two types of CeO2 nanoparticles and quaternary ammonium surfactant CTAB loaded SiO2 nanocontainers, onto the surface of a knitted polyester textile and assess their antiviral activity against two coronaviruses, porcine transmissible gastroenteritis virus (TGEV) and severe acute respiratory syndrome virus (SARS CoV-2). Antiviral testing was carried out in small droplets in semi-dry conditions and in the presence of organic soiling, to mimic aerosol deposition of viruses onto the textiles. In such conditions, SARS CoV-2 stayed infectious at least for 24 h and TGEV infected cells even after 72h of semi-dry deposition suggesting that textiles exhibiting sufficient antiviral activity before or at 24 h, can be considered promising. The antiviral efficacy of nanomaterial-deposited textiles was compared with the activity of the same nanomaterials in colloidal form and with positive control textiles loaded with copper nitrate and CTAB. Our results indicated that after deposition onto the textile, CeO2 nanoparticles lost most of their antiviral activity, but antiviral efficacy of CTAB-loaded SiO2 nanocontainers was retained also after deposition. Copper nitrate deposited textile that was used as a positive control, showed relatively high antiviral activity as expected. However, as copper was effectively washed away from the textile already during 1 h, the use of copper for creating antiviral textiles would be impractical. In summary, our results indicated that antiviral activity of textiles cannot be predicted from antiviral efficacy of the deposited compounds in colloid and attention should be paid on prolonged efficacy of antivirally coated textiles.

Coronaviral Main Protease Induces LPCAT3 Cleavage and Endoplasmic Reticulum (ER) Stress.

Zoonotic coronaviruses infect mammals and birds, causing pulmonary and gastrointestinal infections. Some animal coronaviruses, such as the porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV), lead to severe diarrhea and animal deaths. Gastrointestinal symptoms were also found in COVID-19 and SARS patients. However, the pathogenesis of gastrointestinal symptoms in coronavirus diseases remains elusive. In this study, the main protease-induced LPCAT3 cleavage was monitored by exogenous gene expression and protease inhibitors, and the related regulation of gene expression was confirmed by qRT-PCR and gene knockdown. Interestingly, LPCAT3 plays an important role in lipid absorption in the intestines. The Mpro of coronaviruses causing diarrhea, such as PEDV and MERS-CoV, but not the Mpro of HCoV-OC43 and HCoV-HKU1, which could induce LPCAT3 cleavage. Mutagenesis analysis and inhibitor experiments indicated that LPCAT3 cleavage was independent of the catalytic activity of Mpro. Moreover, LPCAT3 cleavage in cells boosted CHOP and GRP78 expression, which were biomarkers of ER stress. Since LPCAT3 is critical for lipid absorption in the intestines and malabsorption may lead to diarrhea in coronavirus diseases, Mpro-induced LPCAT3 cleavage might trigger gastrointestinal symptoms during coronavirus infection.

Non-coding RNAs derived from the foot-and-mouth disease virus genome trigger broad antiviral activity against coronaviruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of a potentially severe respiratory disease, the coronavirus disease 2019 (COVID-19), an ongoing pandemic with limited therapeutic options. Here, we assessed the anti-coronavirus activity of synthetic RNAs mimicking specific domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome (ncRNAs). These molecules are known to exert broad-spectrum antiviral activity in cell culture, mice and pigs effectively triggering the host innate immune response. The ncRNAs showed potent antiviral activity against SARS-CoV-2 after transfection in human intestinal Caco-2 and lung epithelium Calu-3 2B4 cells. When the in vivo efficacy of the FMDV ncRNAs was assessed in K18-hACE2 mice, administration of naked ncRNA before intranasal SARS-CoV-2 infection significantly decreased the viral load and the levels of pro-inflammatory cytokines in the lungs compared with untreated infected mice. The ncRNAs were also highly efficacious when assayed against common human HCoV-229E and porcine transmissible gastroenteritis virus (TGEV) in hepatocyte-derived Huh-7 and swine testis ST cells, respectively. These results are a proof of concept of the pan-coronavirus antiviral activity of the FMDV ncRNAs including human and animal divergent coronaviruses and potentially enhance our ability to fight future emerging variants.

Characterization and Evaluation of the Pathogenicity of a Natural Gene-Deleted Transmissible Gastroenteritis Virus in China

Porcine transmissible gastroenteritis virus is the major pathogen that causes fatal diarrhea in newborn piglets. In this study, a TGEV strain was isolated from the small intestine of diarrhea piglets in Sichuan Province, China, and designated SC2021. The complete genomic sequence of TGEV SC2021 was 28561 bp, revealing a new natural deletion TGEV strain. Based on phylogenetic analyses, TGEV SC2021 belonged to the Miller cluster and was closely related to CN strains. The newborn piglets orally challenged with TGEV SC2021 showed typical watery diarrhea. In addition, macro and micropathological changes in the lungs and intestines were observed. In conclusion, we isolated a new natural deletion virus strain and confirmed that the virus strain has high pathogenicity in newborn piglets. Moreover, macroscopic and microscopic lesions were observed in the lungs and intestines of all TGEV SC2021-infected piglets. In summary, we isolated a new natural deletion TGEV strain and demonstrated that the natural deletion strain showed high pathogenicity in newborn piglets. These data enrich the diversity of TGEV strains and help us to understand the genetic evolution and molecular pathogenesis of TGEV.

IFITM3 is a host restriction factor that inhibits porcine transmissible gastroenteritis virus infection

Interferon-induced transmembrane proteins (IFITMs) play an important role in the innate immune response triggered by viral infection. Transmissible gastroenteritis virus (TGEV) causes severe diarrhea, vomiting and dehydration in piglets, resulting in huge economic losses to the swine industry. In this study, we showed that IFITM3 inhibits the replication of TGEV and interferes with the binding of TGEV to PK15 cells. Moreover, the inhibitory effect of IFITM3 on TGEV circumvents the upregulation of inflammatory cytokines. Subsequently, we found that the M22A mutant loses part of the antiviral effect of IFITM3 on TGEV; in contrast, the K24A mutant enhances the antiviral effect of IFITM3. Notably, our data shows a synergistic effect between IFITM3 and CQ, which further amplifies the antiviral effect against TGEV.

A Monoclonal Antibody Targeting C-Terminal Domain of Transmissible Gastroenteritis Virus Spike Protein.

The structure and function of the C-terminus domain (CTD) of porcine transmissible gastroenteritis virus (TGEV) spike protein remain largely unknown, thereby a specific monoclonal antibody (MAb) allows us to fully understand this domain. In this study, we developed a murine MAb against CTD of TGEV spike protein, as evidenced by the results of indirect fluorescent assay, Western blotting, and fluorescence-activated cell sorter. Further study showed that the MAb is able to exclusively recognize a 12-residue peptide (FKNVSDGVIYSV) derived from CTD of TGEV spike protein. This MAb can be used to elucidate the potential function of CTD of TGEV spike in virus attachment and entry, and warrants further intensive investigation.

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.

Inactivation of two SARS-CoV-2 virus surrogates by electron beam irradiation on large yellow croaker slices and their packaging surfaces

The detection of infectious SARS-CoV-2 in food and food packaging associated with the cold chain has raised concerns about the possible transmission pathway of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in foods transported through cold-chain logistics and the need for novel decontamination strategies. In this study, the effect of electron beam (E-beam) irradiation on the inactivation of two SARS-CoV-2surrogate, viruses porcine epidemic diarrhea virus (PEDV) and porcine transmissible gastroenteritis virus (TGEV), in culture medium and food substrate, and on food substrate were investigated. The causes of virus inactivation were also investigated by transmission electron microscopy (TEM) and Quantitative Real-time PCR (QRT-PCR). Samples packed inside and outside, including virus-inoculated large yellow croaker and virus suspensions, were irradiated with E-beam irradiation (2, 4, 6, 8, 10 kGy) under refrigerated (0 °C)and frozen (−18 °C) conditions. The titers of both viruses in suspension and fish decreased significantly (P < 0.05) with increasing doses of E-beam irradiation. The maximum D10 value of both viruses in suspension and fish was 1.24 kGy. E-beam irradiation at doses below 10 kGy was found to destroy the spike proteins of both SARS-CoV-2 surrogate viruses by transmission electron microscopy (TEM) and negative staining of thin-sectioned specimens, rendering them uninfectious. E-beam irradiation at doses greater than 10 kGy was also found to degrade viral genomic RNA by qRT-PCR. There were no significant differences in color, pH, TVB-N, TBARS, and sensory properties of irradiated fish samples at doses below 10 kGy. These findings suggested that E-beam irradiation has the potential to be developed as an efficient non-thermal treatment to reduce SARS-CoV-2 contamination in foods transported through cold chain foods to reduce the risk of SARS-CoV-2 infection in humans through the cold chain.