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Nanoceria (cerium dioxide nanoparticles, CeO2 ) has a broad range of biological properties including antiviral activity. The hypothesis was that nanoceria can efficacy against coronavirus (coronavirus of porcine transmissible gastroenteritis) and potentially can target SARS-CoV-2. Transmissible gastroenteritis coronavirus (TGEV) is the etiologic agent of porcine transmissible gastroenteritis (PTG), a highly contagious pig intestinal disease. The aim of the study was to determine the antiviral activity of CeO2 nanoparticles on the model of porcine coronavirus – TGEV. Methods. We used a highly pathogenic virus strain D52-5 (BRE79 ), of TGEV. We evaluated antiviral activity of CeO2 nanoparticles on the experimental model of porcine coronavirus (transmissible gastroenteritis virus) in transplantable line of porcine embryonic kidney cells (PEK) culture. Results. The criterion for evaluating the inhibitory activity of antiviral drugs in different in vitro systems is the selectivity index (SI) and the reduction of infectious titer by 1.5–2.0 lgTCD50 . Nanoceria effectively inhibited the reproduction of porcine coronavirus with SI index of 83.3. © 2021, Zabolotny Institute of Microbiology and Virology, NAS of Ukraine. All rights reserved.

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.

Coronaviruses occurring in humans and animals are a diverse group of pathogens, characterized by high variability and tropism. The most serious animal diseases caused by coronaviruses include equine coronavirus disease (E-CoV), canine coronavirus disease (C-CoV), rabbit enteric coronavirus disease (Rb-CoV), infectious bronchitis in poultry (IB-CoV), epizootic catarrhal enteritis in ferrets, in pigs: transmissible gastroenteritis coronavirus disease (TGE), human enteric coronavirus disease (HE-CoV), and porcine epidemic diarrhea (PED), in cattle (Bo-CoV): neonatal calf diarrhea, respiratory tract infections, winter dysentery in cattle, in cats: feline enteric coronavirus disease (FE-CoV) and feline infectious peritonitis (FIP-CoV). To date, three major epidemics causing severe respiratory diseases in humans (SARS-CoV-1, MERS-CoV, SARS-CoV-2) have been reported. Due to the high morbidity and mortality rate, coronavirus-induced diseases in animals result in serious economic losses. The current human COVID-19 pandemic demonstrates the need for action to modernize and improve diagnostics in this area, but also to develop innovative immunoprophylactic and therapeutic methods.

Highlights • A multiplex reverse transcription polymerase chain rection (mRT-PCR) was developed for simultaneous detection of four RNA viruses in swine. • The detection limit of mRT-PCR was in agreement with the singleplex RT-PCR. • The mRT-PCR were successfully used to detect clinical specimens. • The mRT-PCR developed is a useful tool for clinical diagnosis of single virus and/or mixed infections in swine., A multiplex reverse transcription polymerase chain rection (mRT-PCR) was developed for simultaneous detection of four RNA viruses in swine. The conserved target sequences directed to classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis coronavirus (TGEV) were selected based on alignments of genomic sequences and then specific primers were designed. The mRT-PCR assay was developed and evaluated for its specificity and sensitivity. The expected product from the single viral template was amplified by mRT-PCR and no spurious PCR amplification occurred from the genomic RNA or DNA of other pathogens. For single virus or different combinations of two viruses the detection limit of mRT-PCR was consistent with a single RT-PCR wtith 1 × 103 copies. For different combinations of the three viruses or four viruses, sensitivity of PEDV detection partially decreased. All of positive clinical specimens by the mRT-PCR were identically confirmed using Taqman RT-qPCR. Therefore, the mRT-PCR is a useful tool for epidemiological studies and laboratory diagnosis of single virus and/or mixed infections in swine.

Transmissible gastroenteritis coronavirus (TGEV) is enteropathogenic coronavirus that causes diarrhea in pigs, and is associated with high morbidity and mortality in sucking piglets. The TGEV membrane (M) protein is a decisive protein for the proliferation of viral proteins, and is associated with virus assembly and budding. To identify the cellular proteins that interact with the TGEV M protein, yeast two-hybrid screening was employed, and seven cellular proteins were identified M-binding partners. Using the GST pull-down approach and a CO-IP assay, the M protein was found to interact with porcine intestinal cells via eukaryotic translation initiation factor 4-alpha (EIF4A2), an essential component of the cellular translational machinery. Additionally, confocal microscopy revealed that EIF4A2 and M were colocalized in the cytoplasm. Furthermore, the function of EIF4A2 in intestinal cells during TGEV infection was examined. A knockdown of EIF4A2 by siRNA markedly decreased M protein proliferation and TGEV replication in target cells. Thus demonstrating that EIF4A2 plays a significant role in TGEV replication. The present study provides mechanistic insight into the interaction between the TGEV M protein and intestinal cells which contributes to the understanding of coronavirus replication and may be useful for the development of novel therapeutic strategies for TGEV infection., Highlights • Yeast two-hybrid system identified seven proteins to interact with TGEV M protein. • EIF4A2 was confirmed to interact with and TGEV M protein via GST-pull down and CO-IP. • Immunofluorescence revealed colocalization of EIF4A2 and M protein in the cytoplasm. • EIF4A2 siRNA knockdown reduced TGEV replication in porcine IECs. • EIF4A2 may be associated with TGEV replication in porcine intestinal cells.

This case study report describes a transmissible gastroenteritis coronavirus (TGEV) infection presented in a commercial pig herd. The clinical signs of infection appeared in newborn piglets, including medium morbidity and low mortality rates. Rectal swabs were collected from five different affected litters for laboratory examinations. Samples from two dead piglets and two euthanized affected piglets were collected for gross and histopathological examinations. All fecal samples were tested TGEV positive by real-time polymerase chain reaction (RT-PCR). Necropsy revealed nonspecific gross lesions. The histopathological examinations revealed villi fused with denuded tips and severe villus atrophy, leading to extensive epithelial flattening in middle and lower small intestine. The architecture pattern of villi presented columnar and cuboidal poorly differentiated enterocytes with mild subepithelial edema. In some enterocytes, pycnotic nuclei were detected. Microscopic examination of brain tissue revealed diffuse gliosis in the area of pia matter with mild congestion of the meningeal and parenchymal vessels and neuronal degeneration. In conclusion, this case study reported an epidemic TGEV infection in piglets, characterized by low mortality and medium morbidity rates accompanied by typical histopathological lesions in small intestine, as well as by coexisting brain lesions, that are described for the first time.

Transmissible gastroenteritis (TGE) is a contagious and infectious disease that is characterized by severe vomiting and diarrhea of swine , especially piglet, and caused by transmissible gastroenteritis coronavirus (TGEV) . TGEV infection provokes mitochondrial damage of porcine intestinal epthelial cell (IPEC), which is responsible for inflammation and cell death. In our previous study, we have demonstrated that circular RNA circEZH2 was down-regulated during TGEV infection and promoted the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) via targeting miR-22 in porcine intestinal epithelial cell line (IPEC-J2). Activation of NF-κB is an important factor for mitochondrial damage. Mitochondrial permeability transition pore (mPTP) opening is a key reason for mitochondrial damage. So, we speculate that circEZH2 may regulate TGEV-induced mPTP opening via NF-kB pathway. In the present study, we found that mPTP opening of IPEC-J2 was occured during TGEV infection and suppressed by circEZH2 via attaching miR-22. Hexokinase 2 (HK2) and interleukin 6 (IL-6) were identified as the targets of miR-22. Silencing HK2 enhanced TGEV-induced mPTP opening, while no effect on NF-κB pathway. Silencing IL-6 promoted TGEV-induced mPTP opening and inhibited NF-κB pathway. Inhibitor of NF-κB increased TGEV-induced mPTP opening. The data revealed that TGEV-induced mPTP opening was regulated via two pathways: circEZH2/miR-22/HK2 axis and circEZH2/miR-22/IL-6/NF-κB axis.

Nanotechnology is actively used for diagnosis, treatment and prophylaxis against coronaviruses. To date, methods for producing antibodies and prototypes of vaccines for four types of coronaviruses have been developed. These are: transmissible gastroenteritis coronavirus, avian coronavirus, severe acute respiratory syndrome coronavirus, and SARSCoV-2 coronavirus. Gold nanoparticles can be used as adjuvants to increase the effectiveness of vaccines by stimulating antigen-presenting cells and ensuring controlled antigen release. Thus, gold nanoparticles, which have adjuvant properties, can be an excellent tool in the design of effective vaccines against infectious diseases.

Tylophorine-based compounds and natural cardiotonic steroids (cardenolides and bufadienolides) are two classes of transmissible gastroenteritis coronavirus inhibitors, targeting viral RNA and host cell factors, respectively We tested both types of compounds against two types of coronaviruses, to compare and contrast their antiviral properties, and with view to their further therapeutic development Examples of both types of compounds potently inhibited the replication of both feline infectious peritonitis virus and human coronavirus OC43 with EC50 values of up to 8 and 16 nM, respectively Strikingly, the tylophorine-based compounds tested inhibited viral yields of HCoV-OC43 to a much greater extent (7–8 log magnitudes of p f u /ml) than the cardiotonic steroids (about 2–3 log magnitudes of p f u /ml), as determined by end point assays Based on these results, three tylophorine-based compounds were further examined for their anti-viral activities on two other human coronaviruses, HCoV-229E and SARS-CoV-2 These three tylophorine-based compounds inhibited HCoV-229E with EC50 values of up to 6 5 nM, inhibited viral yields of HCoV-229E by 6–7 log magnitudes of p f u /ml, and were also found to inhibit SARS-CoV-2 with EC50 values of up to 2 5–14 nM In conclusion, tylophorine-based compounds are potent, broad-spectrum inhibitors of coronaviruses including SARS-CoV-2, and could be used for the treatment of COVID-19 © Copyright © 2020 Yang, Lee, Hsu, Jan, Lin, Chang, Peng, Yang, Liang, Liao, Chao, Pang, Kao, Huang, Lin, Chang, Niu, Wu, Sytwu, Chen and Lee

To the Editors of the Panafrican Medical Journal The COVID 19 pandemic caused from SARS CoV 2 was declared on March 11, 2020 [1]. SARS-CoV2 belongs from the beta Coronavirus family, it is a single-stranded RNA, enveloped virus that is 50-200 nm in diameter [1]. According to a recent revision, the world mortality rate is 12%. The number of scientific publications that has been produced in less than 4 months is very high: typing the keyword “coronavirus COVID 19” appear over 4700 results on the PubMed database. It seems that scientific research is directed more towards finding a drug for the treatment of COVID 19, rather than preventing the spread of the virus. As mentioned in January by a group of Chinese researchers [2] oxidizing solutions such as hydrogen peroxide could be effective in the oral cavity. However, when I typed the keywords “coronavirus hydrogen peroxide” on PubMed, I obtained only 13 results and with “coronavirus COVID 19 hydrogen peroxide” I found 4 results (May, 4, 2020). Hydrogen peroxide, a molecule that consists of hydrogen and oxygen (H2O2), was discovered for the firts time, as a biocidal agent from the early 1800s by chemist Louis-Jacques Thenard (1777-1857). The work of early pioneers in disinfection, such as Benjamin Ward Richardson (1828-96), enhanced the applications of H2O2. Today, its use as a disinfectant is very increased, also as general surface disinfectant in dental field [3]. Hydrogen peroxide (HP) in its pure form is light blue, in its diluted form is a colorless, odorless watersoluble liquid. When concentrated, acts as a strong oxidizing agent [4]. It is used in surgery because it has an antispetic effect, improves hemostasis and wound healing, re-epithelialization, and reduce inflammation [4]. A HP concentration comprised between 3% to 6% is bactericidal, and slowly sporicidal. Higher concentrations comprised between 10% to 30% showed much better sporicidal activity in vitro [4]. Its toxicity is very low, in fact authors reported that ingestion of 3% hydrogen peroxide may cause gastrointestinal irritation and whitening of the mucosa, but these are mostly benign collateral events [4]. The use of HI also against virus infection is well documented in the scientific literature. Goyal et al. studied the effectiveness of a vapour of HI versus different viruses: feline calicivirus; human adenovirus type 1; transmissible gastroenteritis coronavirus of pigs (very similar to SARS-CoV); avian influenza virus (AIV); and swine influenza virus (SwIV). Authors demonstrated that adenovirus, TGEV and AIV died at the lowest vaporized HI volume tested (25 ml). They evaluated that the total exposure time, including injection and aeration, was 2-3 hours, varying with the amount of hydrogen peroxide being vaporized [5]. Holtkamp et al. in an animal study, treated pigs infected with porcine epidemic diarrhea virus (PEDV) with a 1:16 or 1:32 concentration of HI disinfectant. The contact time was 30 min at 20 degrees. They obtained a good result [6]. Rudnick et al. proposed an HI vapour to disinfect surface with H1N1 influenza virus. These virus was deposited in aqueous suspensions on stainless-steel surface at ambient conditions, and then exposed for up to 15 minutes to different concentration between 10 to 90 ppmof HP vapor. After 2.5 minutes, a minimal exposure to 10-ppm HP vapor inactivated 99% of H1N1 virus [7]. Lee et al. showed that HI could be spontaneously produced from pure water through a process of atomizing bulk water into droplets comprised between 1 μm to 20 μm in diameter [8]. Water is a stable and relatively inert molecule in bulk solution. The water have an exceptional behavior: they found in this in vitro work, that water molecules are spontaneously oxidized to form hydrogen peroxide near the water-air interface of micron-sized water droplets. A process that does not require any chemical reagent, catalyst, applied electric potential, or radiation [8]. An hydrogen peroxide spray for decontamination of surfaces in hospitals was successfully tested by Cadnum et al. They observed that a 1.4% solution of hydrogen peroxide (IHP) spray disinfectant could be very effective against Staphilococcus aureus and strains of vancomycin-resistant enterococci [9]. In a very recent review of January 2020 researchers found that the analysis of 22 studies reveals that human coronaviruses such as Severe Acute Respiratory Syndrome (SARS) coronavirus, Middle East Respiratory Syndrome (MERS) coronavirus or endemic human coronaviruses (HCoV) could be inactived by 0.5% hydrogen peroxide solution. Coronavirus can persist on inanimate surfaces like metal, glass or plastic for up to 9 days. Disinfection procedures with 62-71% ethanol, 0.1% sodium hypochlorite or 0,5% HI could be effective to prevent the spread [10]. Could hydrogen peroxide spray solutions, which can be scarcely cytotoxic if inhaled, with extremely low costs, be the subject of clinical studies and usable for the prevention of the spread of SARS CoV 2? I hope this consideration of mine can be a starting point for future research. Competing interests The authors declare no competing interests.

The current state of much of the Wuhan pneumonia virus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) research shows a regrettable lack of data sharing and considerable analytical obfuscation. This impedes global research cooperation, which is essential for tackling public health emergencies and requires unimpeded access to data, analysis tools, and computational infrastructure. Here, we show that community efforts in developing open analytical software tools over the past 10 years, combined with national investments into scientific computational infrastructure, can overcome these deficiencies and provide an accessible platform for tackling global health emergencies in an open and transparent manner. Specifically, we use all SARS-CoV-2 genomic data available in the public domain so far to (1) underscore the importance of access to raw data and (2) demonstrate that existing community efforts in curation and deployment of biomedical software can reliably support rapid, reproducible research during global health crises. All our analyses are fully documented at https://github.com/galaxyproject/SARS-CoV-2.

Highlights • A real-time RT-LAMP assay was established for detection of a novel swine acute diarrhea syndrome coronavirus. • This assay in this study was demonstrated to be specific, sensitive and reproducible for detection of SADS-CoV. • This assay will provide a new method for the diagnosis, surveillance, and pathology studies of SADS-CoV., A novel swine acute diarrhea syndrome Coronavirus (SADS-CoV) that causes severe diarrhea in suckling piglets was identified in southern China in 2017. A simple and rapid detection test was developed for this virus using real-time RT-LAMP based on the conserved N gene of the virus. The method had a detection limit of 1.0 × 101 copies/μL with no cross-reactions with classical swine fever virus, porcine and respiratory syndrome virus NA, porcine and respiratory syndrome virus EU, transmissible gastroenteritis coronavirus, foot and mouth disease virus, porcine epidemic diarrhea virus (S-INDEL and non-S-INDEL), swine influenza virus subtype H1N1, porcine circovirus type 2, seneca valley virus, porcine parvovirus, porcine deltacoronavirus and rotavirus. This method was also reproducible. Twenty of 24 clinical samples were identified as SADS-CoV RNA-positive by the real-time RT-LAMP and the results were consistent with that of the real time RT-PCR method. This new method for detecting SADS-CoV is specific and sensitive for the detection of SADS-CoV.

International audience; AbstractTransmissible gastroenteritis coronavirus (TGEV) is an enteropathogenic coronavirus that causes diarrhea in pigs and is associated with high morbidity and mortality in sucking piglets. S1 is one of two protein domains in the spike (S) glycoprotein and is responsible for enteric tropism, sialic acid recognition, and host receptor binding. Although there has been extensive research on the S1 protein of TGEV, little is known about the intracellular role of TGEV-S1. In the present study, we used yeast two-hybrid screening of a cDNA library from porcine intestinal cells to identify proteins that interact with TGEV-S1. Among 120 positive clones from the library, 12 intracellular proteins were identified after sequencing and a BLAST search. These intracellular proteins are involved in protein synthesis and degradation, biological signal transduction, and negative control of signaling pathways. Using a glutathione-S-transferase (GST) pulldown assay and Co-IP, we found that UBXN1 interacts with the S1 protein. Here, we observed that TGEV infection led to increased UBXN1 expression levels during the late phase of infection in IPEC-J2 cells. Inhibition of UBXN1 in IPEC-J2 cells via siRNA interference significantly decreased the viral titer and downregulated the expression of S1. UBXN1 overexpression significantly increased the viral copy number. Additionally, we provided data suggesting that UBXN1 negatively regulates IFN-β expression after TGEV infection. Finally, our research indicated that UBXN1 plays a vital role in the process of TGEV infection, making it a candidate target for the development of a novel antiviral method.

Porcine deltacoronavirus (PDCoV) was initially documented in Hong Kong and later in the United States, South Korea, and Thailand. To investigate if PDCoV is also present in Taiwan, three swine coronaviruses-PDCoV, porcine epidemic diarrhea virus (PEDV), and transmissible gastroenteritis coronavirus (TGEV)-were tested using real-time reverse transcription polymerase chain reaction (rRT-PCR) in 172 rectal swab samples from piglets exhibiting diarrhea between January 2016 and May 2017 on 68 pig farms in Taiwan. The rRT-PCR results were positive for PDCoV (29/172, 16.9%), PEDV (36/172, 20.9%), TGEV (2/172, 1.2%), and coinfections (16/172, 9.3%). After cloning and sequencing, PDCoV nucleocapsid genes were analyzed. Phylogeny results indicated that the nucleotide sequences of all isolates were like those reported in other countries. To further trace PDCoV in the period of 2011 to 2015, an enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against PDCoV. The results showed that 279 of 1,039 (26.9%) sera were positive for the PDCoV nucleocapsid protein, implying that PDCoV might have existed in Taiwan before 2011.

Current measures to prevent TGEV from entering a naive herd include quarantine and testing for TGEV-seronegative animals. However, TGEV serology is complicated due to the cross-reactivity with PRCV, which circulates subclinically in most swine herds worldwide. Conventional serological tests cannot distinguish between TGEV and PRCV antibodies; however, blocking ELISAs using antigen containing a large deletion in the amino terminus of the PRCV S protein permit differentiation of PRCV and TGEV antibodies. Several commercial TGEV/PRCV blocking ELISAs are available, but performance comparisons have not been reported in recent research. This study demonstrates that the serologic cross-reactivity between TGEV and PRCV affects the accuracy of commercial blocking ELISAs. Individual test results must be interpreted with caution, particularly in the event of suspect results. Therefore, commercial TGEV/PRCV blocking ELISAs should only be applied on a herd basis., This study compared the performances of three commercial transmissible gastroenteritis virus/porcine respiratory coronavirus (TGEV/PRCV) blocking enzyme-linked immunosorbent assays (ELISAs) using serum samples (n = 528) collected over a 49-day observation period from pigs inoculated with TGEV strain Purdue (n = 12), TGEV strain Miller (n = 12), PRCV (n = 12), or with virus-free culture medium (n = 12). ELISA results were evaluated both with “suspect” results interpreted as positive and then as negative. All commercial kits showed excellent diagnostic specificity (99 to 100%) when testing samples from pigs inoculated with virus-free culture medium. However, analyses revealed differences between the kits in diagnostic sensitivity (percent TGEV- or PRCV-seropositive pigs), and all kits showed significant (P

Transmissible gastroenteritis coronavirus (TGEV) is a member of the genus Coronavirus, family Coronaviridae, order Nidovirales. TGEV is an enteropathogenic coronavirus that causes highly fatal acute diarrhoea in newborn pigs. An oral Lactobacillus casei (L. casei) vaccine against anti-transmissible gastroenteritis virus developed in our laboratory was used to study mucosal immune responses. In this L. casei vaccine, repetitive peptides expressed by L. casei (specifically the MDP and tuftsin fusion protein (MT)) were repeated 20 times and the D antigenic site of the TGEV spike (S) protein was repeated 6 times. Immunization with recombinant Lactobacillus is crucial for investigations of the effect of immunization, such as the first immunization time and dose. The first immunization is more important than the last immunization in the series. The recombinant Lactobacillus elicited specific systemic and mucosal immune responses. Recombinant L. casei had a strong potentiating effect on the cellular immunity induced by the oral L. casei vaccine. However, during TGEV infection, the systemic and local immune responses switched from Th1 to Th2-based immune responses. The systemic humoral immune response was stronger than the cellular immune response after TGEV infection. We found that the recombinant Lactobacillus stimulated IL-17 expression in both the systemic and mucosal immune responses against TGEV infection. Furthermore, the Lactobacillus vaccine stimulated an anti-TGEV infection Th17 pathway. The histopathological examination showed tremendous potential for recombinant Lactobacillus to enable rapid and effective treatment for TGEV with an intestinal tropism in piglets. The TGEV immune protection was primarily dependent on mucosal immunity.

Blood testing will determine if a cat's immune system has developed antibodies to the coronavirus and if so, will produce a positive result Staying in a kennel A crowded dog show Any condition of overcrowding Dog with a weakened immune system Puppies Combination with another infection [10] E) Pathology of Canine Coronavirus: Coronavirus is spread through contact with other infected animals Staying in a kennel A crowded dog show Any condition of overcrowding Dog with a weakened immune system Puppies Combination with another infection [8] 3) Transmissible gastroenteritis classification Realm- Riboviria [9] Phylum- incertae sedis [9] Order- Nidovirales [9] Family- Coronaviridae [9] Genus- Alphacoronavirus [9] Subgenus- Tegacovirus [9] Species- Alphacoronavirus 1 [9] Virus- Transmissible gastroenteritis coronavirus [9] A] General Introduction of Transmissible gastroenteritis coronavirus: Once the virus infects the host, it multiplies in the cell lining of the small intestine resulting in the loss of absorptive cells that in turn leads to shortening of villi

Transmissible gastroenteritis virus (TGEV) causes transmissible gastroenteritis (TGE), especially in newborn piglets, which severely threatens the worldwide pig industry. In this study, (+)-catechin was evaluated for its antiviral effect against TGEV in vitro. Viability assays revealed that (+)-catechin treatment exerted a dose-dependent rescue effect in TGEV-infected ST cells, and this result was only obtained with the post-treatment application of (+)-catechin. The viral yields in (+)-catechin-treated cultures were reduced by almost three log10 units. Quantitative real-time PCR analysis of the TGEV genome revealed that TGEV RNA replication was restricted after (+)-catechin treatment. Intracellular reactive oxygen species (ROS) detection showed that (+)-catechin alleviated ROS conditions induced by TGEV infection. Our results showed that (+)-catechin exerts an inhibitory effect on TGEV proliferation in vitro and is involved its antioxidation., Highlights • (+)-Catechin has anti-TGEV effect. • Anti-TGEV effect was working after TGEV infection. • (+)-Catechin could relieve CPE caused by TGEV in ST cells. • TGEV proliferation was decreased after (+)-catechin incubation. • Antioxidation of (+)-catechin is related to its antiviral effect.

To the Editor: Porcine deltacoronavirus (PDCoV) was first reported in Hong Kong in 2012 and included the HKU15-44 and HKU15-155 strains (1). In early 2014, PDCoV was reported in pigs with diarrhea on swine farms in Ohio, USA (2), and later in other states (2–5). In April 2014, PDCoV strain KNU14-04 was reported in pigs in South Korea (6). A retrospective study in 2012 reported PDCoV strain S27 in Sichuan, China (7). Recently PDCoV strain CNJXNI2 has been reported in pigs with diarrhea in Jiangxi, China (8). There are currently 28 complete PDCoV genomes from China, South Korea, and the United States available in GenBank. We report emergence of PDCoV infections on a commercial swine farm in Thailand. In June 2015, we investigated reports of acute diarrhea in piglets, gilts, and sows on a swine farm. An outbreak occurred on a commercial swine farm (3,000 sows) located in the eastern province of Thailand. Clinical signs, including acute watery diarrhea, loss of appetite, and agalactia, were observed in gilts and sows in the breeding and gestation houses. Subsequently, piglets in farrowing houses had clinical signs (depression, fever, watery diarrhea, and severe dehydration). Although clinical signs were detected less frequently in fattening pigs in growth-finishing houses, PDCoVs were later detected from blood samples of fattening pigs. The outbreak lasted 6 weeks (June 10–July 20, 2015). The mortality rate was 27.63% (829/3,000) in sows and 64.27% (2,892/4,500) in piglets but was lower than that usually observed for porcine epidemic diarrhea virus (PEDV) infection. A total of 865 (19.22%) piglets died and were culled during 10 production weeks. Postmortem examination of dead piglets showed emaciated animals and yellow pasty feces. Intestines and colons showed thin walls with a watery content and curdled milk. Histopathologic examination showed shortened and fused villi in the jejunum and ileum. An attenuated and vacuolated cytoplasm in enterocytes was also observed (Technical Appendix Figure 1) (9,10). We examined 30 samples from the affected swine farm. Blood (n = 10), intestine (n = 8), lymph node (n = 2), feces (n = 6), and feed (n = 4) samples were collected for 2 day-old piglets and 17-, 19-, and 20-week-old fattening pigs. A total of 26 samples were positive for PDCoV by reverse transcription PCR (2) (Technical Appendix Table 1). Because sick pigs had clinical signs similar to those of pigs with other swine virus diseases, all samples were tested for transmissible gastroenteritis coronavirus; PEDV; rotaviruses A, B, and C; porcine reproductive and respiratory syndrome virus; and circovirus. All test results were negative. We selected 2 PDCoVs (S5011 and S5015L) for whole-genome sequencing and 14 PDCoVs for sequencing of spike (S), envelope (E), membrane (M), and nucleocapsid (N) genes and the 3′-untranslated region (UTR). Nucleotide sequences obtained were submitted to GenBank (Technical Appendix Table 2). Sequence analysis of the 2 PDCoVs from Thailand showed that their whole genomes had 99.98% nt identity (only 4 nt differences) with each other and highest nucleotide identities with PDCoVs from China (98.43% with AH2004). S gene sequences showed greatest diversity (99.97%–100% nt identities and 99.91%–100% aa identities) for PDCoVs from Thailand and 95.93%–96.68% with other reference PDCoVs, which is consistent with findings of previous report (5). In contrast, E, M, and N genes were conserved (100% nt identities for PDCoVs from Thailand and 99.19%–100% for E genes, 98.28%–99.07% for M genes, and 96.88%–97.81% for N genes with reference PDCoVs) (Technical Appendix Table 3). Phylogenetic analysis of the whole genome of PDCoVs from Thailand showed close relatedness with AH-2004, HKU15-44, S27-2012, and HKU15-155 virus strains from China. However, these viruses from Thailand were in a different subcluster than PDCoVs from the United States (Figure; Technical Appendix Figure 2). PDCoVs identified in this study might represent a new variant of PDCoV because these 2 viruses have unique sequence characteristics: 3-nt (TCT) and 1-nt (A) deletions in the 5′-UTR, 6-nt (AGTTTG) and 9-nt (GAGCCAGTC) deletions in open reading frame 1a/b, and 4-nt (CTCT) insertion in the 3′-UTR (Technical Appendix Table 4). Figure Phylogenetic analysis of whole-genome sequences of porcine deltacoronaviruses (PDCoVs), Thailand. Black circles indicate strains isolated in this study. The tree was constructed by using MEGA version 6.06 (http://www.megasoftware.net/) with the neighbor-joining ... We identified PDCoV on a commercial swine farm in Thailand. Affected pigs had clinical signs of acute watery diarrhea, similar to those of pigs infected with PEDV, and had moderate illness and low mortality rates. PDCoVs were detected in symptomatic piglets, sows, and fattening pigs, although clinical signs in fattening pigs were least severe. Swine farmers and veterinarians should be aware of PDCoV as another causative agent of watery diarrhea in pigs. Similar to PEDV, Wang et al. reported that sequence deletions, insertions, and mutations in PDCoVs in pigs might contribute to variant virus virulence (2). Our findings might assist in development of diagnostic assays for differentiating PDCoVs in Thailand from PDCoVs in other countries. Because PDCoVs from Thailand were highly related to each other, PDCoV might have transmitted into Thailand by a single event. However, verification of this possibility would be difficult. Similar to the situation in the United States, PDCoV might be underdiagnosed in Thailand. Technical Appendix: Methods used and additional information for detection of porcine deltacoronavirus, Thailand, 2015. Click here to view.(709K, pdf)

The porcine epidemic diarrhea virus (PEDV) is a coronavirus (CoV) belonging to the alpha-CoV genus and it causes high mortality in infected sucking piglets, resulting in substantial losses in the farming industry. CoV trigger a drastic reorganization of host cell membranes to promote their replication and egression, but a detailed description of the intracellular remodeling induced by PEDV is still missing. In this study, we examined qualitatively and quantitatively, using electron microscopy, the intracellular membrane reorganization induced by PEDV over the course of an infection. With our ultrastructural approach, we reveal that, as most of CoV, PEDV initially forms double-membrane vesicles (DMVs) and convoluted membranes (CMs), which probably serve as replication/transcription platforms. Interestingly, we also found that viral particles start to form almost simultaneously in both the endoplasmic reticulum and the large virion-containing vacuoles (LVCVs), which are compartments originating from the Golgi, confirming that alpha-CoV assemble indistinguishably in two different organelles of the secretory pathway. Moreover, PEDV virons appear to have an immature and a mature form, similar to another alpha-CoV the transmissible gastroenteritis coronavirus (TGEV). Altogether, our study underlies the similarities and differences between the lifecycle of alpha-CoV and that of viruses belonging to other CoV subfamilies.

Highlights • Porcine precision cut intestinal slices can be used to analyze virus infection. • TGEV strains differ in their ability to infect cells of the jejunal epithelium. • Enterotropism of TGEV is determined by its spike protein S., TGEV is a coronavirus that is still widely spread in pig farming. On molecular level this virus has been studied in detail. However, studying TGEV infection within the complexity of the porcine intestinal epithelium reveals difficulties due to limiting infection models. Here we established a new ex vivo model to analyze the enterotropism of TGEV in porcine intestinal tissue. Precision cut intestinal slices (PCIS) were produced and ATP level was measured to proof vitality of the slices. ATP measurements and HE staining revealed living tissue in culture for up to 24 h. PCIS were infected with three different TGEV strains. TGEV PUR 46-MAD is a commonly used TGEV strain that is known to be attenuated. TGEV Miller was passaged in piglets several times to reveal high infection. Finally, TGEV GFP is a recombinant strain that obtained its main body from TGEV PUR 46-MAD, but its spike protein from TGEV PUR-C11 that showed high mortality in piglets in vivo. Our results were in complete consensus of these statements. TGEV Miller mildly and TGEV GFP extensively infected the cells in the jejunum based on the amount of positive stained epithelial cells. However, for TGEV PUR 46-MAD no nucleocapsid protein was detected in the epithelial cells of the tissue. This shows that differences in TGEV strains and their infectious potential are highly dependent on their S protein.

The Transmissible Gastroenteritis Virus (TGEV) causes severe diarrhea and other symptoms which end up in death in young piglets. We have studied the protective effects of the probiotic Lactobacillus plantarum strain N4(Lp) which metabolic products were added to the swine testis (ST) cells with three different orders using MTT cell proliferation assay and CPE analysis. Metabolic products led to dose-dependent rescue of viability of infected cells in a certain order: pre-treatment, post-infection, co-incubation. Pre-treatment of cells with probiotic metabolic products reduced viral proliferation up to 78% at non-cytotoxic concentration 1/4 dilution. The viral yields in pretreatment groups were reduced by over three log10 units. Quantitative real-time PCR revealed the optimal inhibition of TGEV RNA replication was 24 h apply pretreatment way that up to 71% of N gene. Analysised the composition of metabolic products by GC-MS revealed the major component is sugars. Then Exopolysaccharides (EPS) was extracted and showed inhibition effect that co-incubation with TGEV.

The spike protein S of coronaviruses contains a highly conserved cytoplasmic cysteine-rich motif adjacent to the transmembrane region. This motif is palmitoylated in the Betacoronaviruses MHV and SARS-CoV. Here, we demonstrate by metabolic labeling with [3H]-palmitic acid that the S protein of transmissible gastroenteritis coronavirus (TGEV), an Alphacoronavirus, is palmitoylated as well. This is relevant for TGEV replication as virus growth was compromised by the general palmitoylation inhibitor 2-bromopalmitate. Mutation of individual cysteine clusters in the cysteine-rich motif of S revealed that all cysteines must be replaced to abolish acylation and incorporation of S into virus-like particles (VLP). Conversely, the interaction of S with the M protein, essential for VLP incorporation of S, was not impaired by lack of palmitoylation. Thus, palmitoylation of the S protein of Alphacoronaviruses is dispensable for S–M interaction, but required for the generation of progeny virions., Highlights • We analyzed the palmitoylation of the Alphacoronavirus TGEV spike protein. • The cystein-rich region of the TGEV S protein is palmitoylated. • Palmitoylation is essential for S incorporation into virus-like particles. • Palmitoylation is dispensable for the interaction of the TGEV S with the M protein.

Highlights • Interaction between TGEV N protein and EF1A is found in vitro. • Interaction between TGEV N protein and EF1A is found in vivo. • EF1A plays a role in TGEV replication. • Interaction between TGEV N protein and EF1A was firstly found in this study., Transmissible gastroenteritis coronavirus (TGEV) is an enteropathogenic coronavirus that causes diarrhea in pigs, which is correlated with high morbidity and mortality in suckling piglets. Using the method of GST pull-down with the nucleocapsid (N), N protein was found to interact with swine testes (ST) cells elongation factor 1-alpha (EF1A), an essential component of the translational machinery with an important role in cells. In vitro and in virus-infected cells interaction was then confirmed by co-precipitation. Knockdown of EF1A impairs N protein proliferation and TGEV replication in host cell. It was demonstrated that EF1A plays a role in TGEV replication. The present study thus provides a protein-related information that should be useful for underlying mechanism of coronavirus replication.

Summary Background Surface contamination has been implicated in the transmission of certain viruses, and surface disinfection can be an effective measure to interrupt the spread of these agents. Aim To evaluate the in-vitro efficacy of hydrogen peroxide vapour (HPV), a vapour-phase disinfection method, for the inactivation of a number of structurally distinct viruses of importance in the healthcare, veterinary and public sectors. The viruses studied were: feline calicivirus (FCV, a norovirus surrogate); human adenovirus type 1; transmissible gastroenteritis coronavirus of pigs (TGEV, a severe acute respiratory syndrome coronavirus [SARS-CoV] surrogate); avian influenza virus (AIV); and swine influenza virus (SwIV). Methods The viruses were dried on stainless steel discs in 20- or 40-μL aliquots and exposed to HPV produced by a Clarus L generator (Bioquell, Horsham, PA, USA) in a 0.2-m 3 environmental chamber. Three vaporized volumes of hydrogen peroxide were tested in triplicate for each virus: 25, 27 and 33mL. Findings No viable viruses were identified after HPV exposure at any of the vaporized volumes tested. HPV was virucidal (>4-log reduction) against FCV, adenovirus, TGEV and AIV at the lowest vaporized volume tested (25mL). For SwIV, due to low virus titre on the control discs, >3.8-log reduction was shown for the 25-mL vaporized volume and >4-log reduction was shown for the 27-mL and 33-mL vaporized volumes. Conclusion HPV was virucidal for structurally distinct viruses dried on surfaces, suggesting that HPV can be considered for the disinfection of virus-contaminated surfaces.

Transgenic plants have been tested as an alternative host for the production and delivery of experimentaloral vaccines. Here, we developed transgenic potatoes that express the major antigenic sites A and D of the glycoproteinS from transmissible gastroenteritis coronavirus (TGEV-S 0.7 ) under three expression vector systems. The DNAintegration and mRNA expression level of the TGEV-S 0.7 gene were confirmed in transgenic plants by PCR and northernblot analysis. Antigen protein expression in transgenic potato was determined by western blot analysis. Enzyme-linkedimmunosorbent assay results revealed that based on a dilution series of Escherichia coli-derived antigen, the transgenicline P-2 had TGEV-S 70. protein at levels that were 0.015% of total soluble proteins. We then examined theimmunogenicity of potato-derived TGEV-S 70. antigen in mice. Compared with the wild-type potato treated group andsynthetic antigen treated group, mice treated with the potato-derived antigen showed significantly higher levels ofimmunoglobulin (Ig) G and IgA responses.Keywords : oral administration, potato-derived antigen, transmissible gastroenteritis virus, transgenic plant

In this study, two structural models (denoted as MproST and MproSH) of the main proteinase (Mpro) from the novel coronavirus associated with severe acute respiratory syndrome (SARS‐CoV) were constructed based on the crystallographic structures of Mpro from transmissible gastroenteritis coronavirus (TGEV) (MproT) and human coronavirus HcoV‐229E (MproH), respectively. Various 200 ps molecular dynamics simulations were subsequently performed to investigate the dynamics behaviors of several structural features. Both MproST and MproSH exhibit similar folds as their respective template proteins. These structural models reveal three distinct functional domains as well as an intervening loop connecting domains II and III as found in both template proteins. In addition, domain III of these structures exhibits the least secondary structural conservation. A catalytic cleft containing the substrate binding subsites S1 and the S2 between domains I and II are also observed in these structural models. Although these structures share many common features, the most significant difference occurs at the S2 subsite, where the amino acid residues lining up this subsite are least conserved. It may be a critical challenge for designing anti‐SARS drugs by simply screening the known database of proteinase inhibitors.

Porcine Epidemic Diarrhea Virus (PEDV), Porcine Delta Corona Virus (PDCoV), and Transmissible Gastroenteritis Virus (TGEV) are major threats to swine health and contaminated feed plays a role in virus transmission. The objective of our study was to characterize inactivation of PEDV, PDCoV, and TGEV in various feed ingredient matrices. Samples of complete feed, spray dried porcine plasma, meat meal, meat and bone meal, blood meal, corn, soybean meal, and corn dried distillers grains with solubles were weighed (5 g/sample) into scintillation vials and inoculated with 1 mL of PEDV, PDCoV, or TGEV. Samples were incubated at room temperature for up to 56 days. Aliquots were removed at various time points followed by preparing serial 10-fold dilutions and inoculating in cell cultures to determine the amount of surviving virus. Inactivation kinetics were determined using the Weibull model, which estimates a delta value indicating the time necessary to reduce virus concentration by 1 log. Delta values of various ingredients were compared and analyzed as to their nutrient composition. Soybean meal had the greatest delta value (7.50 days) for PEDV (P < 0.06) as compared with all other ingredients. High delta values (P < 0.001) were observed in soybean meal for PDCoV (42.04 days) and TGEV (42.00 days). There was a moderate correlation between moisture content and the delta value for PDCoV (r = 0.49, P = 0.01) and TGEV (r = 0.41, P = 0.02). There was also a moderate negative correlation between TGEV survival and ether extract content (r = -0.51, P = 0.01). In conclusion, these results indicate that the first log reduction of PDCoV and TGEV takes the greatest amount of time in soybean meal. In addition to this, moisture and ether content appear to be an important determinant of virus survival in feed ingredients.

Background Following the initial isolation of porcine deltacoronavirus (PDCoV) from pigs with diarrheal disease in the United States in 2014, the virus has been detected on swine farms in some provinces of China. To date, little is known about the molecular epidemiology of PDCoV in southern China where major swine production is operated. Results To investigate the prevalence of PDCoV in this region and compare its activity to other enteric disease of swine caused by porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis coronavirus (TGEV), and porcine rotavirus group C (Rota C), 390 fecal samples were collected from swine of various ages from 15 swine farms with reported diarrhea. Fecal samples were tested by reverse transcription-PCR (RT-PCR) that targeted PDCoV, PEDV, TGEV, and Rota C, respectively. PDCoV was detected exclusively from nursing piglets with an overall prevalence of approximate 1.28 % (5/390), not in suckling and fattening piglets. Interestingly, all of PDCoV-positive samples were from 2015 rather than 2012–2014. Despite a low detection rate, PDCoV emerged in each province/region of southern China. In addition, compared to TGEV (1.54 %, 5/390) or Rota C (1.28 %, 6/390), there were highly detection rates of PEDV (22.6 %, 88/390) in those samples. Notably, all five PDCoV-positive piglets were co-infected by PEDV. Furthermore, phylogenetic analysis of spike (S) and nucleocapsid (N) gene sequences of PDCoVs revealed that currently circulating PDCoVs in southern China were more closely related to other Chinese strains of PDCoVs than to those reported in United States, South Korea and Thailand. Conclusions This study demonstrated that PDCoV was present in southern China despite the low prevalence, and supported an evolutionary theory of geographical clustering of PDCoVs. Electronic supplementary material The online version of this article (doi:10.1186/s12985-016-0591-6) contains supplementary material, which is available to authorized users.

A total of 310 bacterial strains isolated from the porcine gastrointestinal tract were tested for their activity against transmissible gastroenteritis (TGE) coronavirus and other enteric pathogens. Based on activity, the strains Probio-38 and Probio-37 were selected as potential probiotics and identified as Lactobacillus plantarum Probio-38 and Lactobacillus salivarius Probio-37 respectively by 16S rRNA gene sequencing. Supernatants of these strains inhibited TGE coronavirus in vitro in ST cells, without any cytopathic effect even after 72 h of incubation. Both the strains exhibited high survival in synthetic gastric juice. The strains were resistant to 5% porcine bile and exhibited antimicrobial activity against all the 13 enteric bacterial pathogens tested. These strains also exhibited resistance to most of the antibiotics analyzed. The inhibition of transmissible gastroenteritis coronavirus and enteric bacterial pathogens as well as the bile tolerance, high survival in gastric juice, and the antibiotic resistance indicate that the two isolated bacterial strains are ideal probiotic candidates for animal application after proper in vivo experiments.

Coronavirus RNA synthesis is performed by a multienzymatic replicase complex together with cellular factors. This process requires the specific recognition of RNA cis-acting signals located at the ends of the viral genome. To identify cellular proteins involved in coronavirus RNA synthesis, transmissible gastroenteritis coronavirus (TGEV) genome ends, harboring essential cis-acting signals for replication, were used as baits for RNA affinity protein purification. Ten proteins were preferentially pulled down with either the 5′ or 3′ ends of the genome and identified by proteomic analysis. Nine of them, including members of the heterogeneous ribonucleoprotein family of proteins (hnRNPs), the poly(A)-binding protein (PABP), the p100 transcriptional co-activator protein and two aminoacyl-tRNA synthetases, showed a preferential binding to the 3′ end of the genome, whereas only the polypyrimidine tract-binding protein (PTB) was preferentially pulled down with the 5′ end of the genome. The potential function of the 3′ end-interacting proteins in virus replication was studied by analyzing the effect of their silencing using a TGEV-derived replicon and the infectious virus. Gene silencing of PABP, hnRNP Q, and glutamyl-prolyl-tRNA synthetase (EPRS) caused a significant 2 to 3-fold reduction of viral RNA synthesis. Interestingly, the silencing of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), initially used as a control gene, caused a 2 to 3-fold increase in viral RNA synthesis in both systems. These data suggest that PABP, hnRNP Q, and EPRS play a positive role in virus infection that could be mediated through their interaction with the viral 3′ end, and that GAPDH has a negative effect on viral infection. © 2009 Elsevier Inc. All rights reserved., This work was supported by grants from the Ministry of Science and Innovation of Spain (BIO2007-60978 and CIT-01000-2007-8), the Community of Madrid (S-SAL-0185-2006), the European Community (Frame VII, RiViGene project, SSPE-CT-2005-022639) and Fort Dodge Veterinaria. C. G. received a contract supported by grants from the Ministry of Science and Innovation of Spain (CIT-01000-2007-8) and the Community of Madrid (S-SAL-0185-2006).

Porcine aminopeptidase N (pAPN) is a cellular membrane protein and a functional receptor for porcine coronaviruses. Here, we describe the heterologous expression of pAPN without signal peptide in BL21(DE3)pLysS host cells. The Escherichia coli (E. coli) harboring the recombinant construct was efficiently induced to express the pAPN protein at a high level. The most optimal expression profile for pAPN expression was investigated. By inoculating a rabbit with the purified pAPN, a high tittered specific antibody was achieved. Biologically, the antibody reacted with either pAPN-expressing E. coli or native pAPN on the surface of swine testis cells. The pAPN and its specific antibody blocked transmissible gastroenteritis coronavirus infection in vitro. Furthermore, the localization of pAPN on the small intestine of swine was analyzed by immunohistochemistry.

Coronaviruses are enveloped viruses containing the largest reported RNA genomes. As a result of their pleomorphic nature, our structural insight into the coronavirion is still rudimentary, and it is based mainly on 2D electron microscopy. Here we report the 3D virion structure of coronaviruses obtained by cryo-electron tomography. Our study focused primarily on the coronavirus prototype murine hepatitis virus (MHV). MHV particles have a distinctly spherical shape and a relatively homogenous size (approximate to 85 nm envelope diameter). The viral envelope exhibits an unusual thickness (7.8 +/- 0.7 nm), almost twice that of a typical biological membrane. Focal pairs revealed the existence of an extra internal layer, most likely formed by the C-terminal domains of the major envelope protein M. In the interior of the particles, coiled structures and tubular shapes are observed, consistent with a helical nucleocapsid model. Our reconstructions provide no evidence of a shelled core. Instead, the ribonucleoprotein seems to be extensively folded onto itself, assuming a compact structure that tends to closely follow the envelope at a distance of approximate to 4 nm. Focal contact points and thread-like densities connecting the envelope and the ribonucleoprotein are revealed in the tomograms. Transmissible gastroenteritis coronavirion tomograms confirm all the general features and global architecture observed for MHV. We propose a general model for the structure of the coronavirion in which our own and published observations are combined.

A recombinant transmissible gastroenteritis coronavirus (rTGEV) in which E gene was deleted (rTGEV-ΔE) has been engineered. This deletion mutant only grows in cells expressing E protein (E+ cells) indicating that E was an essential gene for TGEV replication. Electron microscopy studies of rTGEV-ΔE infected BHK-pAPN-E- cells showed that only immature intracellular virions were assembled. These virions were non-infectious and not secreted to the extracellular medium in BHK-pAPN-E- cells. RNA and protein composition analysis by RNase-gold and immunoelectron microscopy showed that rTGEV-ΔE virions contained RNA and also all the structural TGEV proteins, except the deleted E protein. Nevertheless, full virion maturation was blocked. Studies of the rTGEV-ΔE subcellular localization by confocal and immunoelectron microscopy in infected E- cells showed that in the absence of E protein virus trafficking was arrested in the intermediate compartment. Therefore, the absence of E protein in TGEV resulted in two actions, a blockade of virus trafficking in the membranes of the secretory pathway, and prevention of full virus maturation. © 2007 Elsevier Inc. All rights reserved.

The present study is undertaken to investigate the immune response that was induced by the recombinant spike (S) protein from swine-transmissible gastroenteritis virus (TGEV) expressed in mouse mammary cells. A mammary-specific expression vector pEBS containing the full-length cDNA of S gene was constructed and expressed in the mouse mammary cells (EMT6). The recombinant S protein from culture supernatant of transgenic EMT6 was harvested and immunized BALB/c mice. The results demonstrated recombinant S protein was expressed at high levels in mammary cells by Western blotting and enzyme-linked immunosorbent assay (ELISA) detection. The antibody titer in BALB/c mice following immunization with recombinant S protein was detectable after the first immunization. Maximum titers of antibody (8.86 ± 0.19 ng/ml of serum) were attained after the second immunization. In conclusion, the recombinant S protein expressed in mammary cells was able to elicit substantial immunological response against TGEV. This lays the basis for using mammary gland bioreactor generating edible vaccine.

Porcine deltacoronavirus (PDCoV), also known as porcine coronavirus HKU15, was first detected in North America in early 2014 and associated with enteric disease in pigs, resulting in an urgent need to further investigate the ecology of this virus. While assays detecting nucleic acids were implemented quickly, assays to detect anti-PDCoV antibodies have not been available. In this study, an indirect anti-PDCoV IgG enzyme-linked immunosorbent assay (ELISA) based on the putative S1 portion of the spike protein was developed and utilized to determine the prevalence of anti-PDCoV IgG in U.S. pigs. The diagnostic sensitivity of the PDCoV ELISA was 91% with a diagnostic specificity of 95%. A total of 968 serum samples were tested including samples with confirmed infection with PDCoV, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus or porcine respiratory coronavirus. There was no cross-reactivity with any of the other coronaviruses. Among 355 arbitrarily selected serum samples collected in 2014 and originating from 51 farms across 18 U.S. states, anti-PDCoV IgG antibodies were detected in 8.7% of the samples and in 25.5% of the farms whereas anti-PEDV IgG was detected in 22.8% of the samples and in 54.9% of the farms. In addition, anti-PDCoV IgG antibodies were detected in archived samples collected in 2010, perhaps indicating an earlier undetected introduction into the U.S. pig population. Overall, the obtained data suggest that PDCoV seroprevalence in U.S. pigs is lower compared to PEDV and PDCoV may have been introduced to the U.S. prior to PEDV.

A 32-nucleotide (nt) RNA motif located at the 3= end of the transmissible gastroenteritis coronavirus (TGEV) genome was found to specifically interact with the host proteins glutamyl-prolyl-tRNA synthetase (EPRS) and arginyl-tRNA synthetase (RRS). This RNA motif has high homology in sequence and secondary structure with the gamma interferon-activated inhibitor of translation (GAIT) element, which is located at the 3= end of several mRNAs encoding proinflammatory proteins. The GAIT element is involved in the translation silencing of these mRNAs through its interaction with the GAIT complex (EPRS, heterogeneous nuclear ribonucleoprotein Q, ribosomal protein L13a, and glyceraldehyde 3-phosphate dehydrogenase) to favor the resolution of inflammation. Interestingly, we showed that the viral RNA motif bound the GAIT complex and inhibited the in vitro translation of a chimeric mRNA containing this RNA motif. To our knowledge, this is the first GAIT-like motif described in a positive RNA virus. To test the functional role of the GAIT-like RNA motif during TGEV infection, a recombinant coronavirus harboring mutations in this motif was engineered and characterized. Mutations of the GAIT-like RNA motif did not affect virus growth in cell cultures. However, an exacerbated innate immune response, mediated by the melanoma differentiation-associated gene 5 (MDA5) pathway, was observed in cells infected with the mutant virus compared with the response observed in cells infected with the parental virus. Furthermore, the mutant virus was more sensitive to beta interferon than the parental virus. All together, these data strongly suggested that the viral GAIT-like RNA motif modulates the host innate immune response IMPORTANCE The innate immune response is the first line of antiviral defense that culminates with the synthesis of interferon and proinflammatory cytokines to limit virus replication. Coronaviruses encode several proteins that interfere with the innate immune response at different levels, but to date, no viral RNA counteracting antiviral response has been described. In this work, we have characterized a 32-nt RNA motif located at the 3= end of the TGEV genome that specifically interacted with EPRS and RRS. This RNA motif presented high homology with the GAIT element, involved in the modulation of the inflammatory response. Moreover, the disruption of the viral GAIT-like RNA motif led to an exacerbated innate immune response triggered by MDA5, indicating that the GAIT-like RNA motif counteracts the host innate immune response. These novel findings may be of relevance for other coronaviruses and could serve as the basis for the development of novel antiviral strategies., This work was supported by grants from the Ministry of Science and Innovation of Spain (MCINN) (BIO2010-16705), the European Community’s Seventh Framework Programme (FP7/2007-2013) under the project “EMPERIE” (HEALTH-F3-2009-223498), and the National Institutes of Health (NIH) of The United States (2P01AI060699-06A1). S.M.-J. received a predoctoral fellowship from the National Institutes of Health (ISCIII) of Spain and a contract from the National Institutes of Health (NIH) of The United States. A.N. and S.Z. received contracts from EU and NIH

Transmissible gastroenteritis coronavirus (TGEV) is one of the most destructive agents, responsible for the enteric infections that are lethal for suckling piglets, causing enormous economic loss to the porcine fostering industry every year. Although it has been known that TGEV spiker protein is essential for the viral entry for many years, the detail knowledge of the TGEV fusion protein core is still very limited. Here, we report that TGEV fusion core (HR1-SGGRGG-HR2), in vitro expressed in GST prokaryotic expression system, shares the typical properties of the trimer of coiled-coil heterodimer (six alpha-helix bundle), which has been confirmed by a combined series of biochemical and biophysical evidences including size exclusion chromatography (gel-filtration), chemical crossing, and circular diagram. The 3D homologous structure model presents its most likely structure, extremely similar to those of the coronaviruses documented. Taken together, TGEV spiker protein belongs to the class I fusion protein, characterized by the existence of two heptad-repeat (HR) regions, HR1 and HR2, and the present knowledge about the truncated TGEV fusion protein core may facilitate in the design of the small molecule or polypeptide drugs targeting the membrane fusion between TGEV and its host.

The construction of a set of transmissible gastroenteritis coronavirus (TGEV)-derived replicons as bacterial artificial chromosomes is reported. These replicons were generated by sequential deletion of nonessential genes for virus replication, using a modified TGEV full-length cDNA clone containing unique restriction sites between each pair of consecutive genes. Efficient activity of TGEV replicons was associated with the presence of the nucleoprotein provided either in cis or in trans . TGEV replicons were functional in several cell lines, including the human cell line 293T, in which no or very low cytopathic effect was observed, and expressed high amounts of heterologous protein.

Lactobacillus casei strain Shirota was selected as a bacterial carrier for the development of live mucosal vaccines against coronavirus. A 75 kDa fragment of transmissible gastroenteritis coronavirus (TGEV) spike glycoprotein S was used as the model coronavirus antigen. The S glycoprotein was cloned into a Lactobacillus/E. coli shuttle vector (pLP500) where expression and secretion of the glycoprotein S from the recombinant lactobacilli was detected via immunoblotting. Oral immunization of BALB/c mice with recombinant LcS that constitutively expresses the 75 kDa fragment of the glycoprotein S, induced both local mucosal and systemic immune responses against TGEV. Maximum titers of IgG (8.38+/-0.19 ng/ml of serum) and IgA (64.82+/-2.9 ng/ml of intestinal water) were attained 32 days post oral inturbation. The induced antibodies demonstrated neutralizing effects on TGEV infection.

A protocol for obtaining small amounts of highly pure virus preparations starting from reduced volumes (

The antigenic homology of 26 coronavirus isolates, of which 22 were antigenically related to transmissible gastroenteritis virus (TGEV), was determined with 42 monoclonal antibodies. Type, group, and interspecies specific epitopes were defined. Two group specific MAbs distinguished the enteric TGEV isolates from the respiratory variants. An antigenic subsite involved in neutralization was conserved in porcine, feline, and canine coronavirus. The classification of the human coronavirus 229E in a taxonomic cluster distinct from TGEV group is suggested. © 1990.

Human coronaviruses (HCV) are ubiquitous pathogens which cause respiratory, gastrointestinal, and possibly neurological disorders. To better understand the molecular biology of the prototype HCV-229E strain, the complete nucleotide sequence of the membrane protein (M) gene was determined from cloned cDNA. The open reading frame is preceded by a consensus transcriptional initiation sequence UCUAAACU, identical to the one found upstream of the N gene. The M gene encodes a 225-amino acid polypeptide with a molecular weight (MW) of 25,822, slightly higher than the apparent MW of 19,000-22,000 observed for the unprocessed M protein obtained after in vitro translation and immunoprecipitation. The M amino acid sequence presents a significant degree of homology (38%) with its counterpart of transmissible gastroenteritis coronavirus (TGEV). The M protein of HCV-229E is highly hydrophobic and its hydropathicity profile shows a transmembranous region composed of three major hydrophobic domains characteristic of a typical coronavirus M protein. About 10% (20 amino acids) of the HCV-229E M protein constitutes a hydrophilic and probably external portion. One N-glycosylation and three potential O-glycosylation sites are found in this exposed domain.

The 3' end of the 20-kb genome of the Purdue strain of porcine transmissible gastroenteritis coronavirus (TGEV) was copied into cDNA after priming with oligo(dT) and the double-stranded product was cloned into the PstI site of the pUC9 vector. One clone of 2.0-kb contained part of the poly(A) tail and was sequenced in its entirety using the chemical method of Maxam and Gilbert. Another clone of 0.7 kb also contained part of the poly(A) tail and was sequenced in part to confirm the primary structure of the most 3' end of the genome. Two potential, nonoverlapping genes were identified within the 3'-terminal 1663-base sequence from an examination of open reading frames. The first gene encodes a 382-amino acid protein of 43,426 mol wt, that is the apparent nucleocapsid protein on the basis of size, chemical properties, and amino acid sequence homology with other coronavirus nucleocapsid proteins. It is flanked on its 5' side by at least part of the matrix protein gene. The second encodes a hypothetical 78-amino acid protein of 9101 mol wt that is hydrophobic at both ends. A 3'-proximal noncoding sequence of 276 bases was also determined and a conserved stretch of 9 nucleotides near the poly(A) tail was found to be common among TGEV, the mouse hepatitis coronavirus, and the avian infectious bronchitis coronavirus.

Transmissible gastroenteritis coronavirus (TGEV) contains eight overlapping genes that are expressed from a 3'-coterminal nested set of leader-containing mRNAs. To facilitate the genetic manipulation of the viral genome, genes were separated by duplication of transcription regulating sequences (TRSs) and introduction of unique restriction endonuclease sites at the 5' end of each gene using an infectious cDNA clone. The recombinant TGEV (rTGEV) replicated in cell culture with similar efficiency to the wild-type virus and stably maintained the modifications introduced into the genome. In contrast, the rTGEV replication level in the lungs and gut of infected piglets and virulence were significantly reduced. rTGEV in which gene 7 expression was abrogated (rTGEV-delta7) were recovered from cDNA constructs, indicating that TGEV gene 7 was a nonessential gene for virus replication. Interestingly, in vivo infections with rTGEV-delta7 showed an additional reduction in virus replication in the lung and gut, and in virulence, indicating that TGEV gene 7 influences virus pathogenesis.

Pregnant sows were inoculated with inactivated transmissible gastroenteritis virus and with preparations of virus surface projections and subviral particles derived by detergent treatment of the virus. Neutralising antibody was demonstrated in serum and colostrum from animals that received whole virus or preparations of surface projections whereas subviral particles failed to stimulate neutralising antibody formation. Similar results were obtained with serum from rabbits inoculated with whole virus and structural components. All three preparations stimulated the formation of agglutinating antibodies, as demonstrated by sedimentation analysis and filtration studies with radiolabelled virus. The immunoglobulin classes responsible for neutralising antibody activity in sows inoculated by the intramammary route were examined. In each case where the immunoglobulin class was determined, IgG was found. One sow that received surface projections also had IgA with neutralising activity in her colostrum. In contrast, infection of sows with live whole virus resulted in neutralising antibody of the IgG, IgM and IgA classes.

The antigenic structure of transmissible gastroenteritis (TGE) virus E2 glycoprotein has been defined at three levels: antigenic sites, antigenic subsites and epitopes. Four antigenic sites (A, B, C and D) were defined by competitive radioimmunoassay (RIA) using monoclonal antibodies (MAbs) selected from 9 fusions. About 20% (197) of the hybridomas specific for TGE virus produced neutralizing MAbs specific for site A, which was one of the antigenically dominant determinants. Site A was differentiated in three antigenic subsites: a, b and c, by characterization of 11 MAb resistant (mar) mutants, that were defined by 8, 3, and 3 MAbs, respectively. These subsites were further subdivided in epitopes. A total of 11 epitopes were defined in E2 glycoprotein, eight of which were critical for virus neutralization. Neutralizing MAbs were obtained only when native virus was used to immunize mice, although to produce hybridomas mice immunizations were made with antigen in the native, denatured, or mixtures of native and denatured form. All neutralizing MAbs reacted to conformational epitopes. The antigenic structure of the E2-glycoprotein has been defined with murine MAbs, but the antigenic sites were relevant in the swine, the natural host of the virus, because porcine sera reacted against these sites. MAbs specific for TGE virus site C reacted to non-immune porcine sera. This reactivity was not directed against porcine immunoglobulins. These results indicated that TGE virus contains epitope(s) also present in some non-immunoglobulin component of porcine serum.

The surface glycoprotein S of transmissible gastroenteritis virus (TGEV) has two binding activities. (i) Binding to porcine aminopeptidase N (pAPN) is essential for the initiation of infection. (ii) Binding to sialic acid residues on glycoproteins is dispensable for the infection of cultured cells but is required for enteropathogenicity. By comparing parental TGEV with mutant viruses deficient in the sialic acid binding activity, we determined the contributions of both binding activities to the attachment of TGEV to cultured cells. In the presence of a functional sialic acid binding activity, the amount of virus bound to two different porcine cell lines was increased sixfold compared to the binding of the mutant viruses. The attachment of parental virus was reduced to levels observed with the mutants when sialic acid containing inhibitors was present or when the cells were pretreated with neuraminidase. In virus overlay binding assays with immobilized cell surface proteins, the mutant virus only recognized pAPN. In addition, the parental virus bound to a high-molecular-mass sialoglycoprotein. The recognition of pAPN was sensitive to reducing conditions and was not dependent on sialic acid residues. On the other hand, binding to the sialic acid residues of the high-molecular-mass glycoprotein was observed regardless of whether the cellular proteins had been separated under reducing or nonreducing conditions. We propose that binding to a surface sialoglycoprotein is required for TGEV as a primary attachment site to initiate infection of intestinal cells. This concept is discussed in the context of other viruses that use two different receptors to infect cells.

The stable propagation of a full-length transmissible gastroenteritis coronavirus (TGEV) cDNA in Escherichia coli cells as a bacterial artificial chromosome has been considerably improved by the insertion of an intron to disrupt a toxic region identified in the viral genome. The viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and the intron was efficiently removed during translocation of this RNA to the cytoplasm. The insertion in two different positions allowed stable plasmid amplification for at least 200 generations. Infectious TGEV was efficiently recovered from cells transfected with the modified cDNAs., This work was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (CICYT), La Consejería de Educación y Cultura de la Comunidad de Madrid, Fort Dodge Veterinaria, and the European Communities (Frame V, Key Action 2, Control of Infectious Disease Projects QLRT-1999-00002, QLRT-1999-30739, and QLRT2000-00874). J.M.G. received a fellowship from the Spanish Department of Education and Culture. Z.P., F.A., and E.C. received fellowships from the Biotechnology and Fishery and Agro-Industrial Research Programs of the European Community.

Summary. A 1-step RT-PCR assay, targeting a 730 bp fragment of the nucleocapsid (N) gene of bovine coronavirus (BCV), and a nested PCR assay, targeting a 407 bp fragment of the N gene, were developed to detect BCV in nasal swab and fecal samples of calves experimentally exposed to BCV. Both 1-step RT-PCR and nested PCR recognized cell culture passaged isolates of 10 bovine respiratory coronavirus (BRCV), 5 calf diarrhea (CD) and 8 winter dysentery (WD) strains of BCV, but not transmissible gastroenteritis coronavirus or bovine rotavirus. The sensitivity of the 1-step RT-PCR and nested PCR was compared to that of an antigen-capture ELISA. The lowest detection limit of the 1-step RT-PCR and nested PCR as determined by using tenfold serial dilutions of the BRCV 255 and 440 strains in BCV negative nasal swab suspensions from preexposure gnotobiotic calves was 2 × 104 and 2 × 102 TCID50/0.1 ml for each strain, respectively. The lowest detection limit of the antigen-capture ELISA as determined by using the same serially diluted samples was 1 × 106 TCID50/0.1 ml for each strain. Therefore, the 1-step RT-PCR and nested PCR assays were 50 and 5000 times, respectively more sensitive than the antigen-capture ELISA to detect BRCV in nasal swab suspensions. To investigate in vivo cross-protection between the BRCV and CD or WD strains of BCV and to detect nasal and fecal shedding of BCV using the 1-step RT-PCR, nested PCR and antigen-capture ELISA, 6 colostrum-deprived and two gnotobiotic calves were inoculated with a BRCV, a CD or a WD strain of BCV and then challenged 3–4 weeks later with either BRCV, CD or WD strains of BCV. All calves developed diarrhea after inoculation and BCV antigen (ELISA) or RNA (RT-PCR) was detected in the diarrheic fecal samples or the corresponding nasal swab samples. In addition, low amounts of BCV were also detected only by nested PCR in the fecal and nasal swab samples before and after diarrhea. No respiratory clinical signs were observed during the entire experimental period, but elevated rectal temperatures were detected during diarrhea in the BCV-inoculated calves. All calves recovered from infection with the BRCV, CD, or WD strains of BCV were protected from BCV-associated diarrhea after challenge exposure with either a heterologous or homologous strain of BCV. However, all calves challenged with heterologous BCV strains showed subclinical BCV infection evident by detection of nasal and fecal shedding of BCV RNA detected only by nested PCR. Such results confirm field and experimental data documenting reinfection of the respiratory and enteric tracts of cattle, suggesting that, in closed herds, respiratory or enteric tract reinfections may constitute a source of BCV transmissible to cows (WD) or neonatal or feedlot calves. In addition, the present 1-step RT-PCR and nested PCR assays were highly sensitive to detect BCV in nasal swab and fecal specimens. Therefore, these assays should be useful to diagnose BCV infections in calves and adult cows.