Transmissible gastroenteritis virus induces inflammatory responses via RIG-I/NF-κB/HIF-1α/glycolysis axis in intestinal organoids and in vivo.

Transmissible gastroenteritis virus (TGEV)-induced enteritis is characterized by watery diarrhea, vomiting, and dehydration, and has high mortality in newborn piglets, resulting in significant economic losses in the pig industry worldwide. Conventional cell lines have been used for many years to investigate inflammation induced by TGEV, but these cell lines may not mimic the actual intestinal environment, making it difficult to obtain accurate results. In this study, apical-out porcine intestinal organoids were employed to study TEGV-induced inflammation. We found that apical-out organoids were susceptible to TGEV infection, and the expression of representative inflammatory cytokines was significantly upregulated upon TGEV infection. In addition, retinoic acid-inducible gene I (RIG-I) and the nuclear factor-kappa B (NF-κB) pathway were responsible for the expression of inflammatory cytokines induced by TGEV infection. We also discovered that the transcription factor hypoxia-inducible factor-1α (HIF-1α) positively regulated TGEV-induced inflammation by activating glycolysis in apical-out organoids, and pig experiments identified the same molecular mechanism as the ex vivo results. Collectively, we unveiled that the inflammatory responses induced by TGEV were modulated via the RIG-I/NF-κB/HIF-1α/glycolysis axis ex vivo and in vivo. This study provides novel insights into TGEV-induced enteritis and verifies intestinal organoids as a reliable model for investigating virus-induced inflammation. IMPORTANCE: Intestinal organoids are a newly developed culture system for investigating immune responses to virus infection. This culture model better represents the physiological environment compared with well-established cell lines. In this study, we discovered that inflammatory responses induced by TGEV infection were regulated by the RIG-I/NF-κB/HIF-1α/glycolysis axis in apical-out porcine organoids and in pigs. Our findings contribute to understanding the mechanism of intestinal inflammation upon viral infection and highlight apical-out organoids as a physiological model to mimic virus-induced inflammation.

Swine Enteric Coronaviruses (PEDV, TGEV, and PDCoV) Induce Divergent Interferon-Stimulated Gene Responses and Antigen Presentation in Porcine Intestinal Enteroids.

Swine enteric coronaviruses (SECoVs) including porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV), account for the majority of lethal watery diarrhea in neonatal pigs and pose significant economic and public health burdens in the world. While the three SECoVs primarily infect intestinal epithelia in vivo and cause similar clinical signs, there are evident discrepancies in their cellular tropism and pathogenicity. However, the underlying mechanisms to cause the differences remain unclear. Herein, we employed porcine enteroids that are a physiologically relevant model of the intestine to assess the host epithelial responses following infection with the three SECoVs (PEDV, TGEV, and PDCoV). Although SECoVs replicated similarly in jejunal enteroids, a parallel comparison of transcriptomics datasets uncovered that PEDV and TGEV infection induced similar transcriptional profiles and exhibited a more pronounced response with more differentially expressed genes (DEGs) in jejunal enteroids compared with PDCoV infection. Notably, TGEV and PDCoV induced high levels of type I and III IFNs and IFN-stimulated gene (ISG) responses, while PEDV displayed a delayed peak and elicited a much lesser extent of IFN responses. Furthermore, TGEV and PDCoV instead of PEDV elicited a substantial upregulation of antigen-presentation genes and T cell-recruiting chemokines in enteroids. Mechanistically, we demonstrated that IFNs treatment markedly elevated the expression of NOD-like receptor (NLR) family NLRC5 and major histocompatibility complex class I (MHC-I) molecules. Together, our results indicate unique and common viral strategies for manipulating the global IFN responses and antigen presentation utilized by SECoVs, which help us a better understanding of host-SECoVs interactions.

Epithelial-mesenchymal transition of absorptive enterocytes and depletion of Peyer's patch M cells after PEDV infection.

This study focused on intestinal restitution including phenotype switching of absorptive enterocytes and the abundance of different enterocyte subtypes in weaned pigs after porcine epidemic diarrhea virus (PEDV) infection. At 10 days post-PEDV-inoculation, the ratio of villus height to crypt depth in both jejunum and ileum had restored, and the PEDV antigen was not detectable. However, enterocytes at the villus tips revealed epithelial-mesenchymal transition (EMT) in the jejunum in which E-cadherin expression decreased while expression of N-cadherin, vimentin, and Snail increased. Additionally, there was reduced expression of actin in microvilli and Zonula occludens-1 (ZO-1) in tight junctions. Moreover, the protein concentration of transforming growth factor ß1 (TGFß1), which mediates EMT and cytoskeleton alteration, was increased. We also found a decreased number of Peyer's patch M cells in the ileum. These results reveal incomplete restitution of enterocytes in the jejunum and potentially impaired immune surveillance in the ileum after PEDV infection.

Subcellular localization of the porcine deltacoronavirus nucleocapsid protein.

Porcine deltacoronavirus (PDCoV) has been recently identified as an emerging enteropathogenic coronavirus that mainly infects newborn piglets and causes enteritis, diarrhea and high mortality. Although coronavirus N proteins have multifarious activities, the subcellular localization of the PDCoV N protein is still unknown. Here, we produced mouse monoclonal antibodies against the PDCoV N protein. Experiments using anti-haemagglutinin antibodies and these monoclonal antibodies revealed that the PDCoV N protein is shuttled into the nucleolus in both ectopic PDCoV N-expressing cells and PDCoV-infected cells. The results of deletion mutagenesis experiments demonstrated that the predicted nucleolar localization signal at amino acids 295-318 is critical for nucleolar localization. Cumulatively, our study yielded a monoclonal antibody against the PDCoV N protein and revealed a mechanism by which the PDCoV N protein translocated into the nucleolus. The tolls and findings from this work will facilitate further investigations on the functions of the PDCoV N protein.

Role of Porcine Aminopeptidase N and Sialic Acids in Porcine Coronavirus Infections in Primary Porcine Enterocytes.

Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) have been reported to use aminopeptidase N (APN) as a cellular receptor. Recently, the role of APN as a receptor for PEDV has been questioned. In our study, the role of APN in PEDV and TGEV infections was studied in primary porcine enterocytes. After seven days of cultivation, 89% of enterocytes presented microvilli and showed a two- to five-fold higher susceptibility to PEDV and TGEV. A significant increase of PEDV and TGEV infection was correlated with a higher expression of APN, which was indicative that APN plays an important role in porcine coronavirus infections. However, PEDV and TGEV infected both APN positive and negative enterocytes. PEDV and TGEV Miller showed a higher infectivity in APN positive cells than in APN negative cells. In contrast, TGEV Purdue replicated better in APN negative cells. These results show that an additional receptor exists, different from APN for porcine coronaviruses. Subsequently, treatment of enterocytes with neuraminidase (NA) had no effect on infection efficiency of TGEV, implying that terminal cellular sialic acids (SAs) are no receptor determinants for TGEV. Treatment of TGEV with NA significantly enhanced the infection which shows that TGEV is masked by SAs.

Porcine deltacoronavirus infection alters bacterial communities in the colon and feces of neonatal piglets.

Porcine deltacoronavirus (PDCoV) is a novel enteropathogenic coronavirus that causes watery diarrhea in piglets. Little is known regarding the alteration of the gut microbiota in PDCoV-induced diarrhea piglets. In this study, 5-day-old piglets were experimentally infected with PDCoV strain CH-01, and all piglets developed typical clinical disease, characterized by acute and severe watery diarrhea. Histologic lesions were limited to the villous epithelium of the duodenum and ileum. Gut microbiota profiles in the colon and feces of piglets inoculated with PDCoV were investigated using 16S rRNA sequencing. The results showed that PDCoV infection reduced bacterial diversity and significantly altered the composition of the microbiota from the phylum to the genus level in the colon and feces of piglets. Firmicutes (phylum), Lactobacillaceae (family), and Lactobacillus (genus) were significantly increased (p < .01), while the abundance of Bacteroidetes (phylum) was markedly reduced in the colon and feces of the PDCoV-infected piglets (p < .01) when compared to those of the healthy piglets. Furthermore, microbial function prediction indicated that the changes in the intestinal flora also affected the nucleotide transport and metabolism, defense, translation, and transcription function of the intestinal microbiota. The current study provides new insight into the pathology and physiology of PDCoV.

Characterization and evaluation of the pathogenicity of a natural recombinant transmissible gastroenteritis virus in China.

Porcine transmissible gastroenteritis virus (TGEV) is one of the major etiological agents of viral enteritis and fetal diarrhea in suckling piglets. In this study, a TGEV JS2012 strain was isolated from the feces of piglets in Jiangsu Province, China. The phylogenetic analysis showed that TGEV JS2012 was placed between the Purdue and the Miller clusters. Analysis of recombination confirmed that TGEV JS2012 is a natural recombinant strain between Miller M6 and Purdue 115. Similar to Miller M6, virulent Purdue and China strain TS, in S gene the JS2012 maintained genetic integrity and the characteristics of the TGEV virulent strains. In vivo, TGEV JS2012 caused 100% mortality in newborn piglets, indicating the strong pathogenicity of this isolate. These results reveal that the JS2012 is a novel natural recombinant TGEV with high virulence. Our findings provide valuable information about genetic diversity and infection mechanism of the coronavirus family.

Transmissible gastroenteritis virus targets Paneth cells to inhibit the self-renewal and differentiation of Lgr5 intestinal stem cells via Notch signaling.

Infection with transmissible gastroenteritis virus (TGEV) has been associated with villous atrophy within 48 h, which seriously disrupts intestinal homeostasis. However, the underlying mechanisms remain elusive. In this study, we found that TGEV infection severely disrupted intestinal homeostasis via inhibition of self-renewal and differentiation in Lgr5 intestinal stem cells (ISCs). Profoundly, TGEV-encoded NSP10/NSP16 protein complex-mediated the inactivation of Notch signaling provided a mechanistic explanation for this phenomenon. Initial invasions by TGEV-targeted Paneth cells through aminopeptidase N (APN) receptor, then inducing mitochondrial damage and ROS generation in them, ultimately causing Paneth cell decrease and loss of Notch factors (DII4 and Hes5), which are essential for Lgr5 ISCs self-renewal and differentiation. Interestingly, loss of Notch signaling induced goblet cells differentiation at the cost of absorptive enterocytes and promoted mucins secretion, which accelerated TGEV replication. Therefore, the more differentiation of goblet cells, the greater TGEV infection in jejunum. These results provide a detailed mechanistic pathway by which villous atrophy sharply occurs in TGEV-infected jejunum within 48 h. Thus, the pathogenesis of TGEV can be described as a "bottom up scenario", which is contrary to the traditional "top down" hypothesis. Together, our findings provide a potential link between diarrheal virus infection and crypt cells response that regulates Paneth cells function and Lgr5 ISCs fate and could be exploited for therapeutic application.

Efficacy and immunogenicity of a live L. acidophilus expressing SAD epitope of transmissible gastroenteritis virus as an oral vaccine.

Transmissible gastroenteritis virus (TGEV) causes great economic loss to swine industry worldwide. Vaccination is an important method to control the TGEV infection. In this study, a TGEV oral vaccine was generated by transferring a eukaryotic expression recombinant plasmid carrying the SAD (A and D antigenic sites of the S protein) epitope of TGEV into a swine-origin Lactobacillus acidophilus (L. acidophilus). In orally immunized BALB/c mice, the TGEV L. acidophilus oral vaccine induced significantly higher level of SIgA antibodies specific to TGEV compared with the mice immunized with a commercial inactivated TGEV vaccine and similar levels of IgG specific to TGEV as the inactivated vaccine. Furthermore, the TGEV L. acidophilus oral vaccine induced higher levels of IFN-γ, which suggested that the vaccine was able to induce immune response. In brief, this novel TGEV L. acidophilus oral vaccine could induce high levels of both mucosal and humoral immune responses, which has a potential to be used in the pig industries in the future. Keywords: transmissible gastroenteritis virus (TGEV); live L. acidophilus oral vaccine; SIgA antibody; IgG antibody; IFN-γ; IL-4.

The N-Terminal Domain of Spike Protein Is Not the Enteric Tropism Determinant for Transmissible Gastroenteritis Virus in Piglets.

Transmissible gastroenteritis virus (TGEV) is the etiologic agent of transmissible gastroenteritis in pigs, and the N-terminal domain of TGEV spike protein is generally recognized as both the virulence determinant and enteric tropism determinant. Here, we assembled a full-length infectious cDNA clone of TGEV in a bacterial artificial chromosome. Using a novel approach, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems efficiently and rapidly rescued another recombinant virus with a 224-amino-acid deletion in the N-terminal domain of the TGEV Spike gene (S_NTD224), which is analogous to the N-terminal domain of porcine respiratory coronavirus. S_NTD224 notably affected the TGEV growth kinetics in PK-15 cells but was not essential for recombinant virus survival. In animal experiments with 13 two-day-old piglets, the TGEV recombinant viruses with/without S_NTD224 deletion induced obvious clinical signs and mortality. Together, our results directly demonstrated that S_NTD224 of TGEV mildly influenced TGEV virulence but was not the enteric tropism determinant and provide new insights for the development of a new attenuated vaccine against TGEV. Importantly, the optimized reverse genetics platform used in this study will simplify the construction of mutant infectious clones and help accelerate progress in coronavirus research.

Case Report of Transmissible Gastroenteritis Coronavirus Infection Associated with Small Intestine and Brain Lesions in Piglets.

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.

Impact of TGEV infection on the pig small intestine.

BACKGROUND: Pig diarrhea causes high mortality and large economic losses in the swine industry. Transmissible gastroenteritis virus (TGEV) causes pig diarrhea, with 100% mortality in piglets less than 2 weeks old. No investigation has yet been made of the small intestine of piglets that survived infection by TGEV. METHODS: In this study, we evaluated the impact of TGEV infection on the small intestine of recovered pigs. RESULTS: Histological analyses showed that TGEV infection led to villi atrophy, and reduced villous height and crypt depth. The number of SIgA positive cells, CD3+T cells, and dendritic cells (DCs) in jejunum decreased after TGEV infection in vivo. In contrast, microfold cell (M cell) numbers and cell proliferation increased in infected pigs. TGEV infection also significantly enhanced the mRNA expression levels of cytokine IL-1ß, IL-6, TNF-α, IL-10, and TGF-ß. Additionally, lower gene copy numbers of Lactobacillus, and higher numbers of Enterobacteriaceae, were detected in mucosal scraping samples from TGEV-infected pigs. CONCLUSIONS: TGEV infection damages the small intestine, impairs immune functions, and increases pathogenic bacterial loading, all of which may facilitate secondary infections by other pathogens. These findings help quantify the impact of TGEV infection and clarify the pathogenic mechanisms underlying its effects in pigs.

Proteome profile of swine testicular cells infected with porcine transmissible gastroenteritis coronavirus.

The interactions occurring between a virus and a host cell during a viral infection are complex. The purpose of this paper was to analyze altered cellular protein levels in porcine transmissible gastroenteritis coronavirus (TGEV)-infected swine testicular (ST) cells in order to determine potential virus-host interactions. A proteomic approach using isobaric tags for relative and absolute quantitation (iTRAQ)-coupled two-dimensional liquid chromatography-tandem mass spectrometry identification was conducted on the TGEV-infected ST cells. The results showed that the 4-plex iTRAQ-based quantitative approach identified 4,112 proteins, 146 of which showed significant changes in expression 48 h after infection. At 64 h post infection, 219 of these proteins showed significant change, further indicating that a larger number of proteomic changes appear to occur during the later stages of infection. Gene ontology analysis of the altered proteins showed enrichment in multiple biological processes, including cell adhesion, response to stress, generation of precursor metabolites and energy, cell motility, protein complex assembly, growth, developmental maturation, immune system process, extracellular matrix organization, locomotion, cell-cell signaling, neurological system process, and cell junction organization. Changes in the expression levels of transforming growth factor beta 1 (TGF-ß1), caspase-8, and heat shock protein 90 alpha (HSP90α) were also verified by western blot analysis. To our knowledge, this study is the first time the response profile of ST host cells following TGEV infection has been analyzed using iTRAQ technology, and our description of the late proteomic changes that are occurring after the time of vigorous viral production are novel. Therefore, this study provides a solid foundation for further investigation, and will likely help us to better understand the mechanisms of TGEV infection and pathogenesis.

Effects of virulent and attenuated transmissible gastroenteritis virus on the ability of porcine dendritic cells to sample and present antigen.

Virulent transmissible gastroenteritis virus (TGEV) results in an acute, severe pathology and high mortality in piglets, while attenuated TGEV only causes moderate clinical reactions. Dendritic cells (DCs), through uptake and presentation of antigens to T cells, initiate distinct immune responses to different infections. In this study, an attenuated TGEV (STC3) and a virulent TGEV (SHXB) were used to determine whether porcine DCs play an important role in pathogenetic differences between these two TGEVs. Our results showed that immature and mature monocyte-derived dendritic cells (Mo-DCs) were susceptible to infection with SHXB and STC3. However, only SHXB inhibited Mo-DCs to activate T-cell proliferation by down-regulating the expression of cell-surface markers and the secretion of cytokines in vitro. In addition, after 48 h of SHXB infection, there was the impairment in the ability of porcine intestinal DCs to sample the antigen, to migrate from the villi to the lamina propria and to activate T-cell proliferation in vivo. In contrast, these abilities of intestinal DCs were enhanced in STC3-infected piglets. In conclusion, our results show that SHXB significantly impaired the functions of Mo-DCs and intestinal DCs in vitro and in vivo, while STC3 had the opposite effect. These differences may underlie the pathogenesis of virulent and attenuated TGEV in piglets, and could help us to develop a better strategy to prevent virulent TGEV infection.

Regulation of ROS in transmissible gastroenteritis virus-activated apoptotic signaling.

Transmissible gastroenteritis virus (TGEV), an enteropathogenic coronavirus, causes severe lethal watery diarrhea and dehydration in piglets. Previous studies indicate that TGEV infection induces cell apoptosis in host cells. In this study, we investigated the roles and regulation of reactive oxygen species (ROS) in TGEV-activated apoptotic signaling. The results showed that TGEV infection induced ROS accumulation, whereas UV-irradiated TGEV did not promote ROS accumulation. In addition, TGEV infection lowered mitochondrial transmembrane potential in PK-15 cell line, which could be inhibited by ROS scavengers, pyrrolidinedithiocarbamic (PDTC) and N-acetyl-l-cysteine (NAC). Furthermore, the two scavengers significantly inhibited the activation of p38 MAPK and p53 and further blocked apoptosis occurrence through suppressing the TGEV-induced Bcl-2 reduction, Bax redistribution, cytochrome c release and caspase-3 activation. These results suggest that oxidative stress pathway might be a key element in TGEV-induced apoptosis and TGEV pathogenesis.

Diagnosis to detect porcine transmissible gastroenteritis virus (TGEV) by optical and transmission electron microscopy techniques / Diagnóstico para detectar el virus de la gastroenteritis porcina transmisible por técnicas de microscopía óptica y electrónica de transmisión

The porcine transmissible gastroenteritis is a highly severe contagious disease, caused by virus of the Coronaviridae family, genus Coronavirus. Its epizootic shape can reach a rate of up to 100 percent mortality in piglets under two weeks of age as a result of severe dehydration. In this study fragments of small intestine and stool samples were collected from 75 autopsied pigs from properties. The samples of the fragments were frozen and sent to the Laboratory of Electron Microscopy, Instituto Biológico, SP, Brazil, for histological and transmission electron microscopic analyses. According to histological H&E technique, atrophy, villous necrosis and destruction of the enterocytes were observed in 35 (46.6 percent) out of the 75 fragments of the small intestine samples. On the immunohistochemistry technique 19 (25.3 percent) fragments were positively stained with DAB in the Ag-Ac reaction (MabTGEV). In 19 (25.3 percent) positive samples analyzed by in situ hybridization, a brown stain of enterocytes was observed, mainly in the epithelial cells of the villi. By the negative staining technique, we visualized enveloped, pleomorphic coronavirus particles, with typical radial projections resembling solar corona, with 140 nm diameter in 21 samples (28 percent) of the small intestine fragments and in 16 (21.3 percent) stool samples. In the ultrathin sections of 21 (28 percent) samples of small intestine, complete coronavirus particles with 80 nm diameter were seen among the microvilli and in the cytoplasm of epithelial cells. Immature particles with 60 nm diameter, budding from cell membrane and from a rough endoplasmic reticulum and also inside the vacuoles were visualized. In 19 (25.3 percent)...

La gastroenteritis transmisible porcina se caracteriza por ser una enfermedad altamente contagiosa y aguda, causada por virus de la familia Coronaviridae, género Coronavirus. Su forma epizoótica puede alcanzar una tasa de hasta 100 percent de mortalidad en lechones con menos de dos semanas de edad, como resultado de deshidratación severa. En este trabajo se recogieron 75 fragmentos de intestino delgado y muestras de heces de 75 cerdos autopsiados. Las muestras se congelaron y se enviaron al Laboratorio de Microscopía Electrónica, del Instituto Biológico, SP, Brasil, para el análisis histológico y por microscopía electrónica de transmisión. Por la técnica histológica de H&E fue observado atrofia y necrosis de la vellosidades además de la destrucción de los enterocitos en 35 (46,6 ppor ciento) de las 75 muestras de fragmentos del intestino delgado. Por inmunohistoquímica 19 (25,3 por ciento) de los fragmentos se tiñeron positivamente por el DAB en la reacción Ag-Ac (MabTGEV). En 19 (25,3 por ciento) muestras positivas mediante hibridación in situ, se observó tinción marrón de los enterocitos, principalmente en las células epiteliales de las vellosidades. Por la técnica de coloración negativa, se observaron partículas de coronavirus, encapsuladas, pleomórficas con proyecciones radiales típicas, en forma de corona solar, midiendo alrededor de 140 nm de diámetro en 21 (28 por ciento) muestras de fragmentos del intestino delgado y en 16 (21,3 por ciento muestras de heces. En cortes ultra finos de 21 (28 por ciento) fragmentos de intestino delgado fueron visualizadas partículas de coronavirus completas con 80 nm de diámetro entre las microvellosidades y en el citoplasma de las células epiteliales y partículas inmaduras de 60 nm de diámetro brotando de las membranas celulares y del retículo endoplasmático rugoso y en el interior de las vacuolas. En 19 (25,3 por ciento)...

Inhibition of porcine transmissible gastroenteritis virus (TGEV) replication in mini-pigs by shRNA.

Transmissible gastroenteritis virus (TGEV) is the causative agent of porcine transmissible gastroenteritis (TGE), characterized by high mortality and severely retarded growth in piglets that dramatically affects the porcine industry. Previously, we have identified two shRNA-expressing plasmids pEGFP-U6/P1 and pEGFP-U6/P2 that target RNA-dependent RNA polymerase (RdRP) gene of TGEV with more than 95% of virus inhibition in vitro. In this study, inhibition of the TGEV replication by pEGFP-U6/P1 and pEGFP-U6/P2 was tested in mini-pigs. SPF mini-pigs at 25 days old were injected with the shRNA-expressing plasmids and then infected with TGEV. The results from the analyses of clinical signs, histopathology, indirect immunofluorescence (IIF) and RT-PCR show that the two shRNA-expressing plasmids could significantly decrease the quantity of TGEV in different organs and protect mini-pigs from TGEV infection. These findings illustrate the prospect for TGEV-specific shRNAs to be new anti-TGEV agents.

Experimental infection of piglets with transmissible gastroenteritis virus: a comparison of three strains (Korean, Purdue and Miller).

Eighty-four colostrum-deprived piglets aged 1 day were inoculated with either a Korean strain of transmissible gastroenteritis virus (TGEV) or one of two American strains (Purdue and Miller). The purpose was to compare, by morphometric analysis and in-situ hybridization, the Korean strain with the two American strains in respect of pathogenicity and viral distribution over a period of 72 h. The progression of infection in pigs infected with the Korean strain was much slower than that in pigs infected with either of the two American strains. At 6 h post-inoculation (hpi), the mean score of the villous height/crypt depth (VH/CD) ratio of pigs inoculated with the Purdue or Miller strain was significantly less than that of pigs inoculated with the Korean strain or uninfected control pigs. At 12-72 hpi, however, the VH/CD ratio of all infected pigs was significantly less than that of control pigs. In-situ hybridization showed significant differences between the Korean and American strains in terms of the amount of TGEV nucleic acid at 6, 12 and 60 hpi. TGEV nucleic acid was detected in the duodenum, jejunum and ileum of pigs inoculated with the Purdue or Miller strain but only in the jejunum and ileum of those inoculated with the Korean strain. The results suggested that the Korean strain was less virulent than the two American strains.

Transmissible gastroenteritis coronavirus induces programmed cell death in infected cells through a caspase-dependent pathway.

In this report, we show that apoptosis (or programmed cell death) is induced in different cell lines infected with a coronavirus, the porcine transmissible gastroenteritis virus (TGEV). Kinetic analysis of internucleosomal DNA cleavage by agarose gel electrophoresis and flow cytometry or cytometric monitoring of the mitochondrial transmembrane potential showed that, for ST cells infected with TGEV, the first overt signs of apoptosis appeared from 10 to 12 h postinfection on. They preceded morphological changes characteristic of cells undergoing apoptosis, as observed by light and electron microscopy. The tripeptide pan-ICE (caspase) inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone blocked TGEV-induced apoptosis with no effect on virus production. The thiol agent pyrrolidine dithiocarbamate inhibited apoptosis, suggesting that TGEV infection may lead to apoptosis via cellular oxidative stress. The effect of TGEV infection on activation of NF-kappaB, a transcription factor known to be activated by oxidative stress, was examined. NF-kappaB DNA binding was shown to be strongly and quickly induced by TGEV infection. However, transcription factor decoy experiments showed that NF-kappaB activation is not critical for TGEV-induced apoptosis.

The coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment.

Aminopeptidase N is a species-specific receptor for transmissible gastroenteritis virus (TGEV), which infects piglets, and for the 229E virus, which infects humans. It is not known whether these coronaviruses are endocytosed before fusion with a membrane of the target cell, causing a productive infection, or whether they fuse directly with the plasma membrane. We have studied the interaction between TGEV and a cell line (MDCK) stably expressing recombinant pig aminopeptidase N (pAPN). By electron microscopy and flow cytometry, TGEV was found to be associated with the plasma membrane after adsorption to the pAPN-MDCK cells. TGEV was also observed in endocytic pits and apical vesicles after 3 to 10 min of incubation at 38 degrees C. The number of pits and apical vesicles was increased by the TGEV incubation, indicating an increase in endocytosis. After 10 min of incubation, a distinct TGEV-pAPN-containing population of large intracellular vesicles, morphologically compatible with endosomes, was found. A higher density of pAPN receptors was observed in the pits beneath the virus particles than in the surrounding plasma membrane, indicating that TGEV recruits pAPN receptors before endocytosis. Ammonium chloride and bafilomycin A1 markedly inhibited the TGEV infection as judged from virus production and protein biosynthesis analyses but did so only when added early in the course of the infection, i.e., about 1 h after the start of endocytosis. Together our results point to an acid intracellular compartment as the site of fusion for TGEV.