Bile acid-sensitive human norovirus strains are susceptible to sphingosine-1-phosphate receptor 2 inhibition.

Human noroviruses (HuNoVs) are a diverse group of RNA viruses that cause endemic and pandemic acute viral gastroenteritis. Previously, we reported that many HuNoV strains require bile or bile acid (BA) to infect human jejunal intestinal enteroid cultures. BA was not essential for the replication of a pandemic-causing GII.4 HuNoV strain. We found the hydrophobic BA glycochenodeoxycholic acid (GCDCA) promotes the replication of the BA-dependent strain GII.3 in jejunal enteroids. Furthermore, we found that inhibition of the G-protein-coupled BA receptor, sphingosine-1-phosphate receptor 2 (S1PR2), by JTE-013, reduced GII.3 infection dose-dependently and inhibited GII.3 cellular uptake in enteroids. Herein, we sought to determine whether S1PR2 is required for other BA-dependent HuNoV strains, the BA-independent GII.4, and whether S1PR2 is required for BA-dependent HuNoV infection in HIEs from other small intestinal segments. We found a second S1PR2 inhibitor, GLPG2938, reduces GII.3 infection dose-dependently, and an S1PR2 agonist (CYM-5520) enhances GII.3 replication in the absence of GCDCA. GII.3 replication also is abrogated in the presence of JTE-013 and CYM-5520. JTE-013 inhibition of S1PR2 in jejunal HIEs reduces GI.1, GII.3, and GII.17 (BA-dependent) but not GII.4 Sydney (BA-independent) infection, providing additional evidence of strain-specific differences in HuNoV infection. Finally, GII.3 infection of duodenal, jejunal, and ileal lines derived from the same individual is reduced with S1PR2 inhibition, indicating a common mechanism of BA-dependent infection among multiple segments of the small intestine. Our results support a model where BA-dependent HuNoVs exploit BA effects on S1PR2 to infect the entire small intestine.IMPORTANCEHuman noroviruses (HuNoVs) are important viral human pathogens that cause both outbreaks and sporadic gastroenteritis. These viruses are diverse, and many strains are capable of infecting humans. Our previous studies have identified strain-specific requirements for hydrophobic bile acids (BAs) to infect intestinal epithelial cells. Moreover, we identified a BA receptor, sphingosine-1-phosphate receptor 2 (S1PR2), required for infection by a BA-dependent strain. To better understand how various HuNoV strains enter and infect the small intestine and the role of S1PR2 in HuNoV infection, we evaluated infection by additional HuNoV strains using an expanded repertoire of intestinal enteroid cell lines. We found that multiple BA-dependent strains, but not a BA-independent strain, all require S1PR2 for infection. In addition, BA-dependent infection requires S1PR2 in multiple segments of the small intestine. Together, these results indicate that S1PR2 has value as a potential therapeutic target for BA-dependent HuNoV infection.

N-glycoproteomic analyses of human intestinal enteroids, varying in histo-blood group geno- and phenotypes, reveal a wide repertoire of fucosylated glycoproteins.

Human noroviruses, globally the main cause of viral gastroenteritis, show strain specific affinity for histo-blood group antigens (HBGA) and can successfully be propagated ex vivo in human intestinal enteroids (HIEs). HIEs established from jejunal stem cells of individuals with different ABO, Lewis and secretor geno- and phenotypes, show varying susceptibility to such infections. Using bottom-up glycoproteomic approaches we have defined and compared the N-linked glycans of glycoproteins of seven jejunal HIEs. Membrane proteins were extracted, trypsin digested, and glycopeptides enriched by hydrophilic interaction liquid chromatography and analyzed by nanoLC-MS/MS. The Byonic software was used for glycopeptide identification followed by hands-on verifications and interpretations. Glycan structures and attachment sites were identified from MS2 spectra obtained by higher-energy collision dissociation through analysis of diagnostic saccharide oxonium ions (B-ions), stepwise glycosidic fragmentation of the glycans (Y-ions), and peptide sequence ions (b- and y-ions). Altogether 694 unique glycopeptides from 93 glycoproteins were identified. The N-glycans encompassed pauci- and oligomannose, hybrid- and complex-type structures. Notably, polyfucosylated HBGA-containing glycopeptides of the four glycoproteins tetraspanin-8, carcinoembryonic antigen-related cell adhesion molecule 5, sucrose-isomaltase and aminopeptidase N were especially prominent and were characterized in detail and related to donor ABO, Lewis and secretor types of each HIE. Virtually no sialylated N-glycans were identified for these glycoproteins suggesting that terminal sialylation was infrequent compared to fucosylation and HBGA biosynthesis. This approach gives unique site-specific information on the structural complexity of N-linked glycans of glycoproteins of human HIEs and provides a platform for future studies on the role of host glycoproteins in gastrointestinal infectious diseases.

SARS-CoV-2 Spike protein triggers gut impairment since mucosal barrier to innermost layers: From basic science to clinical relevance.

Studies have reported the occurrence of gastrointestinal (GI) symptoms, primarily diarrhea, in COVID-19. However, the pathobiology regarding COVID-19 in the GI tract remains limited. This work aimed to evaluate SARS-CoV-2 Spike protein interaction with gut lumen in different experimental approaches. Here, we present a novel experimental model with the inoculation of viral protein in the murine jejunal lumen, in vitro approach with human enterocytes, and molecular docking analysis. Spike protein led to increased intestinal fluid accompanied by Cl- secretion, followed by intestinal edema, leukocyte infiltration, reduced glutathione levels, and increased cytokine levels [interleukin (IL)-6, tumor necrosis factor-α, IL-1ß, IL-10], indicating inflammation. Additionally, the viral epitope caused disruption in the mucosal histoarchitecture with impairment in Paneth and goblet cells, including decreased lysozyme and mucin, respectively. Upregulation of toll-like receptor 2 and toll-like receptor 4 gene expression suggested potential activation of local innate immunity. Moreover, this experimental model exhibited reduced contractile responses in jejunal smooth muscle. In barrier function, there was a decrease in transepithelial electrical resistance and alterations in the expression of tight junction proteins in the murine jejunal epithelium. Additionally, paracellular intestinal permeability increased in human enterocytes. Finally, in silico data revealed that the Spike protein interacts with cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride conductance (CaCC), inferring its role in the secretory effect. Taken together, all the events observed point to gut impairment, affecting the mucosal barrier to the innermost layers, establishing a successful experimental model for studying COVID-19 in the GI context.

CD32+CD4+ T Cells Sharing B Cell Properties Increase With Simian Immunodeficiency Virus Replication in Lymphoid Tissues.

CD4 T cell responses constitute an important component of adaptive immunity and are critical regulators of anti-microbial protection. CD4+ T cells expressing CD32a have been identified as a target for HIV. CD32a is an Fcγ receptor known to be expressed on myeloid cells, granulocytes, B cells and NK cells. Little is known about the biology of CD32+CD4+ T cells. Our goal was to understand the dynamics of CD32+CD4+ T cells in tissues. We analyzed these cells in the blood, lymph nodes, spleen, ileum, jejunum and liver of two nonhuman primate models frequently used in biomedical research: African green monkeys (AGM) and macaques. We studied them in healthy animals and during viral (SIV) infection. We performed phenotypic and transcriptomic analysis at different stages of infection. In addition, we compared CD32+CD4+ T cells in tissues with well-controlled (spleen) and not efficiently controlled (jejunum) SIV replication in AGM. The CD32+CD4+ T cells more frequently expressed markers associated with T cell activation and HIV infection (CCR5, PD-1, CXCR5, CXCR3) and had higher levels of actively transcribed SIV RNA than CD32-CD4+T cells. Furthermore, CD32+CD4+ T cells from lymphoid tissues strongly expressed B-cell-related transcriptomic signatures, and displayed B cell markers at the cell surface, including immunoglobulins CD32+CD4+ T cells were rare in healthy animals and blood but increased strongly in tissues with ongoing viral replication. CD32+CD4+ T cell levels in tissues correlated with viremia. Our results suggest that the tissue environment induced by SIV replication drives the accumulation of these unusual cells with enhanced susceptibility to viral infection.

Damage to intestinal barrier integrity in piglets caused by porcine reproductive and respiratory syndrome virus infection.

Porcine reproductive and respiratory syndrome (PRRS) induces respiratory disease and reproductive failure accompanied by gastroenteritis-like symptoms. The mechanism of intestinal barrier injury caused by PRRSV infection in piglets has yet to be investigated. An in vivo PRRSV-induced model was established in 30-day-old piglets by the intramuscular injection of 2 mL of 104 TCID50/mL PRRSV for 15 days. Observations of PRRSV replication and histology were conducted in the lungs and intestine, and goblet cell counts, relative MUC2 mRNA expression, and tight junction protein, proinflammatory cytokine, TLR4, MyD88, IκB and p-IκB expression were measured. PRRSV replicated in the lungs and small intestine, as demonstrated by absolute RT-qPCR quantification, and the PRRSV N protein was detected in the lung interstitium and jejunal mucosa. PRRSV infection induced both lung and gut injury, markedly decreased villus height and the villus to crypt ratio in the small intestine, and obviously increased the number of goblet cells and the relative expression of MUC2 mRNA in the jejunum. PRRSV infection aggravated the morphological depletion of tight junction proteins and increased IL-1ß, IL-6, IL-8 and TNF-α expression by activating the NF-κB signalling pathway in the jejunum. PRRSV infection impaired intestinal integrity by damaging physical and immune barriers in the intestine by inducing inflammation, which may be related to the regulation of the gut-lung axis. This study also provides a new hypothesis regarding the pathogenesis of PRRSV-induced diarrhoea.

Rotavirus induces intercellular calcium waves through ADP signaling.

Rotavirus causes severe diarrheal disease in children by broadly dysregulating intestinal homeostasis. However, the underlying mechanism(s) of rotavirus-induced dysregulation remains unclear. We found that rotavirus-infected cells produce paracrine signals that manifested as intercellular calcium waves (ICWs), observed in cell lines and human intestinal enteroids. Rotavirus ICWs were caused by the release of extracellular adenosine 5'-diphosphate (ADP) that activated P2Y1 purinergic receptors on neighboring cells. ICWs were blocked by P2Y1 antagonists or CRISPR-Cas9 knockout of the P2Y1 receptor. Blocking the ADP signal reduced rotavirus replication, inhibited rotavirus-induced serotonin release and fluid secretion, and reduced diarrhea severity in neonatal mice. Thus, rotavirus exploited paracrine purinergic signaling to generate ICWs that amplified the dysregulation of host cells and altered gastrointestinal physiology to cause diarrhea.

Alterations in Intestinal Innate Mucosal Immunity of Weaned Pigs During Porcine Epidemic Diarrhea Virus Infection.

In the small intestine, localized innate mucosal immunity is critical for intestinal homeostasis. Porcine epidemic diarrhea virus (PEDV) infection induces villus injury and impairs digestive function. Moreover, the infection might comprise localized innate mucosal immunity. This study investigated specific enterocyte subtypes and innate immune components of weaned pigs during PEDV infection. Four-week-old pigs were orally inoculated with PEDV IN19338 strain (n = 40) or sham-inoculated (n = 24). At day post inoculation (DPI) 2, 4, and 6, lysozyme expression in Paneth cells, cellular density of villous and Peyer's patch microfold (M) cells, and the expression of polymeric immunoglobulin receptor (pIgR) were assessed in the jejunum and ileum by immunohistochemistry, and interleukin (IL)-1ß and tumor necrosis factor (TNF)-α were measured in the jejunum by ELISA. PEDV infection led to a decrease in the ratios of villus height to crypt depth (VH-CD) in jejunum at DPI 2, 4, and 6 and in ileum at DPI 4. The number of villous M cells was reduced in jejunum at DPI 4 and 6 and in ileum at DPI 6, while the number of Peyer's patch M cells in ileum increased at DPI 2 and then decreased at DPI 6. PEDV-infected pigs also had reduced lysozyme expression in ileal Paneth cells at DPI 2 and increased ileal pIgR expression at DPI 4. There were no significant changes in IL-1ß and TNF-α expression in PEDV-infected pigs compared to controls. In conclusion, PEDV infection affected innate mucosal immunity of weaned pigs through alterations in Paneth cells, villous and Peyer's patch M cells, and pIgR expression.

Application of RNAscope technology to studying the infection dynamics of a Chinese porcine epidemic diarrhea virus variant strain BJ2011C in neonatal piglets.

The highly virulent porcine epidemic diarrhea virus (PEDV) variants cause the death of mainly neonatal piglets, but how the viruses spread within the gastro-intestinal tract in a temporal and spatial manner has remained poorly characterized but is critical to understand the viral pathogenesis. In this study, we used the Chinese PEDV epidemic strain BJ2011C as a model organism and took advantage of the newly developed RNAscope in situ hybridization technology to investigate the tempo-spatial infection dynamics in neonatal piglets. We found that the PEDV strain BJ2011C could quickly colonize the small intestine, which occurred in just 6 h post infection, with virus shedding starting at 6 hpi and peaking at 24 hpi. Jejunum was the first target tissue for infection and then ileum, followed by infrequent infection of duodenum. In these tissues, the virus nucleic acids were mainly present in the villous epithelial cells but not in crypt cells. Interestingly, the viral RNAs were not detectable by RNAscope in large intestines although tissue damages could be discerned by H & E staining. Overall, our results provide useful information about spread dynamics and tissue preference of PEDV epidemic strain BJ2011C.

Expression profile analysis of 5-day-old neonatal piglets infected with porcine Deltacoronavirus.

BACKGROUND: Porcine deltacoronavirus (PDCoV) is a novel coronavirus that can cause diarrhea in nursing piglets. This study was aimed to investigate the roles of host differentially expressed genes on metabolic pathways in PDCoV infections. RESULTS: Twenty thousand six hundred seventy-four differentially expressed mRNAs were identified in 5-day-old piglets responded to PDCoV experimental infections. Many of these genes were correlated to the basic metabolism, such as the peroxisome proliferator-activated receptor (PPAR) signaling pathway which plays a critical role in digestion. At the same time, in the PPAR pathway genes of fatty acid-binding protein (FABP) family members were observed with remarkably differential expressions. The differential expressed genes were associated with appetite decrease and weight loss of PDCoV- affected piglets. DISCUSSION: Fatty acid-binding protein 1 (FABP1) and fatty acid-binding protein 3 (FABP3) were found to be regulated by PDCoV. These two genes not only mediate fatty acid transportation to different cell organelles such as mitochondria, peroxisome, endoplasmic reticulum and nucleus, but also modulate fatty acid metabolism and storage as a signaling molecule outside the cell. Therefore, it can be preliminarily concluded that PPAR differential expression caused by PDCoV was mostly associated with weight loss and death from emaciation. CONCLUSIONS: The host differentially expressed genes were associated with infection response, metabolism signaling and organismal systems signaling pathways. The genes of FABP family members in the PPAR signaling pathway were the most highly altered and played important roles in metabolism. Alteration of these genes were most likely the reason of weight loss and other clinical symptoms. Our results provided new insights into the metabolic mechanisms and pathogenesis of PDCoV infection. METHODS: Animal experiment, Determination of viral growth by real-time RT-PCR, Histopathology, Immunohistochemical staining, Microarray analysis.

Porcine Intestinal Enteroids: a New Model for Studying Enteric Coronavirus Porcine Epidemic Diarrhea Virus Infection and the Host Innate Response.

Porcine epidemic diarrhea virus (PEDV), a member of the group of alphacoronaviruses, is the pathogen of a highly contagious gastrointestinal swine disease. The elucidation of the events associated with the intestinal epithelial response to PEDV infection has been limited by the absence of good in vitro porcine intestinal models that recapitulate the multicellular complexity of the gastrointestinal tract. Here, we generated swine enteroids from the intestinal crypt stem cells of the duodenum, jejunum, or ileum and found that the generated enteroids are able to satisfactorily recapitulate the complicated intestinal epithelium in vivo and are susceptible to infection by PEDV. PEDV infected multiple types of cells, including enterocytes, stem cells, and goblet cells, and exhibited segmental infection discrepancies compared with ileal enteroids and colonoids, and this finding was verified in vivo Moreover, the clinical isolate PEDV-JMS propagated better in ileal enteroids than the cell-adapted isolate PEDV-CV777, and PEDV infection suppressed interferon (IFN) production early during the infection course. IFN lambda elicited a potent antiviral response and inhibited PEDV in enteroids more efficiently than IFN alpha (IFN-α). Therefore, swine enteroids provide a novel in vitro model for exploring the pathogenesis of PEDV and for the in vitro study of the interplay between a host and a variety of swine enteric viruses.IMPORTANCE PEDV is a highly contagious enteric coronavirus that causes significant economic losses, and the lack of a good in vitro model system is a major roadblock to an in-depth understanding of PEDV pathogenesis. Here, we generated a porcine intestinal enteroid model for PEDV infection. Utilizing porcine intestinal enteroids, we demonstrated that PEDV infects multiple lineages of the intestinal epithelium and preferably infects ileal enteroids over colonoids and that enteroids prefer to respond to IFN lambda 1 over IFN-α. These events recapitulate the events that occur in vivo This study constitutes the first use of a primary intestinal enteroid model to investigate the susceptibility of porcine enteroids to PEDV and to determine the antiviral response following infection. Our study provides important insights into the events associated with PEDV infection of the porcine intestine and provides a valuable in vitro model for studying not only PEDV but also other swine enteric viruses.

Supplementation of Vitamin E Protects Chickens from Newcastle Disease Virus-Mediated Exacerbation of Intestinal Oxidative Stress and Tissue Damage.

BACKGROUND/AIMS: Newcastle disease virus (NDV) causes a highly devastating and contagious disease in poultry, which is mainly attributed to extensive tissue damages in the digestive, respiratory and nervous systems. However, nature and dynamics of NDV-induced oxidative stresses in the intestine of chickens remain elusive. METHODS: In this study, we examined the magnitude of intestinal oxidative stress and histopathological changes caused by the virulent NDV infection, and explored the protective roles of vitamin E (vit. E) in ameliorating these pathological changes. For these purposes, chickens were divided into four groups namely i) non supplemented and non-challenged (negative control, CON); ii) no supplementation of vit. E but challenged with ZJ1 (positive control, NS+CHA); iii) vit. E supplementation at the dose of 50 IU/day/Kg body weight and ZJ1 challenge (VE50+CHA); and 4) vit. E supplementation at the dose of 100 IU/day/Kg body weight and ZJ1 challenge (VE100+CHA). In all groups, we analyzed concentrations of glutathione (GSH), malondialdehyde (MDA), nitric oxide (NO), total antioxidant capacity (T-AOC), and activity of glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) using biochemical methods. The virus loads were determined by quantitative RT-PCR and antibody titers by hemagglutination inhibition assays. We also examined the histopathological changes in the duodenal and jejunal mucosa at 3 and 5-day post infection (dpi) with NDV. RESULTS: A significant elevation in the NO level was observed in NDV challenged chickens compared to the CON chickens at 2 dpi. The MDA contents were significantly increased whereas GSH was significantly decreased in NDV-challenged chickens compared to control. Furthermore, activities of GST, CAT, SOD, as well as the TOAC were markedly decreased in challenged chickens in comparison with control. Virus copy numbers were higher in NDV infected NS+CHA group compared to other groups. Severe histopathological changes including inflammation, degeneration and broken villi were observed in the intestine of NDV challenged chickens. However, all these malfunctions of antioxidant system and pathological changes in the intestine were partially or completely reversed by the vit. E supplementation. CONCLUSIONS: Our results suggest that NDV infection causes oxidative stress and histopathological changes in the duodenum and jejunum of chickens, which can be partially or fully ameliorated by supplementation of vit. E. Additionally, these findings suggest that oxidative stress contributes to the intestinal damages in NDV infected chickens. These findings will help to understand the pathogenesis of NDV and further investigation of therapeutic agents for control of Newcastle disease.

Fatal swine acute diarrhoea syndrome caused by an HKU2-related coronavirus of bat origin.

Cross-species transmission of viruses from wildlife animal reservoirs poses a marked threat to human and animal health 1 . Bats have been recognized as one of the most important reservoirs for emerging viruses and the transmission of a coronavirus that originated in bats to humans via intermediate hosts was responsible for the high-impact emerging zoonosis, severe acute respiratory syndrome (SARS) 2-10 . Here we provide virological, epidemiological, evolutionary and experimental evidence that a novel HKU2-related bat coronavirus, swine acute diarrhoea syndrome coronavirus (SADS-CoV), is the aetiological agent that was responsible for a large-scale outbreak of fatal disease in pigs in China that has caused the death of 24,693 piglets across four farms. Notably, the outbreak began in Guangdong province in the vicinity of the origin of the SARS pandemic. Furthermore, we identified SADS-related CoVs with 96-98% sequence identity in 9.8% (58 out of 591) of anal swabs collected from bats in Guangdong province during 2013-2016, predominantly in horseshoe bats (Rhinolophus spp.) that are known reservoirs of SARS-related CoVs. We found that there were striking similarities between the SADS and SARS outbreaks in geographical, temporal, ecological and aetiological settings. This study highlights the importance of identifying coronavirus diversity and distribution in bats to mitigate future outbreaks that could threaten livestock, public health and economic growth.

Cellular hnRNP A1 Interacts with Nucleocapsid Protein of Porcine Epidemic Diarrhea Virus and Impairs Viral Replication.

The nucleocapsid (N) protein is a major structural component of porcine epidemic diarrhea virus (PEDV), which is predicted to be a multifunctional protein in viral replication. Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a cellular protein participating in the splicing of pre-mRNA in the nucleus and translation regulation in the cytoplasm. According to our previous proteomic study about PEDV infection in vivo, hnRNP A1 was thought to be a cellular factor influencing PEDV replication. In this report, PEDV N protein was discovered to colocalize with cellular hnRNP A1 in perinuclear region of PEDV infected cells. Co-immunoprecipitation (CO-IP) results clearly demonstrated that PEDV N protein could bind to human hnRNP A1. Replication of PEDV was inhibited by silencing the expression of hnRNP A1 in CCL-81 cells, suggesting the positive effect of hnRNP A1 on PEDV infection.

Porcine epidemic diarrhea virus reduces feed efficiency in nursery pigs.

Porcine epidemic diarrhea virus (PEDV) infects enterocytes and in nursery pigs, results in diarrhea, anorexia, and reduced performance. Therefore, the objective of this study was to determine how PEDV infection influenced growth performance and repartitioning of amino acids and energy in nursery pigs. A total of 32 barrows and gilts, approximately 1 wk post-wean (BW = 8.46 ± 0.50 kg), and naïve for PEDV were obtained, weighed, and allotted based on sex and BW to one of two treatments: 1) Control, PEDV naïve and 2) PEDV-inoculated (PEDV) with eight pens of two pigs each per treatment. On day post-inoculation (dpi) 0, PEDV pigs were inoculated via intragastric gavage with PEDV isolate (USA/Iowa/18984/2013). Pig and feeder weights were recorded at dpi -7, 0, 5, and 20 in order to calculate ADG, ADFI, and G:F. Eight pigs per treatment were euthanized on dpi 5 and 20, and tissues and blood were collected. At dpi 5, all PEDV pigs were PCR positive for PEDV in feces. Overall, PEDV pigs tended (P < 0.10) to increase ADFI, which resulted in reduced (P < 0.05) feed efficiency. At dpi 5, PEDV pigs had reduced (P < 0.05) villus height and increased (P < 0.05) stem cell proliferation in the jejunum compared with Control pigs. Pigs inoculated with PEDV had increased (P < 0.05) serum haptoglobin and increased insulin-to-glucose ratios compared with Control pigs at dpi 5. Markers of muscle proteolysis were not different (P > 0.05) between treatments within dpi; however, at dpi 5, 20S proteasome activity was increased (P < 0.05) in longissimus dorsi of PEDV pigs compared with Control pigs. Liver and jejunum gluconeogenic enzyme activities were not different (P > 0.05) between treatments within dpi. Overall, PEDV-inoculated pigs did recover the absorptive capacity that was reduced during PEDV infection by increasing proliferation of intestinal stem cells. However, the energy and nutrients needed to recover the epithelium may be originating from available luminal nutrients instead of muscle proteolysis and gluconeogenesis. This study provides insight into the effects of an enteric coronavirus on postabsorptive metabolism in nursery pigs.

Pathogenesis of Human Norovirus Genogroup II Genotype 4 in Post-Weaning Gnotobiotic Pigs.

Norovirus is the most common cause of acute gastroenteritis. Its pathogenesis is poorly understood owing to the difficulty of establishing viral infection in animal models. Here, post-weaning gnotobiotic pigs were infected with human norovirus genogroup II genotype 4 (HuNoV GII.4) to investigate the pathogenesis and replication of the virus. Three groups of four pigs were infected with 1 × 105, 1 × 106, or 1 × 107 genomic equivalent (GE) copies of HuNoV GII.4. Four pigs were used as negative controls. Blood and rectal swab samples were collected after viral infection, and gross legions were examined after necropsy. Diarrhea was induced in 25% and 75% of pigs infected with 1 × 106 and 1 × 107 GE copies, respectively. Viral shedding was detected in 50%, 75%, and 50% of pigs infected with 1 × 105, 1 × 106, and 1 × 107 GE copies, respectively. Viremia was detected in 25% of pigs infected with either 1 × 106 or 1 × 107 GE copies. When gross lesions of gastroenteritis were investigated, the ileum walls of the infected pigs were thinner than those of the controls. Villi atrophy and inflammatory cell infiltration were identified in the ileum of each infected pig. Viral capsid was identified in the jejunum, ileum, colon, spleen, and mesenteric lymph node. Virus replication was newly verified in the spleen and mesenteric lymph nodes by detection of negative-sense viral RNA. In conclusion, HuNoV GII.4 could induce acute gastroenteritis and replicate in the extraintestinal lymphoid tissues in post-weaning gnotobiotic pigs. Therefore, such pigs would be a suitable animal model for studying the pathogenesis and replication of HuNoV.

Effects of porcine epidemic diarrhea virus infection on nursery pig intestinal function and barrier integrity.

The pig intestinal epithelium can be compromised by pathogens leading to reduced integrity and function. Porcine epidemic diarrhea virus (PEDV), recently detected in North America, exemplifies intestinal epithelial insult. Although several studies have investigated the molecular aspects and host immune response to PEDV, there are little data on the impact of PEDV on pig intestinal physiology. The objective of this study was to investigate the longitudinal impact of PEDV on nursery pig intestinal function and integrity. Fifty recently-weaned, 5-week-old barrows and gilts (BW=9.92±0.49kg) were sorted based on body weight (BW) and sex into two treatments: 1) Control or 2) PEDV inoculated. At 2, 5, 7, and 14days post inoculation (dpi), 4 pigs per treatment were euthanized and jejunum sections collected. PEDV antigen was detected in inoculated pigs by immunohistochemistry in 50% (2/4) at dpi 2, 100% (4/4) at dpi 5, and none at later time points. PEDV-infected pigs had reduced (P<0.05) villus height and decreased transepithelial resistance compared with controls. Total acidic mucins, particularly sialomucin, were reduced in PEDV pigs at dpi 2 and then increased compared with controls at dpi 7 and 14. In addition, PEDV pigs had increased stem cell proliferation (P<0.05) and a numerical increase in DNA fragmentation compared with controls through dpi 7 which coincided with an observed return of digestive function to that of controls. Collectively, these data reveal that PEDV infection results in time-dependent changes not only in intestinal morphology but also barrier integrity and function.

Gastrointestinal microbial population of turkey (Meleagris gallopavo) affected by hemorrhagic enteritis virus.

Hemorrhagic enteritis (HE) is an acute viral disease that affects avian species, particularly turkeys, compromising their commercial production and having a negative effect on animal welfare. Turkey adenovirus 3 (TAdV-3), is the main causal agent of the disease. In this study, we considered 3 groups of turkeys to achieve 2 purposes: 1) A preliminary investigation on the microbiota content in the 4 parts of healthy turkey's intestine (group A), namely duodenum, jejunum, ileum, and ceca was done; 2) an investigation on the relationship between natural infections with TAdV-3 and the intestinal microbiota in the jejunum, where HE mostly develops, comparing group A with animals with molecular positivity for the virus and with clinical signs of HE (group B) and animals with molecular positivity for the virus but without clinical signs (group C). Massive sequencing of the hypervariable V1-V2 regions of 16S rRNA gene and QIIME 1.9.1 software analysis was performed, and operation taxonomic units (OTUs) were classified into 4 abundant phyla: Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. The microbial population of small intestine was distributed almost homogeneously in the healthy turkeys, and Firmicutes was the prevalent phylum (79.85% in duodenum, 89.57% in jejunum and 99.28% in ileum). As compared with small intestine, ceca microbial community was much more heterogeneous: Firmicutes (48.03%), Bacteroidetes (33.60%) and Proteobacteria (12.32%). In the natural infections of HEV, the main bacterial families were Bacteroidaceae (Bacteroidetes) and Peptostreptococcaceae (Firmicutes), uniquely detected in group B and C. Also Clostridiaceae (Firmicutes) was detected, uniquely in group B.

Dynamic Modulation of Expression of Lentiviral Restriction Factors in Primary CD4+ T Cells following Simian Immunodeficiency Virus Infection.

Although multiple restriction factors have been shown to inhibit HIV/SIV replication, little is known about their expression in vivo Expression of 45 confirmed and putative HIV/SIV restriction factors was analyzed in CD4+ T cells from peripheral blood and the jejunum in rhesus macaques, revealing distinct expression patterns in naive and memory subsets. In both peripheral blood and the jejunum, memory CD4+ T cells expressed higher levels of multiple restriction factors compared to naive cells. However, relative to their expression in peripheral blood CD4+ T cells, jejunal CCR5+ CD4+ T cells exhibited significantly lower expression of multiple restriction factors, including APOBEC3G, MX2, and TRIM25, which may contribute to the exquisite susceptibility of these cells to SIV infection. In vitro stimulation with anti-CD3/CD28 antibodies or type I interferon resulted in upregulation of distinct subsets of multiple restriction factors. After infection of rhesus macaques with SIVmac239, the expression of most confirmed and putative restriction factors substantially increased in all CD4+ T cell memory subsets at the peak of acute infection. Jejunal CCR5+ CD4+ T cells exhibited the highest levels of SIV RNA, corresponding to the lower restriction factor expression in this subset relative to peripheral blood prior to infection. These results illustrate the dynamic modulation of confirmed and putative restriction factor expression by memory differentiation, stimulation, tissue microenvironment and SIV infection and suggest that differential expression of restriction factors may play a key role in modulating the susceptibility of different populations of CD4+ T cells to lentiviral infection.IMPORTANCE Restriction factors are genes that have evolved to provide intrinsic defense against viruses. HIV and simian immunodeficiency virus (SIV) target CD4+ T cells. The baseline level of expression in vivo and degree to which expression of restriction factors is modulated by conditions such as CD4+ T cell differentiation, stimulation, tissue location, or SIV infection are currently poorly understood. We measured the expression of 45 confirmed and putative restriction factors in primary CD4+ T cells from rhesus macaques under various conditions, finding dynamic changes in each state. Most dramatically, in acute SIV infection, the expression of almost all target genes analyzed increased. These are the first measurements of many of these confirmed and putative restriction factors in primary cells or during the early events after SIV infection and suggest that the level of expression of restriction factors may contribute to the differential susceptibility of CD4+ T cells to SIV infection.

Comparative Proteome Analysis of Porcine Jejunum Tissues in Response to a Virulent Strain of Porcine Epidemic Diarrhea Virus and Its Attenuated Strain.

Porcine epidemic diarrhea virus (PEDV), a predominant cause of acute enteric infection, leads to severe dehydrating diarrhea and mortality in piglets all over the world. A virulent PEDV YN13 strain, isolated in our laboratory, was attenuated to yield an attenuated PEDV strain YN144. To better understand the pathogenesis mechanism and the virus-host interaction during infection with both PEDV YN13 and YN144 strains, a comparative proteomic analysis was carried out to investigate the proteomic changes produced in the primary target organ, using isobaric tags for relative and absolute quantitation (iTRAQ) labeling, followed by liquid chromatography tandem-mass spectrometry (LC-MS/MS). A total of 269 and 301 differently expressed proteins (DEPs) were identified in the jejunum tissues of the piglets inoculated with YN13 and YN144, respectively. Bioinformatics analysis revealed that these proteins were involved in stress responses, signal transduction, and the immune system. All of these involved interferon-stimulated genes (ISGs) which were up-regulated in jejunums by both of the PEDV-infected groups. Based on the comparative analysis, we proposed that different changes induced by YN13 and YN144 in heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), eukaryotic initiation factor 4G1 (eIF4G1), and some members in the heat shock protein (HSP) family, may be responsible for differences in their pathogenicity.

Alteration of Polymeric Immunoglobulin Receptor and Neonatal Fc Receptor Expression in the Gut Mucosa of Immunodeficiency Virus-Infected Rhesus Macaques.

Polymeric immunoglobulin receptors (pIgR) and neonatal Fc receptors (FcRn) are crucial immunoglobulin (Ig) receptors for the transcytosis of immunoglobulins, that is IgA, IgM and IgG, the levels of which in mucosal secretions were altered in both HIV- and SIV-infected individuals. To gain an insight into the changes of pIgR and FcRn expression after immunodeficiency virus (SHIV/SIV) infection, real-time RT-PCR methods were established and the mRNA levels of pIgR and FcRn in normal and SHIV/SIV-infected rhesus macaques were quantitatively examined. It was found that the levels of pIgR mRNA were within a range of 10(7) copies per million copies of GAPDH mRNA in the gut mucosa of rhesus macaques, which were up to 55 times higher than that in the oral mucosa, the highest among the non-gut tissues examined. Levels of FcRn mRNA were generally lower than that of pIgR, and the levels of FcRn mRNA in the gut mucosa were also lower than that in most non-gut tissues examined. Notably, the levels of pIgR mRNA in the duodenal mucosa were positively correlated with that of IL-17A in normal rhesus macaques. Both pIgR and FcRn mRNA levels were significantly reduced in the duodenal mucosa during acute SHIV infection and in the jejunum and caecum during chronic SHIV/SIV infection. These data expanded our knowledge on the expression of pIgR and FcRn in the gastrointestinal tract of rhesus macaques and demonstrated altered expression of pIgR and FcRn in SHIV/SIV, and by extension HIV infections, which might have contributed to HIV/AIDS pathogenesis.