Effects of Hericium erinaceus polysaccharide on immunity and apoptosis of the main immune organs in Muscovy duck reovirus-infected ducklings.

To investigate the effects of Hericium erinaceus polysaccharide (HEP) on immunity in Muscovy duck reovirus (MDRV)-infected ducklings and explore its mechanism of action, an MDRV contact-infection model was established. Then, we investigated the influence of HEP on morphology of main immune organs in MDRV-infected ducklings by HE staining, while antioxidant capacity (T-AOC, MDA), serum protein levels (TP, ALB, GLO), complement levels (C3, C4) and antibody levels (IgA, IgM, IgG) were detected. Apoptotic indexes (apoptosisi rate and FAS-L) were also quantified by TUNEL method and immunohistochemical staining. Meanwhile, FADD and CytC (apoptosis-related genes), were tested by quantitative RT-PCR. Results showed that HEP could reduce the injuries of immune organs caused by MDRV. Additionally, HEP markedly diminished MDA (p < 0.01), while significantly increased T-AOC, TP, ALB, GLO, C3, C4, IgA, IgM and IgG (p < 0.01 or p < 0.05). Then, HEP shifted apoptosis time to an early MDRV-infected stage and reduced apoptosis at later MDRV-infected stage. This was associated with changes of FADD and CytC. Collectively, our data suggested that HEP could reduce the immunesuppression by many ways, such as decreasing organs' injuries, improving antioxidant capacity, serum proteins levels, antibody levels and complement levels, while diminish the apoptosis by lowering the FADD and CytC.

Magnolol protects Ctenopharyngodon idella kidney cells from apoptosis induced by grass carp reovirus.

Many natural products from medicinal plants are small molecular weight compounds with enormous structural diversity and show various biological activities. Magnolol is a biphenol compound rich in the stem bark of Magnolia officinalis Rehd et Wils., and is able to suppress viral replication in GCRV-infected grass carp (Ctenopharyngodon idella) kidney (CIK) cells in the previous study. In this study, in vivo studies demonstrated that magnolol was efficient to restrain the replication of GCRV and repair the low level of superoxide dismutase and total antioxidant capacity in serum at the non-toxic concentration in vivo. Furthermore, magnolol inhibited CIK cell apoptosis induced by GCRV and kept the normal cellular morphological structure, reflecting in the protection of CIK cells from cell swelling, the formation of apoptotic bodies, the disappearance of cellular morphology and nuclear fragmentation. Reverse transcript quantitative polymerase chain reaction (RT-qPCR) showed that magnolol facilitated the expression of apoptosis-inhibiting gene bcl-2, while suppressed the expression of apoptosis-promoting gene bax in GCRV-infected cells. Besides, RT-qPCR and enzyme activity assays proved that magnolol suppressed the expression of caspase 3, caspase 8 and caspase 9. Moreover, interactions between magnolol and proteins were predicted by using the STITCH program, which revealed that ten proteins including caspase 3, were involved in the apoptosis pathway, p53 signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway and toll-like receptor signaling pathway. Further assays were performed to test the effect of magnolol on apoptosis pathway, which showed that magnolol dramatically inhibited the activity of caspase 3 rather than those of caspase 8 and caspase 9. Collectively, the present study revealed that magnolol heightened the resistance of grass carp against GCRV infection and refrained GCRV-induced apoptosis, which may be attributed to the direct interaction of magnolol with caspase 3. The present results make a contribution to understanding the mechanisms by which small-molecule drugs possess antiviral activities, and lay a foundation for the development of broad-spectrum antiviral compounds in aquaculture industry.

Magnolol and honokiol from Magnolia officinalis enhanced antiviral immune responses against grass carp reovirus in Ctenopharyngodon idella kidney cells.

Medicinal plants have been widely used for a long history. Exploration of pharmacologically active compounds from medicinal plants present a broad prevalent of application. By examining viral mRNA expression in GCRV-infected Ctenopharyngodon idella kidney (CIK) cells treated with thirty kinds of plant extracts, we identified Magnolia officinalis Rehd et Wils. was able to preferably suppress viral replication. Further studies demonstrated that the main ingredients of magnolia bark, namely, magnolol and honokiol presented protective pharmacological function when treated GCRV-infected CIK cells with a concentration of 2.00 µg/ml and 1.25 µg/ml, respectively. Furthermore, reverse transcript quantitative polymerase chain reaction (RT-qPCR) and western blot showed that both magnolol and honokiol were efficient to restrain the replication of GCRV in CIK cells at non-toxic concentration (2.51 ± 0.51 µg/ml for magnolol, and 3.18 ± 0.61 µg/ml for honokiol). Moreover, it was found that magnolol and honokiol promoted the expression of immune-related genes. Magnolol obviously significantly increased the expression of interferon (IFN) regulatory factor (IRF)7 rather than that of IRF3 in the GCRV-infected cells, leading to the activation of type I IFN (IFN-I). Simultaneously, magnolol drastically facilitated the expression of interleukin (IL)-1ß, but failed to induce the molecules in nuclear factor (NF)-κB pathways. Differently, honokiol strikingly motivated not only the expression of IL-1ß, but also those of tumor necrosis factor α (TNFα) and NF-κB. Interestingly, though honokiol motivated the expression of IFN-ß promoter stimulator 1 (IPS-1), IRF3 and IRF7, it failed to up-regulate the expression of IFN-I, indicating that honokiol enhanced the host innate antiviral response to GCRV infection via NF-κB pathways. Collectively, the present study revealed that magnolol and honokiol facilitated the expression of innate immune-related genes to strengthen the innate immune signaling responses to resist GCRV infection, which contributed to understanding the mechanisms by which small-molecule drugs possessed antiviral activities. In addition, these results lay a foundation for the development of broad-spectrum antiviral compounds in aquaculture industry.

A specific CpG oligodeoxynucleotide induces protective antiviral responses against grass carp reovirus in grass carp Ctenopharyngodon idella.

CpG oligodeoxynucleotides (ODNs) show strong immune stimulatory activity in vertebrate, however, they possess specific sequence feature among species. In this study, we screened out an optimal CpG ODN sequence for grass carp (Ctenopharyngodon idella), 1670A 5'-TCGAACGTTTTAACGTTTTAACGTT-3', from six published sequences and three sequences designed by authors based on grass carp head kidney mononuclear cells and CIK (C. idella kidney) cells proliferation. VP4 mRNA expression was strongly inhibited by CpG ODN 1670A in CIK cells with GCRV infection, showing its strong antiviral activity. The mechanism via toll-like receptor 9 (TLR9)-mediated signaling pathway was measured by real-time quantitative RT-PCR, and TLR21 did not play a role in the immune response to CpG ODN. The late up-regulation of CiRIG-I mRNA expression indicated that RIG-I-like receptors (RLRs) signaling pathway participated in the immune response to CpG ODN which is the first report on the interaction between CpG and RLRs. We also found that the efficient CpG ODN can activates interferon system. Infected with GCRV, type I interferon expression was reduced and type II interferon was induced by the efficient CpG ODN in CIK cells, especially IFNγ2, suggesting that IFNγ2 played an important role in response to the efficient CpG ODN. These results provide a theoretical basis and new development trend for further research on CpG and the application of CpG vaccine adjuvant in grass carp disease control.

Effect of functional feeds on fatty acid and eicosanoid metabolism in liver and head kidney of Atlantic salmon (Salmo salar L.) with experimentally induced heart and skeletal muscle inflammation.

Heart and Skeletal Muscle Inflammation (HSMI) is an emerging viral disease caused by a novel Atlantic salmon reovirus (ASRV) affecting farmed fish. Primary symptoms associated with HSMI include myocardial and skeletal muscle necrosis indicating a severe inflammatory process. Recently, we applied the concept of clinical nutrition to moderate the long-term inflammatory process associated with HSMI in salmon subjected to experimental ASRV challenge. The use of functional feeds with lower lipid (hence energy) content reduced the inflammatory response to ASRV infection and the severity of associated heart lesions. The aim of the present study was to elucidate possible mechanisms underpinning the observed effects of the functional feeds, focussing on eicosanoid and fatty acid metabolism in liver and head kidney. Here we show that liver was also a site for histopathological lesions in HSMI showing steatosis reflecting impaired lipid metabolism. This study is also the first to evaluate the expression of a suite of key genes involved in pathways relating diet and membrane phospholipid fatty acid compositions, and the inflammatory response after ASRV infection. The expression of hepatic Δ6 and Δ5 desaturases was higher in fish fed the functional feeds, potentially increasing their capacity for endogenous production and availability of anti-inflammatory EPA. Effects on mobilization of lipids and changes in the LC-PUFA composition of membrane phospholipids, along with significant changes in the expression of the genes related to eicosanoid pathways, showed the important role of the head kidney in inflammatory diseases caused by viral infections. The results from the present study suggest that clinical nutrition through functional feeding could be an effective complementary therapy for emerging salmon viral diseases associated with long-term inflammation.

Molecular cloning and immune responsive expression of a ribonuclease III orthologue involved in RNA interference, dicer, in grass carp Ctenopharyngodon idella.

In this study, the dicer gene (designated as cidicer) was identified and characterized from grass carp Ctenopharyngodon idella. The complementary DNA (cDNA) of cidicer contained an open reading frame (ORF) of 5646 nucleotides (nts) encoding a putative protein of 1881 amino acids (aa). The deduced Dicer protein contained all known functional domains identified in other organisms. Tissue tropism analysis indicated that cidicer is abundantly expressed in brain, gill, head kidney, liver, spleen, heart, muscle and intestine. In the C. idella kidney (CIK) cells, messenger RNA (mRNA) expression of cidicer was significantly up-regulated at 24 h (6·36-fold, P < 0·01) after grass carp reovirus (GCRV) infection, and its transcriptional expression level was also transiently induced to a high level (6·54-fold, P < 0·01) at 2 h post-stimulation of synthetic double-stranded polyinosinic-polycytidylic potassium salt [poly(I:C)]. In vivo analysis further showed that the expression of cidicer mRNA in the liver was induced to a significantly high level at 12 h (8·46-fold, P < 0·01), and then dropped to normal level at 72 h post-challenge with GCRV. The transcriptional expression pattern of cidicer in the spleen tissue was similar to that of liver tissue upon GCRV challenge. These results collectively implied that the identified cidicer was an inducible gene responding to viral infection both in vitro and in vivo, and the data would shed light on the interaction between RNA interference (RNAi) antiviral pathway and aquareovirus infection.

Identification of a retinoic acid-inducible gene I from grass carp (Ctenopharyngodon idella) and expression analysis in vivo and in vitro.

RIG-I (retinoic acid inducible gene-I) is a key mediator of antiviral immunity, able to couple detection of infection by RNA and DNA viruses to the induction of interferons. In the present study, a RIG-I gene from grass carp Ctenopharyngodon idella (CiRIG-I) was isolated and characterized. The full-length cDNA of CiRIG-I was of 3198 bp and encoded a polypeptide of 947 amino acids with an estimated molecular mass of 108,730 Da and a predicted isoelectric point of 5.85, including six main overlapping structural domains: two CARDs (caspase activation and recruitment domain), one ResIII (conserved restriction domain of bacterial type III restriction enzyme), one DEXDc (DEAD/DEAH box helicase domain), one HELICc (helicase superfamily c-terminal domain) and one RD (regulatory domain). The CiRIG-I mRNA was widespread expression in the tested 15 tissues by semi-quantitative RT-PCR (sqRT-PCR) assay. The CiRIG-I expressions in spleen and liver were significantly induced following grass carp reovirus (GCRV) infection. CiRIG-I mRNA expression was rapidly and significantly up-regulated in vitro after GCRV infection, and the CiRIG-I transcripts were also significantly enhanced in vitro post the synthetic double stranded RNA polyinosinic-polycytidylic potassium salt (poly(I:C)) stimulation. These results collectively suggested that CiRIG-I was an inducible protein, involved in the antiviral innate immune defense to GCRV in grass carp, and laid the foundation for the further mechanism research of RIG-I in fishes.

Identification and expression profiling analysis of grass carp Ctenopharyngodon idella LGP2 cDNA.

LGP2 (laboratory of genetics and physiology 2), a homologue of RIG-I (Retinoic acid inducible gene-I) and MDA5 (Melanoma differentiation associated gene 5) without the CARD (caspase activation and recruitment domain) required for signaling, plays a pivotal role in modulating signaling by RIG-I and MDA5 for interferon (IFN) synthesis. In this study, a novel LGP2 gene from grass carp Ctenopharyngodon idella (designated as CiLGP2) was isolated and characterized. The full-length cDNA of CiLGP2 was of 2920 bp with five instability motifs (ATTTA). The open reading frame was of 2043 bp and encoded a polypeptide of 680 amino acids, including five main overlapping structural domains: two DEXDc (DEAD/DEAH box helicase domain), one ResIII (conserved restriction domain of bacterial type III restriction enzyme), one HELICc (helicase superfamily c-terminal domain) and one RD (regulatory domain). There was one more alpha-helix in the RD, compared with that in human. The CiLGP2 mRNA was ubiquitous expression in the tested tissues, was high level in spleen, skin, heart and intestine tissues, and was up-regulated by grass carp reovirus (GCRV) injection by semi-quantitative RT-PCR (sqRT-PCR) assay. The CiLGP2 expression in spleen was significantly up-regulated at 12 h (14.5 folds, P < 0.05), reached the crest at 24 h (19.0 folds, P < 0.05), and then dropped a little at 48 h (10.4 folds) post-injection of GCRV and kept this level in the following test period (P < 0.05). In liver, the temporal expression of CiLGP2 mRNA was significantly increased at 24 h (3.8 times, P < 0.05), reached peak at 48 h (10.7 times, P < 0.05), and then decreased a little bit at 72 h (5.8 times, P < 0.05) and kept this high level by the end of the test (P < 0.05). These results collectively suggested that CiLGP2 was a novel member of RLR gene family, engaging in the early stage of antiviral innate immune defense in grass carp, and laid the foundation for the further mechanism research of LGP2 in fishes.

Enteric reovirus infection stimulates peanut-specific IgG2a responses in a mouse food allergy model.

IgE-mediated food allergies are an important cause of life-threatening hypersensitivity reactions. Orally administered peanut antigens mixed with the mucosal adjuvant cholera toxin (CT) induce a strong peanut extract (PE)-specific serum IgE response that is correlated with T-helper type 1 (Th1) and type 2 (Th2)-like T-cell responses. This study was conducted to determine if respiratory enteric orphan virus (reovirus), a non-pathogenic virus that induces robust Th1-mediated mucosal and systemic responses could modulate induction of PE-specific allergic responses when co-administered with PE. Young mice were orally exposed to PE mixed with CT, reovirus, or both CT and reovirus. As expected, CT promoted PE-specific serum IgE, IgG1, and IgG2a and intestinal IgA production as well as splenic Th1- and Th2-associated cytokine recall responses. Reovirus did not alter PE-specific serum IgE and IgG1 levels, but substantially increased the PE-specific IgG2a response when co-administered with PE with or without CT. Additionally, reovirus significantly decreased the percentage of the Peyer's patch CD8+ T-cells and Foxp3+CD4+ T-regulatory cells when co-administered with PE. These results demonstrate that an acute mucosal reovirus infection and subsequent Th1 immune response is capable of modulating the Th1/Th2 controlled humoral response to PE. The reovirus-mediated increase in the PE-specific IgG2a antibody response may have therapeutic implications as increased levels of non-allergenic PE-specific IgG2a could block PE antigens from binding to IgE-sensitized mast cells.

Docosahexaenoic acid-enriched fish oil consumption modulates immunoglobulin responses to and clearance of enteric reovirus infection in mice.

We hypothesized that consumption of the (n-3) PUFA, docosahexaenoic acid (DHA), modulates the mucosal immune response to enteric infection with respiratory enteric orphan virus (reovirus), a model intestinal pathogen. Mice were fed either AIN-93G control diet, containing 10 g/kg corn oil and 60 g/kg high oleic acid safflower oil, or AIN-93G, containing 10 g/kg corn oil and 60 g/kg DHA-enriched fish oil, for 4 wk and then orally gavaged with reovirus strain Type 1 Lang, (T1/L). Reovirus-specific IgA antibody was first detectable in the feces of mice fed a control diet at 6 d postinfection (PI) and was further elevated at 8 and 10 d PI. IgA responses in DHA-fed mice were similar at 6 and 8 d PI but greater at 10 d PI (P < 0.05). Both reovirus-specific serum IgA and IgG(2a) were comparably induced in mice fed control or DHA diets. Reovirus-specific IgA and IgG(2a) secretion by ex vivo Peyer's patch, lamina propria, and spleen cultures derived from control and DHA groups were comparable. Although both groups carried similar numbers of reovirus plaque forming units per intestine, DHA-fed mice shed nearly 10 times more viral RNA in feces than control mice at 2, 4, and 6 d PI (P < 0.05). However, viral RNA was not detectable in either group at 8 and 10 d. Taken together, these data suggest that DHA consumption did not markedly alter mucosal or systemic Ig responses to reovirus but delayed clearance of the virus from the intestinal tract.

Diarrhoea in dairy calves reduced by feeding colostrum from cows vaccinated with rotavirus.

Forty-two dairy calves remained with their dams for two days after birth, and then were removed to a calf rearing shed. Calves were allocated to three groups for the next 14 days, and received twice daily either whole milk, whole milk with a 10 per cent supplement of pooled normal bovine colostrum or whole milk with 10 per cent supplement of colostrum from cows vaccinated with rotavirus. A natural outbreak of diarrhoea occurred, affecting 28 of the 42 calves. Feeding immune colostrum delayed the onset of diarrhoea, and reduced its incidence, duration and severity. Live weight gains were consequently improved. The group fed normal colostrum had diarrhoea intermediate in severity between that of control calves and those fed immune colostrum. The aetiology of the diarrhoea was complex, with calves excreting rotavirus, enteropathogenic Escherichia coli and cryptosporidia.

[Persistence of transplacental antibodies against rotavirus in children less than 6 months of age]. / Persistencia de anticuerpos transplacentarios contra rotavirus en niños menores de seis meses de edad.

The persistence of antibodies against rotavirus was studied in the sera of 54 recently born infants up to six months of age; likewise, in the sera of their mothers. Serum positivities were found to be similar in both, showing 96% for those with antibodies and 94.4% for the latter. The percentage of infants with antibodies dropped gradually to the 4th month of age and since then rises in the titre of antibodies began to appear in four infants, which indicated there had been rotavirus infections. These studies lead to believe that in spite of the presence of serum antibodies, rotavirus multiply in the enteric tract and do not bring about serum antibodies, but perhaps, they stimulate the production of coproantibodies, which do not allow in the future the implantation of these viruses in the intestine.

Distribution of antibodies to rotavirus in serum and lacteal secretions of naturally infected swine and their suckling pigs.

Rotavirus antibodies were demonstrated in lacteal secretions and sera of 20 parturient sows and in sera of their newborn by an enzyme-linked immunosorbent assay blocking technique, using bovine rotavirus cell culture antigen and monospecific antibody to bovine rotavirus. Antibodies to rotavirus occur in the 3 immunoglobulin (Ig) classes IgM, IgA, and IgG in lacteal secretions. High and long-persisting antibody activity was mainly associated with the IgA class. The IgM and IgG decreased to undetectable concentrations in most sows during the 14-day investigation period. Serum antibodies of newborn pigs nursing their dams also decreased rapidly during this time. The heterologous enzyme-linked immunosorbent assay blocking technique was a reliable and rapid procedure for the demonstration of rotavirus antibodies.

Development of immunity to porcine rotavirus in piglets protected from disease by bovine colostrum.

Bovine colostrum with rotavirus-neutralizing activity was fed for 10 days to two groups of piglets, one of which was inoculated intranasally with a rotavirus of porcine origin. A third group, which did not receive colostrum, was also inoculated with the virus, and these piglets developed diarrhea, excreted rotavirus in the feces, and died 6 days after infection. In contrast, the infected piglets fed with bovine colostrum remained healthy, although they developed antibody to rotavirus. Twenty-seven days after the primary inoculation, piglets in the colostrum-fed groups were inoculated intranasally with virus. Those in the previously unexposed group became clinically ill and excreted rotavirus, whereas those which had experienced a previous subclinical infection (the colostrum-fed, virus-inoculated group) remained healthy. It was concluded that bovine colostrum protected piglets from the clinical effects of a porcine rotavirus and that these animals developed an immunity which prevented subsequent disease.

Effects of antibodies, trypsin, and trypsin inhibitors on susceptibility of neonates to rotavirus infection.

Levels of antirotaviral secretory immunoglobulin A were measured by enzyme-linked immunosorbent assay in colostrum and milk samples collected daily for the first 5 days postpartum from 49 mothers breast-feeding their infants. The trypsin-inhibitory capacity of these lacteal secretion samples was assessed by their ability to inhibit the hydrolysis of alpha-N-benzoyl-DL-arginine-p-nitroanilide by trypsin. Stools passed by these breast-fed infants and by an additional 43 bottle-fed infants were pooled by individual and examined by electron microscopy for rotavirus. Stool trypsin levels were estimated with the gelatin hydrolysis test. Breast-fed infants were significantly less likely to become infected with rotavirus and showed significantly lower stool tryptic activity than did bottle-fed infants. Breast-fed infants who did not excrete rotavirus over the 5-day period received milk of significantly higher antirotaviral secretory immunoglobulin A or trypsin-inhibitory capacity or both than breast-fed infants who were infected with rotavirus. A case of probable maternal rotavirus infection during pregnancy, producing greatly elevated lacteal antirotaviral secretory immunoglobulin A levels lasting for 2 years, was detected. Results of this study suggest that both antibodies and trypsin inhibitors in human milk can be associated with the protection of neonates against rotavirus infection in the first 5 days of life.

Probable role of viruses in calfhood diseases.

Bovine enteroviruses, bovine viral diarrhea virus, rotavirus (formerly called reovirus-like agent), coronavirus-like agent, bovine adenovirus, and bovine parainfluenza-3 virus have been isolated from calves suffering from neonatal disease. The experimental disease produced by these viruses is not necessarily severe or fatal, but under farm and ranch conditions, each probably serves as an added to mortality from neonatal disease. After initial losses following the introduction of a virus into a herd, the subsequent losses will be limited because the cow will produce antibodies to protect the fetus during gestation. Antibodies will also be concentrated in colostrum to protect the calf at birth. However, colostrum must be fed immediately after birth, before the calf becomes infected.

Studies on rotavirus infection and diarrhoea in young calves.

The occurrence of diarrhoea in calves was monitored during the first three weeks of life. Calves fed amounts of colostrum sufficient to produce serum Ig levels in excess of 30 mg per ml did not develop diarrhoea, whereas calves fed less colostrum did. Rotaviruses and mycoplasma-like particles were observed in the faeces of calves with and without diarrhoea. The epidemiology of rotavirus infection in calves is discussed.