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Grass carp reovirus II (GCRV II) causes severe hemorrhagic disease with high mortality in grass carp, Cyenopharyngodon idellus. DNA vaccination has been proven to be a very effective method in conferring protection against fish viruses. However, DNA vaccines for GCRV II have not yet been conducted on grass carp. In the current work, we vaccinated grass carp with a DNA vaccine consisting of the segment 6 (pC-S6; encoding VP4) or 10 (pC-S10; encoding NS38) of GCRV II and comparatively analyzed the immune responses induced by these two vaccines. The protective efficacy of pC-S6 and pC-S10, in terms of relative percentage survival (RPS), was 59.9% and 23.1% respectively. This suggests that pC-S6 and pC-S10 DNA vaccines could increase the survival rate of grass carp against GCRV, albeit with variations in immunoprotective effect. Immunological analyses indicated the following. First, post-vaccination (pv), both pC-S6 and pC-S10 up-regulated the expression of interferon (IFN-1), Mx1, IL-1β, and TNF-α. However, CD4 and CD8α were up-regulated in the case of pC-S6 but not pC-S10. Second, comparing non-vaccinated and pC-S10-vaccinated fish, the T cell response related genes, such as CD4, CD8α, and GATA3, were elevated in pC-S6-vaccinated fish at 48 h post-challenge (pc). Third, pC-S6 and pC-S10 induced similar patterns of specific antibody response pv. However, only anti-VP4 IgM in the sera of surviving fish infected with GCRV was significantly increased pc compared with that pre-challenge. Taken together, these results indicate that pC-S6 promotes both innate (IFN-1 and Mx1 induction) and adaptive (T cell and specific antibody response) immunity pv and that the induction of a memory state promptly primes the immune response upon later encounters with the virus, whereas pC-S10 only induces the type I IFN-related response pv and a lower inflammatory response pc., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The grass carp (Ctenopharyngodon idella) hemorrhagic disease, caused by grass carp reovirus (GCRV), is one of the most severe infectious diseases in aquaculture. Given that antiviral therapies are currently limitedly available, vaccination remains the most effective means for the prevention of viral diseases, such as GCRV. A reovirus strain, which was temporarily named GCRV-HN14, was recently isolated from grass carp in Henan province, China. The S11 gene fragment of GCRV-HN14 was speculated to encode viral structural protein VP35, which has no equivalent gene in other aquareviruses but has antigenic epitopes. In this study, the recombinant plasmid pET-32a-vp35 was constructed to express recombinant VP35 proteins in prokaryotic cells, which was used to create a novel subunit vaccine. The immune protection of recombinant VP35 protein was evaluated by a series of experiments in grass carp. Results showed that the number of white blood cells (WBC) in the peripheral blood increased significantly to 7.92 ± 0.72 × 10 7 /ml 5 days after vaccination (P < 0.05). The number of neutrophils and monocytes in WBC were significantly higher than those of the control 3 days after vaccination (P < 0.05) and maximally got to 12.22 ± 1.28% and 18.70 ± 1.78%, respectively. Owing to the significant increase in the number of lymphocytes (92.37 ± 2.10%; P < 0.01), the percentages of neutrophils and monocytes declined significantly (14 dpi; P < 0.01). Serum antibody levels induced by recombinant VP35 protein significantly increased 7 days post immunization and continued to increase until 5 weeks post vaccination. The mRNA expression levels of type I interferon (designated as IFN1), immunoglobulin M, Toll-like receptor 22 and major histocompatibility complex class I were up-regulated significantly in the head kidneys and spleens of immunized fish (P < 0.01). Grass carp immunized by recombinant VP35 protein showed that the relative percentage of survival was about 60% after it was challenged with GCRV. Overall, the results suggested that recombinant VP35 protein can induce immunity and protect grass carp against GCRV infection. Thus, it can be used as a subunit vaccine., (Copyright © 2018. Published by Elsevier Ltd.)

The barbel chub Squaliobarbus curriculus is an important commercial fish species in China, and has shown significant resistance to grass carp reovirus (GCRV). In this study, the cDNA sequence of interferon regulatory factors 3 (IRF3) from Squaliobarbus curriculus, designated as ScIRF3, was cloned, and its effect against GCRV was investigated. The full-length 1837 base pair (bp) cDNA of ScIRF3 contained a complete open reading frame of 1374 bp and encoded a putative polypeptide of 457 amino acid residues. The ScIRF3 protein contained conserved domains, including an N-terminal DNA-binding domain, a C-terminal IRF association domain, and a serine-rich domain. Phylogenetic analysis showed that ScIRF3 was closely clustered with IRF3s from Carassius auratus and Ctenopharyngodon idellus. Quantitative real-time polymerase chain reaction analysis showed that the expression levels of ScIRF3 in Squaliobarbus curriculus were the highest in the spleen and lowest in the muscle. After GCRV infection, expression levels of both ScIRF3 and type I interferon (IFN) were initially up-regulated and subsequently down-regulated in the spleen and intestine. Correlation analysis showed that the expression level of type I IFN is significantly positively correlated with that of ScIRF3 (Pearson correlation coefficient: 0.883, P: 0.004) in the intestine. The expression level of type I IFN was also significantly up-regulated and the GCRV titer was significantly decreased (P < .05) in GCRV-infected ScIRF3-overexpressing Ctenopharyngodon idellus kidney cells. These results indicate that ScIRF3 may play a role in the type I IFN immune response against GCRV in Squaliobarbus curriculus and can also inhibit GCRV replication in Ctenopharyngodon idellus kidney cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objective: Oncolytic viruses (OVs) represent promising, proinflammatory cancer treatments. Here, we explored whether OV-induced innate immune responses could simultaneously inhibit HCV while suppressing hepatocellular carcinoma (HCC). Furthermore, we extended this exemplar to other models of virus-associated cancer., Design and Results: Clinical grade oncolytic orthoreovirus (Reo) elicited innate immune activation within primary human liver tissue in the absence of cytotoxicity and independently of viral genome replication. As well as achieving therapy in preclinical models of HCC through the activation of innate degranulating immune cells, Reo-induced cytokine responses efficiently suppressed HCV replication both in vitro and in vivo. Furthermore, Reo-induced innate responses were also effective against models of HBV-associated HCC, as well as an alternative endogenous model of Epstein-Barr virus-associated lymphoma. Interestingly, Reo appeared superior to the majority of OVs in its ability to elicit innate inflammatory responses from primary liver tissue., Conclusions: We propose that Reo and other select proinflammatory OV may be used in the treatment of multiple cancers associated with oncogenic virus infections, simultaneously reducing both virus-associated oncogenic drive and tumour burden. In the case of HCV-associated HCC (HCV-HCC), Reo should be considered as an alternative agent to supplement and support current HCV-HCC therapies, particularly in those countries where access to new HCV antiviral treatments may be limited., Competing Interests: Competing interests: MC is an employee of Oncolytics Biotech, Calgary, Canada. SG and AM have received research grants from Oncolytics., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Infection with Grass carp reovirus (GCRV) is becoming unprecedentedly widespread in grass carp (Ctenopharyngodon idella) aquaculture industry, yet the management of GCRV infection still remains a challenge. Therefore, it is of importance to develop effective means against GCRV. As a delivery system of viral antigens, surface displaying of heterologous proteins on bacteria using anchoring motifs has successfully been implemented in human and veterinary vaccines research. In this study, a novel vaccine (BL21/InpN/vp7) was developed based on surface displaying a major capsid protein (vp7) of GCRV using the anchoring motif of N-terminal unique domain of ice-nucleation protein (InpN) on Escherichia coli BL21 (DE3) vaccine. Then the grass carp were immunized by surface displaying BL21/InpN/vp7 vaccine against GCRV using both intraperitoneal injection and bath immunization and their immune responses were tested. The results revealed that some non-specific immune parameters (acid phosphatase (ACP), alkaline phosphatase (AKP) and total antioxidant capacity (T-AOC)) were strongly increased in grass carp post injection inoculation (vp7 dose ranged from 10 to 20 μg). The specific antibody levels against GCRV and the transcriptional of immune-related genes (TNF-α, IL-1β, MHCI and IgM) were also significantly enhanced in grass carp by injection inoculation (vp7 dose ranged from 5 to 20 μg). On the other hand, only the highest dose of bath vaccination significantly induced the production of specific antibody and up-regulated transcriptions of several immune-related genes (IgM and MHCI) in grass carp. The lower cumulative mortality of grass carp in vaccinated groups after GCRV challenge clearly demonstrated that surface displayed vp7 vaccine could protect fish against GCRV infection. The relative percentage survival (RPS) value in injection vaccinated group (88.89%) was much higher compared to bath group (18.89%), which was in consistent with the production of specific serum antibodies, non-specific immune response and immune related genes expression. To sum up, our results indicated the surface display of heterologous antigenic proteins on E. coli BL21 (DE3) using the anchoring motif of ice-nucleation protein may provide a promising approach to the vaccine development of aquatic animals and suggested its potential to be used as vaccine to fight against GCRV infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Human LSm14A is a key component of processing body (P-body) assembly that mediates interferon-β (IFN-β) production by sensing viral RNA or DNA. To the best of our knowledge, we are the first to report duck LSm14A (duLSm14A) cloning from duck embryo fibroblasts (DEFs). Full-length duLSm14A encoded 461 amino acids and was highly homologous with chicken and swan goose sequences. More interestingly, the duLSm14A mRNA was extensively expressed in all the studied tissues. In DEFs, duLSm14A was localized in the cytoplasm as P-body-like dots. Expression of duLSm14A induced IFN-β through the activation of interferon regulatory factor-1 and nuclear factor-κB in DEFs. Furthermore, knockdown of duLSm14A by small interfering RNA notably decreased poly(I:C)- or duck reovirus-induced IFN-β production. The present study results indicate that the duLSm14A is an essential sensor that mediates duck innate immunity against viral infections., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Grass carp reovirus (GCRV) is one of the most pathogenic aquareovirus and can cause lethal hemorrhagic disease in grass carp (Ctenopharyngodon idella). However, management of GCRV infection remains a challenge. Therefore, it is necessary to find effective means for the control of its infection. The uses of bacterial ghost (BG, non-living bacteria) as carriers for DNA delivery have received considerable attentions in veterinary and human vaccines studies. Nevertheless, there is still no report about intramuscular administration of bacterial ghost-based DNA vaccines in fish. In the current study, a novel vaccine based on Escherichia coli DH5α bacterial ghost (DH5α-BG), delivering a major capsid protein gene (vp7) of grass carp reovirus encoded DNA vaccine was developed to enhance the efficacy of a vp7 DNA vaccine against GCRV in grass carp. The grass carp was injected intramuscularly by different treatments -i) naked pcDNA-vp7 (containing plasmid 1, 2.5 and 5 μg, respectively), ii) DH5α-BG/pcDNA-vp7 (containing plasmid 1, 2.5 and 5 μg, respectively) and iii) naked pcDNA, DH5α-BG or phosphate buffered saline. The immune responses and disease resistance of grass carp were assessed in different groups, and results indicated that the antibody levels, serum total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, acid phosphatase (ACP) activity and alkaline phosphatase (AKP) activity and immune-related genes were significantly enhanced in fish immunized with DH5α-BG/pcDNA-vp7 vaccine (DNA dose ranged from 2.5 to 5 μg). In addition, the relative percentage survival were significantly enhanced in fish immunized with DH5α-BG/pcDNA-vp7 vaccine and the relative percentage survival reached to 90% in DH5α-BG/pcDNA-vp7 group than that of naked pcDNA-vp7 (42.22%) at the highest DNA dose (5 μg) after 14 days of post infection. Moreover, the level of pcDNA-vp7 plasmid was higher in DH5α-BG/pcDNA-vp7 groups than naked pcDNA-vp7 groups in muscle and kidneys tissues after 21 days. Overall, those results suggested that DH5α bacterial ghost based DNA vaccine might be used as a promising vaccine for aquatic animals to fight against GCRV infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Future growth in aquaculture relies strongly on the control of diseases and pathogens. Vaccination has been a successful strategy for obtaining control of bacterial diseases in fish, but for viral diseases, vaccine development has been more challenging. Effective long-term protection against viral infections is not yet fully understood for fish, and in addition, optimal tools to monitor adaptive immunity are limited. Assays that can detect specific antibodies produced in response to viral infection in fish are still in their early development. Multiplex bead based assays have many advantages over traditional assays, since they are more sensitive and allow detection of multiple antigen-specific antibodies simultaneously in very small amounts of plasma or serum. In the present study, a bead based assay have been developed for detection of plasma IgM directed against Piscine orthoreovirus (PRV), the virus associated with the disease Heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon. Using recombinant PRV proteins coated on beads, antibodies targeting the structural outer capsid protein μ1 and the non-structural protein μNS were detected. Results from a PRV cohabitation challenge trial indicated that the antibody production was initiated approximately two weeks after the peak phase of PRV infection, coinciding with typical HSMI pathology. Thereafter, the antibody production increased while the epicardial inflammation became less prominent. In conclusion, the novel assay can detect PRV-specific antibodies that may play a role in viral defence. The bead-based immunoassay represents a valuable tool for studies on HSMI and possibly other diseases in aquaculture., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Non-self recognition is a common phenomenon among organisms; it often leads to innate immunity to prevent the invasion of parasites and maintain the genetic polymorphism of organisms. Fungal vegetative incompatibility is a type of non-self recognition which often induces programmed cell death (PCD) and restricts the spread of molecular parasites. It is not clearly known whether virus infection could attenuate non-self recognition among host individuals to facilitate its spread. Here, we report that a hypovirulence-associated mycoreovirus, named Sclerotinia sclerotiorum mycoreovirus 4 (SsMYRV4), could suppress host non-self recognition and facilitate horizontal transmission of heterologous viruses. We found that cell death in intermingled colony regions between SsMYRV4-infected Sclerotinia sclerotiorum strain and other tested vegetatively incompatible strains was markedly reduced and inhibition barrage lines were not clearly observed. Vegetative incompatibility, which involves Heterotrimeric guanine nucleotide-binding proteins (G proteins) signaling pathway, is controlled by specific loci termed het (heterokaryon incompatibility) loci. Reactive oxygen species (ROS) plays a key role in vegetative incompatibility-mediated PCD. The expression of G protein subunit genes, het genes, and ROS-related genes were significantly down-regulated, and cellular production of ROS was suppressed in the presence of SsMYRV4. Furthermore, SsMYRV4-infected strain could easily accept other viruses through hyphal contact and these viruses could be efficiently transmitted from SsMYRV4-infected strain to other vegetatively incompatible individuals. Thus, we concluded that SsMYRV4 is capable of suppressing host non-self recognition and facilitating heterologous viruses transmission among host individuals. These findings may enhance our understanding of virus ecology, and provide a potential strategy to utilize hypovirulence-associated mycoviruses to control fungal diseases.

Necroptosis, a regulated form of necrotic cell death, requires the activation of the RIP3 kinase. Here, we identify that infection of host cells with reovirus can result in necroptosis. We find that necroptosis requires sensing of the genomic RNA within incoming virus particles via cytoplasmic RNA sensors to produce type I interferon (IFN). While these events that occur prior to the de novo synthesis of viral RNA are required for the induction of necroptosis, they are not sufficient. The induction of necroptosis also requires late stages of reovirus infection. Specifically, efficient synthesis of double-stranded RNA (dsRNA) within infected cells is required for necroptosis. These data indicate that viral RNA interfaces with host components at two different stages of infection to induce necroptosis. This work provides new molecular details about events in the viral replication cycle that contribute to the induction of necroptosis following infection with an RNA virus. IMPORTANCE An appreciation of how cell death pathways are regulated following viral infection may reveal strategies to limit tissue destruction and prevent the onset of disease. Cell death following virus infection can occur by apoptosis or a regulated form of necrosis known as necroptosis. Apoptotic cells are typically disposed of without activating the immune system. In contrast, necroptotic cells alert the immune system, resulting in inflammation and tissue damage. While apoptosis following virus infection has been extensively investigated, how necroptosis is unleashed following virus infection is understood for only a small group of viruses. Here, using mammalian reovirus, we highlight the molecular mechanism by which infection with a dsRNA virus results in necroptosis., (Copyright © 2017 American Society for Microbiology.)

The anti-tumour effects associated with oncolytic virus therapy are mediated significantly through immune-mediated mechanisms, which depend both on the type of virus and the route of delivery. Here, we show that intra-tumoral oncolysis by Reovirus induced the priming of a CD8+, Th1-type anti-tumour response. By contrast, systemically delivered Vesicular Stomatitis Virus expressing a cDNA library of melanoma antigens (VSV-ASMEL) promoted a potent anti-tumour CD4+ Th17 response. Therefore, we hypothesised that combining the Reovirus-induced CD8+ T cell response, with the VSV-ASMEL CD4+ Th17 helper response, would produce enhanced anti-tumour activity. Consistent with this, priming with intra-tumoral Reovirus, followed by an intra-venous VSV-ASMEL Th17 boost, significantly improved survival of mice bearing established subcutaneous B16 melanoma tumours. We also show that combination of either therapy alone with anti-PD-1 immune checkpoint blockade augmented both the Th1 response induced by systemically delivered Reovirus in combination with GM-CSF, and also the Th17 response induced by VSV-ASMEL. Significantly, anti-PD-1 also uncovered an anti-tumour Th1 response following VSV-ASMEL treatment that was not seen in the absence of checkpoint blockade. Finally, the combination of all three treatments (priming with systemically delivered Reovirus, followed by double boosting with systemic VSV-ASMEL and anti-PD-1) significantly enhanced survival, with long-term cures, compared to any individual, or double, combination therapies, associated with strong Th1 and Th17 responses to tumour antigens. Our data show that it is possible to generate fully systemic, highly effective anti-tumour immunovirotherapy by combining oncolytic viruses, along with immune checkpoint blockade, to induce complementary mechanisms of anti-tumour immune responses.

A vertically integrated monitoring program was set up for breeders hatched in 2013 and their offspring to detect differences in performance related to the reovirus vaccination schedule. Within the same organization in Belgium, 17 breeder flocks were vaccinated with one dose of live and one dose of inactivated reovirus vaccine, while 14 flocks received two doses of inactivated vaccine without live priming. The hatchability of the eggs produced by these birds was examined. Further, the daily growth, feed conversion, mortality, slaughterhouse condemnation, production index, and antibiotic use were monitored in 110 broiler flocks derived from the breeders. All gathered data were examined statistically. In eggs obtained from breeders vaccinated twice with inactivated reovirus vaccine, a significant 2.88% higher hatchability rate was observed. The progeny broiler flocks of these breeders showed a significant 18.2% lower mortality during the fattening period. Although not statistically significant, the slaughterhouse condemnation rate was 10.1% lower as well. The results may indicate that-under the epidemiologic conditions of this study-double administration of inactivated reovirus vaccine in broiler breeders can at least contribute to higher hatchability of breeder eggs and lower broiler mortality.

Purpose: In addition to their direct cytopathic effects, oncolytic viruses are capable of priming antitumor immune responses. However, strategies to enhance the immunotherapeutic potential of these agents are lacking. Here, we investigated the ability of the multi-tyrosine kinase inhibitor and first-line metastatic renal cell carcinoma (RCC) agent, sunitinib, to augment the antitumor immune response generated by oncolytic reovirus., Experimental Design: In vitro, oncolysis and chemokine production were assessed in a panel of human and murine RCC cell lines after exposure to reovirus, sunitinib, or their combination. In vivo, the RENCA syngeneic murine model of RCC was employed to determine therapeutic and tumor-specific immune responses after treatment with reovirus (intratumoral), sunitinib, or their combination. Parallel investigations employing the KLN205 syngeneic murine model of lung squamous cell carcinoma (NSCLC) were conducted for further validation., Results: Reovirus-mediated oncolysis and chemokine production was observed following RCC infection. Reovirus monotherapy reduced tumor burden and was capable of generating a systemic adaptive antitumor immune response evidenced by increased numbers of tumor-specific CD8 + IFNγ-producing cells. Coadministration of sunitinib with reovirus further reduced tumor burden resulting in improved survival, decreased accumulation of immune suppressor cells, and the establishment of protective immunity upon tumor rechallenge. Similar results were observed for KLN205 tumor-bearing mice, highlighting the potential broad applicability of this approach., Conclusions: The ability to repurpose sunitinib for augmentation of reovirus' immunotherapeutic efficacy positions this novel combination therapy as an attractive strategy ready for clinical testing against a range of histologies, including RCC and NSCLC. Clin Cancer Res; 22(23); 5839-50. ©2016 AACR., (©2016 American Association for Cancer Research.)

Interferons (IFNs) play crucial roles in the immune response of defense against viral infection and bacteria invasion. In the present study, we systematically identified and characterized the IFNs, their regulatory factors (Interferon Regulatory Factors, IRFs) and receptors (Cytokine Receptor Family B, CRFBs) in grass carp (Ctenopharyngodon idella). Grass carp IFNs can be classified into type I IFN (IFN-I) and type II IFN (IFN-II) like other teleosts. IFN-I consist of two groups with two (group I) or four (group II) cysteines in the mature peptide and can be further divided into three subgroups (IFN-a, -c and -d), containing four members: IFN1, IFN2, IFN3, IFN4 in grass carp. IFN-II contain two members, IFNγ2 with the similarity to mammalian IFNγ and a cyprinid specific IFNγ1 (IFNγ-rel) molecule. mRNA expression analyses of IFNs discovered that IFN1 and IFN-II were sustainably expressed in many tissues, while other IFN members were transiently expressed in specific tissues and time points. In the immune response, IFN transcriptions are primarily regulated through multiple IRFs after grass carp reovirus (GCRV) challenge. IRF family possess thirteen members in grass carp, which can be further divided into four subfamilies (IRF-1, -3, -4 and -5 subfamily), each of them plays different roles in the innate and adaptive immunity via various signaling pathways to interact with IFNs (mainly IFN-I). IFNs have to bind receptors (CRFBs) to perform their functions. CRFBs as IFN receptors contain six members in grass carp. The structure and expression characterizations of IFNs, IRFs and CRFBs were analyzed using bioinformatics tools. These results might provide basic data for the further functional research of IFN system, and deeply understand fish immune mechanisms against virus infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Grass carp (Ctenopharyngodon idella) hemorrhagic disease, caused by grass carp reovirus (GCRV), is emerging as a serious problem in grass carp aquaculture. There is no available antiviral therapy and vaccination is the primary method of disease control. In the present study, the immunological effects and protective efficacy of an inactivated HuNan1307 vaccine in grass carp were evaluated. The GCRV isolate HuNan1307 was produced by replication onto the grass carp PSF cell line, and inactivated with 1% β-propiolactone for 60 h at 4 °C. Grass carp were injected with inactivated GCRV vaccine, followed by challenge with the isolate HuNan1307. The results showed that the minimum dosage of the inactivated vaccine was 10(5.5) TCID50/0.2 mL to induce immune protection. All grass carp immunized with the inactivated vaccine produced a high titer of serum antibodies and GCRV-specific neutralizing antibody. Moreover, the inactivated vaccine injection increased the expression of 6 immune-related genes in the spleen and head kidney, which indicated that a immune response was induced by the HuNan1307 vaccine. In addition, grass carp immunized with the inactivated vaccine showed a survival rate above 80% after the viral challenge, equal to that of grass carp immunized with a commercial attenuated vaccine, and the protection lasted at least for one year. The data in this study suggested that the inactivated HuNan1307 vaccine may represent an efficient method to induce immunity against GCRV infection and the induced disease in grass carp., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

MicroRNAs (miRNAs) are a novel class of small, non-coding endogenous RNAs that play critical regulatory roles in many metabolic activities in eukaryotes. Reports of the identification of miRNAs in Sogatella furcifera (white-backed planthopper), the insect that acts as the only confirmed vector of the southern rice black-streaked dwarf virus (SRBSDV), are limited. In this study, a total of 382 miRNAs were identified in S. furcifera, including 106 conserved and 276 novel miRNAs, using high-throughput sequencing based on two small RNA libraries from viruliferous and non-viruliferous S. furcifera, and these miRNAs belonged to 52 conserved miRNA families and 58 S. furcifera-specific families, respectively. Comparison with miRNAs from 26 insect species and five other species in miRBase showed that more than half of the conserved miRNA families are highly conserved in Hexapoda, while other miRNAs are only conserved in non-dipterans. Furthermore, 4 117 target genes predicted for the 382 identified miRNAs could be categorized into 45 functional groups annotated by Gene Ontology. Compared with non-viruliferous cells, eight up-regulated miRNAs and four down-regulated miRNAs were identified in cells inoculated with SRBSDV, among which miR-14 and miR-n98a may be involved in the immune response to SRBSDV infection. Analyses of the identified miRNAs will provide insights into the roles of these miRNAs in the regulation and expression of genes involved in the metabolism, development and viral infection of S. furcifera., (© 2016 The Authors Insect Science published by John Wiley & Sons Australia, Ltd on behalf of Institute of Zoology, Chinese Academy of Sciences.)

Grass carp reovirus strain 109 (GCReV-109) was previously isolated from a grass carp (Ctenopharyngodon idellus) with hemorrhagic disease, and its complete genome has been sequenced. However, the infectivity of GCReV-109 has not been studied, and the viral protein VP33, encoded on genome segment S11, had no detectable sequence homology to other known reovirus proteins. In this study, we characterized GCReV-109 infections in vivo and in vitro, as well as the VP33 protein. Infectivity analysis showed that GCReV-109 caused severe hemorrhagic disease and 100% mortality at dilutions up to 10(-4) in rare minnows (Gobiocypris rarus) by 8 days postinfection, but no visible cytopathic effect was observed in GCReV-109-infected subcultured grass carp muscle (GCM) cells. To confirm that GCReV-109 could be propagated in GCM cells, three virus genome segments were detected by RT-PCR, and large numbers of virus particles were observed by transmission electron microscopy in samples from the infected GCM cells. The suspension of GCReV-109-infected GCM cells was pathogenic to rare minnows. VP33 protein was expressed and purified for generation of an anti-VP33 antiserum. In western blot analysis of purified GCReV-109 particles, the antiserum specifically recognized a protein band (approximately 33 kDa). This revealed that VP33 is a major structural protein of GCReV-109 that might have immunogenic properties. The protective efficacy of the anti-VP33 antiserum against GCReV-109 infection was tested. The death of infected fish was delayed and the mortality fell to 10% when fish were treated with the anti-VP33 antiserum, suggesting that it might be useful for the prevention and control of fish reoviral disease.

The gut microbiota has a crucial role in the growth, development and environmental adaptation in the host insect. The objective of our work was to investigate the microbiota of the healthy silkworm Bombyx mori gut and changes after the infection of B. mori cypovirus (BmCPV). Intestinal contents of the infected and healthy larvae of B. mori of fifth instar were collected at 24, 72 and 144 h post infection with BmCPV. The gut bacteria were analyzed by pyrosequencing of the 16S rRNA gene. 147(135) and 113(103) genera were found in the gut content of the healthy control female (male) larvae and BmCPV-infected female (male) larvae, respectively. In general, the microbial communities in the gut content of healthy larvae were dominated by Enterococcus, Delftia, Pelomonas, Ralstonia and Staphylococcus, however the abundance change of each genus was depended on the developmental stage and gender. Microbial diversity reached minimum at 144 h of fifth instar larvae. The abundance of Enterococcus in the females was substantially lower and the abundance of Delftia, Aurantimonas and Staphylococcus was substantially higher compared to the males. Bacterial diversity in the intestinal contents decreased after post infection with BmCPV, whereas the abundance of both Enterococcus and Staphylococcus which belongs to Gram-positive were increased. Therefore, our findings suggested that observed changes in relative abundance was related to the immune response of silkworm to BmCPV infection. Relevance analysis of plenty of the predominant genera showed the abundance of the Enterococcus genus was in negative correlation with the abundance of the most predominant genera. These results provided insight into the relationship between the gut microbiota and development of the BmCPV-infected silkworm.

Monoclonal antibodies (mAbs) play an important role in detection of aquareoviruses. Three mAbs against grass carp reovirus (GCRV) were prepared. Isotyping revealed that all three mAbs were of subclass IgG2b. Western blot assay showed that all three mAbs reacted with GCRV 69 kDa protein (the putative VP5). In addition to the 69 kDa protein of GCRV, mAb 4B6 also recognize a 54 kDa protein. All three mAbs were used for detecting aquareovirus by Western blot assay and indirect immunofluorescence assay (IFA). All of them reacted with GCRV, and mAb 4A3 could also react with turbot Scophthalmus maximus reovirus (SMReV) and largemouth bass Microptererus salmonides reovirus (MsReV). Viral antigens were only observed in the cytoplasm of infected cells. Finally, syncytia formation was observed with light microscopy and fluorescence microscopy using fluorescein labelled 4A3 mAb at various times post-infection. Syncytia were observed at 36 hr post-infection (hpi) by light microscopy and at 12 hpi by fluorescence microscopy. The immunofluorescence based assay allowed earlier detection of virus than observation of virus-induced cytopathic effect (CPE) assay in inoculated cell cultures. The sensitivity and specificity of these mAbs may be useful for diagnosis and monitoring of aquareoviruses.

VP7, an outer capsid protein of grass carp reovirus (GCRV), was expressed and displayed on the surface of Saccharomyces cerevisiae for developing an efficient vaccine against hemorrhagic disease of grass carp. The result of flow cytometry analysis indicated that protein VP7 could be displayed on the surface of yeast cells after inducing with galactose. The expression of VP7 was confirmed by western blot analysis and further visualized with confocal microscopy. The specific antibodies against VP7 generated from mice were detectable from all immune groups except the control group, which was immunized with untransformed yeast cells. The displaying VP7 on glycosylation-deficient strain EBYΔMnn9 was detected to induce a relatively low level of specific antibody amongst the three strains. However, the antiserum of EBYΔM9-VP7 showed relative high capacity to neutralize GCRV. Further neutralization testing assays indicated that the neutralizing ability of antiserum of the EBYΔM9-VP7 group appeared concentration dependent, and could be up to 66.7% when the antiserum was diluted to 1:50. This result indicates that appropriate gene modification of glycosylation in a yeast strain has essential effect on the immunogenicity of a yeast-based vaccine.

To reduce the lethal hemorrhagic disease caused by grass carp reovirus (GCRV) and improve the production of grass carp, efficient and economic prophylactic measure against GCRV is the most pressing desired for the grass carp farming industry. In this work, a novel SWCNTs-pEGFP-vp5 DNA vaccine linked vp5 recombinant in the form of plasmid pEGFP-vp5 and ammonium-functionalized SWCNTs by a chemical modification method was prepared to enhance the efficacy of a vp5 DNA vaccine against GCRV in juvenile grass carp. After intramuscular injection (1, 2.5 and 5 μg) and bath administration (1, 10, and 20 mg/L), the ability of the different immune treatments to induce transgene expression was analyzed. The results showed that higher levels of transcription and expression of vp5 gene could be detected in muscle tissues of grass carp in SWCNTs-pEGFP-vp5 treatment groups compare with naked pEGFP-vp5 treatment groups. Moreover, antibody levels, immune-related genes, and relative percentage survival were significantly enhanced in fish immunized with SWCNTs-pEGFP-vp5 vaccine. In addition, we found that a good immune protective effect was observed in bath immunization group; which at a concentration of 20 mg/L could reach the similar relative percentage survival (approximately 100%) in injection group at a dose of 5 μg. All these results indicated that ammonium-functionalized SWCNTs could provide extensive application prospect to aquatic vaccine and might be used to vaccinate fish by intramuscular injection or bath administration method., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Unlabelled: Numerous viruses are transmitted in a persistent manner by insect vectors. Persistent viruses establish their initial infection in the midgut epithelium, from where they disseminate to the midgut visceral muscles. Although propagation of viruses in insect vectors can be controlled by the small interfering RNA (siRNA) antiviral pathway, whether the siRNA pathway can control viral dissemination from the midgut epithelium is unknown. Infection by a rice virus (Southern rice black streaked dwarf virus [SRBSDV]) of its incompetent vector (the small brown planthopper [SBPH]) is restricted to the midgut epithelium. Here, we show that the siRNA pathway is triggered by SRBSDV infection in continuously cultured cells derived from the SBPH and in the midgut of the intact insect. Knockdown of the expression of the core component Dicer-2 of the siRNA pathway by RNA interference strongly increased the ability of SRBSDV to propagate in continuously cultured SBPH cells and in the midgut epithelium, allowing viral titers in the midgut epithelium to reach the threshold (1.99 × 10(9) copies of the SRBSDV P10 gene/μg of midgut RNA) needed for viral dissemination into the SBPH midgut muscles. Our results thus represent the first elucidation of the threshold for viral dissemination from the insect midgut epithelium. Silencing of Dicer-2 further facilitated the transmission of SRBSDV into rice plants by SBPHs. Taken together, our results reveal the new finding that the siRNA pathway can control the initial infection of the insect midgut epithelium by a virus, which finally affects the competence of the virus's vector., Importance: Many viral pathogens that cause significant global health and agricultural problems are transmitted via insect vectors. The first bottleneck in viral infection, the midgut epithelium, is a principal determinant of the ability of an insect species to transmit a virus. Southern rice black streaked dwarf virus (SRBSDV) is restricted exclusively to the midgut epithelium of an incompetent vector, the small brown planthopper (SBPH). Here, we show that silencing of the core component Dicer-2 of the small interfering RNA (siRNA) pathway increases viral titers in the midgut epithelium past the threshold (1.99 × 10(9) copies of the SRBSDV P10 gene/μg of midgut RNA) for viral dissemination into the midgut muscles and then into the salivary glands, allowing the SBPH to become a competent vector of SRBSDV. This result is the first evidence that the siRNA antiviral pathway has a direct role in the control of viral dissemination from the midgut epithelium and that it affects the competence of the virus's vector., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

IPS-1, as the sole adaptor of RIG-I and MDA5, plays a central role in innate antiviral immunity. In this study, we investigated potential roles of IPS-1 in innate immunity and the domain-requirement of IPS-1 for its signaling in grass carp (Ctenopharyngodon idella). Overexpression experiment showed that CiIPS-1 mediated IFN-I signal possibly dependent on CiIRF7 but not CiIRF3. Post GCRV challenge, CiIPS-1 could enhance antiviral immune responses. CARD and TM domains were crucial for antiviral function of CiIPS-1, and TRAF motif played an assistant role. PRO domain seemed as a negative regulator but was pivotal for the initiation of CiIFN-I and CiMx1. Post viral/bacterial PAMPs stimulation, CiIPS-1-mediated signaling was tightly controlled. CARD domain of CiIPS-1 could significantly elicit poly I:C/LPS/PGN-mediated signaling. PRO domain negatively regulated CiIRF7 and CiIFN-I but was indispensable for inductions of CiMx1 and CiIL-1β. TRAF motif and TM domain regulated the signaling presumably in a cooperative fashion. Post poly I:C stimulation, TRAF motif negatively regulated CiIRF7, CiIFN-I and CiIL-1β at a relative early time while TM domain functioned at a relative late time. TRAF motif was indispensable for the production of CiMx1, while TM domain slightly negatively regulated the expression. Post LPS and PGN stimulation, TRAF motif excited an assistant and persistent negative role on CiIFN-I, CiIRF7 and CiIL-1β induction, but was crucial for induction of CiMx1. TM domain slightly negatively regulated LPS- and PGN-triggered signaling. Taken together, CiIPS-1 not only exerted important functions in antiviral immune response but also participated in viral/bacterial PAMPs-triggered immune response which was tightly controlled to prevent harmful effects resulting from excessive activation. This study provided novel insights into the pivotal role of IPS-1 in innate immunity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Unlabelled: The lepidopteran innate immune response against RNA viruses remains poorly understood, while in other insects several studies have highlighted an essential role for the exo-RNAi pathway in combating viral infection. Here, by using deep-sequencing technology for viral small-RNA (vsRNA) assessment, we provide evidence that exo-RNAi is operative in the silkworm Bombyx mori against both persistent and pathogenic infection of B. mori cytoplasmic polyhedrosis virus (BmCPV) which is characterized by a segmented double-stranded RNA (dsRNA) genome. Further, we show that Dicer-2 predominantly targets viral dsRNA and produces 20-nucleotide (nt) vsRNAs, whereas an additional pathway is responsive to viral mRNA derived from segment 10. Importantly, vsRNA distributions, which define specific hot and cold spot profiles for each viral segment, to a considerable degree overlap between Dicer-2-related (19 to 21 nt) and Dicer-2-unrelated vsRNAs, suggesting a common origin for these profiles. We found a degenerate motif significantly enriched at the cut sites of vsRNAs of various lengths which link an unknown RNase to the origins of vsRNAs biogenesis and distribution. Accordingly, the indicated RNase activity may be an important early factor for the host's antiviral defense in Lepidoptera., Importance: This work contributes to the elucidation of the lepidopteran antiviral response against infection of segmented double-stranded RNA (dsRNA) virus (CPV; Reoviridae) and highlights the importance of viral small-RNA (vsRNA) analysis for getting insights into host-pathogen interactions. Three vsRNA pathways are implicated in antiviral defense. For dsRNA, two pathways are proposed, either based on Dicer-2 cleavage to generate 20-nucleotide vsRNAs or based on the activity of an uncharacterized endo-RNase that cleaves the viral RNA substrate at a degenerate motif. The analysis also indicates the existence of a degradation pathway that targets the positive strand of segment 10., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

In Vietnam, the two main viruses that cause disease in rice are the Rice grassy stunt virus (RGSV) and the Rice ragged stunt virus (RRSV). Outbreaks of these two viruses have dramatically decreased rice production in Vietnam. Because natural resistance genes are unknown, an RNAi strategy may be an alternative method to develop resistance to RGSV and RRSV. However, this strategy will be efficient only if putative silencing suppressors encoded by the two viruses are neutralized. To identify these suppressors, we used the classical green fluorescent protein (GFP) agroinfiltration method in Nicotiana benthamiana. Then, we investigated the effects of viral candidate proteins on GFP expression and GFP siRNA accumulation and their interference with the short- or long-range signal of silencing. RGSV genes s2gp1, s5gp2, and s6gp1 and RRSV genes s5gp1, s6gp1, s9gp1, and s10gp1 were selected for viral silencing suppressor investigation according to their small molecular weight, the presence of cysteines, or the presence of a GW motif in related protein products. We confirmed that protein p6 of RRSV displays mild silencing suppressor activity and affects long-range silencing by delaying the systemic silencing signal. In addition, we identified two new silencing suppressors that displayed mild activity: p2 of RGSV and p9 of RRSV.

Characterization of the transcriptomic response to infection is an effective approach to understanding the immune mechanisms. Herein we challenged grass carp (Ctenopharyngodon idella) with grass carp reovirus (GCRV) and sequenced four cDNA libraries obtained from head-kidney and spleen by using Illumina Miseq. As a result, we gained a total of 21.52 Gb clean data with 107.96 million reads, and de novo assembled 55,199 unigenes with an average length of 1,470 bp. Comparative transcriptome analysis reveals that 217 unigenes are differentially expressed (fold-change of at least 4) between resistant and susceptible fish in both head-kidney and spleen, and of which 36 unigenes were validated by RT-qPCR experiment. The expression profile of immune-related genes demonstrates that the immune response of spleen is more intense than that of head-kidney. Remarkably, 11,811 unigenes contain multiple transcripts, of which 322 unigenes possess notably differentially expressed transcripts between the four transcriptomic datasets. Furthermore, the splicing transcripts of IL-12p40 and IL-1R1 are firstly found to play diverse roles in the antiviral response of fishes. This study provides a complete transcriptome dataset of C. idella, which is valuable for the studies of immune complexity and, moreover, throws light on the regulatory role of AS in antiviral immunity.

We developed adenovirus serotype 5 (Ad5) vectors displaying the sigma 1 protein from reovirus as mucosal vaccines. Ad5-sigma retargets to JAM-1 and sialic acid, but has 40-fold reduced gene delivery when compared to Ad5. While weaker at transduction, Ad5-sigma generates stronger T cell responses than Ad5 when used for mucosal immunization. In this work, new Ad5-fiber-sigma vectors were generated by varying the number of fiber β-spiral shaft repeats (R) between the fiber tail and sigma. Increasing chimera length led to decreasing insertion of these proteinsAd5 virions. Ad-R3 and R14 vectors effectively targeted JAM-1 in vitro while R20 did not. When wereused to immunize mice by the intranasal route, Ad5-R3-sigma produced higher serum and vaginal antibody responses than Ad5. These data suggest optimized Ad-sigma vectors may be useful vectors for mucosal vaccination., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A suicidal DNA vaccine based on a Semliki Forest virus (SFV) replicon was evaluated for the development of a vaccine against novel duck reovirus (NDRV). The sigma C gene of NDRV was cloned and inserted into pSCA1, an SFV DNA-based replicon vector. After transfected into BHK-21 cells, the antigenicity of the sigma C protein was confirmed using an indirect immunofluorescence and Western blot assay. Immunogenicity was studied in ducklings. The results showed that NDRV-specific antibodies, neutralizing antibodies, IFN-γ and IL-4 were well induced in ducklings. Furthermore, all of the ducklings were protected against challenge with wild-type NDRV., (Copyright © 2015 Elsevier B.V. All rights reserved.)

High-mobility group box (HMGB) proteins, a family of chromatin-associated nuclear proteins, play amazingly multifaceted roles in the immune system of mammals. Thus far, little is known about the nucleocytoplasmic distribution of HMGBs in teleosts. The present study systematically investigated the dynamic localization of all six HMGB proteins in Ctenopharyngodon idella kidney (CIK) cells. Under basal conditions, all HMGBs exclusively localized to the nucleus. Grass carp reovirus (GCRV), polyinosinic-polycytidylic (poly(I∶C)) potassium salt and lipopolysaccharide (LPS) challenge evoked the nuclear export of HMGBs to various degrees: GCRV challenge induced the highest nuclear export of CiHMGB2b, and poly(I∶C) and LPS evoked the highest nucleocytoplasmic shuttling of CiHMGB1b. Overall, the nucleocytoplasmic shuttling of CiHMGB2a and CiHMGB3b was rarely induced by these challenges. Dynamic imaging uncovered that the nucleocytoplasmic GCRV-induced relocation of CiHMGB2b occurred in cells undergoing karyotheca rupture, apoptosis or proliferation. Western blot analyses were used to examine HMGB-EGFP fusion proteins in whole cell lysates, cytosol, nuclear fractions and culture medium. Further investigation demonstrated the nuclear retention of N-terminal HMG-boxes and the nucleocytoplasmic distribution of the C-terminal acidic tails. Comparative analyses of the dynamic relocation of full-length, truncated or chimeric HMGBs confirmed that the intramolecular interaction between HMG-boxes and C-tail domains mediated the nucleocytoplasmic translocation of HMGBs. These results not only provide an overall understanding of the subcellular localization of HMGBs, but also reveal the induction mechanism of the nucleocytoplasmic translocation of HMGBs by GCRV challenge, which lays a foundation for further studies on the interactions among pathogens, HMGBs and pattern recognition receptors in the innate immunity of teleosts.

Human natural killer (NK) cells play an important role in anti-viral immunity. However, studying their activation kinetics during infection is highly problematic. A clinical trial of a therapeutic virus provided an opportunity to study human NK cell activation in vivo in a controlled manner. Ten colorectal cancer patients with liver metastases received between one and five doses of oncolytic reovirus prior to surgical resection of their tumour. NK cell surface expression of the interferon-inducible molecules CD69 and tetherin peaked 24-48 h post-infection, coincident with a peak of interferon-induced gene expression. The interferon response and NK cell activation were transient, declining by 96 h post-infection. Furthermore, neither NK cell activation nor the interferon response were sustained in patients undergoing multiple rounds of virus treatment. These results show that reovirus modulates human NK cell activity in vivo and suggest that this may contribute to any therapeutic effect of this oncolytic virus. Detection of a single, transient peak of activation, despite multiple treatment rounds, has implications for the design of reovirus-based therapy. Furthermore, our results suggest the existence of a post-infection refractory period when the interferon response and NK cell activation are blunted. This refractory period has been observed previously in animal models and may underlie the enhanced susceptibility to secondary infections that is seen following viral infection., (© 2014 British Society for Immunology.)

LGP2 (laboratory of genetics and physiology 2), RIG-I (retinoic acid inducible gene-I) and MDA5 (melanoma differentiation associated gene 5) constitute the RLR (RIG-I-like receptor) family. LGP2 plays a pivotal role in modulating signaling of RIG-I and MDA5 in innate immune responses. In this study, three representative overexpression vectors were constructed and transfected into C. idella kidney (CIK) cell line to research functional characterizations of CiLGP2 (C. idella LGP2). CiLGP2 overexpression led to the induction of CiRIG-I transcripts. After GCRV challenge, CiLGP2 enhanced CiMDA5 and CiIPS-1 to reinforce the immune response, however, impaired the expression of CiRIG-I. Meanwhile, antiviral activity assays showed that overexpression of CiLGP2 or its domains could inhibit GCRV replication and protect cells from death. Besides, CiLGP2 lingeringly induced CiRIG-I mRNA expression and inhibited CiMDA5 transcripts post poly(I:C) simulation. As a result, CiLGP2 suppressed the RLR-mediated signaling pathway against poly(I:C). Furthermore, CiLGP2 played active roles in RLR signaling response to bacterial PAMPs (LPS and PGN) stimulation. CiLGP2 altered the expression pattern of CiIPS-1 after LPS treatment, while it significantly enhanced the RLR signaling pathway against PGN stimulation. These results collectively suggested that CiLGP2 played a strikingly broad regulation in RLR mediated innate immune responses in C. idella, responding to not only the dsRNA virus or synthetic dsRNA but also bacterial PAMPs, which contribute to the understanding of C. idella LGP2 and RLR signaling pathways. In addition, these results lay a foundation for the further functional mechanism research of LGP2 in fishes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The uses of single walled carbon nanotubes (SWCNTs) as carriers for DNA delivery have received considerable attention in cell studies. DNA vaccination of fish has been shown to elicit durable transgene expression, but no reports exist on intramuscular administration of SWCNTs-DNA vaccine electrostatic complexes which prepared through non-covalent conjugation. In this study, we injected grass carp intramuscularly with a plasmid vector containing a major capsid protein gene (vp7) of grass carp reovirus as a) naked pcDNA-vp7, b) SWCNTs-pcDNA-vp7, c) empty plasmid vector, or phosphate buffered saline. After intramuscular administration, the ability of the different immune treatments to induce transgene expression was analyzed. The results indicated that higher levels of transcription and expression of the vp7 gene could be detected in muscle tissues of grass carp 28 days intramuscular injection in SWCNTs-pcDNA-vp7 treatment groups compare with naked pcDNA-vp7 treatment groups. Moreover, the serum respiratory burst activity, complement activity, lysozyme activity, superoxide dismutase activity, immune-related genes, antibody levels and relative percentage survival were significantly enhanced in fish immunized with SWCNTs-pcDNA-vp7 vaccine. The data in this study suggested that SWCNTs were promising carriers for plasmid DNA vaccine and might be used to vaccinate fish by intramuscular approach., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: To investigate the impact of the injection of inactivated vaccine for hemorrhage of Grass carp on the expressions of main immune-related genes in spleen including immunoglobulin M (IgM), major histocompatibility complexI(MHC I), interferon I(IFN1), complement 3 (C3), interleukin-1β (IL-1β) and intelectin genes., Methods: Ctenopharyngoden idellus kidney (CIK) cells were inoculated with Grass carp reovirus (GCRV). Then inactivated vaccine was prepared by inactivating virus suspension with formaldehyde. Vaccine was 50- and 100-times diluted with normal saline. The experiments included four groups: undiluted group, 50-times diluted group, 100-times diluted group and normal saline control group. Then healthy Grass carps with the average body mass (20 ± 5) g were intraperitoneally injected with vaccine or normal saline separately for each group. Spleens were collected at different time points after injection (0, 6, 12, 24, 72 hours) and total RNA extraction was performed immediately. Real-time quantitative PCR (qRT-PCR) was used to detect mRNA expressions of the six immune-related genes., Results: Compared with normal saline control group, inactivated vaccine injection increased mRNA expressions of the six immune-related genes in the spleen of Grass carp, indicating that the vaccine stimulated the Grass carp to generate non-specific and specific immune responses. In the immunized groups, relative expressions of the genes intelectin, MHCI, IL-1β, C3 were all up-regulated at first and then declined as time went on. Relative expression of IFNIgene reached the peak at 6 hours post-injection and then decreased gradually to the normal level. Differently, relative expression of IgM gene increased continuously within 0-72 hours post-injection., Conclusion: The expressions of the six major immune-related genes were all up-regulated in the spleen of Grass carp injected with different doses of inactivated vaccine for hemorrhage of Grass carp.

Ajuba belongs to the LIM domain proteins, which are involved in the assembly of the extracellular matrix and, along with associated proteins, regulate target genes that connect the extracellular matrix and the cytoskeleton. In the present study, we characterized the entire cDNA sequence of the Ajuba gene from grass carp (gcAjuba). The gcAjuba cDNA contained an open reading frame (ORF) of 2121 bp encoding a polypeptide of 706 amino acids with an estimated molecular mass of 75.966 kDa and three LIM domains in the C-terminal. The transcriptional level of gcAjuba was significantly up-regulated following the stimulation of virus in vitro. Sub-cellular location of gcAjuba and GCRV-JX01 NS26 proteins did not overlap in the cytoplasm and no direct interaction between gcAjuba and the protein NS26 was detected by co-immunoprecipitation (CO-IP) test in grass carp kidney cells. Based on these results, the gcAjuba is determined to be an immediately inducible gene responding to viral infection and in vivo association of gcAjuba with NS26 could not be confirmed, which has been suggested by yeast two-hybrid assay in previous report., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Global fish production from aquaculture has rapidly grown over the past decades, and grass carp shares the largest portion. However, hemorrhagic disease caused by grass carp reovirus (GCRV) results in tremendous loss of grass carp (Ctenopharyngodon idella) industry. During the past years, development of molecular biology and cellular biology technologies has promoted significant advances in the understanding of the pathogen and the immune system. Immunoprophylaxis based on stimulation of the immune system of fish has also got some achievements. In this review, authors summarize the recent progresses in basic researches on GCRV; viral nucleic acid sensors, high-mobility group box proteins (HMGBs); pattern recognition receptors (PRRs), Toll-like receptors (TLRs) and retinoic acid inducible gene I- (RIG-I-) like receptors (RLRs); antiviral immune responses induced by PRRs-mediated signaling cascades of type I interferon (IFN-I) and IFN-stimulated genes (ISGs) activation. The present review also notices the potential applications of molecule genetic markers. Additionally, authors discuss the current preventive and therapeutic strategies (vaccines, RNAi, and prevention medicine) and highlight the importance of innate immunity in long term control for grass carp hemorrhagic disease.

Melanoma differentiation-associated gene 5 (MDA5) plays a crucial role in recognizing intracellular viral infection, activating the interferon regulatory factor pathways as well as inducing antiviral response. While the antiviral regulatory mechanism of MDA5 remains unclear. In the present study, CiMDA5 (Ctenopharyngodon idella MDA5) against grass carp reovirus (GCRV) would be initially revealed from the perspective of DNA methylation, a pivotal epigenetic modification. Two CpG islands (CGIs) were predicted located in the first exon of CiMDA5, of which the first CpG island was 427 bp in length possessed 29 candidate CpG loci and 34 CpA loci, and the second one was 130 bp in length involving 7 CpG loci as well as 10 CpA loci. By bisulfite sequencing PCR (BSP), the methylation statuses were detected in spleen of 70 individuals divided into resistant/susceptible groups post challenge experiment, and the resistance-association analysis was performed with Chi-square test. Quantitative real-time RT-PCR (qRT-PCR) was carried out to explore the relationship between DNA methylation and gene expression in CiMDA5. Results indicated that the methylation levels of CpA/CpG sites at +200, +202, +204, +207 nt, which consisted of a putative densely methylated element (DME), were significantly higher in the susceptible group than those in the resistant group. Meanwhile, the average transcription of CiMDA5 was down-regulated in the susceptible individuals compared with the resistant individuals. Evidently, the DNA methylation may be the negative modulator of CiMDA5 antiviral expression. Collectively, the methylation levels of CiMDA5 demonstrated the tight association with the resistance against GCRV and the negative-regulated roles in mRNA expression. This study first discovered the resistance-associated gene modulated by DNA methylation in teleost, preliminary revealed the underlying regulatory mechanism of CiMDA5 transcription against GCRV as well as laid a theoretical foundation on molecular nosogenesis of hemorrhagic diseases in C. idella., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Sterile alpha and Toll/IL-1R motif containing 1 (SARM1) negatively regulates TRIF-dependent TLR signaling in mammals. However, its immune function remains unclear in teleost. Here, a grass carp Ctenopharyngodon idella SARM1 (CiSARM1) gene and its two novel splice variants (CiSARM1s1 and CiSARM1s2) were identified. CiSARM1s1 and CiSARM1s2 are generated by intron retention mechanism, and they only retain N-terminal HEAT/armadillo motifs. In C. idella kidney (CIK) cells, CiSARM1 and CiSARM1s1 are located in mitochondria, whereas CiSARM1s2 distributes in the whole cell. All the three transcripts are ubiquitously expressed in 15 investigated tissues. They were responsive to GCRV in vivo and in vitro and to viral/bacterial PAMPs in vitro, implying they participate in both antiviral and antibacterial immune responses. By overexpression experiment, CiSARM1 and its two isoforms affected each other's expression in CIK cells. CiSARM1 inhibited GCRV-triggered IFN-I response by affecting the expressions of CiTRIF, CiMyD88, CiIPS-1, CiTRAF6, CiTBK1, CiIRF3 and CiIRF7 in TRIF-, MyD88- and IPS-1-dependent pathways; CiSARM1s1 and CiSARM1s2 inhibited GCRV-triggered IFN-I production through suppressing the expressions of CiMyD88, CiIPS-1, CiTRAF6, CiTBK1, CiIRF3 and CiIRF7 in MyD88- and IPS-1-dependent pathways. Moreover, antiviral activity assays indicated that all the three genes promote GCRV-induced cell death. These results were further verified by RNAi experiments. Thus, CiSARM1 and its two splice variants jointly prevent excessive activation of the host immune response. These findings uncover the regulatory mechanisms of SARM1 in teleost and lay a foundation for further functional and evolutionary researches on SARM1., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

TWEAK and APRIL are important members of the TNF superfamily, which play a crucial role in several diseases. Here, we describe the identification of grass carp (Ctenopharyngodon idella) homologs of TWEAK and APRIL (designated gcTWEAK and gcAPRIL, respectively) and their response to Aeromonas hydrophila and Aquareovirus infection. The gcTWEAK cDNA sequence contains 2273 bases with an open reading frame of 753 bases encoding 250-amino acid residues. The gcTWEAK protein contains a predicted transmembrane domain, a putative furin protease cleavage site, 3 conserved cysteine residues, and a typical TNF homology domain. The gcAPRIL cDNA sequence contains 1408 bases with an open reading frame of 747 bases encoding 248-amino acid residues. The gcAPRIL protein contains a predicted transmembrane domain, a putative furin protease cleavage site, 2 conserved cysteine residues, and a typical TNF homology domain corresponding to other, known APRIL homologs. Reverse transcription-polymerase chain reaction analysis shows that both gcTWEAK and gcAPRIL transcripts are predominantly expressed in the skin, spleen, and head kidney, and they are significantly upregulated in most immune tissues by A. hydrophila and Aquareovirus infections. Our results demonstrate that liver is the most responsive tissue against bacterial infection, whereas gill is the most responsive tissue against viral infection. The association of increased gcTWEAK and gcAPRIL expression after bacterial and viral infections suggests that they play a potentially important role in the immune system of fish.

Grass carp reovirus (GCRV), the most pathogenic aquareovirus, can cause fatal hemorrhagic disease in fingerling and yearling grass carp. Vaccination by injection is by far the most effective method of combating disease. However it is labor intensive, costly and not feasible to vaccinate large numbers of the fish. Thus, an efficient and economic strategy for the prevention of GCRV infection becomes urgent. Here, functionalized single-walled carbon nanotubes (SWCNTs) as carrier were used to manufacture SWCNTs-VP7 subunit vaccine with chemical modification. Different developmental stages of grass carps were immunized by VP7/SWCNTs-VP7 subunit vaccine against GCRV by intramuscular injection and bath immunization. The results indicate that better immune responses of grass carp immunized with the SWCNTs-VP7 subunit vaccine were induced in comparison with VP7 subunit vaccine alone. Immunization doses/concentrations are significantly reduced (about 5-8 times) to prevent GCRV infection in different developmental stages of grass carp with injection or bath treatment when SWCNTs carrier was used. A good immune protective effect (relative percentage survival greater than 95%) is observed in smaller size fish (0.2 g) with SWCNTs-VP7 bath immunization. In addition, serum respiratory burst activity, complement activity, lysozyme activity, superoxide dismutase activity, alkaline phosphatase activity, immune-related genes and antibody levels were significantly enhanced in fish immunized with vaccine. This study suggested that functionalized SWCNTs was the promising carrier for recombinant subunit vaccine and might be used to vaccinate fish by bath approach., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The present study was undertaken to isolate some compounds from methanol extract of Polygala tenuifolia and evaluate their immunostimulatory properties and antiviral activity using grass carp Ctenopharyngodon idella kidney (CIK) cells and GCRV. By applying insecticidal bioassay-guided, chromatography techniques and successive recrystallization, two purified compounds were obtained. The changes of expression of selected immune genes (Mx1, IL-1β, TNFα, MyD88 and IgM) in C. idella kidney cell lines were evaluated after exposure to these isolated compounds. The results showed that compound 1 and 2 up-regulated to varying degrees of Mx1, IL-1β, TNFα, and MyD88 in C. idella kidney cells. WST-8 kit assay verified the two compounds has no toxic effects on CIK cell, and furthermore, have in vitro antivirus activity. Especially, that there is keeping 79% cell viability when exposure to compound 2 (100 mg L(-1)). According to in vivo insecticidal assays against Dactylogyrus intermedius, compound 2 exhibited higher efficacy than compound 1, which was found to be 87.2% effective at the concentrations of 5 mg L(-1) and safe to goldfish (Carassius auratus). Besides, the purified compounds were identified by spectral data as: (1) 1,5-Anhydro-D-glucitol and (2) 3,4,5-trimethoxy cinnamic acid. Overall, the results indicate that bath administration of these compounds modulates the immune related genes in C. idella kidney cells and to some extent, eliminate the virus and parasitic infections., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Mammalian cells possess mechanisms to detect and defend themselves from invading viruses. In the cytosol, the RIG-I-like receptors (RLRs), RIG-I (retinoic acid-inducible gene I; encoded by DDX58) and MDA5 (melanoma differentiation-associated gene 5; encoded by IFIH1) sense atypical RNAs associated with virus infection. Detection triggers a signalling cascade via the adaptor MAVS that culminates in the production of type I interferons (IFN-α and β; hereafter IFN), which are key antiviral cytokines. RIG-I and MDA5 are activated by distinct viral RNA structures and much evidence indicates that RIG-I responds to RNAs bearing a triphosphate (ppp) moiety in conjunction with a blunt-ended, base-paired region at the 5'-end (reviewed in refs 1, 2, 3). Here we show that RIG-I also mediates antiviral responses to RNAs bearing 5'-diphosphates (5'pp). Genomes from mammalian reoviruses with 5'pp termini, 5'pp-RNA isolated from yeast L-A virus, and base-paired 5'pp-RNAs made by in vitro transcription or chemical synthesis, all bind to RIG-I and serve as RIG-I agonists. Furthermore, a RIG-I-dependent response to 5'pp-RNA is essential for controlling reovirus infection in cultured cells and in mice. Thus, the minimal determinant for RIG-I recognition is a base-paired RNA with 5'pp. Such RNAs are found in some viruses but not in uninfected cells, indicating that recognition of 5'pp-RNA, like that of 5'ppp-RNA, acts as a powerful means of self/non-self discrimination by the innate immune system.

To evaluate the presence of a new type of reovirus (designated R4) in humans, we determined the prevalence of specific antibodies using a neutralization assay and ELISA. The sera from 97 healthy people and 219 patients in our hospital with measles, hand-foot-and-mouth disease, liver diseases, and diarrhoea were investigated. Although the study population was limited, our data suggested that R4 is widespread in the human population. A significantly higher level of R4-specific antibody in patients than in healthy people is worthy of consideration, since it poses a risk for aggravation of the extant illness by the reovirus.

Hemorrhagic disease of the grass carp, Ctenopharyngodon idella, is a fatal disease in fingerlings and yearlings caused by a reovirus, GCRV. RNA-seq data from four diseased grass carp tissues (gill, intestine, liver and spleen) were obtained at 2h before and six times after (2h, 24h, 48h, 72h, 96h and 120h) GCRV challenge. A total of 7.25±0.18 million (M) clean reads and 3.53±0.37M unique reads were obtained per RNA-seq analysis. Compared with controls, there were 9060 unique differentially expressed genes (DEGs) in the four tissues at the six time points post-GCRV challenge. Hierarchical clustering analysis of the DEGs showed that the data from the six time points fell into three branches: 2h, 24h/48h, and 72h/96h/120h. Singular (SEA) and modular enrichment analyses of DEGs per RNA-seq dataset were performed based on gene ontology. The results showed that immune responses occurred in all four tissues, indicating that GCRV probably does not target any tissue specifically. Moreover, during the course of disease, disturbances were observed in lipid and carbohydrate metabolism in each of the organs. SEA of DEGs based on the Kyoto Encyclopedia of Genes and Genomes database was also performed, and this indicated that the complement system and cellular immunity played an important role during the course of hemorrhagic disease. The qPCR of pooled samples of duplicate challenge experiment were used to confirm our RNA-seq approach., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Type I interferon responses are considered the primary means by which viral infections are controlled in mammals. Despite this view, several pathogens activate antiviral responses in the absence of type I interferons. The mechanisms controlling type I interferon-independent responses are undefined. We found that RIG-I like receptors (RLRs) induce type III interferon expression in a variety of human cell types, and identified factors that differentially regulate expression of type I and type III interferons. We identified peroxisomes as a primary site of initiation of type III interferon expression, and revealed that the process of intestinal epithelial cell differentiation upregulates peroxisome biogenesis and promotes robust type III interferon responses in human cells. These findings highlight the importance of different intracellular organelles in specific innate immune responses.

Stimulator of interferon gene (STING), an important adapter responsible for RLR pathway, plays a pivotal role in both viral RNA- and DNA-triggered induction of IFNs in mammals. To understand the roles of STING in piscine immune system, STING gene (CiSTING) was identified from grass carp (Ctenopharyngodon idella). The genomic sequence of CiSTING was of 8548 base pairs (bp), including 899 bp 5' flank region, 7 exons and 6 introns. Promoter region was predicted and promoter activity was verified. The CiSTING cDNA was of 1358 bp with an open reading frame of 1185 bp, encoding a polypeptide of 394 amino acids with a signal peptide and three transmembrane motifs in the N-terminal region. mRNA expression of CiSTING was widespread in fifteen tissues investigated, and was up-regulated by GCRV in vivo and in vitro. Meanwhile, the transcription of CiSTING was inhibited at early stage, and then up-regulated at late phase upon poly(I:C) or PGN stimulation in vitro. Interestingly, CiSTING had little impact on LPS in vitro. In CiSTING over-expression cells, CiTBK1, CiIRF3 and CiIRF7 were significantly up-regulated post GCRV or viral/bacterial PAMPs stimulation. In addition, post GCRV or PGN stimulation, the transcription of CiIFN-I was remarkably inhibited while CiMx1 was up-regulated; as for poly(I:C) stimulation, mRNA expressions of CiIFN-I and CiMx1 were inhibited at early stage while enhanced at late phrase; after LPS stimulation, both CiIFN-I and CiMx1 were inhibited. Furthermore, antiviral activity of CiSTING was manifested by the inhibition of GCRV yield. Taken together, these results demonstrated that CiSTING may be involved in board innate immune responses via the TBK1-IRF3/IRF7 cascade, responding to not only dsRNA analogue in an IFN-dependent pathway, but also virus and bacterial PAMPs in an IFN-independent pathway. This study provided novel insights into the essential role of STING in innate immunity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Rice dwarf virus (RDV) is the causal agent of rice dwarf disease, which limits rice production in many areas of south East Asia. Transcriptional changes of rice in response to RDV infection have been characterized by Shimizu et al. and Satoh et al.. Both studies found induction of defense related genes and correlations between transcriptional changes and symptom development in RDV-infected rice. However, the same rice cultivar, namely Nipponbare belonging to the Japonic subspecies of rice was used in both studies., Methods: Gene expression changes of the indica subspecies of rice, namely Oryza sativa L. ssp. indica cv Yixiang2292 that show moderate resistance to RDV, in response to RDV infection were characterized using an Affymetrix Rice Genome Array. Differentially expressed genes (DEGs) were classified according to their Gene Ontology (GO) annotation. The effects of transient expression of Pns11 in Nicotiana benthaminana on the expression of nucleolar genes were studied using real-time PCR (RT-PCR)., Results: 856 genes involved in defense or other physiological processes were identified to be DEGs, most of which showed up-regulation. Ribosome- and nucleolus related genes were significantly enriched in the DEGs. Representative genes related to nucleolar function exhibited altered expression in N. benthaminana plants transiently expressing Pns11 of RDV., Conclusions: Induction of defense related genes is common for rice infected with RDV. There is a co-relation between symptom severity and transcriptional alteration in RDV infected rice. Besides ribosome, RDV may also target nucleolus to manipulate the translation machinery of rice. Given the tight links between nucleolus and ribosome, it is intriguing to speculate that RDV may enhance expression of ribosomal genes by targeting nucleolus through Pns11.

While several studies have been conducted to investigate the stability of dsRNA in the extracellular medium (hemolymph, gut content, saliva), little is known regarding the persistence of dsRNA once it has been introduced into the cell. Here, we investigate the stability of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) genomic dsRNA fragments after transfection into Bombyx-derived Bm5 cells. Using RT-PCR as a detection method, we found that dsRNA could persist for long periods (up to 8 days) in the intracellular environment. While the BmCPV genomic dsRNA was processed by the RNAi machinery, its presence had no effects on other RNAi processes, such as the silencing of a luciferase reporter by dsLuc. We also found that transfection of BmCPV genomic dsRNA could not establish a viral infection in the Bm5 cells, even when co-transfections were carried out with dsRNAs targeting Dicer and Argonaute genes, suggesting that the neutralization by RNAi does not play a role in the establishment of an in vitro culture system. The mechanism of the dsRNA stability in Bm5 cells is discussed, as well as the implications for the establishment for an in vitro culture system for BmCPV., (Copyright © 2014 Elsevier Ltd. All rights reserved.)