Rhabdovirus Infection Research Articles

Innate immune response of snakehead fish to Indian strain of snakehead rhabdovirus (SHRV-In) infection and the infectivity potential of the virus to other freshwater fishes

A new virus known as snakehead rhabdovirus (SHRV-In) was discovered in South India in striped snakehead (Channa striata) that had hemorrhagic patches and cutaneous ulcerations. The virus is the most potentially harmful pathogen of snakehead because it could cause 100% mortality within 5 days. The goal of the current investigation was to evaluate the infectivity of rhabdovirus in freshwater fishes and to analyze the immune response in snakehead fish after challenge with SHRV-In. The infectivity study of SHRV-In against three freshwater fish such as tilapia, grass carp and loach showed that the virus could not induce mortality in any of them. Snakehead fish challenged with SHRV-In showed significant (p < 0.05) changes in haematological parameters such as red blood cell (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), white blood cell (WBC), total platelet (PLT) counts, mean platelet volume (MPV) and immunological markers such as respiratory burst, superoxide dismutase, catalase activity and myeloperoxidase activity at 6, 12, 24 and 48 hpi. Real time PCR was executed to examine the expression profile of innate immune genes such as IRF-7, IL-8 and IL-12 in Snakehead fish at 6, 12, 24 and 48 h post SHRV-In infection. Immune gene expression of IRF-7, IL-8 and IL-12 were up-regulated in the spleen when compared to kidney at 6 and 12 hpi. However, the expression level of all the genes was down-regulated at 24 and 48 hpi. The down regulation of innate immune genes after 24 hpi in these tissues may be the result of increased multiplication of SHRV-In by interfering with the immune signaling pathway.

A novel protein encoded by circVPS13D attenuates antiviral innate immunity by targeting MAVS in teleost fish.

The expression of circVPS13D was upregulated with SCRV invasion, which proved that circVPS13D was involved in the regulation of the antiviral immune response. Our study revealed that the existence of circVPS13D promoted the replication of SCRV. Functionally, circVPS13D negatively regulates the antiviral responses of fish. Mechanistically, we confirmed that circVPS13D inhibited RLRs antiviral signaling pathway via the encoded protein VPS13D-170aa by targeting MAVS. Our study provided novel insights into the roles of protein-coding circRNAs and supported VPS13D-170aa as a negative regulator in the antiviral immune responses of teleost fish.

Eukaryotic translation elongation factor 1 alpha (eEF1A) inhibits Siniperca chuatsi rhabdovirus (SCRV) infection through two distinct mechanisms.

Although a virus can regulate many cellular responses to facilitate its replication by interacting with host proteins, the host can also restrict virus infection through these interactions. In the present study, we showed that the host eukaryotic translation elongation factor 1 alpha (eEF1A), an essential protein in the translation machinery, interacted with two proteins of a fish rhabdovirus, Siniperca chuatsi rhabdovirus (SCRV), and inhibited virus infection via two different mechanisms: (i) inhibiting the formation of crucial viral protein complexes required for virus transcription and replication and (ii) promoting the ubiquitin-proteasome degradation of viral protein. We also revealed the functional regions of eEF1A that are involved in the two processes. Such a host protein inhibiting a rhabdovirus infection in two ways is rarely reported. These findings provided new information for the interactions between host and fish rhabdovirus.

Rhabdovirus encoded glycoprotein induces and harnesses host antiviral autophagy for maintaining its compatible infection

ABSTRACT Macroautophagy/autophagy has been recognized as a central antiviral defense mechanism in plant, which involves complex interactions between viral proteins and host factors. Rhabdoviruses are single-stranded RNA viruses, and the infection causes serious harm to public health, livestock, and crop production. However, little is known about the role of autophagy in the defense against rhabdovirus infection by plant. In this work, we showed that Rice stripe mosaic cytorhabdovirus(RSMV) activated autophagy in plants and that autophagy served as an indispensable defense mechanism during RSMV infection. We identified RSMV glycoprotein as an autophagy inducer that interacted with OsSnRK1B and promoted the kinase activity of OsSnRK1B on OsATG6b. RSMV glycoprotein was toxic to rice cells and its targeted degradation by OsATG6b-mediated autophagy was essential to restrict the viral titer in plants. Importantly, SnRK1-glycoprotein and ATG6-glycoprotein interactions were well-conserved between several other rhabdoviruses and plants. Together, our data support a model that SnRK1 senses rhabdovirus glycoprotein for autophagy initiation, while ATG6 mediates targeted degradation of viral glycoprotein. This conserved mechanism ensures compatible infection by limiting the toxicity of viral glycoprotein and restricting the infection of rhabdoviruses. Abbreviations: AMPK: adenosine 5‘-monophosphate (AMP)-activated protein kinase; ANOVA: analysis of variance; ATG: autophagy related; AZD: AZD8055; BiFC: bimolecular fluorescence complementation; BYSMV: barley yellow striate mosaic virus; Co-IP: co-immunoprecipitation; ConA: concanamycin A; CTD: C-terminal domain; DEX: dexamethasone; DMSO: dimethyl sulfoxide; G: glycoprotein; GFP: green fluorescent protein; MD: middle domain; MDC: monodansylcadaverine; NTD: N-terminal domain; OE: over expression; Os: Oryza sativa; PBS: phosphate-buffered saline; PtdIns3K: class III phosphatidylinositol-3-kinase; qRT-PCR: quantitative real-time reverse-transcription PCR; RFP: red fluorescent protein; RSMV: rice stripe mosaic virus; RSV: rice stripe virus; SGS3: suppressor of gene silencing 3; SnRK1: sucrose nonfermenting1-related protein kinase1; SYNV: sonchus yellow net virus; TEM: transmission electron microscopy; TM: transmembrane region; TOR: target of rapamycin; TRV: tobacco rattle virus; TYMaV: tomato yellow mottle-associated virus; VSV: vesicular stomatitis virus; WT: wild type; Y2H: yeast two-hybrid; YFP: yellow fluorescent protein.

Myricetin inhibits Micropterus salmoides rhabdovirus infection by damping inflammation through the NF-κB pathway

Largemouth bass have been plagued by diseases elicited by bacteria, parasites, and viruses, particularly viral diseases, which have caused significant economic losses in recent years. And there is a lack of approved antiviral drugs in the aquaculture industry. Micropterus salmoides Rhabdovirus (MSRV) is the causative agent of Micropterus salmoides Rhabdovirus disease, which mostly affects largemouth bass fry. By using experimental infection, transcriptome sequencing, and inhibitor treatment, the inflammatory response mediated by mitogen-activated protein kinase (MAPK), phosphatidylinositol3-kinase (PI3K), and nuclear factor kappa-B (NF-κB) pathways was verified as one of the pathological processes of MSRV infection. Further, Myricetin, arteannuin, naringenin, ursolic acid, andrographolide, and resveratrol were selected to evolute their anti-inflammatory activity based on the promoter of NF-κB and found myricetin, naringenin, andrographolide, and resveratrol significantly inhibited NF-κB 1 promoter activity. Myricetin was found to damp MSRV caused-inflammation and inhibit MSRV infection by suppressing NF-κB signaling. Besides, myricetin improves the survival rate of largemouth bass after MSRV infection. In conclusion, myricetin presented anti-MSRV activity by targeting the NF-κB inflammatory pathway and can be further developed as a potential preventative drug of rhabdovirus disease.

Untargeted LC-MS metabolomics reveals the metabolic responses in olive flounder subjected to hirame rhabdovirus infection.

Hirame novirhabdovirus (HIRRV), which mainly infects the olive flounder (Paralichthys olivaceus), is considered to be one of the most serious viral pathogens threatening the global fish culture industry. However, little is known about the mechanism of host-pathogen interactions at the metabolomic level. In this study, in order to explore the metabolic response of olive flounder to HIRRV infection, liquid chromatography mass spectrometry (LC-MS) was used to detect the changes of endogenous compounds of the olive flounder after HIRRV infection. A total of 954 unique masses were obtained, including 495 metabolites and 459 lipids. Among them, 7 and 173 qualified differential metabolites were identified at 2 days and 7 days post-infection, respectively. Distinct metabolic profiles were observed along with viral infection. At the early stage of infection, only a few metabolites were perturbed. Among them, the level of inosine and carnosine were increased and the potential antiviral ability of these two metabolites was further confirmed by exogenous addition experiment. At the late stage of HIRRV infection, the metabolic profiles changed remarkably. The changes in amino acids and nucleotides especially the 7-methylguanine also accelerated the amplification of viral particles. And the down-regulation of glutathione (GSH) implied an elevated level of ROS (reactive oxygen species) that attenuated the immune system of flounders. HIRRV also induced the accumulation of purine and reduction of pyrimidine, and elevated LPC and LPE levels. The unbalanced purine/pyrimidine and altered lipid profile may be beneficial for the replication and infection of HIRRV at the late stage of infection. These findings provide new insights into the pathogenic mechanism of HIRRV infection in olive flounder.

Oxygen-Sensing Protein Cysteamine Dioxygenase from Mandarin Fish Involved in the Arg/N-Degron Pathway and Siniperca chuatsi Rhabdovirus Infection.

Mammalia cysteamine (2-aminoethanethiol) dioxygenase (ADO) controls the stability of the regulator of G protein signaling 4 (RGS4) through the Cys branch of the Arg/N-degron pathway, thereby affecting the response of the body to hypoxia. However, the oxygen-sensing function of ADO remains unknown in teleost fish. Mandarin fish (Siniperca chuatsi) is one of the most important freshwater economic fishes in China. As the scale of the rearing density continues to increase, hypoxia has become an important factor threatening the growth of mandarin fish. Herein, the molecular characterization, the oxygen-sensing enzyme function, and the role in virus infection of ADO from mandarin fish (scADO) were explored. Bioinformation analysis results showed that scADO had all the molecular foundations for achieving thiol dioxygenase function: three histidine residues coordinated with Fe(II), PCO/ADO domain, and a "jelly roll" β-barrel structure. The expression pattern analysis showed that scAdo was highly expressed in the immune-related tissues, liver, and kidneys and responded to hypoxia on the expression level. Protein degradation experiment results revealed that scADO could lead to the degradation of RGS4 protein through the Cys branch of the Arg/N-degron pathway. Furthermore, the expression levels of scADO responded to fish virus infection. scADO could significantly promote the replication of Siniperca chuatsi rhabdovirus, and this was associated with its thiol dioxygenase activity. These findings not only demonstrate scADO as an oxygen-sensing protein in teleost fish, but are also of considerable importance for clarifying the contribution of the mechanism of hypoxia to the outbreaks of fish viruses.

Morphogenesis of Bullet-Shaped Rabies Virus Particles Regulated by TSG101.

Viral protein assembly and virion budding are tightly regulated to enable the proper formation of progeny virions. At this late stage in the virus life cycle, some enveloped viruses take advantage of the host endosomal sorting complex required for transport (ESCRT) machinery, which contributes to the physiological functions of membrane modulation and abscission. Bullet-shaped viral particles are unique morphological characteristics of rhabdoviruses; however, the involvement of host factors in rhabdovirus infection and, specifically, the molecular mechanisms underlying virion formation are not fully understood. In the present study, we used a small interfering RNA (siRNA) screening approach and found that the ESCRT-I component TSG101 contributes to the propagation of rabies virus (RABV). We demonstrated that the matrix protein (M) of RABV interacts with TSG101 via the late domain containing the PY and YL motifs, which are conserved in various viral proteins. Loss of the YL motif in the RABV M or the downregulation of host TSG101 expression resulted in the intracellular aggregation of viral proteins and abnormal virus particle formation, indicating a defect in the RABV assembly and budding processes. These results indicate that the interaction of the RABV M and TSG101 is pivotal for not only the efficient budding of progeny RABV from infected cells but also for the bullet-shaped virion morphology. IMPORTANCE Enveloped viruses bud from cells with the host lipid bilayer. Generally, the membrane modulation and abscission are mediated by host ESCRT complexes. Some enveloped viruses utilize their late (L-) domain to interact with ESCRTs, which promotes viral budding. Rhabdoviruses form characteristic bullet-shaped enveloped virions, but the underlying molecular mechanisms involved remain elusive. Here, we showed that TSG101, one of the ESCRT components, supports rabies virus (RABV) budding and proliferation. TSG101 interacted with RABV matrix protein via the L-domain, and the absence of this interaction resulted in intracellular virion accumulation and distortion of the morphology of progeny virions. Our study reveals that virion formation of RABV is highly regulated by TSG101 and the virus matrix protein.

DDX3X Is Hijacked by Snakehead Vesiculovirus Phosphoprotein To Facilitate Virus Replication via Stabilization of the Phosphoprotein.

Asp-Glu-Ala-Asp (DEAD) box helicase 3 X-linked (DDX3X) plays important regulatory roles in the replication of many viruses. However, the role of DDX3X in rhabdovirus replication has seldomly been investigated. In this study, snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus, was used to study the role of DDX3X in rhabdovirus replication. DDX3X was identified as an interacting partner of SHVV phosphoprotein (P). The expression level of DDX3X was increased at an early stage of SHVV infection and then decreased to a normal level at a later infection stage. Overexpression of DDX3X promoted, while knockdown of DDX3X using specific small interfering RNAs (siRNAs) suppressed, SHVV replication, indicating that DDX3X was a proviral factor for SHVV replication. The N-terminal and core domains of DDX3X (DDX3X-N and DDX3X-Core) were determined to be the regions responsible for its interaction with SHVV P. Overexpression of DDX3X-Core suppressed SHVV replication by competitively disrupting the interaction between full-length DDX3X and SHVV P, suggesting that full-length DDX3X-P interaction was required for SHVV replication. Mechanistically, DDX3X-mediated promotion of SHVV replication was due not to inhibition of interferon expression but to maintenance of the stability of SHVV P to avoid autophagy-lysosome-dependent degradation. Collectively, our data suggest that DDX3X is hijacked by SHVV P to ensure effective replication of SHVV, which suggests an important anti-SHVV target. This study will help elucidate the role of DDX3X in regulating the replication of rhabdoviruses. IMPORTANCE Growing evidence has suggested that DDX3X plays important roles in virus replication. In one respect, DDX3X inhibits the replication of viruses, including hepatitis B virus, influenza A virus, Newcastle disease virus, duck Tembusu virus, and red-spotted grouper nervous necrosis virus. In another respect, DDX3X is required for the replication of viruses, including hepatitis C virus, Japanese encephalitis virus, West Nile virus, murine norovirus, herpes simplex virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Because DDX3X has rarely been investigated in rhabdovirus replication, this study aimed at investigating the role of DDX3X in rhabdovirus replication by using the fish rhabdovirus SHVV as a model. We found that DDX3X was required for SHVV replication, with the mechanism that DDX3X interacts with and maintains the stability of SHVV phosphoprotein. Our data provide novel insights into the role of DDX3X in virus replication and will facilitate the design of antiviral drugs against rhabdovirus infection.

The m6A Reader YTHDF2 Modulates Antiviral and Antibacterial Activity by Suppressing METTL3 Methylation-Modified STING in Fish.

At present, N6-methyladenosine (m6A) modification has been proven to participate in a wide range of gene expression regulation, such as stability, translation, splicing, and output, among others, which has attracted much attention. Unlike mammals, however, the role of m6A in innate immunity of lower invertebrates has not yet been studied. In this study, we found that the total m6A level of Miichthys miiuy increased during Siniperca chuatsi rhabdovirus and Vibrio anguillarum infection, suggesting that m6A may play an important role in the immune process against pathogens in fish. In addition, our study shows that stimulator of IFN genes (STING) plays a dual immune function against viruses and bacteria in fish, and through degrading STING by identifying its m6A methylation site modified by methyltransferase-like 3 (METTL3), YTH domain family 2 (YTHDF2) can weaken the IRF3 and NF-κB-driven signaling pathway, thus weakening the innate immunity and promoting the infection of Siniperca chuatsi rhabdovirus and V. anguillarum to the M. miiuy. Although there have been reports on m6A modification of STING in mammals, it is still unclear whether there is also m6A modification in lower vertebrates, especially in fish. Therefore, our study provides a reference for filling the gap of m6A modification between fish and mammals.

Review on the Antiviral Organic Agents against Fish Rhabdoviruses

Fish rhabdoviruses are harmful single-stranded RNA viruses with high mortality rates which cause considerable economic losses in aquaculture. It is imperative to explore and develop new antiviral compounds against them. In recent years, in addition to inorganic antiviral substances, more than 50 different organic compounds have been confirmed to be effective in the prevention and treatment of rhabdovirus infection and its dissemination in fish. The main types of extracts or agents and their trial designs are here considered for review. This review reveals the reported antiviral activities of extracts from organisms, proteins, lipids, polysaccharides, nucleic acids, coumarin derivatives, arctigenin derivatives, and other antiviral organic molecules against fish rhabdoviruses, respectively. Additionally, their antiviral mechanisms of action include direct virucidal effects, inhibiting virus-induced host cell apoptosis, the blocking of the viral replication cycle, affecting gene expression and innate antiviral immune responses, and so on. This review also gives perspectives on how to comprehensively explore the potential applications of the candidate molecules, which lay the foundation for the future development of new compounds or strategies for the prevention and control of fish rhabdoviruses in aquaculture.

FTO promotes innate immunity by controlling NOD1 expression via m6A-YTHDF2 manner in teleost.

N6-methyladenosine (m6A), the most abundant internal mRNA modification in eukaryotes, plays a vital role in regulating innate immunity. However, its underlying mechanism remains largely unknown, especially in lower vertebrates. The results of the present study show that fat-mass- and obesity-associated protein (FTO), also known as a m6A demethylase, improved the innate immunity and prevented Siniperca chuatsi rhabdo virus and Vibrio anguillarum infection in miiuy croaker. Significantly, FTO-promoted immunity was dependent on its m6A demethylase activity. In terms of mechanism, NOD1 has abundant methylation modification in its CDS and 3'UTR regions, and FTO can reduce its methylation level, thus avoiding its degradation by YTHDF2. In summary, our results indicate that the FTO-mediated m6A modification in NOD1 mRNA promotes innate immunity by activating the NOD-like receptor pathway, which provides a molecular mechanism for the regulation of immune response via RNA methylation in teleost.

Long-term exposure to azoxystrobin induces immunodeficiency in fish that are vulnerable to subsequent rhabdovirus infection

Azoxystrobin (AZ) is one of the most widely used strobilurin fungicides in the world, and its residue has seriously endangered aquatic ecological security. Our previous data showed that AZ exposure may reduce the resistance of fish to rhabdovirus infection in aquatic environment. Here, we further reported a potential long-term adverse effect of AZ exposure on the antiviral and immunosuppressive recovery in fish, and observed that mitochondrial dynamic balance was disturbed by AZ in which excessive mitochondrial fission occurred in response to decreased ATP levels. When a recovery operation was performed in AZ-exposed cells and fish, infectivity of our model virus, spring viraemia of carp virus (SVCV), was significantly decreased in vitro (using the epithelioma papulosum cyprini [EPC] fish cell line) and in vivo (using zebrafish) in a time-dependent manner. Also, the expression of eight innate antiviral immune genes (IFNs, ISG15, MX1, RIG-I, IRF3, Nrf2 and HO-1) showed a similar change to SVCV replication between the longer exposure period and the expression recovery. Additionally, AZ facilitated horizontal transmission of SVCV in a static cohabitation challenge model, predicting the increase of the potential for the viral outbreak. Therefore, our data suggest that long-term effect of AZ on irreparable impairment in fish made AZ residue potentially greater for ecological risks.

Screening the potential part of the G protein antigen is an achievable strategy to improve the immune effect of DNA vaccine against MSRV infection

DNA vaccines, as an effective prophylactic technology to induce both humoral and cellular immune responses, have already been widely studied to prevent and control viral and bacterial infections in aquaculture. To find a more effective and safer way to control Micropterus salmoides rhabdovirus (MSRV) infection in largemouth bass, two different DNA vaccines expressing partial (pcDNA3.1-G2) and full-length (pcDNA3.1-G) of the MSRV G protein were developed and injected intramuscularly with different doses. The immune effect was comprehensively compared and evaluated by detecting immune-related parameters including serum antibody levels, immune-related physiological indexes, immune-related gene expression and relative survival rates in this study. The results showed that compared with the pcDNA3.1-G vaccine, the pcDNA3.1-G2 vaccine induced higher serum antibody levels, a lower nonspecific immune response in serum (ACP, SOD and T-AOC activities), higher immune-related gene expression and a higher relative survival rate. Moreover, the immune effect of pcDNA3.1-G2-vaccinated fish showed gradually higher with the increasing pcDNA3.1-G2 concentration, especially in pcDNA3.1-G2 (10μg/per fish) group, the relative survival rate reached to 82.5%, which was significant higher (p < 0.05) than pcDNA3.1-G (10μg/per fish) group. This study indicated that screening the potential core part of an antigen is an achievable strategy to improve the immunogenicity and immunoprotective effect of DNA vaccine.

Transcriptome and 16S rRNA analysis revealed the response of largemouth bass (Micropterus salmoides) to Rhabdovirus infection.

To better understand the response of largemouth bass (Micropterus salmoides) to Micropterus salmoides rhabdovirus (MSRV) infection, we investigated the intestinal bacterial flora and transcriptome profile of fish at 72 hours post-infection (hpi). Total of 1574 differentially expressed genes (DEGs) were identified in largemouth bass spleen following MSRV infection, including 573 upregulated and 1001 downregulated genes. KEGG and GO enrichment analysis revealed that upregulated genes were enriched in certain antiviral related signaling pathway, including NOD-like receptor (NLR), RIG-I like receptors (RLR) and regulation of the interferon (IFN)-γ-mediated signaling pathway, whereas some immune-related DEGs enriched in focal adhesion (FA) and ECM-receptor interaction(ECM-RI) were downregulated, as well as genes associated with metabolic processes, such as peroxisome proliferator-activated receptors (PPAR), adipocytokine signaling pathway, Glycerolipid and Retinol metabolism. Furthermore, the principal component analysis (PCA) and phylogenetic analysis revealed that MSRV infection significantly affected the microbiota of largemouth bass intestine; the LEfSe analysis showed that relative abundances of Streptococcus were significantly increased, while the content of Akkermansia, Enterococcus and Lactobacillus were remarkably decreased in the fish intestine following MSRV infection. Additionally, a high correlation was determined between the expressions of interferon-related upregulated genes and the relative abundance of Streptococcus by redundancy analysis (RDA). These results collectively illustrated that intestinal microbiota composition might be associated with the immune-related gene expression in largemouth bass in response to MSRV infection.

Evaluation on the antiviral activity of artemisinin against rhabdovirus infection in common carp

The spring viremia of carp virus (SVCV) is a serious deadly virus that results in high mortality and significant economic losses in the aquaculture industry of carp species. The present study was conducted to evaluate the antiviral effect of 8 natural compounds against SVCV in epithelioma papulosum cyprinid (EPC) cells and common carp (Cyprinus carpio). Herein, artemisinin was identified to inhibit SVCV replication obviously, with the highest inhibition ratio on SVCV nucleoprotein gene expression at 100 μM in cells. Artemisinin not only maintained normal cell morphology and reduced intracellular nuclear damage in SVCV-infected cells, but also disrupted SVCV infection by affecting viral attachment and entry. In vivo experiments revealed that artemisinin post-incubation by feeding significantly increased common carp survival by 33.3%, and significantly decreased the viral loads. Meanwhile, artemisinin induced up-regulation of antiviral-related genes, such as type I IFN (IFN1), IFN-stimulated gene product 15 (ISG15), Mx1, and viperin, implying an immunoregulatory capacity. Taking together, artemisinin may promise in the prevention and treatment of SVCV infection in aquaculture.

Quinoline, with the active site of 8-hydroxyl, efficiently inhibits Micropterus salmoides rhabdovirus (MSRV) infection in vitro and in vivo.

Micropterus salmoides rhabdovirus (MSRV) is an significant pathogen that causes high mortality and related economic losses in bass aquaculture. There is no effective or approved therapy to date. In this study, we evaluated the anti-MSRV effects of 22 quinoline derivatives in grass carp ovary (GCO) cells. Among these compounds, 8-hydroxyquinoline exhibited valid inhibition in decreasing MSRV nucleoprotein gene expression levels of 99.3% with a half-maximal inhibitory concentrations (IC50 ) value of 4.66μM at 48h. Moreover, 8-hydroxyquinoline significantly enhanced a protective effect in GCO cells by reducing the cytopathic effect (CPE). By comparing the anti-MSRV activity of 22 quinoline derivatives, we found that 8-hydroxyquinoline possessed the efficient active site of 8-hydroxyl and inhibited MSRV infection in vitro. For in vivo studies, 8-hydroxyquinoline via intraperitoneal injection exhibited an antiviral effect in MSRV-infected largemouth bass by substantially enhancing the survival rate by 15.0%. Importantly, the viral loads in the infected largemouth bass notably reduced in the spleen on the third days post-infection. Overall, 8-hydroxyquinoline was considered to be an efficient agent against MSRV in aquaculture.

Earlier Activation of Interferon and Pro-Inflammatory Response Is Beneficial to Largemouth Bass (Micropterus salmoides) against Rhabdovirus Infection

In order to understand the immune response of largemouth bass against Micropterus salmoides Rhabdovirus (MSRV), assisting disease resistance breeding, three largemouth bass breeding varieties Micropterus salmoides “Youlu No 3” (U3), “Youlu No 1” (U1) and “Zhelu No 1” (P1) were challenged intraperitoneally with MSRV. Serum and tissues were sampled to study the changes in non-specific immune parameters, viral loads, and transcript levels of immune-related genes, and the cumulative mortality rate was recorded daily for 14 days. The results showed that the cumulative mortality rates in the U1, P1, and U3 groups were 6.66% ± 2.89%, 3.33% ± 2.89%, and 0, respectively. The higher mortality may attribute to the increased viral loads after infection in the liver (2.79 × 105 and 2.38 × 105 vs. 1.3 × 104 copies/mg), spleen (2.14 × 105 and 9.40 × 104 vs. 4.21 × 103 copies/mg), and kidney (3.59 × 104 and 8.40 × 103 vs. 2.42 × 103 copies/mg) in the U1 and P1 groups compared to the U3 group. The serum non-specific immune parameters (lysozyme, catalase, and acid phosphatase) were found to be increased significantly in the U3 group. In addition, the transcripts of interferon-related genes (IFN-γ, IRF3, and IRF7) and pro-inflammatory-related genes (TNF-α and IL-1β) exhibited up-regulation and peaked at 6 h post infection in the U3 group, which also exhibited up-regulation but peaked at 12–24 h post infection in the U1 and P1 groups. In conclusion, these findings indicate that earlier activation of interferon and pro-inflammatory response is beneficial to largemouth bass against MSRV infection. This experiment may provide an insight into understanding the immune mechanism of largemouth bass against MSRV infection and contributes to molecular-assisted selection.

Innate response of rainbow trout gill epithelial (RTgill-W1) cell line to ultraviolet-inactivated VHSV and FliC and rhabdovirus infection

Gills reportedly play a crucial role in induction of an antiviral immune response in fish. We investigated the expression of innate response genes in the rainbow trout gill epithelial cell line RTgill-W1 36 h after pretreatment with ultraviolet-inactivated viral hemorrhagic septicemia virus (UV-VHSV), flagellin C protein from Edwardsiella tarda (FliC), VHSV and SVCV using an Agilent 4 × 44k cGRASP salmonid microarray. RTgill-W1 cells pretreated with UV-VHSV, triggered an independent gene expression profile from those treated with a recombinant flagellin C protein from Edwardsiella tarda. In addition, exposure of RTgill-W1 cells to live viruses spring viremia of carp virus and viral hemorrhagic septicemia virus induced a less robust transcriptional change of 24 and 22 gene probes, respectively, when compared to 123 genes for UV-VHSV. Further the pretreatment of RTgill-W1 cells with (UV-VHSV) significantly reduced VHSV genome copy number at 6 d post infection (dpi) relative to the FliC-treated and untreated control. A quantitative PCR was used to study the transcriptional modulation of a set of 25 innate immune-related genes highlighted by the microarray data and a panel of 7 established antiviral genes in the protected cells. Notably, the expression of ifn1, ifn2, mx1 and mx3 were expressed more in untreated cells than in UV-VHSV-treated cells where virus replication was inhibited. The results from this study shed light on the mechanisms and pathways used by teleost gill epithelium innate immunity in combating viral and bacterial infection.

MicroRNA-181b-2 and MicroRNA-21-1 Negatively Regulate NF-κB and IRF3-Mediated Innate Immune Responses via Targeting TRIF in Teleost.

Upon recognition of bacterial or viral components by Toll-like receptors (TLRs), cells could be activated to induce a series of reactions to produce inflammatory cytokines, type I interferon (IFN), and IFN stimulating genes (ISG). MicroRNAs (miRNAs) are an important regulatory molecules that are widely involved in the regulatory networks of mammalian inflammation and immune responses; however, in lower vertebrates, the regulatory network of miRNA-mediated immune responses is poorly understood. Here, we report two miRNAs form Miichthys miiuy, namely, miR-181b-2 and miR-21-1, that play a negative role in host antiviral and antibacterial immunity. We found that miR-181b-2 and miR-21-1 are abundantly expressed in gram-negative bacteria, as well as RNA rhabdovirus infection. Inducible miR-181b-2 and miR-21-1 suppress the production of inflammatory cytokines and type I IFN by targeting TRIF, thereby avoiding excessive inflammation. We further revealed that miR-181b-2 and miR-21-1 modulate antibacterial and antiviral immunity through the TRIF-mediated NF-κB and IRF3 signaling pathways. The overall results indicate that miR-181b-2 and miR-21-1 act as negative feedback regulators and participate in host antibacterial and antiviral immune responses; this finding could provide information for a deeper understanding of the resistance of lower vertebrates to the invasion of pathogens and to avoidance of excessive immunity.