Junfa Yuan

Spring viraemia of carp virus: recent advances.

Spring viraemia of carp is an environmentally and economically important disease affecting cyprinids, primarily common carp (Cyprinus carpio). The causative agent of this disease is Spring viraemia of carp virus (SVCV) - a member of the genus Vesiculovirus of the family Rhabdoviridae. The disease is presently endemic in Europe, America and several Asian countries, where it causes significant morbidity and mortality in affected fish. SVCV infection is generally associated with exophthalmia; abdominal distension; petechial haemorrhage of the skin, gills, eyes and internal organs; degeneration of the gill lamellae; a swollen and coarse-textured spleen; hepatic necrosis; enteritis; and pericarditis. The SVCV genome is composed of linear, negative-sense, ssRNA containing five genes in the order 3-N-P-M-G-L-5, encoding a nucleoprotein, phosphoprotein, matrix protein, glycoprotein and RNA-dependent RNA polymerase, respectively. Fully sequenced SVCV strains exhibit distinct amino acid substitutions at unique positions, which may contribute to as-yet unknown strain-specific characteristics. To advance the study of SVCV and the control of spring viraemia of carp disease in the future, this review summarizes our current understanding of SVCV in terms of its genomic characteristics, genetic diversity and pathogenesis, and provides insights into antiviral immunity against SVCV, diagnosis of SVCV and vaccination strategies to combat SVCV.

Spring viraemia of carp virus induces autophagy for necessary viral replication

Outbreaks of spring viraemia of carp virus (SVCV) in several carp species and other cultivated fish can cause significant mortality and jeopardize the billion‐dollar worldwide fish industry. Spring viraemia of carp virus, also known as Rhabdovirus carpio, is a bullet‐shaped RNA virus that enters and amplifies in gill epithelium and later spreads to internal organs. Young fish under stressed conditions (spring cold water, etc.) are more vulnerable to SVCV‐induced lethality because of their lack of a mature immune system. Currently, the host response of SVCV remains largely unknown. Here, we observed that autophagy is activated in SVCV‐infected epithelioma papulosum cyprini (EPC) cells. We demonstrated that the SVCV glycoprotein, rather than viral replication, activates the autophagy pathway. In addition, SVCV utilized the autophagy pathway to facilitate its own genomic RNA replication and to enhance its titres in the supernatants. Autophagy promoted the survival of SVCV‐infected cells by eliminating damaged mitochondrial DNA generated during viral infection. We further showed that SVCV induces autophagy in EPC cells through the ERK/mTOR signalling pathway. Our results reveal a connection between autophagy and SVCV replication and propose autophagy suppression as a novel means to restrict SVCV viral replication.

Proteomic analysis of epithelioma papulosum cyprini cells infected with spring viremia of carp virus

Spring viremia of carp (SVC), caused by spring viremia of carp virus (SVCV) is an important disease due to its drastic effects on carp fisheries in many countries. To better understand molecular responses to SVCV infection, two dimensional electrophoresis (2-DE) and MALDI-TOF/TOF were performed to investigate altered proteins in epithelioma papulosum cyprini cells (EPCs). Differentially expressed proteins in mock-infected EPCs and SVCV-infected EPCs were compared. A total of 54 differentially expressed spots were successfully identified (33 up-regulated spots and 21 down-regulated spots) which include cytoskeleton proteins, macromolecular biosynthesis-associated proteins, stress response proteins, signal transduction proteins, energy metabolism, and ubiquitin proteasome pathway-associated proteins. Moreover, 7 corresponding genes of the differentially expressed proteins were quantified using real time RT-PCR to examine their transcriptional profiles. The presence of four selected cellular proteins (beta-actin, gamma1-actin, heat shock cognate 71 kDa protein and annexin A2) associated with the spring viremia of carp virus (SVCV) particles was validated by Western blot assay. This study provides dynamic and useful protein-related information to further understand the underlying pathogenesis of SVCV infection.

Haematopoietic necrosis of cultured Prussian carp, Carassius gibelio (Bloch), associated with Cyprinid herpesvirus 2

The members of the Alloherpesviridae are widespread among fish and frogs and have been grouped into four genera: Batrachovirus, Cyprinivirus, Ictalurivirus and Salmonivirus (Davison et al. 2009). Among them, the members of the Cyprinivirus can cause devastating diseases in fish culture. There are four known species in Cyprinivirus, named Anguillid herpesvirus 1 (AngHV-1) in European eel, Anguilla anguilla of the, Cyprinid herpesvirus 1 (CyHV-1) and Cyprinid herpesvirus 3 (CyHV-3) in common carp, Cyprinus carpio L., and koi carp (a variety of Cyprinus carpio), and Cyprinid herpesvirus 2 (CyHV-2) in goldfish, Carassius auratus (L.) (Hanson, Dishon & Kotler 2011). Recently, complete genomes of CyHV-1, CyHV-2 and CyHV-3 have been determined, and the structures of the genomes have been compared in detail (Davison et al. 2013). Besides the differences of the genomic structures, the three cyprinid herpesviruses also differ in the host range and clinical signs (Waltzek et al. 2005; Davison et al. 2013). Although both CyHV-1 and CyHV-3 infect common carp and koi carp, in comparison with waxy epidermal growth on the CyHV-1-infected carp, CyHV-3 infection results in carp nephritis and gill necrosis (Davison et al. 2013). Interestingly, common carp and koi carp are not susceptible to CyHV-2, which infects goldfish and produces herpesviral haematopoietic necrosis disease with lesions occurring in haematopoietic tissues, including kidney and spleen (Jung & Miyazaki 1995). In 1995, CyHV-2 was first reported as a severe pathogen of goldfish in Japan (Jung & Miyazaki 1995); thereafter, there have been a number of outbreaks in goldfish in other countries and areas, including USA (Groff et al. 1998; Goodwin et al. 2006a), Taiwan (Chang et al. 1999), Australia (Stephens, Raidal & Jones 2004) and the UK (Jeffery et al. 2007). Recently, our surveillance of CyHV-2 in goldfish cultured in China suggested that CyHV-2 has a wide geographic distribution in China (Li et al. 2013). Besides goldfish, most recently it has been reported that CyHV-2 caused massive mortality of wild-ranging Prussian carp, Carassius gibelio (Bloch), in the Elbe River in the Czech Republic (Danek et al. 2012). Despite many outbreaks of herpesviral haematopoietic necrosis diseases, there is no report in domestic cultured Prussian carp. Here, we report that CyHV-2 infection can cause massive mortality of Prussian carp cultured in domestic ponds in China. Prussian carp is an important commercial fish species, which has been cultured for more than 30 years in China. Its annual product has increased to more than 2 billion kg in China (Gui & Zhou 2010; Wang et al. 2011). During May to June 2012, the first epizootic was observed in Prussian carp cultured together with silver carp, Hypophthalmichthys molitrix (Valenciennes), and bighead carp, Aristichthys nobilis Correspondence L J Li, Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China (e-mail: lilijuan@mail.hzau.edu.cn) and J F Yuan, Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China (e-mail: jfyuan@mail.hzau.edu.cn)

Transcriptome analysis of epithelioma papulosum cyprini cells after SVCV infection

BackgroundSpring viraemia of carp virus (SVCV) has been identified as the causative agent of spring viraemia of carp (SVC) and it has caused significant losses in the cultured common carp (Cyprinus carpio) industry. The molecular mechanisms that underlie the pathogenesis of the disease remain poorly understood. In this study, deep RNA sequencing was used to analyse the transcriptome and gene expression profile of EPC cells at progressive times after SVCV infection. This study addressed the complexity of virus–cell interactions and added knowledge that may help to understand SVCV.ResultsA total of 33,849,764 clean data from 36,000,000 sequence reads, with a mean read length 100 bp, were obtained. These raw data were assembled into 88,772 contigs. Of these contigs, 19,642 and 25,966 had significant hits to the NR and Uniprot databases where they matched 17,642 and 13,351 unique protein accessions, respectively. At 24 h post SVCV infection (1.0 MOI), a total of 623 genes were differentially expressed in EPC cells compared to non-infected cells, including 288 up-regulated genes and 335 down-regulated genes. These regulated genes were primarily involved in pathways of apoptosis, oxidative stress and the interferon system, all of which may be involved in viral pathogenesis. In addition, 8 differentially expressed genes (DEGs) were validated by quantitative PCR.ConclusionsOur findings demonstrate previously unrecognised changes in gene transcription that are associated with SVCV infection in vitro, and many potential cascades identified in the study clearly warrant further experimental investigation. Our data provide new clues to the mechanism of viral susceptibility in EPC cells.

Identification and Characterization of MicroRNAs in Snakehead Fish Cell Line upon Snakehead Fish Vesiculovirus Infection

MicroRNAs (miRNAs) play important roles in mediating multiple biological processes in eukaryotes and are being increasingly studied to evaluate their roles associated with cellular changes following viral infection. Snakehead fish Vesiculovirus (SHVV) has caused mass mortality in snakehead fish during the past few years. To identify specific miRNAs involved in SHVV infection, we performed microRNA deep sequencing on a snakehead fish cell line (SSN-1) with or without SHVV infection. A total of 205 known miRNAs were identified when they were aligned with the known zebrafish miRNAs, and nine novel miRNAs were identified using MiRDeep2 software. Eighteen and 143 of the 205 known miRNAs were differentially expressed at three and 24 h post-infection (poi), respectively. From the differentially-expressed miRNAs, five were randomly selected to validate their expression profiles using quantitative reverse transcription polymerase chain reaction (qRT-PCR), and their expression profiles were consistent with the microRNA sequencing results. In addition, the target gene prediction of the SHVV genome was performed for the differentially-expressed host miRNAs, and a total of 10 and 58 differentially-expressed miRNAs were predicted to bind to the SHVV genome at three and 24 h poi, respectively. The effects of three selected miRNAs (miR-130-5p, miR-214 and miR-216b) on SHVV multiplication were evaluated using their mimics and inhibitors via qRT-PCR and Western blotting. The results showed that all three miRNAs were able to inhibit the multiplication of SHVV; whereas the mechanisms underlying the SHVV multiplication inhibited by the specific miRNAs need to be further characterized in the future.

MicroRNA profile analysis of Epithelioma papulosum cyprini cell line before and after SVCV infection

MicroRNAs (miRNAs) play significant roles in regulating almost all of the biological processes in eukaryotes. An accumulating body of evidence shows that miRNAs are associated with cellular changes following viral infection. Spring viremia of carp virus (SVCV) is the pathogen of Spring viremia of carp (SVC), which results in heavy losses in the cultured common carp (Cyprinus carpio) industry in many countries. To study the involvement of miRNAs during SVCV infection, we adopted the Solexa sequencing technology to sequence small RNA libraries from the Epithelioma papulosum cyprini (EPC) cell line before and after infection with SVCV. In this study, a total of 161 conserved and 26 novel miRNAs were identified. Subsequently, the expression patterns of these miRNAs were compared between the uninfected (control library, M) and SVCV-infected (infection library, E) libraries. In addition, to verify the Solexa sequencing results, the expression patterns of 14 randomly selected miRNAs were validated by qRT-PCR. The targets of the significantly differentially expressed miRNAs were then predicted, and the miRNAs that could directly target the SVCV genome were also predicted. No miRNA encoded by SVCV itself was detected. To the best of our knowledge, this study presents the first miRNA profiling assessment in association with fish rhabdovirus infection, and the data presented lay a foundation for further investigations to determine the roles of miRNAs in regulating the molecular mechanism during SVCV infection.

Up-regulation of nuclear factor E2 – Related factor 2 upon SVCV infection

Nuclear factor E2 - related factor 2 (Nrf2) is a crucial transcription factor that regulates the basal and inducible expression of many antioxidant response element (ARE)-dependent genes, including heme oxygenase-1 (HO-1) and superoxide dismutase 1 (SOD1). The Nrf2/ARE pathway has been regarded as a critical switch in the initiation of cellular defence systems for surviving oxidative insults and viral infection. In this study, the Nrf2 gene of EPC cells, which is originally derived from Pimephales promelas, was cloned, and an investigation on the interactions between Nrf2 and spring viraemia of carp virus (SVCV) was performed. These results demonstrated that the virus facilitated the nuclear accumulation of Nrf2 and up-regulated its transcriptional and protein profiles in EPC cells. In addition, exogenous activation of Nrf2 conferred EPC cells with a higher cellular total antioxidant capacity via an increase in the expression of HO-1 and SOD1, but did not suppress the replication of SVCV.

Up-regulation of nuclear factor E2-related factor 2 (Nrf2) represses the replication of SVCV

Generation of reactive oxygen species (ROS) and failure to maintain an appropriate redox balance contribute to viral pathogenesis. Nuclear factor E2-related factor 2 (Nrf2) is an important transcription factor that plays a pivotal role in maintaining intracellular homoeostasis and coping with invasive pathogens by coordinately activating a series of cytoprotective genes. Previous studies indicated that the transcription and expression levels of Nrf2 were up-regulated in SVCV-infected EPC cells with the unknown mechanism(s). In this study, the interactions between the Nrf2-ARE signalling pathway and SVCV replication were investigated, which demonstrated that SVCV infection induced accumulation of ROS as well as protein carbonyl groups and 8-OHdG, accompanied by the up-regulation of Nrf2 and its downstream genes. At the same time, the activation of Nrf2 with D, l-sulforaphane (SFN) and CDDO-Me could repress the replication of SVCV, and knockdown of Nrf2 by siRNA could promote the replication of SVCV. Taken together, these observations indicate that the Nrf2-ARE signal pathway activates a passive defensive response upon SVCV infection. The conclusions presented here suggest that targeting the Nrf2 pathway has potential for combating SVCV infection.

A novel L-type lectin was required for the multiplication of WSSV in red swamp crayfish (Procambarus clakii)

n Abstractn n L-type lectins are involved in glycoproteins secretory pathways and are associated with many immune responses. There is growing evidence that L-type lectins are also involved in viral replication. In this study, a novel L-type lectin (named as PcL-lectin) was identified from red swamp crayfish (Procambarus clakii). Gene sequencing and phylogenetic tree analysis results showed that the PcL-lectin was a kind of endoplasmic reticulum Golgi intermediate compartment-53 (ERGIC-53). The expression level of PcL-lectin was significantly down regulated in crayfish after challenged with white spot syndrome virus (WSSV). Recombinant PcL-lectin protein facilitated the replication of WSSV in crayfish. In addition, WSSV replication was decreased when endogenous PcL-lectin was knocked down by RNA interference in crayfish. Furthermore, PcL-lectin may interact with VP24, an envelope protein of WSSV. Our results suggest that PcL-lectin may be required for the multiplication of WSSV, and will pave a new way for the developing of strategies against WSSV infection.n n