Small RNA transcriptome profiling reveals an essential role for miR-133b-3p_3 and miR-146b_1 in spring viraemia of carp virus infection in common carp (Cyprinus carpio L.).

Spring viraemia of carp (SVC) is an infectious disease responsible for severe economic losses for various cyprinid species, particularly common carp (Cyprinus carpio carpio). The causative agent is the Rhabdovirus carpio or SVC virus (SVCV), a member of the Sprivivirus genus, within the Rhabdoviridae family. Phylogenetically, SVCV is divided into four genogroups (SVCV a, SVCV b, SVCV c and SVCV d), which have a reasonable correlation with the geographical distribution of the virus. In the late twentieth century, the disease was widespread in Serbian aquaculture and caused massive deaths in common carp. This study aimed to molecularly characterize the circulating SVCV isolates in Serbia over a 17-year period. The genetic relationships between 21 SVCV isolates from common carp and rainbow trout in Serbia between 1992 and 2009 were determined based on the partial nucleotide sequence of the glycoprotein gene (G gene). The phylogenetic analysis showed that the dominant SVCV isolates in Serbia belong to the SVCV d genogroup, with only one isolate belonging to genogroup SVCV b. The SVCV strains circulating in Serbia exhibited high homogeneity, as several isolates shared 100% similarity within these genogroups. Most Serbian isolates belonged to SVCV d1 and d2 subgroups, with one isolate notably different and included in a new subgroup SVCV d5. Understanding the SVCV genetic variants circulating in Serbia would be helpful in future epizootic investigations.

Spring viremia of carp virus (SVCV) is a highly lethal virus in common carp (Cyprinus carpio) and other cyprinid fish species. The aim of the present study was to develop an in vivo therapeutic measure against SVCV using artificial microRNA (AmiRNA) targeting the SVCV P gene transcript. Three candidates of AmiRNAs (AmiR-P1, -P2, and -P3) were selected, and their ability to downregulate SVCV P gene transcript was analyzed by both synthesized AmiRNA mimics and AmiRNA-expressing vector system, in which AmiR-P3 showed the strongest inhibitory activity among the three candidates. To overcome in vivo limitation of miRNA mimics or plasmid-based miRNA expression systems, we rescued recombinant snakehead rhabdoviruses (SHRVs) expressing SVCV P gene-targeting AmiRNA (rSHRV-AmiR-P3) or control AmiRNA (rSHRV-AmiR-C) using reverse genetic technology. The successful expression of AmiR-P3 and AmiR-C in cells infected with the rescued viruses was verified by quantitative PCR. To evaluate the availability of rSHRV-AmiR-P3 for in vivo control of SVCV, zebrafish (Danio rerio) were (i) infected with either rSHRV-AmiR-C or rSHRV-AmiR-P3 followed by SVCV infection or (ii) infected with SVCV followed by either rSHRV-AmiR-C or rSHRV-AmiR-P3 infection. Fish infected with rSHRVs before and after SVCV infection showed significantly higher survival rates than fish infected with SVCV alone. There was no significant difference in survival rates between groups of fish infected with rSHRV-AmiR-C and rSHRV-AmiR-P3 before SVCV infection; however, fish infected with SVCV followed by infection with rSHRV-AmiR-P3 showed significantly higher survival rates than fish infected with rSHRV-AmiR-C. These results suggest that rSHRV-AmiR-P3 has therapeutic potential against SVCV in fish when administered after SVCV infection, and rSHRVs expressing artificial microRNAs targeting SVCV transcripts could be used as a tool to control SVCV infection in fish for a therapeutic purpose.

Viral infection-induced changes in the expression profile of non-RLR DExD/H-box RNA helicases (DDX1, DDX3, DHX9, DDX21 and DHX36) in zebrafish and common carp

Experimental infection of six North American fish species with the North Carolina strain of spring Viremia of Carp Virus

Expression analysis of Igs and mucosal immune responses upon SVCV infection in common carp (Cyprinus carpio L.)

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 viremia of carp virus (SVCV) is highly lethal in common carp (Cyprinus carpio) and other cyprinid fish species. In this study, we evaluated the effect of the SVCV phosphoprotein (P protein) on the virulence of snakehead rhabdovirus (SHRV) by generating a recombinant SHRV expressing the SVCV P protein. The SVCV P gene was inserted between the N and P genes of the SHRV genome, resulting in the generation of rSHRV-A-Psvcv, which contains not only its own P gene but also the P gene of SVCV. In preliminary experiments, we determined the minimum titer of SHRV that was lethal in zebrafish (Danio rerio). We then conducted experiments using titers below this threshold to assess the in vivo virulence of rSHRV-A-Psvcv in zebrafish. Under these conditions, no mortality was observed in fish infected with rSHRV-A-eGFP, a modified SHRV containing the enhanced green fluorescent protein (eGFP) gene between the N and P genes, which was used as a control. However, fish infected with rSHRV-A-Psvcv exhibited mortality accompanied by hemorrhage and ophthalmia. In a minigenome assay, the SVCV P protein failed to induce reporter protein expression when co-expressed with the SHRV N and L proteins, suggesting that the SVCV P protein is unable to form a ribonucleoprotein complex with SHRV N and L proteins. Therefore, the enhanced virulence of rSHRV-A-Psvcv resulting from the addition of the SVCV P gene can probably be attributed to the intrinsic properties of the SVCV P protein itself. Epithelioma papulosum cyprini (EPC) cells infected with rSHRV-A-Psvcv exhibited significantly lower type I interferon (IFN) responses compared to those infected with rSHRV-A-eGFP, suggesting that the SVCV P protein expressed by the recombinant SHRV inhibits type I IFN responses. Thus, the immune-suppressive activity of the SVCV P protein might be one of the causes of the enhanced virulence of rSHRV-A-Psvcv.

Spring viremia of carp virus (SVCV) represents a significant threat to cyprinids, particularly common carp (Cyprinus carpio). The disease caused by this virus is characterized by tissue necrosis and petechial hemorrhages. However, the pathogenesis of SVCV infection remains poorly understood. Pyroptosis, a recently identified form of programmed cell death, plays a crucial role in host-pathogen interactions and provides a novel approach for studying inflammation-related diseases. This study demonstrates that SVCV induces gasdermin Eb-dependent pyroptosis through activation of NLRP3 and initiation of cellular inflammatory death. This process results in the production of active caspase-B (p20), mature interleukin-1β, and lactate dehydrogenase release. The gasdermin Eb-dependent pyroptosis induced by SVCV is inhibited by treatment with either an NLRP3 inhibitor or a caspase-B inhibitor. Mechanistic investigations reveal that the SVCV-G protein plays a critical role in inducing pyroptosis, while the host-interacting protein, voltage-dependent anion channel 2 (VDAC2), is essential for inflammasome activation by maintaining NLRP3 protein stability. In vivo experiments show that DIDS, a VDAC2 inhibitor, reduces SVCV-induced pyroptosis and NLRP3 inflammasome activation, thereby alleviating inflammation and tissue damage in zebrafish. Furthermore, zebrafish larvae with VDAC2 gene knockdown exhibit reduced cellular damage from SVCV infection, resulting in increased survival. These findings elucidate a mechanism by which SVCV activates the NLRP3 inflammasome, inducing inflammation and pyroptosis, and provide novel insights into the pathogenesis of SVCV infection.

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

The complete genome of spring viraemia of carp virus (SVCV) strain A-1 isolated from cultured common carp (Cyprinus carpio) in China was sequenced and characterized. Reverse transcription-polymerase chain reaction (RT-PCR) derived clones were constructed and the DNA was sequenced. It showed that the entire genome of SVCV A-1 consists of 11,100 nucleotide base pairs, the predicted size of the viral RNA of rhabdoviruses. However, the additional insertions in bp 4633-4676 and bp 4684-4724 of SVCV A-1 were different from the other two published SVCV complete genomes. Five open reading frames (ORFs) of SVCV A-1 were identified and further confirmed by RT-PCR and DNA sequencing of their respective RT-PCR products. The 5 structural proteins encoded by the viral RNA were ordered 3'-N-P-M-G-L-5'. This is the first report of a complete genome sequence of SVCV isolated from cultured carp in China. Phylogenetic analysis indicates that SVCV A-1 is closely related to the members of the genus Vesiculovirus, family Rhabdoviridae.

Our study demonstrates the first application of the salmonid alphavirus-based replicon vector system (pSAV) as a DNA vaccine in a non-salmonid fish species, in common carp (Cyprinus carpio) against spring viraemia of carp virus (SVCV). SAV replicon encoding the glycoprotein of the SVCV was used as a DNA-layered plasmid, and its efficacy was compared with a previously described conventional DNA vaccine construct (pcDNA3.1 based vector) and with a control group (pcDNA3.1-empty-plasmid) in an SVCV challenge at a water temperature of 14 ± 1 °C. Vaccine prototypes were administered intramuscularly at a dose of 0.1 µg/g of fish (n = 25 per group). The DNA-layered SAV replicon resulted in 88% survival, compared to around 50% in all other groups. The DNA-layered pSAV vaccination induced the innate immune genes at the injection site, and increased IgM upregulation was also observed. Our preliminary results show that the SAV-based replicon construct may serve as a potential vaccine candidate for the protection of non-salmonid fish in the future provided that further clinical and field trials confirm its efficiency.

Tripartite motif-containing protein 32 (TRIM32) belongs to the tripartite motif (TRIM) family, which consists of a large number of proteins containing a RING (Really Interesting New Gene) domain, one or two B-box domains, and coiled coil motif followed by different C-terminal domains. The TRIM family is known to be implicated in multiple cellular functions, including antiviral activity. However, it is presently unknown whether TRIM32 of common carp (Cyprinus carpio) has the antiviral effect. In this study, the sequence, expression, and antiviral function of TRIM32 homolog from common carp were analyzed. The full-length coding sequence region of trim32 was cloned from common carp. The results showed that the expression of TRIM32 (mRNA) was highest in the brain, remained stably expressed during embryonic development, and significantly increased following spring viraemia of carp virus (SVCV) infection. Transient overexpression of TRIM32 in affected Epithelioma papulosum cyprinid cells led to significant decrease of SVCV production as compared to the control group. These results suggested a potentially important role of common carp TRIM32 in enhancing host immune response during SVCV infection both in vivo and in vitro.

Spring viraemia of carp (SVC) is an infectious disease responsible for severe economic losses for various cyprinid species, particularly common carp (Cyprinus carpio carpio). The causative agent is the SVC virus (SVCV), a member of the Sprivivirus genus, Rhabdoviridae family, and a List 1 pathogen notifiable by the World Organization for Animal Health. This study describes the diagnosis of an SVCV pathogen isolated in October 2015 from wild common carp inhabiting a natural lagoon in central Mexico. While neither an epidemic nor fish mortalities were reported, the collected killed specimens exhibited clinical signs of disease (e.g., exopthalmia, moderate abdominal distension and haemorrhaging, as well as internal haemorrhages and adhesions). Histological results of injuries were consistent with the pathology caused by SVCV. This finding was supported by the isolation of a virus in EPC and BF‐2 cells and subsequent RT‐PCR confirmation of SVCV. The phylogenetic analyses of partial SVCV glycoprotein gene sequences classified the isolates into the Ia genogroup. These findings make this the first report of SVCV detection in Mexico, extending the southern geographical range of SVCV within North America. However, since this pathogen was detected in fish inhabiting a natural body of water without tributaries or effluents, it is difficult to estimate the risk of SVCV for other wild/feral cohabitating cyprinid species in the lagoon. The status of this virus is also unknown for other bodies of water within this region.

ROS induced by spring viraemia of carp virus activate the inflammatory response via the MAPK/AP-1 and PI3K signaling pathways

Objetivo. Realizar el aislamiento del virus de la viremia primaveral de la carpa (SVCV) en ejemplares de carpa común (Cyprinus carpio), evaluar su crecimiento en diferentes tipos de células así como la supervivencia viral a diferentes temperaturas. Materiales y métodos. Diez carpas de entre 400-500 gramos de una laguna del centro de México fueron procesadas para el diagnóstico de SVCV mediante aislamiento en cultivo de células y RT-PCR semi-anidado. El virus obtenido se inoculó en células EPC, BF-2, CHSE-214 y RTG-2 para determinar diferencias de crecimiento de SVCV. Además, se evaluó la supervivencia del virus conservado a temperatura ambiente (TA 20-25°C), refrigeración (REF 4°C) y congelación (CONG -80°C) hasta once meses. Los órganos internos se procesaron para análisis histológico. Resultados. Los peces analizados no presentaron signos externos sugestivos de enfermedad, pero internamente e histopatológicamente se observaron lesiones sugestivas de infección sistémica. SVCV fue aislado en células EPC y BF-2 y confirmado por RT-PCR semi-anidado. SVCV únicamente indujo CPE en células EPC y BF-2 y fue negativo en RTG-2 y CHSE-214. El virus conservado a TA perdió viabilidad después de cuatro meses post-infección (mpi), siendo total a seis mpi; mientras REF y CONG fueron estables durante los once meses de estudio. Conclusiones. La infección subclínica por SVCV fue confirmada en carpas que presentaron lesiones histológicas asociadas a esta infección. SVCV únicamente causó CPE en células EPC y BF-2 y el virus conservó su viabilidad a 4ºC y -80°C hasta once meses; mientras que a TA se perdió en seis meses.