“OLD” AND “NEW” BACTERIA ASSOCIATED WITH GRANULOMAS IN AQUARIUM FISH

Cichlids include several fish species having a high economic value in the field of aquaculture. The ornamental fish export trade is mostly based on fish from the african Lake Malawi. Despite their huge economic importance, management of ornamental fisheries is challenged by a paucity of information on the status of the exploited fish stock. The possibility of guaranteeing healthy animals is of paramount importance and has several implications, both for commercial and sanitary reasons. Grossly, cutaneous nodules and black spots are pathological findings frequently encountered in fish, suggesting a meandering disease without a specific etiologic association. Ornamental fish species are plagued by mycobacteriosis, which is quite classically associated with granulomas. This work focuses on debilitated ornamental cichlids presenting cutaneous nodules and black spots and sampled during routinary managing activities held in an aquarium commercial facility; the fish underwent pathological analysis and the presence of pathogens was investigated through a molecular approach. In particular, the presence of lymphocystis disease virus (LCDV), typically associated with cutaneous nodular disease, was excluded.Histologically the granulomas were localized in the spleen, sometimes extending to the other visceral organs. Bacterial Heat-Shock Protein 65 PCR products were detected in tissues associated to granulomas and molecular investigation identified Mycobacterium spp. in two samples and Cutibacterium acnes in seven samples. Variably sized round “Hamazaki-Wesenberg-like” bodies were immunolabeled with C. acnes antibody within macrophages forming the granuloma in the spleen. C. acnes has been recently detected by Next Generation Sequencing in the microbiome of internal organs of fish. The role of C. acnes within internal fish tissues deserves attention; its role as potential granulomatogenous agent, is taken in consideration.

Genome Sequence of Lymphocystis Disease Virus 2 LCDV-JP_Oita_2018, Isolated from a Diseased Japanese Flounder (Paralichthys olivaceus) in Japan

Here, we present the genome sequence of lymphocystis disease virus 2 LCDV-JP_Oita_2018 (genus Lymphocystivirus , family Iridoviridae ), which was isolated from a diseased Japanese flounder ( Paralichthys olivaceus ) in Japan. The LCDV-JP_Oita_2018 genome was assembled into a circular contig of 186,627 bp, with 140 predicted protein-coding genes and a GC content of 27%.

Evaluation of Gilthead Seabream (Sparus aurata) Immune Response after LCDV-Sa DNA Vaccination

Lymphocystis disease is the main viral pathology reported in gilthead seabream. Its etiological agent is Lymphocystis disease virus 3 (LCDV-Sa), genus Lymphocystivirus, family Iridoviridae. There are no effective treatments or vaccines for LCDV control, thus the main aim of this study was to develop a DNA vaccine, and to evaluate both the protection conferred against LCDV-Sa infection and the immune response in vaccinated fish. The vaccine was constructed by cloning the mcp gene (ORF LCDVSa062R) into pcDNA3.1/NT-GFP-TOPO. Two independent vaccination trials were conducted. In the first one, 5–7 g fish were intramuscularly injected with the vaccine (pcDNA-MCP) or the empty-plasmid, and the distribution and expression of the vaccine was investigated. Furthermore, vaccinated fish were challenged with LCDV-Sa in order to access the protective capacity of the vaccine. In the second trial, 70–100 g fish were vaccinated as specified, and the immune response was evaluated analyzing the expression of 23 immune-related genes and the production of specific antibodies. The results showed that the vaccine triggers an immune response characterized by the overexpression of genes relating to the inflammatory process, but not the innate antiviral immunity relating to type I IFN (interferon), and also induces the production of specific neutralizing antibodies, which could explain the protection against LCDV-Sa in vaccinated fish.

Characterization of Viral Insulins Reveals White Adipose Tissue Specific Effects in Mice

ABSTRACTMembers of the insulin/IGF superfamily are well conserved across the evolutionary tree. We recently showed that four viruses in the Iridoviridae family possess genes that encode proteins highly homologous to human insulin/IGF-1. Using chemically synthesized single chain (sc), i.e. IGF-1-like, forms of the viral insulin/IGF-1 like peptides (VILPs), we previously showed that they can stimulate human receptors. Because these peptides possess potential cleavage sites to form double chain (dc), i.e. more insulin-like, VILPs, in this study, we have characterized dc forms of VILPs for Grouper iridovirus (GIV), Singapore grouper iridovirus (SGIV) and Lymphocystis disease virus-1 (LCDV-1). GIV and SGIV dcVILPs bind to both isoforms of human insulin receptor (IR-A, IR-B) and to the IGF1R, and for the latter show higher affinity than human insulin. These dcVILPs stimulate IR and IGF1R phosphorylation and post-receptor signaling in vitro and in vivo. Both GIV and SGIV dcVILPs stimulate glucose uptake in mice. In vivo infusion experiments in awake mice revealed that while insulin (0.015 nmol/kg/min) and GIV dcVILP (0.75nmol/kg/min) stimulated a comparable glucose uptake in heart, skeletal muscle and brown adipose tissue, GIV dcVILP stimulated ~2 fold higher glucose uptake in white adipose tissue (WAT) compared to insulin. This was associated with increased Akt phosphorylation and glucose transporter type 4 (GLUT4) gene expression compared to insulin. Taken together, these results show that GIV and SGIV dcVILPs are active members of the insulin superfamily with unique characteristics. Elucidating the mechanism of tissue specificity for GIV dcVILP will help us to better understand insulin action, design new analogues that specifically target the tissues, and provide new insights into their potential role in disease.

Lymphocystis Disease Virus (Iridoviridae) Enters Flounder (Paralichthys olivaceus) Gill Cells via a Caveolae-Mediated Endocytosis Mechanism Facilitated by Viral Receptors

In previous research, voltage-dependent anion channel protein 2 (VDAC2) and the receptor of activated protein C kinase 1 (RACK1) in flounder (Paralichthys olivaceus) were confirmed as functional receptors for lymphocystis disease virus (LCDV) entry; however, the underlying mechanism of VDAC2- and RACK1-mediated LCDV entry remains unclear. In this study, we elucidated the endocytosis pathway of LCDV entry into flounder gill (FG) cells by treatment with specific inhibitory agents, siRNAs, and co-localization analysis. LCDV entry was significantly inhibited by the disruption of caveolae-mediated endocytosis, dynamin, and microtubules, and the knockdown of caveoline-1 and dynamin expression, but was not inhibited by the disruption of clathrin-mediated endocytosis, micropinocytosis, or low-pH conditions. The disruption of caveolae-mediated and clathrin-mediated endocytosis was verified by the internalization of cholera toxin subunit B (CTB) and transferrin, respectively. Confocal immunofluorescence assay demonstrated that LCDV was co-localized with VDAC2 and RACK1, CTB was co-localized with VDAC2 and RACK1 and partially with LCDV, but transferrin was not co-localized with LCDV, VDAC2, or RACK1, indicating that LCDV utilized the same pathway as CTB, i.e., caveolae-mediated endocytosis. This was different from the pathway of transferrin, which used clathrin-mediated endocytosis. Furthermore, caveolin-1 was co-localized with LCDV, VDAC2, and RACK1, suggesting that caveolin-1 was involved in LCDV entry. These results revealed for the first time that LCDV entered into FG cells via caveolae-mediated endocytosis facilitated by VDAC2 and RACK1 receptors, relying on dynamin and microtubules in a pH-independent manner, which provided new insight into the molecular mechanisms of LCDV entry and potential for the development of antiviral agents, expanding our understanding of iridovirus infection.

Complete genome sequence and analysis of a novel lymphocystivirus detected in whitemouth croaker (Micropogonias furnieri): lymphocystis disease virus 4

A novel lymphocystivirus causing typical signs of lymphocystis virus disease in whitemouth croaker (Micropogonias furnieri) on the coast of Uruguay was detected and described recently. Based on genetic analysis of some partially sequenced core genes, the virus seemed to differ from previously described members of the genus Lymphocystivirus. In this study, using next-generation sequencing, the whole genome of this virus was sequenced and analysed. The complete genome was found to be 211,086 bp in size, containing 148 predicted protein-coding regions, including the 26 core genes that seem to have a homologue in every iridovirus genome sequenced to date. Considering the current species demarcation criteria for the family Iridoviridae (genome organization, G+C content, amino acid sequence similarity, and phylogenetic relatedness of the core genes), the establishment of a novel species (“Lymphocystis disease virus 4”) in the genus Lymphocystivirus is suggested.