Molecular characterization of lymphocystis disease virus in Indian glass fish: first report from the Andaman Islands.

Here, we report the first detection of lymphocystis disease virus (LCDV) in Indian glass fish in the Andaman Islands, India. Microscopic examination revealed the presence of whitish clusters of nodules on the fish's skin, fins, and eyes. The histopathology of the nodules revealed typical hypertrophied fibroblasts. Molecular characterization of the major capsid protein (MCP) gene of the virus showed a significant resemblance to known LCDV sequences from Korea and Iran, with 98.92% and 97.85% sequence identity, respectively. Phylogenetic analysis confirmed that the MCP gene sequence of the virus belonged to genotype V. This study represents the first documented case of LCDV in finfish from the Andaman Islands, emphasizing the necessity for continued monitoring and research on the health of aquatic species in this fragile ecosystem.

Diversity, evolution, and emergence of fish viruses.

The production of aquatic animals has more than doubled over the last 50 years and is anticipated to continually increase. While fish are recognized as a valuable and sustainable source of nutrition, particularly in the context of human population growth and climate change, the rapid expansion of aquaculture coincides with the emergence of highly pathogenic viruses that often spread globally through aquacultural practices. Here, we provide an overview of the fish virome and its relevance for disease emergence, with a focus on the insights gained through metagenomic sequencing, noting potential areas for future study. In particular, we describe the diversity and evolution of fish viruses, for which the majority have no known disease associations, and demonstrate how viruses emerge in fish populations, most notably at an expanding domestic-wild interface. We also show how wild fish are a powerful and tractable model system to study virus ecology and evolution more broadly and can be used to identify the major factors that shape vertebrate viromes. Central to this is a process of virus-host co-divergence that proceeds over many millions of years, combined with ongoing cross-species virus transmission.

Extracellular traps in skin lesions infected with lymphocystis disease virus in black rockfish (Sebastes schlegelii).

The lymphocystis disease (LCD), caused by Lymphocystis disease virus (LCDV), is a benign and self-limiting disease described in a many freshwater and marine fish species. Hypertrophic fibroblasts and extensive aggregation of inflammatory cells are characteristics of LCD. In the present study, small animal imaging and ultrastructural investigations were carried out on the lymphocystis nodules of black rockfish (Sebastes schlegelii) naturally infected with lymphocystis iridovirus, to assess pathology, and the exudate with particular attention to the formation of extracellular traps (ETs) in vivo. Ex vivo were examined by nodules sections and primary cells stimulation. By histopathological analysis, the nodules contained infiltrated inflammatory cells and extensive basophilic fibrillar filaments at the periphery of the hypertrophied fibroblasts. ETs were assessed in nodules samples using indirect immunofluorescence to detect DNA and myeloperoxidase. Moreover, LCDV was able to infect peritoneal cells of black rockfish in vitro and induce the formation of ETs within 4 h. In summary, this study proved that ETs are involved in the response to LCDV infection and may be involved in formation of lymphoid nodules. Taken together, the findings provide a new perspective to determine the impact factors on the growth of nodules.

Growth and replication of infectious bursal disease virus in the fish cell line as an experimental vaccine.

Recently, several attempts have been made to replace egg-based with cell-based vaccines to prevent and control Infectious Bursal Disease Virus (IBDV). This study aimed to evaluate a new fish cell line (M99) for culturing and replicating IBDV. After observing complete cytopathic effects (CPE) on the M99 cell line, virus titers were determined using the TCID50 test, and the presence of the virus was confirmed using an RT-PCR test. Subsequently, 135 broiler chickens (14 days old) were randomly divided into three equal groups for immune response measurements: G1: immunized with a commercial vaccine, G2: immunized with an experimental vaccine, and G3: control. Antibody responses, bursal index, and histopathological evaluations were examined on different days after immunization. Based on the results, CPE of the virus was noticeable from the first passage, becoming complete by the third passage. The infectious titer of the virus was log106.9. Antibody titer measured 21 days after immunization in both vaccinated groups were significantly differed from the control group (p < 0.05). The results obtained from examining the bursal index and histopathological evaluations showed no significant difference between the studied groups at different times. Overall, this research is the first report on the successful cultivation of infectious bursal virus on a permanent cell line of fish origin, with the advantages of tolerance to a wide temperature range (26-40 degrees Celsius). Therefore, this cell line has potential for use to attenuate, cultivate, and adapt other pathogens to cold temperatures in future studies.

Establishment and characterization of a kidney cell line from hybrid snakehead (male Channa argus × female Channa maculata) and its susceptibility to hybrid snakehead rhabdovirus (HSHRV).

Hybrid snakehead (male Channa argus × female Channa maculata) is an emerging fish breed with increasing production levels. However, infection with hybrid snakehead rhabdovirus (HSHRV) critically affects hybrid snakehead farming. In this study, a fish cell line called CAMK, derived from the kidneys of hybrid snakehead, was established and characterized. CAMK cells exhibited the maximum growth rate at 28 °C in Leibovitz's-15 medium supplemented with 10% fetal bovine serum(FBS). Karyotyping revealed diploid chromosomes in 54% of the cells at the 50th passage (2n = 66), and 16S rRNA sequencing validated that CAMK cells originated fromhybrid snakehead, and the detection of kidney-specific antibodies suggested that it originated from kidney. .The culture was free from mycoplasma contamination, and the green fluorescent protein gene was effectively transfected into CAMK cells, indicating their potential use for in vitro gene expression investigations. Furthermore, qRT-PCR and immunofluorescence analysis revealed that HSHRV could replicate in CAMK cells, indicating that the cells were susceptible to the virus. Transmission electron microscopy revealed that the viral particles had bullet-like morphology. The replication efficiency of HSHRV was 107.33 TCID50/mL. Altogether, we successfully established and characterized a kidney cell line susceptible to the virus. These findings provide a valuable reference for further genetic and virological studies.

Deep mining of the Sequence Read Archive reveals major genetic innovations in coronaviruses and other nidoviruses of aquatic vertebrates.

Virus discovery by genomics and metagenomics empowered studies of viromes, facilitated characterization of pathogen epidemiology, and redefined our understanding of the natural genetic diversity of viruses with profound functional and structural implications. Here we employed a data-driven virus discovery approach that directly queries unprocessed sequencing data in a highly parallelized way and involves a targeted viral genome assembly strategy in a wide range of sequence similarity. By screening more than 269,000 datasets of numerous authors from the Sequence Read Archive and using two metrics that quantitatively assess assembly quality, we discovered 40 nidoviruses from six virus families whose members infect vertebrate hosts. They form 13 and 32 putative viral subfamilies and genera, respectively, and include 11 coronaviruses with bisegmented genomes from fishes and amphibians, a giant 36.1 kilobase coronavirus genome with a duplicated spike glycoprotein (S) gene, 11 tobaniviruses and 17 additional corona-, arteri-, cremega-, nanhypo- and nangoshaviruses. Genome segmentation emerged in a single evolutionary event in the monophyletic lineage encompassing the subfamily Pitovirinae. We recovered the bisegmented genome sequences of two coronaviruses from RNA samples of 69 infected fishes and validated the presence of poly(A) tails at both segments using 3'RACE PCR and subsequent Sanger sequencing. We report a genetic linkage between accessory and structural proteins whose phylogenetic relationships and evolutionary distances are incongruent with the phylogeny of replicase proteins. We rationalize these observations in a model of inter-family S recombination involving at least five ancestral corona- and tobaniviruses of aquatic hosts. In support of this model, we describe an individual fish co-infected with members from the families Coronaviridae and Tobaniviridae. Our results expand the scale of the known extraordinary evolutionary plasticity in nidoviral genome architecture and call for revisiting fundamentals of genome expression, virus particle biology, host range and ecology of vertebrate nidoviruses.

Acipenserid herpesvirus 2 genome and partial validation of a qPCR for its detection in white sturgeon Acipenser transmontanus.

White sturgeon Acipenser transmontanus is the primary species used for caviar and sturgeon meat production in the USA. An important pathogen of white sturgeon is acipenserid herpesvirus 2 (AciHV-2). In this study, 4 archived isolates from temporally discrete natural outbreaks spanning the past 30 yr were sequenced via Illumina and Oxford Nanopore Technologies platforms. Assemblies of approximately 134 kb were obtained for each isolate, and the putative ATPase subunit of the terminase gene was selected as a potential quantitative PCR (qPCR) target based on sequence conservation among AciHV-2 isolates and low sequence homology with other important viral pathogens. The qPCR was repeatable and reproducible, with a linear dynamic range covering 5 orders of magnitude, an efficiency of approximately 96%, an R2 of 0.9872, and an analytical sensitivity of 103 copies per reaction after 35 cycles. There was no cross-reaction with other known viruses or closely related sturgeon species, and no inhibition by sturgeon DNA. Clinical accuracy was assessed from white sturgeon juveniles exposed to AciHV-2 by immersion. Viral culture (gold standard) and qPCR were in complete agreement for both cell culture negative and cell culture positive samples, indicating that this assay has 100% relative accuracy compared to cell culture during an active outbreak. The availability of a whole-genome sequence for AciHV-2 and a highly specific and sensitive qPCR assay for detection of AciHV-2 in white sturgeon lays a foundation for further studies on host-pathogen interactions while providing a specific and rapid test for AciHV-2 in captive and wild populations.

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

IMPORTANCE: 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.

Cytokine Receptor-Like Factor 3 Negatively Regulates Antiviral Immunity by Promoting the Degradation of TBK1 in Teleost Fish.

The cytokine receptor-like factor 3 (Crlf3) belongs to the orphan class I cytokine receptors and is identified as a neuroprotective erythropoietin receptor. In previous studies of Crlf3, few focused on its role in innate immunity. Therefore, this study explored the regulatory role of Crlf3 in innate immunity. TANK-binding kinase 1 (TBK1) is a vital adaptor protein for the activation of the RLRs-MVAS-IRF3 antiviral signaling axis; thus, its expression and activity must be tightly regulated to maintain immune homeostasis and avoid undesirable effects. Here, we report that Crlf3 is a negative regulator of type I interferon production. The expression of Crlf3 is induced by poly(I·C) or Siniperca chuatsi rhabdovirus (SCRV) treatment. Silencing of Crlf3 enhanced poly(I·C)- and SCRV-induced type I interferon production, whereas overexpression of Crlf3 suppressed type I interferon production. Mechanistically, Crlf3 interacted with TBK1 via its N domain and then inhibited type I interferon production by promoting TBK1 proteasomal degradation through K48-linked polyubiquitination. Our study shows that Crlf3 is a key factor for viral escape from innate antiviral immunity in fish and provides a new perspective on mammalian resistance to viral invasion. IMPORTANCE The expression of Crlf3 was upregulated with SCRV invasion, which proved that Crlf3 was involved in the regulation of the antiviral immune response. In this study, we found that the existence of Crlf3 promoted the replication of SCRV. Therefore, it is reasonable to believe that SCRV evades innate immune attack with the assistance of Crlf3. In addition, we report that Crlf3 negatively regulates interferon (IFN) induction by promoting the degradation of TBK1 in fish. We showed that Crlf3 is evenly distributed in the cytoplasm and interacts with TBK1. Further studies showed that Crlf3 specifically mediates K48-linked ubiquitination of TBK1 and promotes TBK1 degradation, resulting in a marked inhibition of retinoic acid-inducible gene I (RIG-I) downstream signaling.

Aquatic Flaviviruses.

Flaviviruses are positive-sense single-stranded RNA viruses, including some well-known human pathogens such as Zika, dengue, and yellow fever viruses, which are primarily associated with mosquito and tick vectors. The vast majority of flavivirus research has focused on terrestrial environments; however, recent findings indicate that a range of flaviviruses are also present in aquatic environments, both marine and freshwater. These flaviviruses are found in various hosts, including fish, crustaceans, molluscs, and echinoderms. Although the effects of aquatic flaviviruses on the hosts they infect are not all known, some have been detected in farmed species and may have detrimental effects on the aquaculture industry. Exploration of the evolutionary history through the discovery of the Wenzhou shark flavivirus in both a shark and crab host is of particular interest since the potential dual-host nature of this virus may indicate that the invertebrate-vertebrate relationship seen in other flaviviruses may have a more profound evolutionary root than previously expected. Potential endogenous viral elements and the range of novel aquatic flaviviruses discovered thus shed light on virus origins and evolutionary history and may indicate that, like terrestrial life, the origins of flaviviruses may lie in aquatic environments.

Viromes of Freshwater Fish with Lacustrine and Diadromous Life Histories Differ in Composition.

Viruses that infect fish are understudied, yet they provide important evolutionary context to the viruses that infect terrestrial vertebrates. We surveyed gill tissue meta-transcriptomes collected from two species of native freshwater fish from Aotearoa New Zealand-Retropinna retropinna and Gobiomorphus cotidianus. A total of 64 fish were used for gill tissue meta-transcriptomic sequencing, from populations with contrasting life histories-landlocked (i.e., lacustrine) and diadromous-on the South Island and Chatham Islands. We observed that both viral richness and taxonomic diversity were significantly associated with life history and host species, with lacustrine R. retropinna characterised by higher viral alpha diversity than diadromous R. retropinna. Additionally, we observed transcripts of fish viruses from 12 vertebrate host-associated virus families, and phylogenetically placed eight novel RNA viruses and three novel DNA viruses in the Astroviridae, Paramyxoviridae, Orthomyxoviridae, Rhabdoviridae, Totiviridae, Poxviridae, Alloherpesviridae, and Adintoviridae in their evolutionary contexts. These results represent an important survey of the viruses that infect two widespread native fish species in New Zealand, and provide insight useful for future fish virus surveys.

Reservoirs of Red-Spotted Grouper Nervous Necrosis Virus (RGNNV) in Squid and Shrimp Species of Northern Alboran Sea.

The production of the aquaculture industry has increased to be equal to that of the world fisheries in recent years. However, aquaculture production faces threats such as infectious diseases. Betanodaviruses induce a neurological disease that affects fish species worldwide and is caused by nervous necrosis virus (NNV). NNV has a nude capsid protecting a bipartite RNA genome that consists of molecules RNA1 and RNA2. Four NNV strains distributed worldwide are discriminated according to sequence homology of the capsid protein encoded by RNA2. Since its first description over 30 years ago, the virus has expanded and reassortant strains have appeared. Preventive treatments prioritize the RGNNV (red-spotted grouper nervous necrosis virus) strain that has the highest optimum temperature for replication and the broadest range of susceptible species. There is strong concern about the spreading of NNV in the mariculture industry through contaminated diet. To surveil natural reservoirs of NNV in the western Mediterranean Sea, we collected invertebrate species in 2015 in the Alboran Sea. We report the detection of the RGNNV strain in two species of cephalopod mollusks (Alloteuthis media and Abralia veranyi), and in one decapod crustacean (Plesionika heterocarpus). According to RNA2 sequences obtained from invertebrate species and reported to date in the Mediterranean Sea, the strain RGNNV is predominant in this semienclosed sea. Neither an ecosystem- nor host-driven distribution of RGNNV were observed in the Mediterranean basin.

PI3K/AKT/p53 pathway inhibits infectious spleen and kidney necrosis virus infection by regulating autophagy and immune responses.

The PI3K/AKT/p53 signaling pathway is activated by various types of cellular stimuli or pathogenic infection, and then regulates fundamental cellular functions to combat these stimulations. Here, we studied the meaningful roles of PI3K/AKT/p53 in regulating cellular machine such as autophagy, immune responses, as well as antiviral activity in Chinese perch brain (CPB) cells infected by infectious spleen and kidney necrosis virus (ISKNV), which is an agent caused devastating losses in mandarin fish (Siniperca chuatsi) industry. We found that ISKNV infection induced up-regulation of host PI3K/AKT/p53 axis, but inhibited autophagy in CPB cells. Interestingly, activation of PI3K/AKT/p53 axis factors trough agonists or overexpression dramatically decreased host autophagy level, inhibited ISKNV replication, and elevated the expression of immune-related genes in CPB cells. In contrast, suppression of PI3K/AKT/p53 pathway by inhibitors or small interfering RNA (siRNA)-mediated gene silence increased the autophagy and ISKNV replication, but down-regulated immune responses in CPB cells. All these results indicate that PI3K/AKT/p53 pathway plays an important role in anti-ISKNV infection and can be used as a new target for controlling ISKNV disease.

Discovery of novel fish papillomaviruses: From the Antarctic to the commercial fish market.

Fish papillomaviruses form a newly discovered group broadly recognized as the Secondpapillomavirinae subfamily. This study expands the documented genomes of the fish papillomaviruses from six to 16, including one from the Antarctic emerald notothen, seven from commercial market fishes, one from data mining of sea bream sequence data, and one from a western gull cloacal swab that is likely diet derived. The genomes of secondpapillomaviruses are ∼6 kilobasepairs (kb), which is substantially smaller than the ∼8 kb of terrestrial vertebrate papillomaviruses. Each genome encodes a clear homolog of the four canonical papillomavirus genes, E1, E2, L1, and L2. In addition, we identified open reading frames (ORFs) with short linear peptide motifs reminiscent of E6/E7 oncoproteins. Fish papillomaviruses are extremely diverse and phylogenetically distant from other papillomaviruses suggesting a model in which terrestrial vertebrate-infecting papillomaviruses arose after an evolutionary bottleneck event, possibly during the water-to-land transition.

Singapore Grouper Iridovirus Disturbed Glycerophospholipids Homeostasis: Cytosolic Phospholipase A2 Was Essential for Virus Replication.

Singapore grouper iridovirus (SGIV), belonging to genus Ranavirus, family Iridoviridae, causes great economic losses in the aquaculture industry. Previous studies demonstrated the lipid composition of intracellular unenveloped viruses, but the changes in host-cell glyceophospholipids components and the roles of key enzymes during SGIV infection still remain largely unknown. Here, the whole cell lipidomic profiling during SGIV infection was analyzed using UPLC-Q-TOF-MS/MS. The lipidomic data showed that glycerophospholipids (GPs), including phosphatidylcholine (PC), phosphatidylserine (PS), glycerophosphoinositols (PI) and fatty acids (FAs) were significantly elevated in SGIV-infected cells, indicating that SGIV infection disturbed GPs homeostasis, and then affected the metabolism of FAs, especially arachidonic acid (AA). The roles of key enzymes, such as cytosolic phospholipase A2 (cPLA2), 5-Lipoxygenase (5-LOX), and cyclooxygenase (COX) in SGIV infection were further investigated using the corresponding specific inhibitors. The inhibition of cPLA2 by AACOCF3 decreased SGIV replication, suggesting that cPLA2 might play important roles in the process of SGIV infection. Consistent with this result, the ectopic expression of EccPLA2α or knockdown significantly enhanced or suppressed viral replication in vitro, respectively. In addition, the inhibition of both 5-LOX and COX significantly suppressed SGIV replication, indicating that AA metabolism was essential for SGIV infection. Taken together, our results demonstrated for the first time that SGIV infection in vitro disturbed GPs homeostasis and cPLA2 exerted crucial roles in SGIV replication.

Functionality of the putative surface glycoproteins of the Wuhan spiny eel influenza virus.

A panel of influenza virus-like sequences were recently documented in fish and amphibians. Of these, the Wuhan spiny eel influenza virus (WSEIV) was found to phylogenetically cluster with influenza B viruses as a sister clade. Influenza B viruses have been documented to circulate only in humans, with certain virus isolates found in harbor seals. It is therefore interesting that a similar virus was potentially found in fish. Here we characterize the putative hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins of the WSEIV. Functionally, we show that the WSEIV NA-like protein has sialidase activity comparable to B/Malaysia/2506/2004 influenza B virus NA, making it a bona fide neuraminidase that is sensitive to NA inhibitors. We tested the functionality of the HA by addressing the receptor specificity, stability, preferential airway protease cleavage, and fusogenicity. We show highly specific binding to monosialic ganglioside 2 (GM2) and fusogenicity at a range of different pH conditions. In addition, we found limited antigenic conservation of the WSEIV HA and NA relative to the B/Malaysia/2506/2004 virus HA and NA. In summary, we perform a functional and antigenic characterization of the glycoproteins of WSEIV to assess if it is indeed a bona fide influenza virus potentially circulating in ray-finned fish.

Evidence for a Novel Antiviral Mechanism of Teleost Fish: Serum-Derived Exosomes Inhibit Virus Replication through Incorporating Mx1 Protein.

Exosomes are associated with cancer progression, pregnancy, cardiovascular diseases, central nervous system-related diseases, immune responses and viral pathogenicity. However, study on the role of exosomes in the immune response of teleost fish, especially antiviral immunity, is limited. Herein, serum-derived exosomes from mandarin fish were used to investigate the antiviral effect on the exosomes of teleost fish. Exosomes isolated from mandarin fish serum by ultra-centrifugation were internalized by mandarin fish fry cells and were able to inhibit Infectious spleen and kidney necrosis virus (ISKNV) infection. To further investigate the underlying mechanisms of exosomes in inhibiting ISKNV infection, the protein composition of serum-derived exosomes was analyzed by mass spectrometry. It was found that myxovirus resistance 1 (Mx1) was incorporated by exosomes. Furthermore, the mandarin fish Mx1 protein was proven to be transferred into the recipient cells though exosomes. Our results showed that the serum-derived exosomes from mandarin fish could inhibit ISKNV replication, which suggested an underlying mechanism of the exosome antivirus in that it incorporates Mx1 protein and delivery into recipient cells. This study provided evidence for the important antiviral role of exosomes in the immune system of teleost fish.

Discovery and Genetic Characterization of Novel Paramyxoviruses Related to the Genus Henipavirus in Crocidura Species in the Republic of Korea.

Paramyxoviruses, negative-sense single-stranded RNA viruses, pose a critical threat to human public health. Currently, 78 species, 17 genera, and 4 subfamilies of paramyxoviruses are harbored by multiple natural reservoirs, including rodents, bats, birds, reptiles, and fish. Henipaviruses are critical zoonotic pathogens that cause severe acute respiratory distress and neurological diseases in humans. Using reverse transcription-polymerase chain reaction, 115 Crocidura species individuals were examined for the prevalence of paramyxovirus infections. Paramyxovirus RNA was observed in 26 (22.6%) shrews collected at five trapping sites, Republic of Korea. Herein, we report two genetically distinct novel paramyxoviruses (genus: Henipavirus): Gamak virus (GAKV) and Daeryong virus (DARV) isolated from C. lasiura and C. shantungensis, respectively. Two GAKVs and one DARV were nearly completely sequenced using next-generation sequencing. GAKV and DARV contain six genes (3'-N-P-M-F-G-L-5') with genome sizes of 18,460 nucleotides and 19,471 nucleotides, respectively. The phylogenetic inference demonstrated that GAKV and DARV form independent genetic lineages of Henipavirus in Crocidura species. GAKV-infected human lung epithelial cells elicited the induction of type I/III interferons, interferon-stimulated genes, and proinflammatory cytokines. In conclusion, this study contributes further understandings of the molecular prevalence, genetic characteristics and diversity, and zoonotic potential of novel paramyxoviruses in shrews.

Special Issue "Emerging Viruses in Aquaculture".

According to the 2018 FAO report on aquaculture, there are 598 species of finfish, molluscs, crustaceans, and other organisms used in aquafarming around the world [...].