Microalgae as fishmeal alternatives in aquaculture: current status, existing problems, and possible solutions.

Fishmeal is an indispensable ingredient for most aquatic animals. However, the finite supply and escalating price of fishmeal seriously limit its use in aquaculture. Thus the development of new, sustainable protein ingredients has been a research focus. Microalgae are potential fishmeal alternatives owing to their high protein content and balanced amino acid profile. Studies suggest that suitable replacement of fishmeal with microalgae is beneficial for fish growth performance, but excessive replacement would induce poor growth and feed utilization. Therefore, this paper aims to review research on the maximum substitutional level of fishmeal by microalgae and propose the main issues and possible solutions for fishmeal replacement by microalgae. The maximum replacement level is affected by microalgal species, fish feeding habits, quality of fishmeal and microalgal meals, and supplemental levels of fishmeal in the control group. Microalgae could generally replace 100%, 95%, 95%, 64.1%, 25.6%, and 18.6% fishmeal protein in diets of carp, shrimp, catfish, tilapia, marine fish, and salmon and trout, respectively. The main issues with fishmeal replacement using microalgae include low production and high production cost, poor digestibility, and anti-nutritional factors. Possible solutions to these problems are recommended in this paper. Overall, microalgae are promising fishmeal alternatives in aquaculture.

Influence of multi-walled carbon nanotubes on the toxicity of ZnO nanoparticles in the intestinal histopathology, apoptosis, and microbial community of common carp.

In recent years, carbon nanotubes (CNTs) have gained tremendous attention due to their widespread application. Previous research indicated that carbon nanomaterials can affect the toxicity of some pollutants. In this study, we investigated the influence of multi-walled CNTs (MWCNTs) on the toxicity of ZnO nanoparticles (ZnONPs) in the intestine of common carp (Cyprinus carpio). After four-week exposure, histopathological observation and TUNEL assay showed concentration ratio-dependent intestinal lesions and apoptosis, with the most severe in the HSC-ZnONPs group (50 mg L-1 ZnONPs and 2.5 mg L-1 MWCNTs), less severe in the ZnONPs group (50 mg L-1 ZnONPs) and the least in the LSC-ZnONPs group (50 mg L-1 ZnONPs and 0.25 mg L-1 MWCNTs). Furthermore, ICP-OES indicated that intercellular zinc accumulation was significantly decreased by the presence of the MWCNTs, which suggested the varied contribution of ZnONPs to intestine injury in different groups. Moreover, 16 s rDNA sequencing revealed that ZnONPs alone and in combination with MWCNTs significantly altered the microbial community diversity and composition of the gut microbiota compared with controls. In addition, the predominant phylum, class, order, family, and genus were significantly different among these groups. In conclusion, the influence of MWCNTs on the toxicity of ZnONPs was related to the concentration and concentration ratio of the mixture.

Biological functions of circRNAs and their advance on skeletal muscle development in bovine.

The development of skeletal muscle in animals is a complex biological process, which are strictly and precisely regulated by many genes and non-coding RNAs. Circular RNA (circRNA) was found as a novel class of functional non-coding RNA with ring structure in recent years, which appears in the process of transcription and is formed by covalent binding of single-stranded RNA molecules. With the development of sequencing and bioinformatics analysis technology, the functions and regulation mechanisms of circRNAs have attracted great attention due to its high stability characteristics. The role of circRNAs in skeletal muscle development have been gradually revealed, where circRNAs were involved in various biological processes, such as proliferation, differentiation, and apoptosis of skeletal muscle cells. In this review, we summarized the current studies advance of circRNAs involved in skeletal muscle development in bovine, and hope to gain a deeper understanding of the functional roles of the circRNAs in muscle growth. Our results will provide some theoretical supports and great helps for the genetic breeding of this species, and aiming at improving bovine growth and development and preventing muscle diseases.

Multi-walled carbon nanotube induced liver injuries possibly by promoting endoplasmic reticulum stress in Cyprinus carpio.

The mass production and discharge of carbon nanotubes (CNTs) to the water environment are of great concern since they threaten the health of organisms in the aquatic ecosystem. CNTs induce multi-organ injuries in fish, but limited literature is available regarding the mechanisms involved. In the present study, juvenile common carp (Cyprinus carpio) were exposed to multi-walled carbon nanotubes (MWCNTs) (0.25 mg L-1 and 2.5 mg L-1) for four weeks. MWCNTs caused dose-dependent alterations in the pathological morphology of liver tissues. Ultrastructural changes manifested as nuclear deformation, chromatin condensation, endoplasmic reticulum (ER) disorderly arrangement, mitochondria vacuolation, and mitochondrial membrane destruction. TUNEL analysis indicated that the apoptosis rate in hepatocytes markedly increased upon exposure to MWCNTs. Moreover, the apoptosis was confirmed by significant upregulation of mRNA levels of apoptosis-related genes (Bcl-2, XBP1, Bax, and caspase3) in MWCNTs-exposure groups, except for Bcl-2 expression which was not significantly changed in HSC groups (2.5 mg L-1 MWCNTs). Furthermore, real-time PCR assay indicated the increased expression of ER stress (ERS) marker genes (GRP78, PERK, and eIF2α) in the exposure groups compared to the control groups, suggesting that the PERK/eIF2α signaling pathway involved in the injuries of the liver tissue. Overall, the results above indicate that MWCNTs induce ERS by activating the PERK/eIF2α pathway in the liver of common carp, and resulted in the initiation of apoptosis procedure.

Identification and functional analysis of miRNAs in skeletal muscle of juvenile and adult largemouth bass, Micropterus salmoides.

MicroRNAs (miRNAs) are considered key regulators to post-transcriptionally regulate gene expression affecting multiple biological activities. However, the developmental process of fish skeletal muscles is regulated by complicated molecular mechanism that has not been completely well-described. In this study, two small RNAs libraries from skeletal muscle of juvenile as well as adult largemouth bass (LMB) were obtained and sequenced using deep sequencing to investigate the development-related miRNAs. We identified an overall number of 486 already recognized miRNAs in addition to 43 novel miRNAs. Comparison of two different skeletal muscle development stages led to the identification of 220 differently expressed miRNAs between juvenile and adult LMB containing 116 up-regulated as well as 104 down-regulated miRNAs. Of them, confirmation of some differently expressed miRNAs was performed via a stem-loop qRT-PCR, which exhibited differently expressed level in juvenile and adult LMB. Furthermore, GO and KEGG enrichment analyses of targets of differently-expressed miRNAs were carried out. Additionally, the analysis of miRNAs-targets interaction network showed that miR-181b-5p_R-1, miR-725 and miR-103 as the nodal miRNAs has over 20 target genes. Moreover, miR-103 could bind the 3'-UTR of actr8, which was validated via dual-luciferase reporter assay. It has been reasonably hypothesized that miR-103 may play a crucial role, which regulate skeletal muscle development of LMB. The present study provides the first identification of miRNA expression profiles at two different skeletal muscle development stages in LMB. Results may be valuable in interpreting the regulatory role miRNAs plays in the growth and developmental process of skeletal muscle and its possible use in LMB breeding.

Toxicity of amine-functionalized single-carbon nanotube (NH2 f-SWCNT) to Channel Catfish (Ietalurus Punetaus): Organ pathologies, oxidative stress, inflammation, and apoptosis.

The rapid development of carbon nanotubes (CNTs) in the field of fish disease control and prevention raises concerns about the toxicity and safe use in fish. This study was performed to assess the effect on histological changes, oxidative stress related markers in response to various concentrations of amine-functionalized single carbon nanotubes (NH2 f-SWCNT) (1, 10 and 100 mg kg-1 fish) in Channel Catfish (Ietalurus Punetaus) for up to 10 days. Moreover, pro-inflammatory cytokine genes and apoptotic genes were analyzed to obtain a better understanding of molecular mechanism of NH2 f-SWCNT induced toxicity. As a result, intraperitoneal (i.p.) administration of NH2 f-SWCNT caused dose-dependent and time-dependent injuries in the sampled tissues. In comparison with the control groups, decrease of catalase (CAT) activity and superoxide dismutase (SOD), and increase of malondialdehyde (MDA) and lactin dehydrogenase (LDH) were observed in all treatments. Real-time PCR assay showed inflammatory response with dose-dependent increase of tumor necrosis factor alpha (TNFα) and transient increase of interleukin 1ß (IL-1ß) in the liver. NH2 f-SWCNT administration induced increase of p38 as well as caspase-3 in all treatments compared to the control groups, indicating the involvement of p38-MAPK cascade and caspase-3 cascade in liver cell apoptosis. Overall, we conclude that NH2 f-SWCNT exert effects by direct injury and indirectly oxidative stress, resulting in inflammation and apoptosis, which provides data for understanding of the biological mechanisms underlying the toxicity of CNTs in fish.

Emerging landscape of circHIPK3 and its role in cancer and other diseases (Review).

Circular RNAs (circRNAs) are a special class of recently re­discovered RNAs, which are covalently closed ring RNA molecules. circRNAs have been reported to possess multiple functions and are considered crucial regulators of several processes, and are therefore gaining increasing attention. In recent years, increasing evidence has shown that circRNAs are implicated in several crucial biological processes via regulation of gene expression, and their dysregulation is also associated with the development of numerous diseases, particularly acting as oncogenic or tumor­suppressor molecules in cancer. Furthermore, circRNAs are involved in cell proliferation, differentiation, apoptosis, invasion and metastasis. In the present review, the biogenesis and functions of circRNAs are described, with a focus on the most recent research advances and the emerging roles of circular homeodomain­interacting protein kinase 3 (circHIPK3) in human diseases. The present review may provide novel avenues for research on the roles of circHIPK3 as a clinical diagnostic and prognostic biomarker, as well as highlighting promising therapeutic targets for certain diseases and cancer.

Rana grylio virus 43R encodes an envelope protein involved in virus entry.

Rana grylio virus (RGV), a member of genus Ranavirus in the family Iridoviridae, is a viral pathogen infecting aquatic animal. RGV 43R has homologues only in Ranavirus and contains a transmembrane (TM) domain, but its role in RGV infection is unknown. In this study, 43R was determined to be associated with virion membrane. The transcripts encoding 43R and the protein itself appeared late in RGV-infected EPC cells and its expression was blocked by viral DNA replication inhibitor, indicating that 43R is a late expressed protein. Subcellular localization showed that 43R-EGFP fusion protein distributed in cytoplasm of EPC cells and that TM domain is essential for its distribution in cytoplasm. 43R-EGFP fusion protein colocalized with viral factories in RGV-infected cells. A recombinant RGV deleting 43R (Δ43R-RGV) was constructed by homologous recombination to investigate its role in virus infection. Compared with wild type RGV, the ability of Δ43R-RGV to induce the cytopathic effect and its virus titers were significantly reduced. Furthermore, it is revealed that 43R deletion significantly inhibited viral entry but did not influence viral DNA replication by measuring and comparing the DNA levels of RGV and Δ43R-RGV in the infected cells at the early stage of infection. RGV neutralization with anti-43R serum reduced the virus titer. Therefore, these data showed that RGV 43R is a late gene that encodes an envelope protein involved in RGV entry.

Protective Immunity Induced by DNA Vaccination against Ranavirus Infection in Chinese Giant Salamander Andrias davidianus.

Andrias davidianus ranavirus (ADRV) is an emerging viral pathogen that causes severe systemic hemorrhagic disease in Chinese giant salamanders. There is an urgent need for developing an effective vaccine against this fatal disease. In this study, DNA vaccines containing the ADRV 2L gene (pcDNA-2L) and the 58L gene (pcDNA-58L) were respectively constructed, and their immune protective effects were evaluated in Chinese giant salamanders. In vitro and in vivo expression of the vaccine plasmids were confirmed in transfected cells and muscle tissues of vaccinated Chinese giant salamanders by using immunoblot analysis or RT-PCR. Following ADRV challenge, the Chinese giant salamanders vaccinated with pcDNA-2L showed a relative percent survival (RPS) of 66.7%, which was significant higher than that in Chinese giant salamanders immunized with pcDNA-58L (RPS of 3.3%). Moreover, the specific antibody against ADRV was detected in Chinese giant salamanders vaccinated with pcDNA-2L at 14 and 21 days post-vaccination by indirect enzyme-linked immunosorbent assay (ELISA). Transcriptional analysis revealed that the expression levels of immune-related genes including type I interferon (IFN), myxovirus resistance (Mx), major histocompatibility complex class IA (MHCIA), and immunoglobulin M (IgM) were strongly up-regulated after vaccination with pcDNA-2L. Furthermore, vaccination with pcDNA-2L significantly suppressed the virus replication, which was seen by a low viral load in the spleen of Chinese giant salamander survivals after ADRV challenge. These results indicated that pcDNA-2L could induce a significant innate immune response and an adaptive immune response involving both humoral and cell-mediated immunity that conferred effective protection against ADRV infection, and might be a potential vaccine candidate for controlling ADRV disease in Chinese giant salamanders.

Identification of the conserved and novel microRNAs by deep sequencing and prediction of their targets in Topmouth culter.

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that play important roles in regulation of almost all biological processes in organisms. Topmouth culter is an economically important fish species in the Chinese freshwater polyculture system for its delicacy and high economic value. However, to date, no miRNAs have been reported in Topmouth culter species. In this study, the liver, muscle, spleen and kidney of Topmouth culter are sampled and used for construction of a small RNA library for deep sequencing. A total of 273 conserved miRNAs/miRNAs-star and 8 novel miRNAs/miRNAs-star were discovered, these miRNAs belong to 86 different miRNA families. There is a strong base bias on miRNA nucleotide compositions at certain positions. U is the dominant nucleotide, particularly at the beginning, middle, and end of conserved miRNAs. The expression of all 8 novel identified miRNAs in Topmouth culter was validated by using stem-loop qRT-PCR. Furthermore, the potential targets were predicted for these miRNAs, which were found to be involved in diverse biological processes. Our study is the first significantly enriches the Topmouth culter miRNA repertoire and provides a reference for further elucidation of complex miRNA-mediated regulatory networks for gene expression in the growth and developmental progression of Topmouth culter.

Cloning, characterization and mRNA expression of interleukin-6 in blunt snout bream (Megalobrama amblycephala).

In the present study, the interleukin-6 gene (IL-6) cDNA in blunt snout bream (Megalobrama amblycephala) was identified and its expression profiles under ammonia stress and bacterial challenge were investigated. The IL-6 sequence consisted of 1045 bp, including a 696 bp ORF which translated into a 232 amino acid (AA) protein. The protein contained a putative signal peptide of 24 AA in length. IL-6 expression analysis showed that the it is differentially expressed in various tissues under normal conditions and the highest IL-6 level was observed in the intestine tissue, followed by the liver, and then in the gills. Under ammonia stress, the IL-6 mRNA level both in spleens and intestine increased significantly (P < 0.05), with the maximum levels attained at 6 h, 12 h (72, 10-fold, respectively). Thereafter, they all significantly decreased (P < 0.01) and returned to the basal value within 48 h. Whereas, in livers it slightly decreased at 3 h firstly (0.5-fold), and then significantly (P < 0.05) increased with the maximum level attained 12 h (3-fold). Further expression analysis showed that the mRNA level of IL-6 in spleens, intestine and livers of blunt snout bream all increased significantly (P < 0.05), with maximum values attained at 6 h, 3 h, 6 h (10, 6, 18-fold, respectively) after Aeromonas hydrophila (A. hydrophila) injection, and then decreased to the basal value within 24 h which suggested that IL-6 was involved in the immune response to A. hydrophila. The cloning and expression analysis of the IL-6 provide theoretical basis to further study the mechanism of anti-adverseness and expression characteristics under stress conditions in blunt snout bream.

Development and application of monoclonal antibodies for detection and analysis of aquareoviruses.

Monoclonal antibodies (mAbs) play an important role in detection of aquareoviruses. Three mAbs against grass carp reovirus (GCRV) were prepared. Isotyping revealed that all three mAbs were of subclass IgG2b. Western blot assay showed that all three mAbs reacted with GCRV 69 kDa protein (the putative VP5). In addition to the 69 kDa protein of GCRV, mAb 4B6 also recognize a 54 kDa protein. All three mAbs were used for detecting aquareovirus by Western blot assay and indirect immunofluorescence assay (IFA). All of them reacted with GCRV, and mAb 4A3 could also react with turbot Scophthalmus maximus reovirus (SMReV) and largemouth bass Microptererus salmonides reovirus (MsReV). Viral antigens were only observed in the cytoplasm of infected cells. Finally, syncytia formation was observed with light microscopy and fluorescence microscopy using fluorescein labelled 4A3 mAb at various times post-infection. Syncytia were observed at 36 hr post-infection (hpi) by light microscopy and at 12 hpi by fluorescence microscopy. The immunofluorescence based assay allowed earlier detection of virus than observation of virus-induced cytopathic effect (CPE) assay in inoculated cell cultures. The sensitivity and specificity of these mAbs may be useful for diagnosis and monitoring of aquareoviruses.

Fish reovirus GCReV-109 VP33 protein elicits protective immunity in rare minnows.

Grass carp reovirus strain 109 (GCReV-109) was previously isolated from a grass carp (Ctenopharyngodon idellus) with hemorrhagic disease, and its complete genome has been sequenced. However, the infectivity of GCReV-109 has not been studied, and the viral protein VP33, encoded on genome segment S11, had no detectable sequence homology to other known reovirus proteins. In this study, we characterized GCReV-109 infections in vivo and in vitro, as well as the VP33 protein. Infectivity analysis showed that GCReV-109 caused severe hemorrhagic disease and 100% mortality at dilutions up to 10(-4) in rare minnows (Gobiocypris rarus) by 8 days postinfection, but no visible cytopathic effect was observed in GCReV-109-infected subcultured grass carp muscle (GCM) cells. To confirm that GCReV-109 could be propagated in GCM cells, three virus genome segments were detected by RT-PCR, and large numbers of virus particles were observed by transmission electron microscopy in samples from the infected GCM cells. The suspension of GCReV-109-infected GCM cells was pathogenic to rare minnows. VP33 protein was expressed and purified for generation of an anti-VP33 antiserum. In western blot analysis of purified GCReV-109 particles, the antiserum specifically recognized a protein band (approximately 33 kDa). This revealed that VP33 is a major structural protein of GCReV-109 that might have immunogenic properties. The protective efficacy of the anti-VP33 antiserum against GCReV-109 infection was tested. The death of infected fish was delayed and the mortality fell to 10% when fish were treated with the anti-VP33 antiserum, suggesting that it might be useful for the prevention and control of fish reoviral disease.

Genome analysis and gene nblA identification of Microcystis aeruginosa myovirus (MaMV-DC) reveal the evidence for horizontal gene transfer events between cyanomyovirus and host.

The genome sequence, genetic characterization and nblA gene function of Microcystis aeruginosa myovirus isolated from Lake Dianchi in China (MaMV-DC) have been analysed. The genome DNA is 169 223 bp long, with 170 predicted protein-coding genes (001L­170L) and a tRNA gene. About one-sixth of these genes have homologues in the host cyanobacteria M. aeruginosa. The genome carries a gene homologous to host nblA, which encodes a protein involved in the degradation of cyanobacterial phycobilisome. Its expression during MaMV-DC infection was confirmed by reverse transcriptase PCR and Western blot detection and abundant expression was companied by the significant decline of phycocyanin content and massive release of progeny MaMV-DC. In addition, expressing MaMV-DC nblA reduced the phycocyanin peak and the phycocyanin to chlorophyll ratio in model cyanobacteria. These results confirm that horizontal gene transfer events have occurred between cyanobacterial host and cyanomyovirus and suggest that MaMV-DC carrying host-derived genes (such as 005L, that codes for NblA) is responsible for more efficient expression of cyanophage genes and release of progeny cyanophage. This study provides novel insight into the horizontal gene transfer in cyanophage and the interactions between cyanophage and their host.

Whole-Genome Analysis of a Novel Fish Reovirus (MsReV) Discloses Aquareovirus Genomic Structure Relationship with Host in Saline Environments.

Aquareoviruses are serious pathogens of aquatic animals. Here, genome characterization and functional gene analysis of a novel aquareovirus, largemouth bass Micropterus salmoides reovirus (MsReV), was described. It comprises 11 dsRNA segments (S1-S11) covering 24,024 bp, and encodes 12 putative proteins including the inclusion forming-related protein NS87 and the fusion-associated small transmembrane (FAST) protein NS22. The function of NS22 was confirmed by expression in fish cells. Subsequently, MsReV was compared with two representative aquareoviruses, saltwater fish turbot Scophthalmus maximus reovirus (SMReV) and freshwater fish grass carp reovirus strain 109 (GCReV-109). MsReV NS87 and NS22 genes have the same structure and function with those of SMReV, whereas GCReV-109 is either missing the coiled-coil region in NS79 or the gene-encoding NS22. Significant similarities are also revealed among equivalent genome segments between MsReV and SMReV, but a difference is found between MsReV and GCReV-109. Furthermore, phylogenetic analysis showed that 13 aquareoviruses could be divided into freshwater and saline environments subgroups, and MsReV was closely related to SMReV in saline environments. Consequently, these viruses from hosts in saline environments have more genomic structural similarities than the viruses from hosts in freshwater. This is the first study of the relationships between aquareovirus genomic structure and their host environments.

Establishment of three cell lines from Chinese giant salamander and their sensitivities to the wild-type and recombinant ranavirus.

Known as lethal pathogens, Ranaviruses have been identified in diseased fish, amphibians (including Chinese giant salamander Andrias davidianus, the world's largest amphibian) and reptiles, causing organ necrosis and systemic hemorrhage. Here, three Chinese giant salamander cell lines, thymus cell line (GSTC), spleen cell line (GSSC) and kidney cell line (GSKC) were initially established. Their sensitivities to ranaviruses, wild-type Andrias davidianus ranavirus (ADRV) and recombinant Rana grylio virus carrying EGFP gene (rRGV-EGFP) were tested. Temporal transcription pattern of ranavirus major capsid protein (MCP), fluorescence and electron microscopy observations showed that both the wild-type and recombinant ranavirus could replicate in the cell lines.

Unraveling the genome structure of cyanobacterial podovirus A-4L with long direct terminal repeats.

The freshwater cyanobacterial virus (cyanophage) A-4L, a podovirus, can infect the model cyanobacterium Anabaena sp. strain PCC 7120 resulting in a high burst size and forming concentric plaques on its lawns. The complete genome sequence of A-4L was determined by the combination of high-throughput sequencing, terminal transferase-mediated polymerase chain reaction and restriction mapping. It contains 41,750 bp with 810 bp direct terminal repeats and 38 potential open reading frames. As compared with other cyanobacterial podoviruses in diverse ecosystems, the A-4L has the longest terminal repeat and shares similar genome organizations with freshwater members. Furthermore, phylogenetic analysis based on concatenated sequences of eight core proteins indicated that freshwater cyanobacterial podoviruses were clustered together and distinct from marine counterparts, suggesting a clear divergence in the cyanobacterial podovirus lineage between freshwater and marine ecosystems. Our findings uncover the unique genome structure of A-4L which contains long direct terminal repeats, and create the first model system to address knowledge gaps in understanding cyanobacterial virus-host interactions at the molecular level.

Rana grylio virus (RGV) envelope protein 2L: subcellular localization and essential roles in virus infectivity revealed by conditional lethal mutant.

Rana grylio virus (RGV) is a pathogenic iridovirus that has resulted in high mortality in cultured frog. Here, an envelope protein gene, 2L, was identified from RGV and its possible role in virus infection was investigated. Database searches found that RGV 2L had homologues in all sequenced iridoviruses and is a core gene of iridoviruses. Western blotting detection of purified RGV virions confirmed that 2L protein was associated with virion membrane. Fluorescence localization revealed that 2L protein co-localized with viral factories in RGV infected cells. In co-transfected cells, 2L protein co-localized with two other viral envelope proteins, 22R and 53R. However, 2L protein did not co-localize with the major capsid protein of RGV in co-transfected cells. Meanwhile, fluorescence observation showed that 2L protein co-localized with endoplasmic reticulum, but did not co-localize with mitochondria and Golgi apparatus. Moreover, a conditional lethal mutant virus containing the lac repressor/operator system was constructed to investigate the role of RGV 2L in virus infection. The ability to form plaques and the virus titres were strongly reduced when expression of 2L was repressed. Therefore, the current data showed that 2L protein is essential for virus infection. Our study is the first report, to our knowledge, of co-localization between envelope proteins in iridovirus and provides new insights into the understanding of envelope proteins in iridovirus.

Genome architecture changes and major gene variations of Andrias davidianus ranavirus (ADRV).

Ranaviruses are emerging pathogens that have led to global impact and public concern. As a rarely endangered species and the largest amphibian in the world, the Chinese giant salamander, Andrias davidianus, has recently undergone outbreaks of epidemic diseases with high mortality. In this study, we isolated and identified a novel ranavirus from the Chinese giant salamanders that exhibited systemic hemorrhage and swelling syndrome with high death rate in China during May 2011 to August 2012. The isolate, designated Andrias davidianus ranavirus (ADRV), not only could induce cytopathic effects in different fish cell lines and yield high viral titers, but also caused severely hemorrhagic lesions and resulted in 100% mortality in experimental infections of salamanders. The complete genome of ADRV was sequenced and compared with other sequenced amphibian ranaviruses. Gene content and phylogenetic analyses revealed that ADRV should belong to an amphibian subgroup in genus Ranavirus, and is more closely related to frog ranaviruses than to other salamander ranaviruses. Homologous gene comparisons show that ADRV contains 99%, 97%, 94%, 93% and 85% homologues in RGV, FV3, CMTV, TFV and ATV genomes respectively. In addition, several variable major genes, such as duplicate US22 family-like genes, viral eukaryotic translation initiation factor 2 alpha gene and novel 75L gene with both motifs of nuclear localization signal (NLS) and nuclear export signal (NES), were predicted to contribute to pathogen virulence and host susceptibility. These findings confirm the etiologic role of ADRV in epidemic diseases of Chinese giant salamanders, and broaden our understanding of evolutionary emergence of ranaviruses.

A conditional lethal mutation in Rana grylio virus ORF 53R resulted in a marked reduction in virion formation.

Rana grylio virus (RGV) is a pathogenic iridovirus that has resulted in high mortality in cultured frog. Here, a recombinant RGV (i53R-RGV-lacIO) containing the inducible lac repressor/operator system was constructed. i53R-RGV-lacIO was a conditional lethal mutant in which the expression of envelope protein 53R was regulated by IPTG. i53R-RGV-lacIO shared characteristics similar to RGV in the presence of IPTG. However, the expression level of 53R, the ability of plaques formation, and the virus titers were strongly reduced in the absence of IPTG. Electron microscopy showed that the number of progeny virus produced by i53R-RGV-lacIO was remarkably reduced without IPTG. Furthermore, over-expression of 53R in vitro could increase titers of i53R-RGV-lacIO in the absence of IPTG. Therefore, the current data suggested that the lac repressor/operator system could regulate gene expression in the recombinant iridovirus. Our study was thought to be the first report of the system in aquatic virus.