A novel circular ssDNA virus of the phylum Cressdnaviricota discovered in metagenomic data from otter clams (Lutraria rhynchaena).

In this study, we present an analysis of metagenome sequences obtained from a filtrate of a siphon tissue homogenate of otter clams (Lutraria rhynchaena) with swollen-siphon disease. The viral signal was mined from the metagenomic data, and a novel circular ssDNA virus was identified. Genomic features and phylogenetic analysis showed that the virus belongs to the phylum Cressdnaviricota, which consists of viruses with circular, single-stranded DNA (ssDNA) genomes. Members of this phylum have been identified in various species and in environmental samples. The newly found virus is distantly related to the currently known members of the phylum Cressdnaviricota.

Metagenome Sequences from the Environment of Diseased Otter Clams, Lutraria rhynchaena, from a Farm in Vietnam.

Otter clam farming in Vietnam has recently encountered difficulties due to swollen-siphon disease. Here, we report the metagenome sequences of microorganisms extracted from the siphon tissue of infected otter clams. The data comprised bacterial and viral sequences which likely include those derived from the disease-causing agent.

Efficacy of Rose Myrtle Rhodomyrtus tomentosa Seed Extract against Acute Hepatopancreatic Necrosis Disease in Pacific Whiteleg Shrimp Penaeus vannamei.

Acute hepatopancreatic necrosis disease (AHPND) is a new emerging bacterial disease that has been recently reported to cause mass mortalities in Pacific whiteleg shrimp Penaeus vannamei. Antibiotics have been used to treat bacterial diseases in shrimp, but most of them have been ineffective and have resulted in drug residues in the harvested shrimp products. In this study, an alternative approach was tested for its efficacy in controlling AHPND. The extract of rose myrtle Rhodomyrtus tomentosa seed, a traditional Vietnamese medicine, was tested for antibacterial effect against three AHPND bacterial strains in vitro (Vibrio parahaemolyticus [VPAHPND ] KC12.020, VPAHPND KC13.14.2, and V. harveyi KC13.17.5) and was further evaluated for its potential efficacy in prevention of AHPND in shrimp in vivo. The in vitro studies showed that the antibacterial activity of the R. tomentosa extract was dose dependent, with the strongest bacterial susceptibility (≥18.0 mm) at a concentration of around 3,500 µg/disc. The in vivo studies showed that after challenge with VPAHPND KC12.020, the survival rates for shrimp in the groups that received feed pellets supplemented with extract at 3.5% or 7.0% (survival ~48.9% and 52.2%, respectively) were significantly higher than the zero survival rate in the positive control group, which received feed without the extract. These results indicate that the use of the R. tomentosa extract as an alternative therapy for control of AHPND in shrimp could help to minimize disease outbreaks. As a result, the extract is further expected to reduce drug/chemical residues in shrimp products.

Draft Genome Sequence of Non-Vibrio parahaemolyticus Acute Hepatopancreatic Necrosis Disease Strain KC13.17.5, Isolated from Diseased Shrimp in Vietnam.

A strain of Vibrio (KC13.17.5) causing acute hepatopancreatic necrosis disease (AHPND) in shrimp in northern Vietnam was isolated. Normally, AHPND is caused by Vibrio parahaemolyticus, but the genomic sequence of the strain indicated that it belonged to Vibrio harveyi. The sequence data included plasmid-like sequences and putative virulence genes.

Bacillus subtilis spores expressing the VP28 antigen: a potential oral treatment to protect Litopenaeus vannamei against white spot syndrome.

The envelope protein VP28 of white spot syndrome virus (WSSV) is considered a candidate antigen for use in a potential vaccine to this important shrimp pathogen (the cause of white spot syndrome, WSS). Here, we used spores of Bacillus subtilis to display VP28 on the spore surface. Trials were conducted to evaluate their ability to protect shrimps against WSSV infection. The gene cotB-vp28 was integrated into the chromosome of the laboratory strain B. subtilis PY79, and expression of CotB-VP28 was detected by Western blotting and immunofluorescence. Expression of CotB-VP28 was equivalent to 1000 molecules per spore. PY79 and CotB-VP28 spores were mixed with pellets for feeding of whiteleg shrimps (Litopenaeus vannamei), followed by WSSV challenge. Superoxidase dismutase (SOD), phenoloxidase activities and mortality rates of the two shrimp groups were evaluated. Groups fed with PY79 and CotB-VP28 spores at day 7 had increased SOD activities of 29% and increased phenoloxidase activities of 15% and 33%, respectively, compared to those of the control group. Fourteen days postchallenge, 35% of vaccinated shrimps had died compared to 49% of those fed naked spores (PY79) and 66% untreated, unchallenged animals. These data suggest that spores expressing VP28 have potential as a prophylactic treatment of WSS.

Involvement of WSSV-shrimp homologs in WSSV infectivity in kuruma shrimp: Marsupenaeus japonicus.

White spot syndrome virus (WSSV) is pathogenic and specific to shrimp, and is capable of producing a persistent infection in the host. Moreover, shrimp are capable of persistently carrying a single or multiple viruses, allowing them to survive for long periods with latent infections. In order to identify genes that are specially involved in the intricate WSSV-shrimp association, we focused on homologs between the WSSV and shrimp genomes. We here investigated whether homologous WssvORFs (WssvORF285, WssvORF332) and their homologs in the kuruma shrimp genome (MjORF16, MjORF18) are important for WSSV infectivity by utilizing dsRNA-mediated RNA interference, and further proposed potential roles of homologous WssvORFs associated with the persistent viral infection stage. Homologous MjORFs were found to be highly up-regulated in several tested tissues upon WSSV infection. Injection of dsRNAs specific to homologous MjORFs, followed by WSSV challenge, led to reduced and delayed shrimp mortality when compared to that of shrimp without dsRNA injection. Silencing of homologous WssvORFs by specific dsRNAs sharply increased shrimp survival. WssvORF332 may function as a latency gene especially associated with the persistent WSSV infection stage while WssvORF285 may be classified into the same group as WssvVP28 and may play a role in virus penetration during the infection. Our results suggest that WSSV-shrimp homologs are involved in WSSV infectivity and support the hypothesis that homologous WssvORFs are related to WSSV latency and pathogenesis.

Engineered virus-encoded pre-microRNA (pre-miRNA) induces sequence-specific antiviral response in addition to nonspecific immunity in a fish cell line: convergence of RNAi-related pathways and IFN-related pathways in antiviral response.

Transfection with synthesized virus-specific small interfering RNAs (siRNAs) efficiently inhibits viral replication in viral-infected fish cell lines, implying the involvement of RNA interference (RNAi)-related pathways in the antiviral response of fish cells. Here, we demonstrate that plasmid expressing virus-encoded pre-microRNAs (pre-miRNAs) can also inhibit viral replication through these pathways. By incorporating sequences encoding miRNAs specific to major capsid protein (MCP) gene of red sea bream iridovirus (RSIV) and a miRNA specific to hirame rhabdovirus (HIRRV) genome into a murine miR-155 pre-miRNA backbone, we were able to intracellularly express viral pre-miRNAs (miR-MCPs and miR-HIRRV) in a fish cell line. The miR-MCPs and miR-HIRRV, delivered as pre-miRNA precursors in transfected cells, inhibited viral replication when these cells were infected with the target virus. Although this may suggest sequence-specific interference, inhibitory effect on viral replication was also observed in cells transfected with a plasmid expressing pre-miRNA targeting beta-galactosidase gene (miR-LacZ) that served as a specificity control. Expression of pre-miRNAs was found to activate interferon (IFN)-related pathways, correlating with upregulation of the antiviral IFN-induced Mx protein. The antiviral effects of viral-miRNAs observed here were partly the result of the antiviral miRNA-related pathways and partly the result of the antiviral IFN-related pathways. We propose that engineered virus-encoded pre-miRNA can engage not only RNAi-related pathways but also IFN-related pathways to induce potent antiviral responses in fish cells.

Inhibition of red seabream iridovirus (RSIV) replication by small interfering RNA (siRNA) in a cell culture system.

Small interfering RNAs (siRNAs), mediators of a process of sequence-specific gene silencing called RNA interference, have been shown to have activity against a wide range of viruses and are considered to be potential antiviral tools. Here, we describe an antiviral activity of a siRNA that targets the major capsid protein (MCP) gene of red seabream iridovirus (RSIV), a marine fish-pathogenic virus, in a cell culture system. Inhibition of RSIV replication was demonstrated by reduced MCP expression level and reduced RSIV titer. MCP-targeted siRNA (siR-MCP) dose-dependently inhibited the expression of MCP gene in cells that either transiently expressed or stably expressed the MCP gene. At 84 and 96h after viral infection, siR-MCP reduced the expression of MCP gene by 55.2% and 97.1%, respectively. Transfection with siR-MCP reduced the production of RSIV particles in supernatants of samples infected with RSIV, while the corresponding mismatched siR-MCP (MsiR-MCP) and nsRNA controls did not exhibit this effect. These results show that MCP-targeted siRNA can effectively and specifically inhibit the expression of the target gene and hinder RSIV replication during an in vitro infection, providing a potential approach for the control of viral diseases in aquaculture.