Effect of temperature on the protective efficacy of a live attenuated vaccine against herpesviral haematopoietic necrosis in goldfish.

The live attenuated vaccine P7-P8 strain against herpesviral haematopoietic necrosis, which is caused by cyprinid herpesvirus 2 (CyHV-2), exhibits high protective efficacy in goldfish at 25°C, the predominant temperature for this disease; however, the effect of water temperature during the vaccination period on efficacy has not been determined. In this study, an in vitro experiment revealed that the vaccine strain grew between 15 and 30°C in the goldfish cell line RyuF-2. Subsequent in vivo efficacy tests were conducted with vaccination temperatures ranging from 15 to 30°C. During the vaccination period, organs were sampled to determine the vaccine growth dynamics. Blood plasma was collected to assess anti-CyHV-2 antibody titres. The protective efficacy of the vaccine at 15, 20, 25, and 30°C after subsequent virulent CyHV-2 challenge resulted in a relative percentage survival of 73.3%, 77.8%, 100%, and 77.8%, respectively, which indicated that the vaccine is effective over this temperature range. The vaccine virus load in the spleen was lowest at 15°C (103.7 DNA copies/mg) and highest at 25°C (106.5 DNA copies/mg). This indicates that the vaccine virus load over 104 DNA copies/mg may elicit sufficient acquired immunity. No significant differences in antibody titre were observed between groups, which suggests that cell-mediated immunity can be fundamentally involved in protection.

Crustacean Genome Exploration Reveals the Evolutionary Origin of White Spot Syndrome Virus.

White spot syndrome virus (WSSV) is a crustacean-infecting, double-stranded DNA virus and is the most serious viral pathogen in the global shrimp industry. WSSV is the sole recognized member of the family Nimaviridae, and the lack of genomic data on other nimaviruses has obscured the evolutionary history of WSSV. Here, we investigated the evolutionary history of WSSV by characterizing WSSV relatives hidden in host genomic data. We surveyed 14 host crustacean genomes and identified five novel nimaviral genomes. Comparative genomic analysis of Nimaviridae identified 28 "core genes" that are ubiquitously conserved in Nimaviridae; unexpected conservation of 13 uncharacterized proteins highlighted yet-unknown essential functions underlying the nimavirus replication cycle. The ancestral Nimaviridae gene set contained five baculoviral per os infectivity factor homologs and a sulfhydryl oxidase homolog, suggesting a shared phylogenetic origin of Nimaviridae and insect-associated double-stranded DNA viruses. Moreover, we show that novel gene acquisition and subsequent amplification reinforced the unique accessory gene repertoire of WSSV. Expansion of unique envelope protein and nonstructural virulence-associated genes may have been the key genomic event that made WSSV such a deadly pathogen.IMPORTANCE WSSV is the deadliest viral pathogen threatening global shrimp aquaculture. The evolutionary history of WSSV has remained a mystery, because few WSSV relatives, or nimaviruses, had been reported. Our aim was to trace the history of WSSV using the genomes of novel nimaviruses hidden in host genome data. We demonstrate that WSSV emerged from a diverse family of crustacean-infecting large DNA viruses. By comparing the genomes of WSSV and its relatives, we show that WSSV possesses an expanded set of unique host-virus interaction-related genes. This extensive gene gain may have been the key genomic event that made WSSV such a deadly pathogen. Moreover, conservation of insect-infecting virus protein homologs suggests a common phylogenetic origin of crustacean-infecting Nimaviridae and other insect-infecting DNA viruses. Our work redefines the previously poorly characterized crustacean virus family and reveals the ancient genomic events that preordained the emergence of a devastating shrimp pathogen.

Role of Marsupenaeus japonicus crustin-like peptide against Vibrio penaeicida and white spot syndrome virus infection.

Crustins are important AMP that has been identified in crustaceans. In this study, the role of Marsupenaeus japonicus crustin-like peptide (MjCRS) was examined in vivo by RNA interference (RNAi) using double-stranded RNA (dsRNA). Tissue expression analysis revealed that MjCRS transcripts are expressed in different tissues tested with the highest expression observed in hemocytes. Treatment with double-stranded RNA specific to MjCRS led to a significant reduction of MjCRS transcripts within the hemocytes. When MjCRS was silenced and subsequently infected with Vibrio penaeicida final mortality was significantly higher compared with PBS and dsGFP treated groups. On the other hand, final mortalities of MjCRS silenced and PBS injected groups were not significantly different after infection with white spot virus, however, both are significantly higher compared with dsGFP treated group. V. penaeicida infection significantly decreased MjCRS expression at 3, 6, 12 and 24h followed by significant increase at 48 h post-infection. On the contrary, white spot infection significantly increased MjCRS expression at 6 and 12h and decreased at 48 h post-infection. dsRNA treatment alone decreased total hemocyte counts (THCs) and subsequent V. penaeicida or white spot virus infection further decreased THCs. VP28 gene expression was both similarly increased in PBS injected group and MjCRS silenced group at 24 and 48 h-post infection. Results suggest that MjCRS is involved in antibacterial defense and might not have critical function against viral infection.

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.