Occurrence and seasonal variation of Perkinsus sp. Infection in wild mollusk populations from coastal waters of Qingdao, northern China.

Perkinsosis has been recognized as one of the major threats to natural and farmed bivalve populations, many of which are of commercial as well as environmental significance. Three Perkinsus species have been identified in China, and the Manila clam (Ruditapes philippinarum) was the most frequently infected species in northern China. Although the occurrence and seasonal variation of Perkinsus spp. have previously been examined, the pathological characteristics of these infections in wild Manila clams and sympatric species in China have seldom been reported. In the present study, the prevalence and intensity of Perkinsus infection in wild populations of Manila clams and 10 sympatric species from three sites were investigated by Ray's fluid thioglycolate medium (RFTM) assay seasonally across a single year. Perkinsus infection was only identified in Manila clams, with a high prevalence (274/284 = 96.48 %) and low intensity (89.8 % with a Mackin value ≤ 2, suggesting generally low-intensity infections) throughout the year. Heavily infected clams were mainly identified in Tianheng in January, which displayed no macroscopic signs of disease. An overview of the whole visceral mass section showed that the trophozoites mostly aggregated in gills and connective tissue of the digestive tract, to a lesser extent in the mantle and foot, and even less frequently in adductor muscle and connective tissues of the gonad. PCR and ITS-5.8S rRNA sequencing of 93 representative RFTM-positive samples revealed a 99.69 to 100 % DNA sequence identity to Perkinsus olseni. Unexpectedly, significantly higher infection intensities were usually identified in January and April when the Condition Index (CI) was relatively high. We propose that factors associated with the anthropogenic harvesting pressure and irregular disturbances should be responsible for the uncommon seasonal infection dynamics of perkinsosis observed in the present study.

A Putative Receptor for Ferritin in Mollusks: Characterization of the Insulin-like Growth Factor Type 1 Receptor.

The ferritin secreted by mammals has been well documented, with the protein capable of localizing to cell membranes and facilitating the delivery of iron to cells through endocytosis. However, the presence of ferritin in the circulatory fluid of mollusks and its functions remain largely unknown. In this study, we aimed to investigate the potential interacting proteins of ferritin in the ark clam (SbFn) through the use of a pull-down assay. Our findings revealed the presence of an insulin-like growth factor type 1 receptor (IGF-1R) in ark clams, which was capable of binding to SbFn and was named SbIGF-1R. SbIGF-1R was found to be composed of two leucine-rich repeat domains (L domain), a cysteine-rich domain, three fibronectin type III domains, a transmembrane domain, and a tyrosine kinase domain. The ectodomain of SbIGF-1R was observed to form a symmetrical antiparallel homodimer in the shape of the letter 'A', with the fibronectin type III domains serving as its 'legs'. The mRNA expression of SbIGF-1R gene was detected ubiquitously in various tissues of the ark clam, with the highest expression levels found in hemocytes, as determined by qRT-PCR. Using a confocal microscopic and yeast two-hybrid assays, the interaction between SbIGF-1R and SbFn was further verified. The results showed that SbFn co-localized with SbIGF-1R on the cell membrane, and their interaction was expected to occur on the FNIII domains of the SbIGF-1R. In conclusion, our findings highlight the identification of a putative receptor, SbIGF-1R, for SbFn, demonstrating the versatility of IGF-1R in ark clams.

A glimpse on metazoan ZNFX1 helicases, ancient players of antiviral innate immunity.

The human zinc finger NFX1-type containing 1 (ZNFX1) is an interferon-stimulated protein associated to the outer mitochondrial membrane, able to bind dsRNAs and interact with MAVS proteins, promoting type I IFN response in the early stage of viral infection. An N-terminal Armadillo (ARM)-type fold and a large helicase core (P-loop) and zinc fingers confer RNA-binding and ATPase activities to ZNFX1. We studied the phylogenetic distribution of metazoan ZNFX1s, ZNFX1 gene expression trends and genomic and protein signatures during viral infection of invertebrates. Based on 221 ZNFX1 sequences, we obtained a polyphyletic tree with a taxonomy-consistent branching at the phylum-level only. In metazoan genomes, ZNFX1 genes were found either in single copy, with up to some tens of exons in vertebrates, or in multiple copies, with one or a few exons and one of them sometimes encompassing most of the coding sequence, in invertebrates like sponges, sea urchins and mollusks. Structural analyses of selected ZNFX1 proteins showed high conservation of the helicase region (P-loop), an overall conserved region and domain architecture, an ARM-fold mostly traceable, and the presence of intrinsically disordered regions of varying length and position. The remarkable over-expression of ZNFX1 in bivalve and gastropod mollusks infected with dsDNA viruses underscores the antiviral role of ZNFX1, whereas nothing similar was found in virus-infected nematodes and corals. Whether the functional diversification reported in the C. elegans ZNFX1 occurs in other metazoan proteins remains to be established.

Paired miRNA and RNA sequencing provides a first insight into molecular defense mechanisms of Scapharca broughtonii during ostreid herpesvirus-1 infection.

Ostreid herpesvirus 1 (OsHV-1) infection caused mortalities with relevant economic losses in bivalve aquaculture industry worldwide. Initially described as an oyster pathogen, OsHV-1 can infect other bivalve species, like the blood clam Scapharca broughtonii. However, at present, little is known about the molecular interactions during OsHV-1 infection in the blood clam. We produced paired miRNA and total RNA-seq data to investigate the blood clam transcriptional changes from 0 to 72 h after experimental infection with OsHV-1. High-throughput miRNA sequencing of 24 libraries revealed 580 conserved and 270 new blood clam miRNAs, whereas no genuine miRNA was identified for OsHV-1. Total 88-203 differently expressed miRNAs were identified per time point, mostly up-regulated and mainly targeting metabolic pathways. Most of the blood clam mRNAs, in contrast, were down-regulated up to 60 h post-injection, with the trend analysis revealing the activation of immune genes only when comparing the early and latest stage of infection. Taken together, paired short and long RNA data suggested a miRNA-mediated down-regulation of host metabolic and energetic processes as a possible antiviral strategy during early infection stages, whereas antiviral pathways appeared upregulated only at late infection.

Parallel analysis of miRNAs and mRNAs suggests distinct regulatory networks in Crassostrea gigas infected by Ostreid herpesvirus 1.

BACKGROUND: Since 2008, the aquaculture production of Crassostrea gigas was heavily affected by mass mortalities associated to Ostreid herpesvirus 1 (OsHV-1) microvariants worldwide. Transcriptomic studies revealed the major antiviral pathways of the oyster immune response while other findings suggested that also small non-coding RNAs (sncRNA) such as microRNAs might act as key regulators of the oyster response against OsHV-1. To explore the explicit connection between small non-coding and protein-coding transcripts, we performed paired whole transcriptome analysis of sncRNA and messenger RNA (mRNA) in six oysters selected for different intensities of OsHV-1 infection. RESULTS: The mRNA profiles of the naturally infected oysters were mostly governed by the transcriptional activity of OsHV-1, with several differentially expressed genes mapping to the interferon, toll, apoptosis, and pro-PO pathways. In contrast, miRNA profiles suggested more complex regulatory mechanisms, with 15 differentially expressed miRNAs (DE-miRNA) pointing to a possible modulation of the host response during OsHV-1 infection. We predicted 68 interactions between DE-miRNAs and oyster 3'-UTRs, but only few of them involved antiviral genes. The sncRNA reads assigned to OsHV-1 rather resembled mRNA degradation products, suggesting the absence of genuine viral miRNAs. CONCLUSIONS: We provided data describing the miRNAome during OsHV-1 infection in C. gigas. This information can be used to understand the role of miRNAs in healthy and diseased oysters, to identify new targets for functional studies and, eventually to disentangle cause and effect relationships during viral infections in marine mollusks.

Influence of temperature on the pathogenicity of Ostreid herpesvirus-1 in ark clam, Scapharca broughtonii.

OsHV-1 is an epidemic pathogen of molluscs, and temperature has been recognized as a decisive environmental factor in its pathogenicity. In recent years, ark clam, Scapharca broughtonii, emerged as a host for OsHV-1. In the north of China, massive summer mortalities of ark clams infected with OsHV-1 have been continuously reported since 2012. However, the interaction between temperature and the pathogenicity of OsHV-1 was unknown in ark clams. In this study, the effect of temperature (10 °C to 18 °C stepped by 2 °C) on the occurrence of OsHV-1 disease in ark clams was analyzed. OsHV-1 infection led to gill erosion but not below the critical low temperature (between 12 °C and 14 °C). However, OsHV-1 persisted for more than 2 weeks at 12 °C post inoculation and replication was reactivated when the temperature was elevated to 18 °C. No significant reduction of OsHV-1 DNA load was found when the temperature descended to 12 °C from 18 °C, while the gill erosion remained unchanged. Ark clams failed to show the capability of effective clearance of OsHV-1 below the critical low temperature. Our results demonstrated that the pathogenicity of OsHV-1 was influenced significantly by temperature. Moreover, high temperature favored infection, which could provide more information to understand summer mortality of ark clams.

In situ hybridization revealed wide distribution of Haliotid herpesvirus 1 in infected small abalone, Haliotis diversicolor supertexta.

Ganglioneuritis was the primary pathologic change in infected abalone associated with Haliotid herpesvirus 1 (HaHV-1) infection, which eventually became known as abalone viral ganglioneuritis (AVG). However, the distribution of HaHV-1 in the other tissues and organs of infected abalone has not been systemically investigated. In the present study, the distribution of HaHV-1-CN2003 variant in different organs of small abalone, Haliotis diversicolor supertexta, collected at seven different time points post experimental infection, was investigated with histopathological examination and in situ hybridization (ISH) of HaHV-1 DNA. ISH signals were first observed in pedal ganglia at 48 h post injection, and were consistently observed in this tissue of challenged abalone. At the same time, increased cellularity accompanied by ISH signals was observed in some peripheral ganglia of mantle and kidney. At the end of infection period, lesions and co-localized ISH signals in infiltrated cells were detected occasionally in the mantle and hepatopancreas. Transmission electron microscope analysis revealed the presence of herpes-like viral particles in haemocyte nuclei of infected abalone. Our results indicated that, although HaHV-1-CN2003 was primarily neurotropic, it could infect other tissues including haemocytes.

A-to-I editing of Malacoherpesviridae RNAs supports the antiviral role of ADAR1 in mollusks.

BACKGROUND: Adenosine deaminase enzymes of the ADAR family are conserved in metazoans. They convert adenine into inosine in dsRNAs and thus alter both structural properties and the coding potential of their substrates. Acting on exogenous dsRNAs, ADAR1 exerts a pro- or anti-viral role in vertebrates and Drosophila. RESULTS: We traced 4 ADAR homologs in 14 lophotrochozoan genomes and we classified them into ADAD, ADAR1 or ADAR2, based on phylogenetic and structural analyses of the enzymatic domain. Using RNA-seq and quantitative real time PCR we demonstrated the upregulation of one ADAR1 homolog in the bivalve Crassostrea gigas and in the gastropod Haliotis diversicolor supertexta during Ostreid herpesvirus-1 or Haliotid herpesvirus-1 infection. Accordingly, we demonstrated an extensive ADAR-mediated editing of viral RNAs. Single nucleotide variation (SNV) profiles obtained by pairing RNA- and DNA-seq data from the viral infected individuals resulted to be mostly compatible with ADAR-mediated A-to-I editing (up to 97%). SNVs occurred at low frequency in genomic hotspots, denoted by the overlapping of viral genes encoded on opposite DNA strands. The SNV sites and their upstream neighbor nucleotide indicated the targeting of selected adenosines. The analysis of viral sequences suggested that, under the pressure of the ADAR editing, the two Malacoherpesviridae genomes have evolved to reduce the number of deamination targets. CONCLUSIONS: We report, for the first time, evidence of an extensive editing of Malacoherpesviridae RNAs attributable to host ADAR1 enzymes. The analysis of base neighbor preferences, structural features and expression profiles of molluscan ADAR1 supports the conservation of the enzyme function among metazoans and further suggested that ADAR1 exerts an antiviral role in mollusks.

Susceptibility of two abalone species, Haliotis diversicolor supertexta and Haliotis discus hannai, to Haliotid herpesvirus 1 infection.

Abalone viral ganglioneuritis (AVG), caused by Haliotid herpesvirus-1 (HaHV-1) infection, has been reported as the main cause of mortality and heavy losses of wild and cultivated abalone in Taiwan and Australia since 2003. HaHV-1 DNA has also been reported in diseased abalone collected in early 2000s in China. However, no data is available about the susceptibility, disease process and pathological changes of HaHV-1 infection in the primary cultivated abalone species in China. In the present study, two cultivated abalone species, Haliotis diversicolor supertexta and Haliotis discus hannai, were challenged with HaHV-1-CN2003 collected in 2003 in China using three different methods. Results showed that H. diversicolor supertexta was highly susceptible to HaHV-1-CN2003 infection and suffered acute mortality using all three challenge methods. H. discus hannai was not susceptible to the viral infection. Histopathology combined with transmission electron microscopy and quantitative PCR analysis revealed that the tropism of HaHV-1-CN2003 includes both neural tissue and haemocytes.

Dual transcriptomic analysis of Ostreid herpesvirus 1 infected Scapharca broughtonii with an emphasis on viral anti-apoptosis activities and host oxidative bursts.

The ark shell, Scapharca (Anadara) broughtonii, is an economically important marine shellfish species in Northwestern Pacific. Mass mortalities of ark shell adults related to Ostreid herpesvirus-1 (OsHV-1) infection have occurred frequently since 2012. However, due to the lack of transcriptomic resource of ark shells, the molecular mechanisms underpinning the virus-host interaction remains largely undetermined. In the present study, we resolved the dual transcriptome changes of OsHV-1 infected ark shell with Illumina sequencing. A total of 44 M sequence reads were generated, of which 67,119 reads were mapped to the OsHV-1 genome. De novo assembly of host reads resulted in 276,997 unigenes. 74,529 (26.90%), 47,653 (17.20%) and 19, 611 (7.07%) unigenes were annotated into GO, KOG and KEGG database, respectively. According to RSEM expression values, we identified 2998 differentially expressed genes (DEGs) between control and challenged groups, which included 2065 up-regulated unigenes and 933 down-regulated unigenes. Further analysis of functional pathways indicated that OsHV-1 could inhibit host cell apoptosis mainly by the up-regulation of inhibitor of apoptosis protein (IAP), and thus facilitating its successful replication. While host hemoglobins could induce oxidative burst by suppressing its peroxidase activity, and thus defense against OsHV-1 infection. Although we reported a narrow expression of the OsHV-1 genome compared to Crassostrea gigas infection, we highlighted several common viral genes highly expressed in the two hosts, suggesting an important functional role. This study offers insights into the pathogenesis mechanisms of OsHV-1 infection in bivalve mollusks of the Arcidae family.

Validation of housekeeping genes for quantitative mRNA expression analysis in OsHV-1 infected ark clam, Scapharca broughtonii.

Ostreid herpesvirus-1 (OsHV-1) presents interspecies transmission among bivalves. Recently, events of mass mortalities of ark clams (Scapharca broughtonii) infected with OsHV-1 have been recorded. To accurately assess the gene responding patterns of ark clams post OsHV-1 infection, constant stable housekeeping genes (HKGs) are needed as internal control to normalize raw mRNA expression data. In this study, ten candidate HKGs were selected, including 18S rRNA (18S), beta-actin (ACT), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), NADH dehydrogenase subunit (NADH), Elongation factor-1a (EF-1a), Elongation factor-1ß (EF-1ß), Elongation factor-1γ (EF-1γ), Ribosomal protein L7 (RL7), Ribosomal protein L15 (RL15) and Ribosomal protein S18 (S18). The expression levels of ten candidate HKGs were analyzed by real-time PCR under given experimental conditions, including various tissues, OsHV-1 challenge, temperature stress and OsHV-1 challenge at different temperature. Their expression stability values were further calculated using two different statistical models (geNorm and NormFinder). The results showed that different tissues presented distinct best pair genes combinations for gene expression analysis under OsHV-1 challenge. RL15 was comparatively more stable than other HKGs under various experimental conditions, while commonly used 18s and ACT seemed to be more greatly influenced by most given experimental conditions in ark clams. This study emphasized the necessity of prior validation of HKGs and would facilitate future gene expression analysis in ark clams or other shellfishes.

Experimental infection of adult Scapharca broughtonii with Ostreid herpesvirus SB strain.

We investigated the susceptibility of ark shell, Scapharca broughtonii, adults to Ostreid herpesvirus SB strain (OsHV-1-SB) through experimental infection by intramuscular injection assays. Results showed the onset of mortality occurred at 3days post injection, one day after the water turbidity became evident in rearing tanks. The mortality curves for the challenged group were similar to those observed at affected hatcheries. Histological lesions, herpesvirus-like particles and high OsHV-1-SB quantities were detected in challenged ark shells. This is the first study to successfully reproduce OsHV-1 disease in Arcoida species, and very few studies in adult bivalves (over 24months old).

Primary culture of Zhikong scallop Chlamys farreri hemocytes as an in vitro model for studying host-pathogen interactions.

Primary cultured cells can be a useful tool in studies on physiology, virology, and toxicology. Hemocytes play an important role in animal rapid response to pathogen invasion. In this study, an appropriate medium for primary culture of hemocytes of the bivalve Chlamys farreri was developed by adding 5% fetal bovine serum and 1% C. farreri serum to Leibovitz L-15 medium. These primary cultured hemocytes were maintained for more than 40 d in vitro and were classified into 3 types: (1) granulocytes containing numerous granules in the cytoplasm, (2) hyalinocytes with no or few granules, (3) a small percentage of macrophage-like cells. Furthermore, the primary cultured hemocytes were observed to be sensitive to bacterial and viral challenges. These hemocytes could phagocytose the bacterium Vibrio anguillarum, and presented cytopathic effects on the extracellular products (ECPs) of V. anguillarum; the mRNA level of QM, which plays an important role in immune response, also significantly increased 12 h after infection. When these hemocytes were challenged with ostreid herpesvirus 1 (OsHV-1), virus particles and empty capsids in the cells infected for 48 h were observed by transmission electron microscopy, and the QM mRNA level increased significantly at 12 h and 24 h following OsHV-1 challenge. This primary culture system is available for C. farreri hemocytes which can be used in the future to study host-pathogen interactions.

Identification and characterization of ostreid herpesvirus 1 associated with massive mortalities of Scapharca broughtonii broodstocks in China.

In the early summer of 2012 and 2013, mass mortalities of blood ark shell (Scapharca [Anadara] broughtonii), broodstocks were reported in several hatcheries on the coast of northern China. Clinical signs including slow response, gaping valves and pale visceral mass were observed in diseased individuals. In response to these reported mortalities, 238 samples were collected from hatcheries at 6 sites. Microscopic changes including lysed connective tissue, dilation of the digestive tubules, eosinophilic inclusion bodies, nuclear chromatin margination and pyknosis were found in affected animals. Transmission electron microscopy (TEM) revealed herpes-like viral particles within the connective tissue of the mantle. Quantative PCR (qPCR) and nested PCR (nPCR) analysis using primers specific for ostreid herpesvirus 1 (OsHV-1) indicated significant higher prevalence of OsHV-1 DNA in cases associated with mass mortalities than those without mass mortalities (p = 0.0012 for qPCR, p < 0.0001 for nPCR). qPCR also indicated that samples associated with mass mortalities carried high viral DNA loads, while the loads in apparently healthy samples were significantly lower (t = 3.15, df = 92, p = 0.002). Sequence analysis of the C2/C6 region of nPCR products revealed 5 newly described variants, which were closely related to each other. Phylogenetic analysis of the 5 virus variants and 48 virus variants reported in previous studies identified 2 main phylogenetic groups, and the 5 virus variants identified here were allocated to a separate subclade. To our knowledge, this is the first report of mass mortalities of bivalve broodstocks associated with OsHV-1 infection.

Complete genome sequence of Ostreid herpesvirus-1 associated with mortalities of Scapharca broughtonii broodstocks.

BACKGROUND: Ostreid herpesvirus-1 (OsHV-1) is the major bivalve pathogen associated with severe mortality events in a wide host range. In the early summer of 2012 and 2013, mass mortalities of blood clam (Scapharca broughtonii) broodstocks associated with a newly described variant of OsHV-1 (OsHV-1-SB) were reported. METHODS: In this study, the complete genome sequence of the newly described variant was determined through the primer walking approach, and compared with those of the other two OsHV-1 variants. RESULTS: OsHV-1-SB genome was found to contain 199, 354 bp nucleotides with 38.5% G/C content, which is highly similar to those of acute viral necrosis virus (AVNV) and OsHV-1 reference type. A total of 123 open reading frames (ORFs) putatively encoding functional proteins were identified; eight of which were duplicated in the major repeat elements of the genome. The genomic organization of OsHV-1-SB could be represented as TRL-UL-IRL-IRS-US-TRS, which is different from that of OsHV-1 reference type and AVNV due to the deletion of a unique region (X, 1.5Kb) between IRL and IRS. The DNA sequence of OsHV-1-SB is 95.2% and 97.3% identical to that of OsHV-1 reference type and AVNV respectively. On the basis of nucleotide sequences of 32 ORFs in OsHV-1-SB and the other nine OsHV-1 variants, results from phylogenetic analysis also demonstrated that OsHV-1-SB is most closely related to AVNV. CONCLUSIONS: The determination of the genome of OsHV-1 with distinguished epidemiological features will aid in our better understanding of OsHV-1 diversity, and facilitate further research on the origin, evolution, and epidemiology of the virus.

Emerging and endemic types of Ostreid herpesvirus 1 were detected in bivalves in China.

Viral infection caused by Ostreid herpesvirus 1 (OsHV-1) is one of the proximate causes of mass mortalities of cultivated bivalves around the world. The emergence and spread of different variants of OsHV-1 accompanied by different epidemiological characteristics have been reported frequently in different countries around the world. In this paper, we present a study of the detection of OsHV-1 DNA and their variations from 1599 samples over 18 species collected in 27 aquaculture sites and two food markets during 2001-2013 in China. All of the samples were examined by a nested PCR assay targeting the C2/C6 fragment of OsHV-1 followed by sequencing. Our results showed 338 individuals (21.1%) of seven species sampled from 14 (14/27=51.9%) sites and the two food markets were positive for viral DNA. Sequencing of 289 PCR products revealed 24 virus types. No shared virus type was found among different countries with 47 types (23 in Japan, 16 in France, 2 in South Korea and 1 in each country of Australia, USA, Ireland, New Zealand, Mexico and China) identified in previous studies. As previously reported, two main phylogenetic groups were identified by phylogenetic analysis based on the 71 virus types; within which 6 separate clades were identified. Our results also demonstrated that two clades were associated with abnormal mortalities of the scallop, Chlamys farrier and the calm, Scapharca broughtonii in China. These findings indicated that cultivated bivalves may face potential threats from OsHV-1 types found in our study.

Localization and Tissue Tropism of Ostreid Herpesvirus 1 in Blood Clam Anadara broughtonii.

OsHV-1 caused detrimental infections in a variety of bivalve species of major importance to aquaculture worldwide. Since 2012, there has been a notable increase in the frequency of mass mortality events of the blood clam associated with OsHV-1 infection. The pathological characteristics, tissue and cellular tropisms of OsHV-1 in A. broughtonii remain unknown. In this study, we sought to investigate the distribution of OsHV-1 in five different organs (mantle, hepatopancreas, gill, foot, and adductor muscle) of A. broughtonii by quantitative PCR, histopathology and in situ hybridization (ISH), to obtain insight into the progression of the viral infection. Our results indicated a continuous increase in viral loads with the progression of OsHV-1 infection, reaching a peak at 48 h or 72 h post-infection according to different tissues. Tissue damage and necrosis, as well as colocalized OsHV-1 ISH signals, were observed primarily in the connective tissues of various organs and gills. Additionally, minor tissue damage accompanied by relatively weak ISH signals was detected in the foot and adductor muscle, which were filled with muscle tissue. The predominant cell types labeled by ISH signals were infiltrated hemocytes, fibroblastic-like cells, and flat cells in the gill filaments. These results collectively illustrated the progressive alterations in pathological confusion and OsHV-1 distribution in A. broughtonii, which represent most of the possible responses of cells and tissues to the virus.

Complete genome sequence of acute viral necrosis virus associated with massive mortality outbreaks in the Chinese scallop, Chlamys farreri.

BACKGROUND: Acute viral necrosis virus (AVNV) is the causative agent of a serious disease resulting in high mortality in cultured Chinese scallops, Chlamys farreri. We have sequenced and analyzed the complete genome of AVNV. RESULTS: The AVNV genome is a linear, double-stranded DNA molecule of 210,993 bp with a nucleotide composition of 38.5% G + C. A total of 123 open reading frames were predicted to encode functional proteins, ranging from 41 to 1,878 amino acid residues. The DNA sequence of AVNV is 97% identical to that of ostreid herpesvirus 1 (OsHV-1), and the amino acid sequences of the encoded proteins of these two viruses are 94-100% identical. The genomic organization of AVNV is similar to that of OsHV-1, and consists of two unique regions (170.4 kb and 3.4 kb, respectively), each flanked by two inverted repeats (7.6 kb and 10.2 kb, respectively), with a third unique region (1.5 kb) situated between the two internal repeats. CONCLUSIONS: Our results indicate that AVNV is a variant of OsHV-1. The AVNV genome sequence provides information useful for understanding the evolution and divergence of OsHV-1 in marine molluscs.

Identification and Characterization of Infectious Pathogens Associated with Mass Mortalities of Pacific Oyster (Crassostrea gigas) Cultured in Northern China.

The Pacific oyster (Crassostrea gigas) aquaculture industry increased rapidly in China with the introduction and promotion of triploid oysters in recent years. Mass mortalities affecting different life stages of Pacific oysters emerged periodically in several important production areas of Northern China. During 2020 and 2021, we conducted a passive two-year investigation of infectious pathogens linked to mass mortality. Ostreid herpesvirus-1 (OsHV-1) was detected to be associated with mass mortalities of hatchery larvae, but not juveniles and adults in the open sea. Protozoan parasites, such as Marteilia spp., Perkinsus spp. and Bonamia spp. were not detected. Bacterial isolation and identification revealed that Vibrio natriegens and Vibrio alginolyticus were the most frequently (9 out of 13) identified two dominant bacteria associated with mass mortalities. Pseudoalteromonas spp. was identified as the dominant bacteria in three mortality events that occurred during the cold season. Further bacteriological analysis was conducted on two representative isolates of V. natriegens and V. alginolyticus, designated as CgA1-1 and CgA1-2. Multisequence analysis (MLSA) showed that CgA1-1 and CgA1-2 were closely related to each other and nested within the Harveyi clade. Bacteriological investigation revealed faster growth, and more remarkable haemolytic activity and siderophore production capacity at 25 °C than at 15 °C for both CgA1-1 and CgA1-2. The accumulative mortalities of experimental immersion infections were also higher at 25 °C (90% and 63.33%) than at 15 °C (43.33% and 33.33%) using both CgA1-1 and CgA1-2, respectively. Similar clinical and pathological features were identified in samples collected during both naturally and experimentally occurring mortalities, such as thin visceral mass, discolouration, and connective tissue and digestive tube lesions. The results presented here highlight the potential risk of OsHV-1 to hatchery production of larvae, and the pathogenic role of V. natriegens and V. alginolyticus during mass mortalities of all life stages of Pacific oysters in Northern China.

The Probiotic Bacillus hwajinpoensis Colonizes the Digestive System of Crassostrea gigas Larvae and Protects Them from Vibrio alginolyticus Infection.

The Pacific oyster Crassostrea gigas is one of the most important cultured marine species around the world. Production of Pacific oysters in China has depended primarily on hatchery produced seeds since 2016, with the successful introduction and development of triploid oysters. However, the seed supply of Pacific oysters is threatened by recurring mass mortality events in recent years. Vibriosis is the most commonly encountered disease associated with intensive oyster culture in hatcheries and nurseries. Vibrio alginolyticus and Bacillus hwajinpoensis were the two strains with pathogenic and probiotic effects, respectively, identified during the Pacific oyster larvae production. To monitor their colonization process in Pacific oyster larvae, green fluorescent protein (GFP) and red fluorescent protein (RFP) were labeled to the pathogenic V. alginolyticus and the probiotic B. hwajinpoensis stain, respectively. The pathogenic and probiotic effects of the two strains during the colonization process were then assessed. Stabile expression of GFP and RFP were observed in corresponding stains, and the capabilities of growth, biofilm formation and in vitro adhesion of GFP- and RFP- tagged stains were not significantly different from those of the wild-type strains. Usage of probiotics of 105 CFU/mL significantly inhibited the growth of pathogenic V. alginolyticus and reduced the mortality of D-sharped larvae. Both the pathogenic and probiotic strains employed a similar route to enter and colonize the oyster larvae, which indicates that competing with pathogens for binding and spreading sites were one of the mechanisms of B. hwajinpoensis to provide the probiotic effects to oyster larvae. In summary, employment of fluorescence-tagged pathogenic and probiotic strains simultaneously provides us with an excellent bioassay model to investigate the potential mechanisms of probiotics.