Mass mortality in Korean bay scallop (Argopecten irradians) associated with Ostreid Herpesvirus-1 µVar.

Since November 2017, mass mortalities of larvae of bay scallop (Argopecten irradians) were reported in hatcheries located at the southern area of Republic of Korea. Over 90% of larvae aged 5-10 days sank to the bottom of the tank and died. The hatcheries could not produce spat, and thus artificial seed production industry incurred huge losses. We identified Ostreid Herpesvirus-1 µVar (OsHV-1 µVar) associated with mass mortality by PCR, sequencing and transmission electron microscopy (TEM). All the samples were positive for OsHV-1 µVar with 99% sequence identity to previously reported OsHV-1 µVar sequences. Partial sequence of ORF-4 of OsHV-1 detected in this study was more closely related to sequences isolated from Europe. This is the first report to confirm the mortality caused by an OsHV-1 infection in the bay scallop.

Identification and characterization of a ferritin gene involved in the immune defense response of scallop Chlamys farreri.

Scallop Chlamys farreri is an important aquaculture species in northern China. However, its mass mortality caused by several pathogens can result in great economic loss and negative impacts to the sustainable development of the scallop industry. Thus, improving the overall understanding of immune response mechanisms involved in host-pathogen interactions is necessary. Ferritins are conserved molecules in organisms that are involved in diverse biological processes, such as mediating host-pathogen responses. In this study, we report a novel ferritin gene from C. farreri (denoted as CfFER). The full length of CfFER is 848 bp and contains a 5'-UTR of 113 bp, a 3'-UTR of 219 bp, and a complete open reading frame (ORF) of 516 bp. The ORF encodes a polypeptide of 171 amino acid residues with a molecular weight of approximately 19.95 kDa and an isoelectric point of 5.07. The CfFER protein exhibited typical ferritin structures, namely, a ferroxidase diiron center, a ferrihydrite nucleation center, and an iron-binding response signature. Phylogenetic analysis revealed that CfFER was closely related to other mollusk ferritin proteins. Expression of CfFER in different tissues was analyzed by quantitative real-time PCR, and results showed that CfFER was ubiquitously expressed in all examined tissues. The highest and lowest expression levels of CfFER were measured in the muscle and hemocyte, respectively. The relative mRNA expression of CfFER in response to bacterial (Vibrio anguillarum) and viral (acute viral necrobiotic virus) challenges sharply increased by ca. 5-fold about12 h post-infection (hpi) and then normalized at 48 hpi. Western blot analysis with polyclonal antibodies generated from the recombinant product of CfFER also demonstrated the presence of ferritin protein in hemocytes. These findings strongly suggest that CfFER is involved in the immune response of C. farreri and protection against pathogen challenge.

Molecular characterization and immune response expression of the QM gene from the scallop Chlamys farreri.

The scallop Chlamys farreri is an important aquaculture species in northern China. However, the sustainable development of the scallop industry is currently threatened by several pathogens that cause mass mortality of this mollusk. Therefore, a complete understanding of the immune response mechanisms involved in host-virus interactions is necessary. This study reports a novel QM gene from C. farreri. This gene was first identified as a putative tumor suppressor gene from human and then confirmed to participate in several functions, including immune response. The QM gene from C. farreri (CfQM) was identified by suppression subtractive hybridization, and its full-length (763 bp) cDNA was obtained through rapid amplification of cDNA ends. The cDNA of CfQM contained a short 5'-UTR of 22 bp and a 3'-UTR of 84 bp. Its ORF comprised 657 nucleotides that encode 218 amino acids with a molecular weight of approximately 28.3 kDa and an isoelectric point of 10.06. The deduced amino acid sequence of CfQM contained a series of conserved functional motifs that belong to the QM family. Phylogenetic analysis revealed that CfQM was closely related to other mollusk QM proteins, and altogether they form a mollusk QM protein subfamily that displays evolutionary conservation from yeast to human. The tissue-specific expression and transcriptional regulation of CfQM were investigated by quantitative real-time PCR in response to bacterial (Vibrio anguillarum) and viral (acute viral necrobiotic virus) challenges. The transcript level of CfQM was high in all of the examined tissues in a constitutive manner. The highest and lowest expression levels of CfQM were measured in the hepatopancreas and hemocyte, respectively. Upon bacterial and viral challenges, the relative mRNA expression of CfQM sharply increased at 6 h post-infection (hpi) and then normalized at 48 hpi. These findings suggest that CfQM can respond to and protect against pathogen challenge. To the best of our knowledge, this study is the first report of the QM gene from scallop. The results presented herein provided new insights into the molecular basis of host-pathogen interactions in C. farreri.

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.

Bioinformatics analysis of hemocyte miRNAs of scallop Chlamys farreri against acute viral necrobiotic virus (AVNV).

The sustainable development of the scallop Chlamys farreri industry in China is hindered by mass mortality mainly caused by a novel pathogen known as acute viral necrosis virus (AVNV). A better understanding of host-virus interactions, especially those at the molecular level, may facilitate the prevention and cure of AVNV infections. MicroRNAs (miRNAs) represent a class of small RNA molecules involved in several biological processes, including mediating host-pathogen responses. In this study, two hemocyte small RNA libraries were constructed from control (control library, CL) and AVNV-infected (infection library, IL) C. farreri for high throughput sequencing using Solexa technology. Acquired data were further used to identify conserved and novel miRNAs, screen differentially expressed miRNAs, and predict their target genes through bioinformatics analysis. Solexa sequencing produced 19,485,719 and 20,594,513 clean reads representing 2,248,814 and 1,510,256 unique small RNAs from CL and IL, respectively. A total of 57 conserved miRNAs were identified in both libraries, among which only two were unique to IL. Novel miRNA prediction using the Crassostrea gigas genome as a reference revealed 11 candidate miRNAs, 10 of which were validated by RT-PCR. Differential expression (p < 0.001) between libraries was observed in 37 miRNAs, among which 30 and 7 miRNAs were up- and downregulated, respectively. Expression differences were further confirmed by qRT-PCR. A sequence homology search against available C. farreri ESTs showed that these differentially expressed miRNAs may target 177 genes involved in a broad range of biological processes including immune defense and stress response. This study is the first to characterize C. farreri miRNAs and miRNA expression profiles in response to AVNV infection by deep sequencing. The results presented here will deepen our understanding of the pathogenesis of AVNV at the molecular level and provide new insights into the molecular basis of host-pathogen interactions in C. farreri.

The protein expression profile in hepatopancreas of scallop Chlamys farreri under heat stress and Vibrio anguillarum challenge.

Heat stress and pathogen infection have been considered as the main causes for mass mortality of cultured scallops during summer. In the present study, the expression profiles of proteins in the hepatopancreas of scallop Chlamys farreri were examined to reveal the possible mechanisms of physiological responses of scallop beneath heat stress and bacterial infection. An earlier occurred and higher mortality was observed in the scallops from combination treated group (28 °C and an injection of Vibrio anguillarum) in comparison to those in heat stress (28 °C) and bacteria challenge (V. anguillarum injection only) group, as well as control (PBS) and blank (untreated) group. The proteins in the hepatopancreas from scallops post 6 h of treatment were analyzed by using 2-D PAGE and ImageMaster 2D Platinum. There were total 1003 spots detected in control group, 1193 spots in heat stress group, 1263 spots in bacteria challenge group, and 1241 spots in the combination group. Fifteen protein spots expressed differentially between the combination treatment group and the bacteria challenge group were successfully identified by mass spectrometry and they were mainly classified as binding and catalytic proteins, such as endoglucanase, methylmalonate-semialdehyde dehydrogenase, xylose isomerase, tryptophanyl-tRNA synthetase, 40s ribosomal protein SA, glutathione S-transferase 4, and Mitochondrial transcription factor A, etc. These results indicated that the mortality of scallops suffered from the combination treatment was probably attributed to the impaired modulation of digestion and metabolism and ruined protein synthesis caused by heat stress together with bacteria infection. These data also provided valuable insights into the possible mechanisms of summer mortality occurrence of scallop at protein level.

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.

A preliminary study of differentially expressed genes of the scallop Chlamys farreri against acute viral necrobiotic virus (AVNV).

The scallop Chlamys farreri is one of the most important aquaculture species in northern coastal provinces. However, the sustainable development of scallop industry is currently threatened by a notorious pathogen named as acute viral necrobiotic virus (AVNV), which often causes mass mortality of the animals. Despite that great attention has been focused on this novel pathogen, little knowledge about the host-virus interactions is available. In this study, suppression subtractive hybridization (SSH) was employed to identify the up-regulated differentially expressed genes in the hemocytes of C. farreri challenged by AVNV. A forward subtracted cDNA library was finally constructed and 288 positive colonies representing differentially genes were screened to perform sequencing. A total of 275 ESTs were used for further analysis using bioinformatics tools after vector screening, among which 167 ESTs could be finally identified, with significant match (E values <1 × 10(-3)) to the deposited genes (proteins) in the corresponding databases. These genes could be classified into ten categories according to their Gene Ontology annotations of biological processes and molecular functions, i.e. cell defense and homeostasis (13.82%), cellular protein metabolic process (14.90), cellular metabolism (13.09%), cytoskeletal or cellular component (5.82%), transcription regulation or RNA processing (2.18%), cell division (meiosis)/apoptosis (2.18%), DNA metabolic process and repair (1.45%), cell adhesion/signaling (1.09%), microsatellite (0.73%), and ungrouped or unknown functions (6.88). The possible biological significance of some novel genes (mainly immune and homeostasis related genes) in the host response to AVNV were discussed. This study is the first global analysis of differentially expressed genes in hemocytes from AVNV-infected C. farreri, and in addition to increasing our understanding of the molecular pathogenesis of this virus-associated scallop disease, the results presented here should provide new insights into the molecular basis of host-pathogen interactions in C. farreri.

Haemocyte protein expression profiling of scallop Chlamys farreri response to acute viral necrosis virus (AVNV) infection.

Acute viral necrosis virus (AVNV) was newly reported as one causative agent responsible for mass mortality of adult Chinese scallop Chlamys farreri, which is widely cultured on northern China coast. Unfortunately, the interaction between virus and host is largely unknown. According to these, this study was undertaken to deeply explore the immune response of haemocyte against AVNV. Two-dimensional gel electrophoresis (2-DE) was introduced to produce protein expression profiles from samples taken at 24h post-infection (hpi) from the haemocytes of C. farreri that were either specific pathogen free or else infected with AVNV. Forty-eight protein spots, which consistently showed either a marked change (≥1.5-fold difference) in accumulated levels or else were highly expressed in haemocytes, were selected for further investigation. In-gel trypsin digestion was conducted followed by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-MS). Matching search was subsequently performed throughout bioinformatics databases. A total of 42 proteins were identified, all of which were classified into eight categories according to their Gene Ontology annotations of biological processes and molecular functions, i.e. cytoskeleton proteins, proteins involved in metabolism, proteins related to calcium homeostasis, chaperone, proteins involved in immunity, proteins involved in transcriptional regulation, proteins related to signal transduction, and ungrouped proteins. The possible biological significance of some observed proteins in the host response to AVNV was discussed. These studies could be served as the first global analysis of differentially expressed proteins in haemocytes from AVNV-infected C. farreri, and in addition to increasing our understanding of the pathogenesis of this virus-associated scallop disease, the results presented here should be useful both for potential biomarkers identification and anti-virus approaches development as well.

Development of a monoclonal antibody specific to granulocytes and its application for variation of granulocytes in scallop Chlamys farreri after acute viral necrobiotic virus (AVNV) infection.

A monoclonal antibody (MAb 6H7) specific to granulocytes of scallop Chlamys farreri was produced by immunising mice with separated granulocytes as an antigen. Characterised using a flow cytometric immunofluorescence assay, MAb 6H7 reacted to granulocytes by 87.1% of total positive haemocytes. At the ultrastructural level, MAb 6H7 demonstrated epitope in cytoplasmic granules of granulocytes. Western blotting analysis indicated that a peptide of 155 kDa was recognised by MAb 6H7. It was therefore used to investigate granulocyte variation in C. farreri after acute viral necrobiotic virus (AVNV) infection using an enzyme-linked immunosorbent assay. The result illustrated that granulocytes varied greatly by AVNV infection, and their amount significantly increased on day 1 post-injection, then decreased on days 2, 3 and 4, thereafter, rebounded and approached to a second peak on day 6, finally went down gradually to the control level on day 8.

Physiological and immune responses of Zhikong Scallop Chlamys farreri to the acute viral necrobiotic virus infection.

The Zhikong Scallop, Chlamys farreri, is one of the most important bivalve mollusks cultured in northern China. However, mass mortality of the cultured C. farreri has posed a serious threat to the maricultural industry in recent years. Acute Viral Necrobiotic Virus (AVNV) is believed as an important etiological agent causing the scallop mass mortalities. To understand the mechanism behind the AVNV associated scallop disease and mortality, we assessed the physiological and immune responses of C. farreri to the virus infection using oxygen consumption rate, ammonium-nitrogen excretion rate, hemocyte copper, zinc superoxide dismutase gene expression, and plasma superoxide dismutase activity and alkaline phosphatase activity as indicators. Scallops challenged by AVNV at 25 degrees C developed typical disease signs 2 days after virus injection. Before the disease manifested, scallop oxygen consumption and NH4+-N excretion rates rose and then fell back. Real-time PCR revealed that the hemocyte cytosol Cu, Zn SOD gene expression was upregulated followed by recovery. The plasma SOD activity, however, augmented consistently following virus injection. Moreover, plasma AKP activity first lowered and then elevated gradually to the highest level at 24 h post virus injection. Scallops challenged by AVNV at 17 degrees C neither developed notable disease nor showed obvious responses that could be associated with the virus infection. While the results suggested a correlation between the elevated seawater temperature and the AVNV infection associated C. farreri mortalities, they also indicated that the viral infection provoked multiple physiological and immune responses in the host scallops.

Loop-mediated isothermal amplification for rapid detection of acute viral necrobiotic virus in scallop Chlamys farreri.

Loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive detection of Acute viral necrobiotic virus (AVNV) in scallop Chlamys farreri was developed and evaluated. Four primers recognizing six targets on distinct AVNV DNA sequences were designed and the LAMP reaction was carried out in a water bath. Reaction temperature and time were optimized at 64 degrees C for 60 mins and LAMP products were detected using agarose gel electrophoresis and visual assessment. Confirmation of the expected LAMP products was performed with MboI restriction enzyme analysis. The detection limit of LAMP assay was as low as 1 fg AVNV DNA and accordingly, this assay was 100 times more sensitive than conventional PCR technique. A comparative evaluation of 20 samples using the LAMP and PCR assays revealed a complete accord in positivity or negativity for AVNV. These results indicate that the LAMP assay is simple, sensitive, specific, and has a great potential for detection of AVNV in the laboratory and field.

Variations of enzyme activities in the haemocytes of scallop Chlamys farreri after infection with the acute virus necrobiotic virus (AVNV).

Acute virus necrobiotic virus (AVNV) is one of the main pathogens for large scale mortality of Chinese scallop Chlamys farreri. In this paper, C. farreri were infected by different dilutions of AVNV supernatant (5(0), 5(-1), 5(-2), 5(-3), 5(-4), 5(-5), 5(-6), 5(-7), respectively), and dead individuals were counted every day for 15 days. Samples from groups of 5(-3) and 5(-5) were taken every day till 15 days and the activities of acid phosphatase (ACP), alkaline phosphatase (ALP), superoxide dismutase (SOD), myeloperoxidase (MPO), phenoloxidase (PO), peroxidase (POD) and catalase (CAT) in haemocytes were measured. The results of virus challenge showed that survival rates of scallops in groups of 5(0) and 5(-1) decreased sharply after the first day and died out completely on the 5th and 4th day, respectively. In other groups (5(-2), 5(-3), 5(-4), 5(-5), 5(-6) and 5(-7)), survival rates decreased gradually till 6 or 7 days, then kept steady till 15 days, and they were dose-dependent, increasing from 12% to 80% as the dose decreased from 5(-2) to 5(-7) viral supernatant. In the control group, survival rate was 88%. Enzyme activities for groups of 5(-3) and 5(-5) illustrated that activities of ACP, SOD, MPO, PO in groups of 5(-3) and 5(-5) were significantly higher than the control group in the first 9 or 10 days, and went back to the control group levels gradually after 10 days. Moreover, their activities in group of 5(-3) varied more than that in the group of 5(-5), especially activities of MPO, PO. Differently, the activities of POD and CAT were reduced or induced by virus infection and showed no regular trends in the experiments. The activity of ALP was not detected.

[Histopathological research and immunofluorescence of AVND virus infection in cultured scallop Chlamys farreri].

The naturally infected scallops Chlamys farreri sampled during mass mortality in summer of 2003 was detected by means of histopathological and MAb-based immunofluorescence assay (IFA). The results of histological examination demonstrated that a series of histopathological changes including cell swelling, basophilic increase, disorder, partial sloughing and excessive sloughing were always observed in epithelia of many different organs, e.g. mantle, gills, stomach, intestine and kidney. Additionally, the infected tissues were applied for in situ detection of the "acute virus necrobiotic disease" (AVND) virus by means of specific MAb-based IFA, and the result demonstrated that this pathological changes or lesions were perfectly coincident with the positive cells (fluorescencing cells) . The positive cells were denser in some local area of epithelia, and exhibited serious pathological lesions, which would reveal the roles of this virus in pathogenesis and further confirm that the AVND virus is the main causative agent of mass mortalities among cultured scallop Chlamys farreri farmed in northern coast of China.