Susceptibility of Goldfish to Cyprinid Herpesvirus 2 (CyHV-2) SH01 Isolated from Cultured Crucian Carp.

Cyprinid herpesvirus 2 (CyHV-2), a member of the Alloherpesviridae family belonging to the genus Cyprinivirus, is a fatal contagious aquatic pathogen that affects goldfish (Carassius auratus) and crucian carp (Carassius carassius). Although crucian carp and goldfish belong to the genus Carassius, it is unclear whether they are susceptible to the same CyHV-2 isolate. In addition, the origin of the crucian carp-derived CyHV-2 virus isolate remains unclear. CyHV-2 SH01 was isolated during herpesviral hematopoietic necrosis disease (HVHN) outbreaks in crucian carp at a local fish farm near Shanghai. CyHV-2 SH01 was confirmed by PCR and Western blot analysis of kidney, spleen, muscle, and blood tissue from the diseased crucian carp. Moreover, histopathological and ultra-pathological analyses revealed pathological changes characteristic of CyHV-2 SH01 infection in the tissues of the diseased crucian carp. In the present study, goldfish and crucian carp were challenged with CyHV-2 SH01 to elucidate viral virulence. We found that CyHV-2 SH01 could cause rapid and fatal disease progression in goldfish and crucian carp 24 h post-injection at 28 °C. Experimental infection of goldfish by injection indicated that the average virus titer in the kidney of the goldfish was 103.47 to 103.59 copies/mg. In addition, tissues exhibited the most prominent histopathological changes (cellular wrinkling and shrinkage, cytoplasmic vacuolation, fusion of the gill lamellae, and hepatic congestion) in CyHV-2 SH01-infected goldfish and crucian carp. Thus, crucian carp and goldfish showed a high sensitivity, with typical symptoms, to HVHN disease caused by CyHV-2 SH01.

Structural and Functional Diversity among Five RING Finger Proteins from Carassius Auratus Herpesvirus (CaHV).

Carassius auratus herpesvirus (CaHV) has been identified as a high-virulence pathogenic virus that infects aquatic animals, but the key factor for virus-host interaction is still unclear. Five Really interesting new genes (RING) finger proteins (39L, 52L, 131R, 136L, and 143R) of CaHV were screened to determine structural diversity. RING finger proteins were also predicted in other known fish herpesviruses, with an arrangement and number similar to CaHV. We performed multifaceted analyses of the proteins, including protein sizes, skeleton structures, subcellular localizations, and ubiquitination activities, to determine their precise roles in virus-host interactions. The five proteins were overexpressed and detected different levels of ubiquitination activities, and 143R showed the highest activity. Then, the prokaryotic expressed and purified full-length proteins (131R and 136L), RING domain isolates (131R12-43 and 136L45-87), and RING domain-deleted mutants (131RΔ12-43 and 136LΔ45-87) were prepared to detect their activities through ubiquitination assays. The results indicate that both full-length proteins and their isolates have activities that catalyze ubiquitination, and the full-length proteins possess higher activity than the isolates, but RING domain-deleted mutants lose their activities. Furthermore, the activities of the five proteins were verified as E3 ubiquitin ligase activity, showing that the RING domains determine the ubiquitination activity. These proteins present different subcellular localization. RING domain-deleted mutants showed similar subcellular localization with their full-length proteins, and all the isolates diffused in whole cells. The current results indicate that the sequence outside the RING domain determines subcellular localization and the level of ubiquitination activity, suggesting that the RING finger proteins of fish herpesviruses might have diverse functions in virus-host interaction.

Susceptibility of Pimephales promelas and Carassius auratus to a strain of koi herpesvirus isolated from wild Cyprinus carpio in North America.

Cyprinid herpesvirus-3 (CyHV-3, syn. koi herpesvirus) is an important pathogen worldwide and a common cause of mass mortality events of wild common carp (Cyprinus carpio) in North America, however, reference strains and genomes obtained from wild carp are not available. Additionally, it is unclear if fishes in North America are susceptible to CyHV-3 infection due to incomplete susceptibility testing. Here we present the first North American type strain and whole-genome sequence of CyHV-3 isolated from wild carp collected from a lake with a history and recent incidence of carp mortality. Additionally, the strain was used in an in-vivo infection model to test the susceptibility of a common native minnow (Pimephales promelas) and goldfish (Carrasius auratus) which is invasive in North America. Detection of CyHV-3 DNA was confirmed in the tissues of a single fathead minnow but the same tissues were negative for CyHV-3 mRNA and samples from exposed fathead minnows were negative on cell culture. There was no detection of CyHV-3 DNA or mRNA in goldfish throughout the experiment. CyHV-3 DNA in carp tissues was reproducibly accompanied by the detection of CyHV-3 mRNA and isolation on cell culture. Additionally, environmental CyHV-3 DNA was detected on all tank filters during the study. These findings suggest that fathead minnows and goldfish are not susceptible to CyHV-3 infection and that detection of CyHV-3 DNA alone in host susceptibility trials should be interpreted with caution.

Identification of the Immediate-Early Genes of Cyprinid Herpesvirus 2.

Cyprinid herpesvirus 2 (CyHV-2), which infects goldfish and crucian carp causing high mortality, is an emerging viral pathogen worldwide. The genome of CyHV-2 is large and comprises double-stranded DNA, including several genes similar to cyprinid herpesvirus 1, ictalurid herpesvirus-1, cyprinid herpesvirus 3, and ranid herpesvirus-1. Genes of DNA viruses are expressed in three temporal phases: immediate-early (IE), early (E), and late (L) genes. Viral IE genes initiate transcription as soon as the virus enters the host, without viral DNA replication. IE gene products enable the efficient expression of E and L genes or regulate the host to initiate virus replication. In the present study, five IE genes of CyHV-2 were identified, including open reading frame (ORF)54, ORF121, ORF141, ORF147, and ORF155. Time course analysis and reverse transcription polymerase chain reaction confirmed five IE genes, thirty-four E genes, and thirty-nine L genes. In addition, all 150 ORFs identified in the CyHV-2 genome are transcribed, and are expressed in chronological order, similar to other herpesviruses. This study is the first to identify the IE genes of CyHV-2, which will provide more information for viral molecular characterization.

Isolation of a Chinook Salmon Bafinivirus (CSBV) in Imported Goldfish Carassius auratus L. in the United Kingdom and Evaluation of Its Virulence in Resident Fish Species.

This is the first record of a fish nidovirus isolated from a consignment of goldfish at the United Kingdom (UK) border. The full-length viral genome was 25,985 nt, sharing a 97.9% nucleotide identity with the Chinook salmon bafinivirus (CSBV) NIDO with two deletions of 537 and 480 nt on the ORF Ia protein. To assess the potential impact on UK fish species, Atlantic salmon, common carp and goldfish were exposed to the virus via an intraperitoneal (IP) injection and bath challenge. Moribundity was recorded in only 8% of IP-injected goldfish. A high viral load, ≈107 of the CSBV PpIa gene, was measured in the kidney of moribund goldfish. Mild histopathological changes were observed in the kidneys of challenged carps. Ultrastructural observations in renal tubule epithelial cells of goldfish showed cylindrical tubes (≈15 nm in diameter) and tubular structures budding spherical virions (≈200 nm in diameter) with external spike-like structures. Negative staining showed both circular and bacilliform virions. Seroconversion was measured in common carp and goldfish but not in Atlantic salmon. This study reinforces the potential risk of novel and emerging pathogens being introduced to recipient countries via the international ornamental fish trade and the importance of regular full health screens at the border inspection posts to reduce this risk.

ß-glucan and anisodamine can enhance the immersion immune efficacy of inactivated cyprinid herpesvirus 2 vaccine in Carassius auratus gibelio.

As one of the most important fish in freshwater aquaculture, gibel carp (Carassius auratus gibelio) is easily susceptible to Cyprinid herpesvirus 2 (CyHV-2). Immersion vaccination has attracted many researchers due to its simple operation in preventing infectious diseases. However, the unavoidable disadvantage is that the immersion vaccine must be used with adjuvants to get a better performance. In this study, gibel carps were vaccinated by a 60 min bath in a ß-propiolactone-inactivated Cyprinid herpesvirus 2, mixed with DTT, ß-glucan, anisodamine and scopolamine, respectively. After immunization, the fishs were challenged by CyHV-2 in 2 weeks. By analyzing pathological section, we found that ß-glucan, anisodamine and scopolamine groups protected the gibel carp compared to the control group, which was consistent with the trend of survival rate. Specifically, ß-glucan group in serum appeared best on lysozyme, TSOD and complement C3. Real time quantitative RT-PCR results demonstrated that in both spleen and head kidney tissues, mRNA expressions of typical Th1 immune response cytokines IL-2 and IFN-γ2 in ß-glucan group and anisodamine group were significantly higher than other groups and the level of immunoglobulins related to systemic immunity (IgM) and mucosal immunity (IgZ) were also enhanced in the immune period. DTT group slightly affected immune gene and serum enzyme activity, while did not show an adjuvant effect on survival rate. In addition, four adjuvant groups could obviously inhibit CyHV-2 replication. This study explored and proved the good efficiency of ß-glucan or anisodamine as immersion immune adjuvant and also provided reference for improving the efficiency of immersion immunity.

Development of two brain cell lines from goldfish and silver crucian carp and viral susceptibility to Cyprinid herpesivirus-2.

Two cell lines were established from silver crucian carp and goldfish brain tissue and used as the biological tool for monitoring viral diseases. Characterization including optimal growth kinetics study, karyotyping, and mitochondrial ribosomal RNA (rRNA) genotyping were performed. The primary cultures of these cells were generated by the explant technique using the medium 199 supplemented with 20 % fetal bovine serum and epidermal/fibroblast growth factors. The cells grew over the range of 15 to 30°C, while the optimal temperature for culture was 30°C. The cell lines were maintained in vitro and could be subcultured over 40 times. Following cryopreservation in liquid nitrogen, thawed cells exhibit viability of > 90 % after a 13-mo period of storage. The chromosome count of two cell lines were determined to be 154 and 110, respectively, which agreed well with triploid crucian carp brain cells and diploid goldfish brain cells. Polymerase chain reaction amplification and sequence analysis indicated 100 % and 94% match with known crucian carp mitochondrial DNA sequences. Cytopathic effect was continuously observed in both cell lines over 10 passages after inoculation with tissue homogenates of sick or died goldfish from cyprinid herpesvirus 2 (CyHV-2) outbreaks. These newly established cell lines could be a diagnostic tool for viral diseases in fish species.

Investigation of bacterial diversity and pathogen abundances in gibel carp (Carassius auratus gibelio) ponds during a cyprinid herpesvirus 2 outbreak.

Cyprinid herpesvirus 2 (CyHV-2) infection is detrimental to gibel carp health and may result in severe economic loss in freshwater aquaculture. However, information regarding the interaction of this pathogen with the aquatic environment is scarce. In this study, quantitative polymerase chain reaction (qPCR) and high-throughput sequencing were used to determine the abundances of pathogens and bacterial community compositions in two aquaculture ponds in Jiangsu Province, China. The results indicate that the concentrations of six selected pathogens were higher in the water than in the sediment and that these concentrations peaked during disease outbreak. In total, 8,326 and 18,244 operational taxonomic units were identified from water and sediment samples, respectively. The dominant phyla were Proteobacteria, Actinobacteria, Cyanobacteria, Bacteroidetes, and Chlorobi in water samples and Proteobacteria, Firmicutes, Actinobacteria, Chloroflexi, and Bacteroidetes in sediment samples. Bacterial communities were similar at the phylum level in different ponds, although significant differences were observed at the genus level. In addition, bacterial diversity was associated with environmental factors (temperature, chemical oxygen demand, NO2- -N, NO3- -N, and NH4+ -N) in the pond where the outbreak occurred. Additionally, CyHV-2 abundance was positively correlated with dissolved oxygen levels and Aeromonas spp. abundance in pond water (p < .01). This study provides comprehensive insight into the mechanisms of interaction between potential pathogens and the freshwater environment of aquaculture ponds during CyHV-2 disease outbreaks. Furthermore, the results from this study can contribute to improvement of the aquatic environment and establishment of disease prevention and control measures.

Persistence of cyprinid herpesvirus 2 in asymptomatic goldfish Carassius auratus (L.) that survived an experimental infection.

Cyprinid herpesvirus 2 (CyHV-2) is the causative agent of herpesviral haematopoietic necrosis (HVHN) in goldfish, Carassius auratus, and Prussian carp, C. auratus gibelio. In this study, we investigated virus persistence in goldfish experimentally infected with CyHV-2. Virus DNA presence in organs was monitored in survivors reared at a virus permissive temperature and also in survivors treated with a non-permissive temperature for 4 days, initiated at three different time points post-infection in order to obtain fish with different virus loads. We detected virus DNA in all organs tested at 51 days post-infection (dpi) and in the spleen, trunk kidney and gills of survivors at 81 dpi, although the virus load in fish influenced the subsequent number of organs that tested positive for virus DNA. In addition, some organs dissected from four out of five asymptomatic survivors tested positive by PCR following incubation in vitro in a medium for 5 days. Following inoculation with the homogenate of PCR-positive kidney incubated in vitro, one of the three inoculated fish died, showing that the detected virus by PCR produced infectious particles. This study suggests that CyHV-2 can establish a persistent infection in some organs, especially the spleen and trunk kidney, and that asymptomatic surviving fish can be a source of infection.

Differential interferon system gene expression profiles in susceptible and resistant gynogenetic clones of gibel carp challenged with herpesvirus CaHV.

Interferon (IFN) system plays a vital role in the first line of defense against viruses. In this study, we first identified multiple transcripts of 15 IFN system genes, including PRRs (TLR2, TLR3, RIG-I, and LGP2), PRR-mediated IFN signal pathway (MyD88, MITA, and MAVS), IFN regulatory factors (IRF1, IRF3, IRF7, and IRF9), IFNs (IFNφ1 and IFNφ3), and ISGs (Mx and viperin), and one transcript of TLR9 in de novo transcriptome assembly data of gibel carp head-kidney. Multiple nucleotide alignments and phylogenetic analysis of common region showed that the transcripts of every of the 15 IFN system genes were classified into two homologs with distinctly divergent sequences, indicating that hexaploid gibel carp may be an allopolyploid. During Carassius auratus herpesvirus (CaHV) infection, gibel carp resistant clone H significantly suppressed CaHV replication with markedly less viral loads than those in highly susceptible clone A+ and moderately resistant clone F. Then, qPCR analyses were performed to reveal their differential and dynamic expression changes during CaHV infection in head kidney, spleen and liver among three gibel carp gynogenetic clones. Through qPCR and hierarchical clustering analysis, 8 genes, such as RIG-Is, LGP2s, IRF1-B, IRF3s, IRF7s, IRF9-B, Mxs, and viperins, were identified as candidate resistant-related genes. They remarkably increased their expression in immune tissues of three clones after CaHV infection. Significantly, the up-regulation folds of these genes in clone A+, F and H were related to their resistance ability to CaHV, progressively increasing from susceptible clone to resistant clone at 1 dpi. The positive correlation to the resistance ability suggested that resistant clone H immediately triggered stronger IFN response. IFNφ3 showed a different dynamic change and was sharply induced in moderately resistant clone F at 3 dpi. The other 5 IFN system genes (TLR2, TLR3, TLR9, MyD88, and MITA) maintained a low expression level after CaHV challenge. Interestingly, the A or B copies/homologs of almost these IFN system genes exhibited differential transcript abundance in immune tissue after CaHV challenge, suggesting A or B homologs might occur dominant or biased expression of homeologs during gibel carp evolution. These data provide candidate resistant-related genes for disease-resistance breeding of gibel carp.

Differential expression of miRNA in Carassius auratus gibelio in response to cyprinid herpesvirus 2 infection.

miRNAs (microRNAs), a small endogenous non-coding RNAs, play crucial roles in post-transcriptional regulator of genes expression in various biological processes. Cyprinid Herpesvirus-2 (CyHV-2) is a highly pathogenic member of the alloherpesviridae that causes acute mass mortalities in populations of Carassius auratus gibelio and Carassius auratus auratus. However, the molecular mechanisms underlying the pathogenicity of CyHV-2 have not been fully determined. Here, miRNA expression profiles were identified via high-throughput sequencing in the kidney of Carassius auratus gibelio infected or uninfected with CyHV-2. The results showed that a total number of 840 known miRNAs and 48 putative novel miRNAs were identified. Then we compared the expression patterns of miRNAs in the two groups, 23 miRNAs were significantly differentially expressed between the uninfected and infected groups. Further, the expressions of 23 miRNAs were validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR), the results showed that the expression patterns were basically the same with the sequencing. Prediction of targets of differentially expressed miRNAs revealed that the miRNAs participated in the regulation of multiple immune-related signaling pathways, including Chemokine signaling pathway, Apoptosis, Jak-STAT signaling pathway and MAPK signaling pathway. Taken together, these data provide insight into the regulatory mechanisms of miRNA and highlight the function of miRNA in the regulation of the immune response during the interaction between host and virus pathogens.

Integrated analysis of mRNA and viral miRNAs in the kidney of Carassius auratus gibelio response to cyprinid herpesvirus 2.

MicroRNAs (miRNAs) are small, non-coding single stranded RNAs that play crucial roles in numerous biological processes. Vertebrate herpesviruses encode multiple viral miRNAs that modulate host and viral genes. However, the roles of viral miRNAs in lower vertebrates have not been fully determined. Here, we used high-throughput sequencing to analyse the miRNA and mRNA expression profiles of Carassius auratus gibelio in response to infection by cyprinid herpesvirus 2 (CyHV-2). RNA sequencing obtained 26,664 assembled transcripts, including 2,912 differentially expressed genes. Based on small RNA sequencing and secondary structure predictions, we identified 17 CyHV-2 encoded miRNAs, among which 14 were validated by stem-loop quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and eight were validated by northern blotting. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of miRNAs-mRNA pairs revealed diverse affected immune signalling pathways, including the RIG-I-like receptor and JAK-STAT pathways. Finally, we presented four genes involved in RIG-I-like pathways, including host gene IRF3, RBMX, PIN1, viral gene ORF4, which are negatively regulated by CyHV-2 encoded miRNA miR-C4. The present study is the first to provide a comprehensive overview of viral miRNA-mRNA co-regulation, which might have a key role in controlling post-transcriptomic regulation during CyHV-2 infection.

Importation of CyHV-2-infected goldfish into the Netherlands.

Cyprinid herpesvirus 2 (CyHV-2) is known as the causative agent of herpesviral haematopoietic necrosis in goldfish Carassius auratus auratus. However, the virus has also been detected in Prussian carp C. gibelio and crucian carp C. carassius from European and Asian countries. To prevent spread of the causative virus to other areas, investigation of the risk factors of spread of this virus is important. In this study, 8 batches of goldfish imported into the Netherlands by airfreight from Asia and the Middle East were investigated for the presence of the virus. CyHV-2 DNA was detected by PCR in the pooled kidneys of 4 of the 8 imported goldfish batches, of which 1 was from a CyHV-2 disease case at a Dutch importer's quarantine facility. Sequence analysis of the CyHV-2 strains from this study and from previous reports showed that there were at least 6 different lengths in the mA region, resulting in tentatively at least 4 genotypes. Virus isolation was positive for only 1 (Amsterdam Schiphol-1 [AMS-1]) of the 8 samples. It was shown that the AMS-1 isolate was highly virulent to Ryukin goldfish after 100.3 TCID50 fish-1 intraperitoneal injection. The viral titre of the AMS-1 isolate for goldfish fin cells at several temperatures was similar to that of a Japanese CyHV-2 isolate. Our results prove that one of the routes of spread of various CyHV-2 strains is through the global trade of apparently healthy infected goldfish.

Distinct herpesvirus resistances and immune responses of three gynogenetic clones of gibel carp revealed by comprehensive transcriptomes.

BACKGROUND: Gibel carp is an important aquaculture species in China, and a herpesvirus, called as Carassius auratus herpesvirus (CaHV), has hampered the aquaculture development. Diverse gynogenetic clones of gibel carp have been identified or created, and some of them have been used as aquaculture varieties, but their resistances to herpesvirus and the underlying mechanism remain unknown. RESULTS: To reveal their susceptibility differences, we firstly performed herpesvirus challenge experiments in three gynogenetic clones of gibel carp, including the leading variety clone A+, candidate variety clone F and wild clone H. Three clones showed distinct resistances to CaHV. Moreover, 8772, 8679 and 10,982 differentially expressed unigenes (DEUs) were identified from comparative transcriptomes between diseased individuals and control individuals of clone A+, F and H, respectively. Comprehensive analysis of the shared DEUs in all three clones displayed common defense pathways to the herpesvirus infection, activating IFN system and suppressing complements. KEGG pathway analysis of specifically changed DEUs in respective clones revealed distinct immune responses to the herpesvirus infection. The DEU numbers identified from clone H in KEGG immune-related pathways, such as "chemokine signaling pathway", "Toll-like receptor signaling pathway" and others, were remarkably much more than those from clone A+ and F. Several IFN-related genes, including Mx1, viperin, PKR and others, showed higher increases in the resistant clone H than that in the others. IFNphi3, IFI44-like and Gig2 displayed the highest expression in clone F and IRF1 uniquely increased in susceptible clone A+. In contrast to strong immune defense in resistant clone H, susceptible clone A+ showed remarkable up-regulation of genes related to apoptosis or death, indicating that clone A+ failed to resist virus offensive and evidently induced apoptosis or death. CONCLUSIONS: Our study is the first attempt to screen distinct resistances and immune responses of three gynogenetic gibel carp clones to herpesvirus infection by comprehensive transcriptomes. These differential DEUs, immune-related pathways and IFN system genes identified from susceptible and resistant clones will be beneficial to marker-assisted selection (MAS) breeding or molecular module-based resistance breeding in gibel carp.

Detection of goldfish haematopoietic necrosis herpes virus (Cyprinid herpesvirus-2) with multi-drug resistant Aeromonas hydrophila infection in goldfish: First evidence of any viral disease outbreak in ornamental freshwater aquaculture farms in India.

This outbreak report details of a mortality event where Cyprinid herpes virus-2 (CyHV-2) was detected in association with multidrug-resistant Aeromonas hydrophila infection in goldfish, Carassius auratus, from commercial farms. The goldfish exhibited large scale haemorrhages on the body, fins and gills, lepidorthosis, necrosed gills, protruded anus and shrunken eyes. White nodular necrotic foci in spleen and kidneys were noticed, along with necrosis and fusion of gill lamellae. Transmission electron microscopy of affected tissues revealed the presence of mature virus particles. Involvement of CyHV-2 was confirmed by PCR, sequencing and observed cytopathic effect in koi carp fin cell line along with experimental infection study. A bacterium isolated from the internal organs of affected fish was found to be pathogenic Aeromonas hydrophila having resistance to more than 10 classes of antibiotics. We postulate that CyHV-2 was the primary etiological agent responsible for this outbreak with secondary infection by A. hydrophila. The experimental infection trials in Labeo rohita and koi carp by intraperitoneal challenge with CyHV-2 tissue homogenates failed to reproduce the disease in those co-cultured fish species. This is the first report of a viral disease outbreak in organised earthen ornamental fish farms in India and bears further investigation.

Herpesviral Hematopoietic Necrosis in Goldfish in Switzerland: Early Lesions in Clinically Normal Goldfish (Carassius auratus).

Cyprinid herpesvirus 2 is a pathogen of goldfish, inducing a disease referred to as herpesviral hematopoietic necrosis. The disease is described so far in Japan, North America, Taiwan, Australia, the United Kingdom, and recently also Italy. Here the authors describe histologic lesions in clinically affected fish in comparison with clinically normal but virus DNA-positive goldfish in Switzerland. While necrosis or enhanced single-cell necrosis in the hematopoietic tissue in the pronephros or mesonephros was evident in dead and sick animals, in clinically normal goldfish, only single-cell necrosis was observed. Virus DNA was demonstrated in dead as well as clinically affected and subclinically infected goldfish by polymerase chain reaction and in situ hybridization. This study identifies the presence of goldfish herpesvirus in Switzerland and highlights the fact that the virus might be more widespread than assumed, as clinically normal goldfish can also carry cyprinid herpesvirus 2, showing histologically similar lesions but of lesser extent and severity.

Proteomic Analysis of Cytoskeleton Proteins in Fish.

In this chapter, we describe laboratory protocols for rearing fish and a simple and efficient method of extracting and identifying pathogen and host proteins that may be involved in entry and replication of commercially important fish viruses. We have used the common carp (Cyprinus carpio L.) and goldfish (Cyprinus auratus) as a model system for studies of proteins involved in viral entry and replication. The chapter describes detailed protocols for maintenance of carp, cell culture, antibody purification of proteins, and use of electrospray-ionization mass spectrometry analysis to screen and identify cytoskeleton and other proteins that may be involved in viral infection and propagation in fish.

Establishment of a novel and highly permissive cell line for the efficient replication of cyprinid herpesvirus 2 (CyHV-2).

Haematopoietic necrosis of gibel carp (Carassius auratus gibelio) is caused by cyprinid herpesvirus 2 (CyHV-2) and has caused huge economic losses in aquaculture worldwide. Currently the isolation and propagation of CyHV-2 in vitro is very difficult due to the lack of permissive cell lines. Studies on the pathogenesis of CyHV-2 have been hampered because the virus has not been extensively characterized. In this study, a novel cell line from the brain of gibel carp, denoted GiCB, has been established and characterized. Sustainable propagation of CyHV-2 in the GiCB cell line has been confirmed by virus infection and titration, PCR, transmission electron microscopy, immunofluorescence assay and fluorescence in situ hybridization. The GiCB cells showed typical cytopathic effect by day 6 post-infection with CyHV-2 including cell shrinkage, rounding, and cell fusion with cytoplasmic vacuolization. The virus titer reached 10(7.5 ± 0.37)TCID50/ml and has been successfully passaged over 50 times in the GiCB cell line. Electron microscopy analysis revealed the complete replication of CyHV-2 in GiCB cells. CyHV-2-infected GiCB cells reacted strongly with polyclonal antibodies against CyHV-2 and CyHV-2 RNA in cells hybridized specifically with the virus RNA probes. Additionally, an experimental infection demonstrated that CyHV-2 produced in GiCB cells caused 100% mortality in gibel carp. All the results provide solid evidence that the GiCB cell line is highly permissive for the isolation and propagation of CyHV-2. This is a significant advancement that will promote additional research on CyHV-2 infection in fish in the future.

Molecular characterisation and prevalence of a new genotype of Cyprinid herpesvirus 2 in mainland China.

Cyprinid herpesvirus 2 (CyHV-2, species Cyprinid herpesvirus 2) has been confirmed as a causative agent of the acute haematopoietic necrosis disease outbreak in farmed goldfish (Carassius auratus L.) and gibel carp (Carassius auratus gibelio Bloch). In this study, we present the genomic characteristics of a variant CyHV-2 strain (SY-C1) isolated from farmed gibel carp in mainland China and its comparative genomics analysis with the CyHV-2 reference strain ST-J1. Overall, the full-length genome of SY-C1 shares 98.8% homology with that of ST-J1. Sequence comparisons between SY-C1 and ST-J1 indicate that the variations include single-nucleotide mutations, insertions, deletions, and rearrangements, which suggested that SY-C1 is different from ST-J1 and represents a new genotype. Therefore, we propose that the identified CyHV-2 can be divided into 2 different genotypes and be named China genotype (C genotype) and Japan genotype (J genotype) according to their isolation loci. Furthermore, epidemiological surveys indicate that the dominant genotype of CyHV-2 circulating in mainland China is closer to the China genotype than the Japan genotype.

Identification, tissue distribution and characterization of two heat shock factors (HSFs) in goldfish (Carassius auratus).

Heat shock proteins (HSPs) are synthesized rapidly in response to a variety of physiological or environmental stressors, whereas the transcriptional activation of HSPs is regulated by a family of heat shock factors (HSFs). In vertebrates, multiple HSFs (HSF1-4) have been reported to have different roles in response to a range of stresses. This paper reports the cDNA cloning of two goldfish (Carassius auratus) HSF gene families, HSF1 and three isoforms of HSF2. Both HSF1 and HSF2s showed high homology to the known HSFs from other organisms, particularly the zebrafish. Different patterns of HSF1 and HSF2 mRNA expression were detected in several goldfish tissues, highlighting their distinct roles. In cadmium (Cd)-treated tissues, the responses of HSP70 showed less difference. However, the increase in HSF1 and HSF2 in these tissues differs considerable. In particular, HSF2 was induced strongly in the heart and liver. On the other hand, in heart tissue, HSF1 showed the smallest increment. These results suggest the potential role of HSF2 in assisting HSF1 in these tissues. In another in vitro experiment of hepatocyte cultures, Cd exposure caused similar patterns of goldfish HSF1 and HSF2 mRNA expression and induction of the HSP70 protein. On the other hand, an examination of the characterization of recombinant proteins showed that HSF1 undergoes a conformation change induced by heat shock above 30 °C and approaches each other in the trimer, whereas HSF2 could not sense thermal stress directly. Furthermore, immune-blot analysis of HSFs showed that both monomers and trimmers of HSF1 were observed in cadmium-induced tissues, whereas HSF2 were all in monomeric. These results show that HSF1 and HSF2 play different roles in the transcription of heat shock proteins.