Largemouth Bass Virus Research Articles

Inhibition of the largemouth bass virus replication by piperine demonstrates potential application in aquaculture

Largemouth bass virus (LMBV) is a systemic viral pathogen that can cause high mortality rates in cultivated largemouth bass. However, no treatment is currently approved. Therapeutic strategies against LMBV infection are urgently needed. In this study, we investigated the antiviral activity of piperine against LMBV in vitro and in vivo. In vitro antiviral activity assay showed that 210.28 μM piperine significantly decreased LMBV major capsid protein (MCP) gene expression in epithelioma papulosum cyprinid (EPC) cells by a maximum inhibitory rate of >95%. Piperine treatment inhibited LMBV replication in a dose-dependent manner, with the half-maximal activity (IC50 ) of 34.61 μM. Moreover, piperine significantly decreased the viral titers and cytopathic effects (CPE), contributing to the protection of infected cells. With regard to the steps of piperine affecting the life cycle of viruses, piperine had a direct inactivating effect on LMBV. During the virus adsorption phase, piperine prevented the adsorption of LMBV to EPC cells. Furthermore, piperine played an antiviral role mainly in the later stages of viral infection (4-8h). To further evaluate the antiviral activity of piperine against LMBV in vivo, largemouth bass as a model organism was carried out in relevant experiments. Intraperitoneal injection of piperine (25 mg/kg) effectively improved the survival rate of LMBV-infected largemouth bass by 20%. In addition, RT-qPCR results of viral replication in liver, spleen, kidney, gill and swim bladder tissues showed that piperine significantly inhibited LMBV replication in vivo, thus protecting largemouth bass from LMBV-induced death. Together, our results suggested that piperine is a therapeutic and preventative agent against LMBV infection.

Production and characterization of monoclonal antibodies against mandarinfish ranavirus and first identification of pyloric caecum as the major target tissue.

Mandarinfish ranavirus (MRV), also known as a variant of largemouth bass virus (LMBV), is an emerging pathogen in mandarinfish aquaculture. In this study, monoclonal antibodies (mAbs) against MRV were produced and characterized, and 7 mAbs were obtained through Western blotting screening and all 7 mAbs specifically recognized MRV/LMBV but not several piscine iridoviruses as ISKNV, GIV and TFV. By LC MS/MS analysis, the recognized viral proteins by seven mAbs were identified as MRV-pORF47L, MRV-pORF55R, MRV-pORF57L, MRV-pORF77L and MRV-pORF78L, respectively, and all five viral proteins are late expression structural proteins by Western blotting. Based on mAb 1C4, immuno-histochemistry and immuno-histo-fluorescence were performed to re-assess the tissue tropism of MRV. The result showed that abundant reactive signals were observed in infected spleen, kidney as well as intestine and pyloric caecum. Real-time quantitative PCR also demonstrated that spleen as well as pyloric caecum and intestines are the major target tissue upon MRV infection. In infected intestines and pyloric caecum, numerous enlarged, multinucleated cells with intracytoplasmic inclusions were identified as the target cells of MRV, suggesting that MRV serves as a digestive tract pathogen to mandarinfish, which may explain why acute infection of MRV can cause the typical clinicopathology featured by severe ascites.

Mesoporous silica nanocarriers loaded with ribavirin against largemouth bass virus

Largemouth bass virus (LMBV) has caused massive mortality in marine and freshwater animals worldwide, resulting in huge economic losses in aquaculture. Currently, rare methods for the treatment of LMBV are available for the lack of specific drugs and the restriction of biofilm barrier. Hence, developing alternative medications and efficient drug delivery strategies for the treatment of LMBV-related diseases is in demand. In this research, the anti-LMBV activity of ribavirin and mesoporous silica nanocarriers loaded with ribavirin (MSNs-R) was evaluated in vitro and in vivo. Ribavirin can efficiently protect EPC cells against LMBV infection, with half inhibitory concentration (IC50) values of 7.16 μM after 48 h of exposure. Similarly, ribavirin demonstrated potent anti-LMBV activity in largemouth bass, with 68.4% inhibition rate of LMBV after 7 days of continuous treatment. Furthermore, MSNs were exploited as drug carriers to enhance the anti-LMBV activity of ribavirin. An intelligent drug delivery system based on MSNs loaded with ribavirin was constructed and characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). MSNs-R was internalized in EPC cells and showed systemic dispersion in zebrafish larvae. And the cumulative survival rate of larvae treated with MSNs-R was 63.3% on the 7th day after infection, while that of the control group and naked ribavirin group was 3.3% and 40.0% respectively. The results to date demonstrated that ribavirin was an effective anti-LMBV drug and that MSNs could be promising candidate drug carriers for the treatment of LMBV-related diseases in aquaculture.

Development of a droplet digital PCR method for the sensitive detection and quantification of largemouth bass ranavirus.

Largemouth bass ranavirus (LMBRaV), also known as largemouth bass virus (LMBV), is a high mortality pathogen in largemouth bass. A rapid, sensitive, specific and convenient diagnosis method is an urgent requirement for the prevention of virus transmission. In the present study, a droplet digital PCR (ddPCR) method based on the major capsid protein (mcp) gene was established to detect and quantify the virus genome copy number. Oligonucleotide primers were designed based on the LMBRaV mcp gene sequence. The specificity and sensitivity of ddPCR assay were analysed. The other aquatic virus including Chinese giant salamander iridovirus (GSIV), Cyprinid herpesvirus II (CyHV-2) and infectious spleen and kidney necrosis virus could not be detected by LMBRaV ddPCR assay. The detection limit of ddPCR assay was 2 ± 0.37 copies/μl DNA sample. And this ddPCR assay had great repeatability and reproducibility. In clinical diagnosis of 50 largemouth bass, 43 positive samples were detected by ddPCR, whereas only 34 positive samples were detected by quantitative PCR (qPCR). This LMBRaV detection assay provided a specific and sensitive method for the rapid diagnosis of LMBRaV infection in largemouth bass as well as quantification of the virus load.

Simultaneous Isolation and Identification of Largemouth Bass Virus and Rhabdovirus from Moribund Largemouth Bass (Micropterus salmoides).

Largemouth bass is an important commercially farmed fish in China, but the rapid expansion of its breeding has resulted in increased incidence of diseases caused by bacteria, viruses and parasites. In this study, moribund largemouth bass containing ulcer foci on body surfaces indicated the most likely pathogens were iridovirus and rhabdovirus members and this was confirmed using a combination of immunohistochemistry, cell culture, electron microscopy and conserved gene sequence analysis. We identified that these fish had been co-infected with these viruses. We observed bullet-shaped virions (100–140 nm long and 50–100 nm in diameter) along with hexagonal virions with 140 nm diameters in cell culture inoculated with tissue homogenates. The viruses were plaque purified and a comparison of the highly conserved regions of the genome of these viruses indicated that they are most similar to largemouth bass virus (LMBV) and hybrid snakehead rhabdovirus (HSHRV), respectively. Regression infection experiments indicated fish mortalities for LMBV-FS2021 and HSHRV-MS2021 were 86.7 and 11.1%, respectively. While co-infection resulted in 93.3% mortality that was significantly (p < 0.05) higher than the single infections even though the viral loads differed by >100-fold. Overall, we simultaneously isolated and identified LMBV and a HSHRV-like virus from diseased largemouth bass, and our results can provide novel ideas for the prevention and treatment of combined virus infection especially in largemouth bass.

Largemouth Bass Virus Infection Induced Non-Apoptotic Cell Death in MsF Cells.

Largemouth bass virus (LMBV), belonging to the genus Ranavirus, causes high mortality and heavy economic losses in largemouth bass aquaculture. In the present study, a novel cell line, designated as MsF, was established from the fin of largemouth bass (Micropterus salmoides), and applied to investigate the characteristics of cell death induced by LMBV. MsF cells showed susceptibility to LMBV, evidenced by the occurrence of a cytopathic effect (CPE), increased viral gene transcription, protein synthesis, and viral titers. In LMBV-infected MsF cells, two or more virus assembly sites were observed around the nucleus. Notably, no apoptotic bodies occurred in LMBV-infected MsF cells after nucleus staining, suggesting that cell death induced by LMBV in host cells was distinct from apoptosis. Consistently, DNA fragmentation was not detected in LMBV-infected MsF cells. Furthermore, only caspase-8 and caspase-3 were significantly activated in LMBV-infected MsF cells, suggesting that caspases were involved in non-apoptotic cell death induced by LMBV in host cells. In addition, the disruption of the mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) generation were detected in both LMBV-infected MsF cells and fathead minnow (FHM) cells. Combined with our previous study, we propose that cell death induced by LMBV infection was cell type dependent. Although LMBV-infected MsF cells showed the characteristics of non-apoptotic cell death, the signal pathways might crosstalk and interconnect between apoptosis and other PCD during LMBV infection. Together, our results not only established the in vitro LMBV infection model for the study of the interaction between LMBV and host cells but also shed new insights into the mechanisms of ranavirus pathogenesis.

Antigenic epitope screening and functional modification of mannose enhance the efficacy of largemouth bass virus subunit vaccines.

Major capsid protein (MCP) can be used as a subunit vaccine against largemouth bass virus (LMBV). However, subunit vaccines usually have low immunogenicity. Here, to identify the major immunogenicity determinant region of the MCP gene, we truncated the MCP of the LMBV gene into four parts (MCP-1, MCP-2, MCP-3 and MCP-4). Enzyme-linked immunosorbent assay (ELISA) was used to identify the antigenicity of these four truncated MCP proteins. Then, the highly antigenic truncated protein was modified with mannose and connected with functionalized single-walled carbon nanotubes (SWCNTs) as carriers. Largemouth basses were immunized by bath immersion, challenged with LMBV on the 28th day after immunization and evaluated for related immune indicators. The results indicated that the MCP-2 protein could induce a higher antibody titre than the other truncated MCP proteins. We found that the levels of immune-related genes (TNF-α, CD40, IgM, IFNγ and IL-10) in the spleen and kidney were significantly increased in the MCP-2 and MCP-2-Man groups. ELISA results showed that the antibody content in the serum increased significantly in the MCP-2 group 7 days post-vaccination and increased with days in all the vaccinated groups, with the highest observed on the 21st day. Notably, the MCP-2-Man vaccine (10mg L-1 ) showed durability of immunoprotection efficacy that could protect largemouth basses from LMBV challenge, and the immune protection rate reached 78.94%. These results suggest that MCP-2 might be the major immunogenicity determinant region of LMBV and that the mannose-modified MCP-2 vaccine can induce stronger adaptive immune responses.

A Novel Sandwich ELASA Based on Aptamer for Detection of Largemouth Bass Virus (LMBV).

Largemouth bass virus (LMBV) is a major viral pathogen in largemouth bass culture, usually causing high mortality and heavy economic losses. Accurate and early detection of LMBV is crucial for diagnosis and control of the diseases caused by LMBV. Previously, we selected the specific aptamers, LA38 and LA13, targeting LMBV by systematic evolution of ligands by exponential enrichment (SELEX). In this study, we further generated truncated LA38 and LA13 (named as LA38s and LA13s) with high specificity and affinities and developed an aptamer-based sandwich enzyme-linked apta-sorbent assay (ELASA) for LMBV diagnosis. The sandwich ELASA showed high specificity and sensitivity for the LMBV detection, without cross reaction with other viruses. The detection limit of the ELASA was as low as 1.25 × 102 LMBV-infected cells, and the incubation time of the lysate and biotin labeled aptamer was as short as 10 min. The ELASA could still detect LMBV infection in spleen lysates at dilutions of 1/25, with good consistency of qRT-PCR. For the fish samples collected from the field, the sensitivity of ELASA was 13.3% less than PCR, but the ELASA was much more convenient and less time consuming. The procedure of ELASA mainly requires washing and incubation, with completion in approximately 4 h. The sandwich ELASA offers a useful tool to rapidly detect LMBV rapidly, contributing to control and prevention of LMBV infection.

Autophagy induced by largemouth bass virus inhibits virus replication and apoptosis in epithelioma papulosum cyprini cells

Autophagy and apoptosis play important roles in the occurrence and development of diseases. Largemouth bass virus (LMBV) is a primary agent that causes infectious skin ulcerative syndrome in largemouth bass and threatens the aquaculture of the species. We investigated the relationship between LMBV and autophagy, as well as the effect of autophagy on apoptosis induced by LMBV. Results showed that LMBV could induce autophagy in epithelioma papulosum cyprinid (EPC) cells. There was also an increase in LC3-II protein and decrease in p62 protein, along with autophagosome-like membranous vesicles and punctate autophagosomes fluorescent spots being observed in EPC cells. Enhancing autophagy inhibited the replication of LMBV and apoptosis in EPC cells while inhibiting autophagy produced the opposite effect. These results offer new insights into the pathogenesis of LMBV and anti-LMBV strategies.

The Effect of Largemouth Bass Virus on Bass Populations in Kansas Impoundments.

Largemouth Bass virus (LMBV) first became a concern in Kansas when it was identified as a potential cause of decreased catch rates at Crawford State Fishing Lake in 2007. The discovery of LMBV in eight additional impoundments from 2008 to 2017 increased concern about the prevalence and effects of LMBV in Kansas. In response, the Kansas Department of Wildlife, Parks, and Tourism tested 25 Largemouth Bass Micropterus salmoides impoundments for the presence of LMBV. The objectives of this study were to quantify the incidence of LMBV and examine differences in population metrics (i.e., body condition, relative abundance, and growth). A total of 1,260 Largemouth Bass were collected by using standard spring electrofishing surveys, and sagittal otoliths were collected from all of the sampled fish to estimate growth rates. Of the 25 study impoundments, 14 tested positive for LMBV. There was no evidence of LMBV effects on body condition, relative abundance of quality-length fish, or growth rates. The initial dates of LMBV infection of Largemouth Bass in these impoundments are unknown. The LMBV-positive populations in Kansas may have been exposed to the virus many years ago, and the fish may be in the process of rebounding from any potential negative effects.

Selection and Characterization of ssDNA Aptamers Targeting Largemouth Bass Virus Infected Cells With Antiviral Activities.

Largemouth bass virus (LMBV) is one of the most devastating viral pathogens in farmed Largemouth bass. Aptamers are novel molecule probes and have been widely applied in the field of efficient therapeutic and diagnostic agents development. LMBV-infected fathead minnow cells (LMBV-FHM) served as target cells in this study, and three DNA aptamers (LBVA1, LBVA2, and LBVA3) were generated against target cells by SELEX technology. The selected aptamers could specifically bind to LMBV-FHM cells, with rather high calculated dissociation constants (Kd) of 890.09, 517.22, and 249.31 nM for aptamers LBVA1, LBVA2, and LBVA3, respectively. Three aptamers displayed efficient antiviral activities in vitro. It indicates that the selected aptamers have great potentials in developing efficient anti-viruses treatments. The targets of aptamers LBVA1, LBVA2, and LBVA3 could be membrane proteins on host cells. The targets of aptamers (LBVA1, LBVA2, and LBVA3) come out on the cells surface at 8, 10, 8 h post-infection. As novel molecular probes for accurate recognition, aptamer LBVA3 could detect LMBV infection in vitro and in vivo, it indicates that the selected aptamers could be applied in the development of rapid detective technologies, which are characterized by high sensitivity, accuracy, and easy operation.

Establishment and characterization of a liver cell line, ALL, derived from yellowfin sea bream, Acanthopagrus latus, and its application to fish virology.

Yellowfin sea bream (Acanthopagrus latus) is an important economic fish, which is seriously threatened by various fish viruses. In this study, a cell line designated as ALL derived from the liver of yellowfin sea bream was developed and characterized. The cell line grew well in Dulbecco's modified Eagle's medium containing 10%-20% foetal bovine serum at 28°C. Amplification of the cytochrome B gene indicated that ALL cells originated from yellowfin sea bream. The modal chromosome number of ALL cells was 48. ALL cells were efficiently transfected with pEGFP-N3 plasmids, indicating the potential application of ALL cells in exogenous gene manipulation studies. ALL cells were susceptive to three main fish viruses, including viral haemorrhagic septicaemia virus (VHSV), red-spotted grouper nervous necrosis virus (RGNNV) and largemouth bass virus (LMBV). The replication of VHSV, RGNNV and LMBV in ALL cells was confirmed by quantitative real-time polymerase chain reaction, virus titre and transmission electron microscopy assays. Moreover, ALL cells could respond to VHSV, RGNNV and LMBV infections, as indicated by the differential expression of antiviral genes involving in the innate immune response. In conclusion, the newly established ALL cell line will be an excellent in vitro platform for the study of the virus-yellowfin sea bream interaction.

Development of a Multiplex Fluorescence in Situ Hybridization Assay to Identify Coinfections in Young-of-the-Year Smallmouth Bass.

Histopathological assessments of young‐of‐the‐year (age‐0) Smallmouth Bass Micropterus dolomieu in the Susquehanna River drainage identified a high prevalence of the myxozoan Myxobolus inornatus. This myxozoan infects the connective tissue of the muscle below the skin but is sometimes observed in the esophagus and buccal cavity. In some instances, shallow infections cause breaks in the skin, which could increase the chance of opportunistic bacterial infections. Several microbial pathogens, including Flavobacterium columnare, Aeromonas spp., and Largemouth Bass virus, have also been cultured from clinically diseased young of year. A multiplex fluorescence in situ hybridization (FISH) assay was developed to determine potential colocalization of M. inornatus, Flavobacterium spp., and Aeromonas spp. infections. With FISH, 75% of age‐0 Smallmouth Bass exhibited M. inornatus infections, 10% had Aeromonas spp. infections, and 5% had Flavobacterium spp. infections, while 3% had coinfections with both bacterial species and M. inornatus. The results of the multiplex FISH assay revealed a low occurrence of coinfections of Flavobacterium spp. and/or Aeromonas spp. with M. inornatus in randomly sampled individuals.

Generation and identification of novel DNA aptamers with antiviral activities against largemouth bass virus (LMBV)

Largemouth bass virus (LMBV) causes high mortality in largemouth bass, and results in serious economic loses in the aquaculture industry. However, there is no effective treatment to control LMBV infection. Aptamers are single-stranded oligonucleotides with high specificity and affinity to various targets, including viruses, viral components, and virus-infected cells. In this study, three novel DNA aptamers (LA38, LB49, and LA13), which specifically targeted LMBV, were identified by selective evolution of ligands by exponential enrichment. These aptamers formed stable stem-loop structures identified by secondary structures and minimum free energy predictions. Moreover, enzyme-linked apta-adsorption assays, dissociation constant (Kd) measurements, and flow cytometry analyses showed that these aptamers specifically bound to LMBV with high affinity at the nanomolar level. Furthermore, these aptamers were nontoxic, and significantly inhibited LMBV infection in vitro and in vivo. The mortality of largemouth bass was reduced approximately 40% after 10 days of infection by LMBV preincubated with LA38 or LB49, compared to that infected with wild or random ssDNA pretreated LMBV. In addition, confocal imaging and flow cytometry analyses indicated that these aptamers only entered cells along with LMBV, suggesting their potential as a delivery system. Presently, this is the first report of specific aptamers targeting iridovirus from freshwater fish. These aptamers have great potential in diagnosis and therapy, and in delivery agents for detecting LMBV infection.

Construction of a DNA vaccine and its protective effect on largemouth bass (Micropterus salmoides) challenged with largemouth bass virus (LMBV)

Largemouth bass virus (LMBV) is the causative agent of a disease causing high mortality rates in largemouth bass during summer. However, there is little information available about the development of vaccines for LMBV disease. Hence, a DNA vaccine, named pCDNA3.1(+)-MCP-Flag, was constructed by inserting the cloned LMBV major capsid protein (MCP) gene into the pCDNA3.1(+)-Flag plasmid. The expression of the recombinant plasmid was confirmed by Western blot (WB) and RT-PCR. The WB result revealed that the MCP protein produced a band of approximately 53 kDa, consistent with the expected result. The RT-PCR results also confirmed that MCP was transcribed in the EPC cells transfected with the recombinant plasmid. The largemouth bass in the DNA vaccine group were immunized with the pCDNA3.1(+)-MCP-Flag plasmid by pectoral fin base injection, and the relative percent survival (RPS) of fish challenged with LMBV was 63%. The relative immunological analyses were as follows. Compared with the PBS and pCDNA3.1(+) groups, the DNA vaccine group showed significantly upregulated expression of IL-1β, IL-8, TNF-α and Mx in the spleen, head kidney and liver. All largemouth bass immunized with the DNA vaccine produced a high titre of LMBV-specific neutralizing antibody during the immunization period. The titre was 1:375 ± 40 and peaked at 14 days post-vaccination. The expression of the recombinant plasmid was analysed in the tissues of the DNA vaccine group by RT-PCR. The recombinant plasmid was expressed in the spleen, head kidney and liver, and MCP protein was successfully expressed after vaccination. In conclusion, the recombinant plasmid expressing LMBV MCP induced significant immune responses in largemouth bass, and might represent a potential LMBV vaccine candidate for largemouth bass.

Naked-eyes detection of Largemouth bass ranavirus in clinical fish samples using gold nanoparticles as colorimetric sensor

Largemouth bass virus (LMBV) infection causes a lethal disease (ulcerative syndrome) in largemouth bass (Micropterus salmoides), which is an economically important freshwater fish species cultured in south-east China. For rapid detection of this virus, a simple and sensitive method was developed using probe-modified gold nanoparticles (AuNPs) as a colorimetric biosensor. A species-specific probe was attached to the surface of AuNPs, and thereby increasing the resistance of AuNPs to NaCl-induced aggregation. The NaCl concentration and ratio of probe to AuNPs in reaction mixture were evaluated, and the assay was optimized to detect only LMBV DNA sequence. Under optimum conditions, a linear relationship was observed between the ratio of A670/A520 and LMBV DNA concentrations over the range from 2.33 to 23.3 ng/μL and as low as 6.99 ng/μL of unamplied virus DNA could be detected only utilizing naked eyes. The entire assay was finished within 30 min, and the detection results were comparable to PCR examination in terms of reliability. Therefore, the visual assay is a potentially useful tool for aquaculture farmers to screen LMBV, and an ideal candidate for prevention and control of ulcerative syndrome in fish.

A Preliminary Study of Largemouth Bass Virus in Mexico.

Disease outbreaks and mortalities caused by largemouth bass virus (LMBV) in largemouth bass ( Micropterus salmoides) have been reported in the US. Blood and mucus samples tested by PCR to assess the presence of LMBV in largemouth bass in northeastern Mexico were negative, and further monitoring is needed.

Laboratory investigation into the role of largemouth bass virus (Ranavirus, Iridoviridae) in smallmouth bass mortality events in Pennsylvania rivers

BackgroundMortality episodes have affected young-of-year smallmouth bass (Micropterus dolomieu) in several river systems in Pennsylvania since 2005. A series of laboratory experiments were performed to determine the potential role of largemouth bass virus (Ranavirus, Iridoviridae) in causing these events.ResultsJuvenile smallmouth bass experimentally infected with the largemouth bass virus exhibited internal and external clinical signs and mortality consistent with those observed during die-offs. Microscopically, infected fish developed multifocal necrosis in the mesenteric fat, liver, spleen and kidneys. Fish challenged by immersion also developed severe ulcerative dermatitis and necrotizing myositis and rarely panuveitis and keratitis. Largemouth bass virus-challenged smallmouth bass experienced greater mortality at 28 °C than at 23 or 11 °C. Co-infection with Flavobacterium columnare at 28 °C resulted in significant increase in mortality of smallmouth bass previously infected with largemouth bass virus. Aeromonas salmonicida seems to be very pathogenic to fish at water temperatures < 23 °C. While co-infection of smallmouth bass by both A. salmonicida and largemouth bass virus can be devastating to juvenile smallmouth bass, the optimal temperatures of each pathogen are 7–10 °C apart, making their synergistic effects highly unlikely under field conditions.ConclusionsThe sum of our data generated in this study suggests that largemouth bass virus can be the causative agent of young-of-year smallmouth bass mortality episodes observed at relatively high water temperature.

Risk Factors Associated with Mortality of Age-0 Smallmouth Bass in the Susquehanna River Basin, Pennsylvania.

Evidence of disease and mortalities of young of the year (age-0) Smallmouth Bass Micropterus dolomieu has occurred during the late spring and summer in many parts of the Susquehanna River watershed since 2005. To better understand contributing factors, fish collected from multiple areas throughout the watershed as well as out-of-basin reference populations (Allegheny and Delaware River basins; experimental ponds, Kearneysville, West Virginia) were examined grossly and histologically for abnormalities. Tissue contaminant concentrations were determined from whole-body homogenates, and water contaminant concentrations were estimated using time-integrated passive samplers at selected sites. Observed or isolated pathogens included bacteria, predominantly motile Aeromonas spp. and Flavobacterium columnare; largemouth bass virus, and parasites, including trematode metacercariae, cestodes, and the myxozoan Myxobolus inornatus. Although these pathogens were found in age-0 Smallmouth Bass from multiple sites, no one pathogen was consistently associated with mortality. Chemicals detected in tissue included polychlorinated biphenyl (PCB) congeners, organochlorine, and current-use pesticides. Pyraclostrobin, PCB congeners 170 and 187, cis-chlordane and trans-nonachlor were detected in all Susquehanna watershed samples but rarely in samples from the reference site. The findings support the idea that there is no single cause for disease of age-0 Smallmouth Bass; rather the cumulative effects of co-infections and potential immunomodulation by environmental stressors during a sensitive developmental life stage may lead to mortality. Identifying the most important risk factors will be necessary for more in-depth analyses of individual stressors and better management of the habitat and fish populations.

Characterization of a virulent ranavirus isolated from marine ornamental fish in India.

A viral agent implicated in the mortality of marine ornamental "Similar Damselfish" (Pomacentrus similis Allen, 1991) was isolated and characterized. The virus grew well in marine and freshwater fish cell lines from seabass and snakehead. The virus was sensitive to chloroform, acidic pH (3.0) and heat treatment at 56°C. Biochemical characterisation indicated that the virus had double stranded DNA genome. Transmission electron microscopic analysis of ultrathin sections of infected cell pellets showed iridovirus-like icosahedral virus particles of 120-130nm. Purified virus had six structural protein bands that ranged from of 44 to 132kDa. PCR analysis confirmed the presence of viral DNA in infected cell cultures and sequence analysis of the major capsid protein gene showed an identity of 99.82% to that of largemouth bass virus. Serum neutralization studies involving the viral agent and koi ranavirus (KIRV) indicated partial homogeneity between the two isolates. Experimental infection of seabass (Lates calcarifer) and similar damselfish (P. similis) fingerlings with the similar damselfish virus showed cumulative mortalities of 68.75 and 93.33%. The biophysical and biochemical properties of the viral agent isolated, serological characteristics, size of major capsid proteins and the sequence similarity of the MCP gene proved that the virus belongs to the genus Ranavirus of the family Iridoviridae. Ability of the virus to grow in marine and freshwater fish cell lines and its pathogenicity to one of the cultivable marine fish shows the wide host range of the virus. This is the first report of ranavirus induced mortality in marine fish in India.