Identification of an iridovirus in Acetes erythraeus (Sergestidae) and the development of in situ hybridization and PCR method for its detection

An iridovirus (tentatively named SIV, sergestid iridovirus) that causes high mortality in the sergestid shrimp, Acetes erythraeus, was found in Madagascar in 2004. Severely affected shrimp exhibit a blue–green opalescence. Histological examination revealed massive cytoplasmic inclusions in the cuticular epithelial cells, connective tissues, ovary and testes. The electron microscopic examination showed paracrystalline arrays of virions at a size of 140 nm, suggesting infection with an iridovirus. A pair of PCR primers were selected from the conserved region of the major capsid protein (MCP)-coding sequence among insect iridoviruses and used to amplify a 1.0 kb fragment from the infected A. erythraeus. This fragment was cloned, sequenced and found to be highly similar (upto 80% similarity in translated amino acids with an E value of 1e−124) to the MCP of invertebrate iridoviruses. This clone was then labeled with digoxigenin-11-dUTP and hybridized to tissue sections of infected A. erythraeus, which reacted positively to the probe. The reacting tissues included epithelial cells, connective tissues, and the germinal cells; the same cells as those with inclusions. A PCR method was also developed from the MCP coding sequence for detecting SIV.     

Histology, ultrastructure, and morphogenesis of a rickettsia-like organism causing disease in the oyster, Crassostrea ariakensis Gould

Moribund specimens of the oyster, Crassostrea ariakensis Gould, aged 2-3 years were collected from Hailing Bay in Yangxi County of Guangdong Province from February to May and November to December in the years 2001, 2002, and 2003. A massive infection by an obligate intracellular prokaryote, specifically a rickettsia-like organism (RLO), was found. Here we report investigations of this RLO in the tissues of the oyster C. ariakensis Gould and describe the histology, ultrastructure, and morphogenesis of this pathogen in C. ariakensis Gould. Light microscopic observations of stained tissues revealed cytoplasmic inclusion bodies typical of prokaryote infection in about 87% (26/30) of the oysters. Most inclusions were observed in epithelial cells and connective tissues of the gill, mantle, and digestive gland of most of the infected oysters. The shape, size, and color of inclusions from different tissues were polymorphic. Electron microscopic examination of digestive gland, gill, and mantle tissues showed that the RLOs were intracytoplasmic. RLOs were often round, dumb-bell-shaped (undergoing binary fission), or occasionally rod-shaped and ranged from approximately 0.58 to 1.20microm in size. The organisms exhibited an ultrastructure characteristic of prokaryotic bacteria-like cells, including a trilaminar cell wall, electron-dense periplasmic ribosome zone, and a DNA nucleoid. Reproductive stages, including transverse binary fission, were observed by TEM. These stages were frequently observed within membrane-bound cytoplasmic vacuoles. Hexagonal phage-like particles in the cytoplasm of RLOs were also observed.     

Histopathological characterization and in situ detection of Callinectes sapidus reovirus

A reovirus (tentatively designated as Callinectes sapidus reovirus, CsRV) was found in the blue crabs C. sapidus collected in Chesapeake Bay in 2005. Histological examination of hepatopancreas and gill from infected crabs revealed eosinophilic to basophilic, cytoplasmic, inclusions in hemocytes and in cells of connective tissue. A cDNA library was constructed from total RNA extracted from hemolymph of infected crabs. One clone (designated as CsRV-28) with a 532-bp insert was 75% identical in nucleotide sequence (and 95% similar in translated amino acid sequence) to the quanylytransferase gene of the Scylla serrata reovirus (SsRV). The insert of CsRV-28 was labeled with digoxigenin-11-dUTP and hybridized to sections of hepatopancreas and gill of infected C. sapidus, this probe reacted to hemocytes and cells in the connective tissue. No reaction was seen in any of the tissues prepared from uninfected crabs. Thus, this in situ hybridization procedure can be used to diagnose CsRV.     

Characterization of an iridovirus detected from cultured turbot Scophthalmus maximus in Korea

Juvenile turbot Scophthalmus maximus that became sick during an outbreak of disease at mariculture facilities at Go-Chang, Korea, in 2003, were examined to identify the cause of the disease. The fish had pale body color, an enlarged abdomen, protruding eyes, an enlarged spleen and kidney, and pale gills and/or liver. Histopathogical examination revealed basophilic enlarged cells in the kidney, spleen, gills, heart, stomach, intestine, liver, pancreas and skin. Hexagonal viral particles with a diameter of 136 to 159 nm were observed in the enlarged cells. A specific 1299 bp fragment of the major capsid protein (MCP) gene of the turbot iridovirus (TBIV) was amplified by PCR. Sequence homology was greater than 93.76% between the MCP gene in TBIV and the same gene in 5 viruses in the tentatively proposed genus Tropivirus (family Iridoviridae): red sea bream iridovirus, sea bass iridovirus, grouper sleepy disease iridovirus, African lampeye iridovirus and dwarf gourami iridovirus. These results suggest that the virus detected from turbot is similar to the proposed genus Tropivirus.     

Phylogenetic analysis of the major capsid protein gene of iridovirus isolates from cultured flounders Paralichthys olivaceus in Korea

In 2003, 13 isolates of iridovirus were obtained from cultured flounders Paralichthys olivaceus during epizootics in Korea. The full open reading frames (ORFs) encoding the major capsid protein (MCP) (1362 bp) from the 13 flounder iridoviruses (FLIVs) were sequenced and the deduced amino acid sequences were phylogenetically analyzed. Phylogenetic analysis of the MCP revealed that all 13 FLIVs were the same species as rock bream iridovirus (RBIV), red sea bream iridovirus (RSIV), and infectious spleen and kidney necrosis virus (ISKNV), and were grouped into an unknown genus which was different from the 2 genera known to infect fish, Ranavirus and Lymphocystivirus. This is the first report on the isolation and phylogenetic analysis of the iridovirus of unknown genus from flounders during epizootics.     

Development of in situ hybridization and RT-PCR assay for the detection of a nodavirus (PvNV) that causes muscle necrosis in Penaeus vannamei

A nodavirus (tentatively named PvNV, Penaeus vannamei nodavirus) that causes muscle necrosis in P. vannamei was found in Belize in 2004. From 2004 to 2006, shrimp samples collected from Belize exhibited clinical signs, white, opaque lesions in the tails and histopathology similar to those of shrimps infected by infectious myonecrosis virus (IMNV). Histological examination revealed multifocal necrosis and hemocytic fibrosis in the skeletal muscle. In addition, basophilic, cytoplasmic inclusions were found in striated muscle, lymphoid organ and connective tissues. However, IMNV was not detected in these shrimps by either RT-PCR or in situ hybridization, suggesting that these lesions may be caused by another RNA virus. Thus, a cDNA library was constructed from total RNA extracted from hemolymph collected from infected shrimp. One clone (designated PvNV-4) with a 928 bp insert was sequenced and found to be similar (69% similarity when comparing the translated amino acid sequences) to the capsid protein gene of MrNV (Macrobrachium rosenbergii nodavirus). The insert of PvNV-4 was labeled with digoxigenin-11-deoxyuridine triphosphate (dUTP) and hybridized to tissue sections of P. vannamei with muscle necrosis collected in Belize and from laboratory bioassays. The samples were positive for PvNV infection. Positively reacting tissues included skeletal muscle, connective tissues, the lymphoid organ, and hemocytes in the heart and gills. In addition, we experimentally infected both P. vannamei and P. monodon with PvNV prepared from Belize samples. A nested RT-PCR assay developed from the PvNV-4 cloned sequence showed that both species are susceptible to PvNV infection.     

Strain variation in an emerging iridovirus of warm-water fishes

Although iridoviruses vary widely within and among genera with respect to their host range and virulence, variation within iridovirus species has been less extensively characterized. This study explores the nature and extent of intraspecific variation within an emerging iridovirus of North American warm-water fishes, largemouth bass virus (LMBV). Three LMBV isolates recovered from three distinct sources differed genetically and phenotypically. Genetically, the isolates differed in the banding patterns generated from amplified fragment length polymorphism analysis but not in their DNA sequences at two loci of different degrees of evolutionary stability. In vitro, the isolates replicated at identical rates in cell culture, as determined by real-time quantitative PCR of viral particles released into suspension. In vivo, the isolates varied over fivefold in virulence, as measured by the rate at which they induced mortality in juvenile largemouth bass. This variation was reflected in the viral loads of exposed fish, measured using real-time quantitative PCR; the most virulent viral strain also replicated to the highest level in fish. Together, these results justify the designation of these isolates as different strains of LMBV. Strain variation in iridoviruses could help explain why animal populations naturally infected with iridovirus pathogens vary so extensively in their clinical responses to infection. The results of this study are especially relevant to emerging iridoviruses of aquaculture systems and wildlife.     

Bacteria in ovarioles of females from maleless families of ladybird beetles Adalia bipunctata L. (Coleoptera: Coccinellidae) naturally infected with Rickettsia,Wolbachia, and Spiroplasma

Ovarioles were found to be infected with Spiroplasma, Wolbachia, and Rickettsia in Adalia bipunctata females with maleless progeny in different natural populations. Ooplasm was infected with few Wolbachia bacteria. In ooplasm infected by Rickettsia, bacteria were present in small foci. Spiroplasmas were found encapsulated into ooplasm from the wider intercellular spaces between epithelial and oocyte cells. The cytoplasm of follicular epithelia infected with Rickettsia was heavily destroyed, but the nucleus was intact and free from bacteria. The essential feature of follicular epithelium cells from Spiroplasma and Wolbachia infected A. bipunctata females was inclusions of three types: crystalline, filaments, and concentric myelin-like lamellae. Observations of smears prepared from ovaries of A. bipunctata from natural populations revealed a low concentration of bacteria within a microscopy field (less 10 bacteria) in more than 90% of specimens, and only a few ovaries were heavily infected. Two different ways of bacterial invasion of the oocyte are suggested: Spiroplasma-like, through the intercellular spaces in the epithelium and Rickettsia-like, through the cytoplasm of follicular epithelium cells. Bacteria were not found in germarium zones and we suggest that each follicle is infected from haemolymph.     

Pathology, isolation, and preliminary molecular characterization of a novel iridovirus from tiger salamanders in Saskatchewan.

All iridovirus was confirmed to be the cause of an epizootic in larval and adult tiger salamanders (Ambystoma tigrinum diaboli) from four separate ponds in southern Saskatchewan (Canada) during the summer of 1997. This organism also is suspected, based on electron microscopic findings, to be the cause of mortality of larval tiger salamanders in a pond over 200 km to the north during the same year. Salamanders developed a generalized viremia which resulted in various lesions including: necrotizing, vesicular and ulcerative dermatitis; gastrointestinal ulceration; and necrosis of hepatic, splenic, renal, lymphoid, and hematopoietic tissues. In cells associated with these lesions, large lightly basophilic cytoplasmic inclusions and vacuolated nuclei with marginated chromatin were consistently found. Virus was isolated from tissue homogenates of infected salamanders following inoculation of epithelioma papilloma cyprini (EPC) cells. The virus, provisionally designated Regina ranavirus (RRV), was initially identified as an iridovirus by electron microscopy. Subsequent molecular characterization, including partial sequence analysis of the major capsid protein (MCP) gene, confirmed this assignment and established that RRV was a ranavirus distinct from frog virus 3 (FV3) and other members of the genus Ranavirus. Intraperitoneal inoculation of 5 x 10(6.23) TCID50 of the field isolate caused mortality in inoculated salamanders at 13 days post infection. Field, clinical, and molecular studies jointly suggest that the etiological agent of recent salamander mortalities is a highly infectious novel ranavirus.     

Early developmental stages of a protozoan parasite, Marteilioides chungmuensis (Paramyxea), the causative agent of the ovary enlargement disease in the Pacific oyster, Crassostrea gigas

A paramyxea, Marteilioides chungmuensis, causes the irregular enlargement of the ovary in the Pacific oyster, Crassostrea gigas in Korea and Japan. The knowledge about the life cycle of the parasite has been limited to the sporulation stages within the oocyte of oysters. In this study, we used the parasite-specific DNA probes and electron microscopy to experimentally infected oysters in a field and successfully clarified early developmental stages of the parasite. The parasite invaded the oysters through the epithelial tissues of the gills, mantle and labial palps. Extrasporogony repeatedly occurred in the connective tissues by binary fusion. The inner cell of the extrasporogonic stage migrated into the gonadal epithelium, invaded the oocyte to start sporulation.     

军曹鱼淋巴囊肿病毒主衣壳蛋白基因全序列分析

军曹鱼(Rachycentron canadum)亦称海鲡,是我国南方沿海一带的重要海水网箱养殖对象。2005年8月,广东省海水网箱养殖的军曹鱼首次暴发类似的淋巴囊肿病,病鱼的口唇、鳃、鳍、尾及体表等处,可看到大小不一的单个或成群的肿瘤,个别网箱的感染率在80%以上,死亡率近30%。病鱼形象丑陋,严重影响其市场价值,造成了较大的经济损失。淋巴囊肿病(Lymphocystis disease)发现于1874年,1965年正式确认该病病原为淋巴囊肿病毒[1],现已知可感染9目34科140种以上鱼类。我国在20世纪90年代陆续在养殖石斑鱼、鲈鱼、牙鲆中发现淋巴囊肿病[2-5],随后对其病原…     

Outbreaks and risks of infectious spleen and kidney necrosis virus disease in freshwater ornamental fishes

We examined the distribution of iridoviruses in 10 freshwater ornamental fish species hatched in Korea and imported from other Asian countries using both 1-step and 2-step polymerase chain reation (PCR). None of the 10 fish species analyzed were free of iridovirus as shown by 2-step PCR positive results, and 3 species yielded 1-step PCR positive results with associated mortality. Cloned PCR amplicons of the adenosine triphosphatase (ATPase) and major capsid protein (MCP) genes in genomic DNA of iridovirus showed the same nucleotide sequences as that of infectious spleen and kidney necrosis virus (ISKNV) isolated from the mandarinfish Siniperca chuatsi. These results indicate the presence of ISKNV disease in various ornamental fish as new host species and that the disease is widespread throughout different Asian countries including Korea, Singapore and China. Such infections were either clinical with associated mortality (and 1-step PCR positive) or asymptomatic in fish that were externally healthy (and only positive in 2-step PCR). Molecular analyses of the K2 region performed on iridovirus samples isolated from freshwater ornamental fishes revealed deletion/insertion of repetitive sequences of various lengths (42 to 339 bp), depending on the ISKNV isolates, without substitutions. Experimental infection of pearl gourami Trichogaster leeri and silver gourami T. microlepis with a tissue homogenate of pearl gourami infected by ISKNV induced 70 and 20% cumulative mortalities in the pearl and silver gourami, respectively.     

Major capsid protein gene sequence analysis of the Santee-Cooper ranaviruses DFV, GV6, and LMBV

The Santee-Cooper ranaviruses doctor fish virus (DFV), guppy virus 6 (GV6), and largemouth bass virus (LMBV) are members of the genus Ranavirus within the family Iridoviridae. The major capsid protein (MCP) is a main structural protein of iridoviruses and supports the differentiation and classification of ranaviruses. Presently the complete sequence of the MCP gene is known for most ranaviruses with the exception of the Santee-Cooper ranaviruses. In the present study, the complete nucleotide sequence of the MCP gene of DFV, GV6, and LMBV was determined. DFV and GV6 are identical within the MCP gene sequence. The identity compared to the corresponding sequence in LMBV amounts to 99.21%. The MCP gene of DFV, GV6, and LMBV exhibits only approximately 78% identity compared to the respective gene of other ranaviruses. Based on the sequence data obtained in the present study, a Rana MCP polymerase chain reaction (PCR) and subsequent restriction fragment length polymorphism (RFLP) analysis were developed to identify and differentiate ranaviruses, including DFV, GV6, and LMBV.     

Characterization of an envelope gene VP19 from Singapore grouper iridovirus

Background

Viral envelope proteins are always proposed to exert important function during virus infection and replication. Vertebrate iridoviruses are enveloped large DNA virus, which can cause great economic losses in aquaculture and ecological destruction. Although numerous iridovirus envelope proteins have been identified using bioinformatics and proteomic methods, their roles in virus infection remained largely unknown.

Methods

Using SMART and TMHMM programs, we investigated the structural characteristics of Singapore grouper iridovirus (SGIV) VP19. A specific antibody against VP19 was generated and the expression profile of VP19 was clarified. The subcellular localization of VP19 in the absence or presence of other viral products was determined via transfection and immune fluorescence assay. In addition, Western blot assay and electron microscopy examination were performed to demonstrate whether SGIV VP19 was an envelope protein or a capsid protein.

Results

Here, SGIV VP19 was cloned and characterized. Among all sequenced iridoviruses, VP19 and its orthologues shared common features, including 19 invariant cysteines, a proline-rich motif and a predicted transmembrane domain. Subsequently, the protein synthesis of VP19 was only detected at the late stage of SGIV infection and inhibited obviously by treating with AraC, confirming that VP19 was a late expressed protein. Ectopic expression of EGFP-VP19 in vitro displayed a punctate pattern in the cytoplasm. In SGIV infected cells, the newly synthesized VP19 protein was initially localized in the cytoplasm in a punctate pattern, and then aggregated into the virus assembly site at the late stage of SGIV infection, suggesting that other viral protein products were essential for VP19’s function during SGIV infection. In addition, Western blot assay and electron microscopy observation revealed that SGIV VP19 was associated with viral envelope, which was different from major capsid protein (MCP).

Conclusion

Taken together, the current data suggested that VP19 represented a conserved envelope protein in iridovirus, and might contribute greatly to virus assembly during virus infection.

     

Extraintestinal gamogony of Aggregata octopiana in the reared common octopus (Octopus vulgaris) (Cephalopoda: Octopodidae)

Aggregata octopiana (Apicomplexa, Aggregatidae) is the most prevalent coccidian in the wild common octopus (Octopus vulgaris), whose heteroxenous life cycle includes gamogony and sporogony undergoing in the octopus digestive tract. In the infected reared octopi, we observed an unusual extraintestinal distribution of the coccidian, with both gamogony and sporogony ongoing in dermal and gill tissue. Oocysts and macrogamonts were embedded in the dermal connective tissue of octopian arms, demarcated by a thin cyst wall or multilayered dark membrane. In gill connective and epithelial tissue all developmental stages were observed, eliciting hemocytic infiltration. Sometimes a complete substitution of the tissue by cysts and developmental stages occurred, resulting in necrosis of gill tissue.     

Comparative genomics of Bacillus thuringiensis phage 0305φ8-36: defining patterns of descent in a novel ancient phage lineage

Background

Members of the familyIridoviridae can cause severe diseases resulting in significant economic and environmental losses. Very little is known about how iridoviruses cause disease in their host. In the present study, we describe the re-analysis of theIridoviridae family of complex DNA viruses using a variety of comparative genomic tools to yield a greater consensus among the annotated sequences of its members.

Results

A series of genomic sequence comparisons were made among, and between theRanavirus andMegalocytivirus genera in order to identify novel conserved ORFs. Of these two genera, theMegalocytivirus genomes required the greatest number of altered annotations. Prior to our re-analysis, the Megalocytivirus species orange-spotted grouper iridovirus and rock bream iridovirus shared 99% sequence identity, but only 82 out of 118 potential ORFs were annotated; in contrast, we predict that these species share an identical complement of genes. These annotation changes allowed the redefinition of the group of core genes shared by all iridoviruses. Seven new core genes were identified, bringing the total number to 26.

Conclusion

Our re-analysis of genomes within theIridoviridae family provides a unifying framework to understand the biology of these viruses. Further re-defining the core set of iridovirus genes will continue to lead us to a better understanding of the phylogenetic relationships between individual iridoviruses as well as giving us a much deeper understanding of iridovirus replication. In addition, this analysis will provide a better framework for characterizing and annotating currently unclassified iridoviruses.     

Fusariosis in the shrimp Penaeus semisulcatus cultured in Israel

The first case of a mycotic infection in shrimp in Israel is reported. Fusarium solani produced a large melanized lesion in a specimen of Penaeus semisulcatus cultured at Eilat, on the Red Sea. Fungal hyphae elicited a strong hemocytic response in cuticular and connective tissues. In the underlying muscle, the inflammatory reaction appeared weaker, suggesting a gradual failure by the host to resist mycelial invasion. Three cases of human keratomycosis by F. solani have been reported in recent years in Israel, suggesting that handling infected shrimp may represent a hazard to aquaculturists.     

Changes in circulating and tissue-infiltrating hemocyte parameters of European flat oysters,Ostrea edulis,naturally infected with Bonamia ostreae

We assayed European flat oyster, Ostrea edulis, hemocyte parameters, circulating and tissue-infiltrating hemocyte densities, circulating hemocyte type distribution and lysosomal enzyme contents, to possibly relate these hematological parameters to Bonamia ostreae infection. Circulating hemocyte densities were not statistically different between infected and uninfected oysters. In contrast, the number of tissue-infiltrating hemocytes increased with infection intensity suggesting a recruitment process at the site of infection and a possibility for cells to migrate from circulatory system to connective tissues. Lysosomal enzymes were localized mainly in granulocytes both infected and uninfected, and mean of alpha-naphtyl butyrate esterase activity decreased with increasing B. ostreae infection level. The main response observed was a change in hemocyte type distribution between uninfected and infected oysters and greater tissue-infiltrating hemocytes with increased infections. These results suggest that the decrease of circulating granulocytes, and, consequently of some cell enzyme activities may be related with B. ostreae infection.