Isolation and partial characterization of a new reovirus in the Chinese mitten crab, Eriocheir sinensis H Milne Edwards.

A second type of freshwater crab reovirus has been isolated from Chinese mitten crab, Eriocheir sinensis H. Milne Edwards, in China; we named it E. sinensis reovirus (EsRV816). The negatively stained virion is a non-enveloped icosahedral particle, 60 ± 5 nm in diameter. Its genome is composed of 10 dsRNA linear pieces exhibiting an electrophoretic pattern of 5/3/2. The largest segment (RNA-1) was cloned and sequenced. The deduced amino acid sequence, corresponding to the RdRp of the virus, showed 26% identity with the RdRp of Operophtera brumata (L.) cypovirus 19 in the genus Cypovirus and 24% identity with RdRp of Nilaparvata lugens (Stal) reovirus in the genus Fijivirus. On the basis of its ultrastructure and physicochemical properties, this virus is quite different from other crab reoviruses, and particularly with another freshwater crab reovirus EsRV905, recently classified in a new genus Cardoreovirus. This virus (EsRV816) possesses all the characters of the members of the reoviridae family and could represent a new genus.

Virus-like particles associated with brown muscle disease in Manila clam, Ruditapes philippinarum, in Arcachon Bay (France).

Recently, Manila clam, Ruditapes philippinarum, populations have suffered mortalities in Arcachon Bay (SW France). Mortality was associated with extensive lesions of the posterior adductor muscle, which become progressively brown and calcified. Ultrastructural observations by transmission electron microscopy revealed tissue degradation with necrotized muscle fibres and granulocytomas. Unenveloped virus-like particles (VLPs) were detected in muscle, granulocytic, epithelial and rectal cells. VLPs were abundant in the extracellular space, in the cytoplasm (free or enclosed in vesicles) and in the nucleoplasm of granulocytes. Nuclei and mitochondria of granulocytes displayed changes which suggested reactive oxygen species production and apoptosis induction. VLPs exhibited an icosahedral structure with a diameter of 25 to 35 nm. These observations suggest that the VLPs could belong to the family Picornaviridae or the Parvoviridae.

Partial susceptibility of the SSN-1 fish cell line to a crustacean virus: a defective replication study.

This study evaluated the possible use of the fish SSN-1 cell line to investigate the development of Macrobrachium rosenbergii nodavirus (MrNV). Cells were incubated with viral particles and cytopathic effects were observed. De novo synthesis of viral capsid proteins was shown by immuno-fluorescence labelling and a sandwich ELISA test. Viral genomic replication was demonstrated by RT-PCR using primers specific to RNA-1 as well as by quantitative RT-PCR (RT-qPCR). Using electron microscopy, only a few empty particles were observed and attempts to isolate complete infectious particles or to re-infect healthy cells (second passage) were unsuccessful. As complete viral particles were rarely observed, it appeared that defaults in MrNV virogenesis might arise resulting in the formation of scarce and non-infectious particles. SSN-1 cells were found to be partially permissive to MrNV infection that induced cell lysis, but key elements for viral infection were lacking such as regulatory factors for gene replication or post-translational modifications.

Experimental vertical transmission of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) from brooders to progeny in Macrobrachium rosenbergii and Artemia.

White tail disease (WTD) is a serious problem in hatcheries and nursery ponds of Macrobrachium rosenbergii in India. Experiments were carried out to determine the possibility of vertical transmission of M. rosenbergii nodavirus (MrNV) and extra small virus (XSV) in M. rosenbergii and Artemia. Prawn broodstock inoculated with MrNV and XSV by oral or immersion challenge survived without any clinical signs of WTD. The brooders spawned 5-7 days after inoculation and the eggs hatched. The survival rate of larvae gradually decreased, and 100% mortality was observed at the post-larvae (PL) stage. Whitish muscle, the typical sign of WTD, was seen in advanced larval developmental stages. The ovarian tissue and fertilized eggs were found to be positive for MrNV/XSV by reverse transcriptase-polymerase chain reaction (RT-PCR) whereas the larval stages showed positive by RT nested PCR (nRT-PCR). In Artemia, reproductive cysts and nauplii derived from challenged brooders were normal and survival rates were within the expected range for normal rearing conditions. The reproductive cysts were found to be positive for MrNV/XSV by RT-PCR whereas the nauplii showed MrNV/XSV-positive by nRT-PCR. The PL of M. rosenbergii fed nauplii derived from challenged Artemia brooders died at 9 days post-inoculum with clinical signs of WTD.

Presumptive detection of yellow head virus by reverse transcriptase-polymerase chain reaction and dot-blot hybridization in Litopenaeus vannamei and L. stylirostris cultured on the Northwest coast of Mexico.

In order to assess the presence of yellow head virus (YHV) in shrimp farms along the Pacific coast of Mexico, 39 samples from 26 randomly chosen farms were analysed by means of reverse transcriptase-polymerase chain reaction (RT-PCR) and dot-blot hybridization. Eleven samples were positive for YHV. The disease was reproduced by means of an infectivity bioassay performed with an extract of pleopods from the positive samples. Cumulative mortality reached 50% in 14 days. Four pairs of primers which amplified several YHV genome regions were designed and used to test dead and surviving shrimp from the bioassay by RT-PCR, resulting in positive results for every expected amplicon. The results of this study provide presumptive evidence of the presence of YHV in Mexican shrimp farms at least during 1999-2000.

Pre-exposure to infectious hypodermal and haematopoietic necrosis virus or to inactivated white spot syndrome virus (WSSV) confers protection against WSSV in Penaeus vannamei (Boone) post-larvae.

Larvae and post-larvae of Penaeus vannamei (Boone) were submitted to primary challenge with infectious hypodermal and haematopoietic necrosis virus (IHHNV) or formalin-inactivated white spot syndrome virus (WSSV). Survival rate and viral load were evaluated after secondary per os challenge with WSSV at post-larval stage 45 (PL45). Only shrimp treated with inactivated WSSV at PL35 or with IHHNV infection at nauplius 5, zoea 1 and PL22 were alive (4.7% and 4%, respectively) at 10 days post-infection (p.i.). Moreover, at 9 days p.i. there was 100% mortality in all remaining treatments, while there was 94% mortality in shrimp treated with inactivated WSSV at PL35 and 95% mortality in shrimp previously treated with IHHNV at N5, Z1 and PL22. Based on viral genome copy quantification by real-time PCR, surviving shrimp previously challenged with IHHNV at PL22 contained the lowest load of WSSV (0-1x10(3) copies microg-1 of DNA). In addition, surviving shrimp previously exposed to inactivated WSSV at PL35 also contained few WSSV (0-2x10(3) copies microg-1 of DNA). Consequently, pre-exposure to either IHHNV or inactivated WSSV resulted in slower WSSV replication and delayed mortality. This evidence suggests a protective role of IHHNV as an interfering virus, while protection obtained by inactivated WSSV might result from non-specific antiviral immune response.

Rapid detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), the pathogenic agents of white tail disease of Macrobrachium rosenbergii (De Man), by loop-mediated isothermal amplification.

A loop-mediated isothermal amplification (LAMP) procedure is described for rapid diagnosis of white tail disease, a viral disease caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), in the giant freshwater prawn, Macrobrachium rosenbergii. This method was more sensitive than conventional RT-PCR for detecting the two viruses. A set of four primers, two outer and two inner, were designed for MrNV detection. An additional pair of loop primers was also used in an accelerated LAMP reaction for detection of XSV. Time and temperature conditions were optimized for detection of the two viruses. The LAMP reaction is highly suited for disease diagnosis in developing countries as amplification of DNA can be detected without the use of agarose gel electrophoresis, by the production of whitish precipitate of magnesium pyrophosphate as a by-product.

Gross signs and histopathology of branchiostegal blister disease (balloon disease): an idiopathic disease of farmed Macrobrachium rosenbergii (De Man).

The giant freshwater prawn, Macrobrachium rosenbergii, is facing increased threat due to disease as its culture becomes more widespread. A disease characterized by the swelling of the branchiostegal region and deformities of the appendages, named balloon disease by farmers, has caused considerable economic loss in the Nellore region of Andhra Pradesh, India. Clinical signs of diseased animals include a voluminous hypertrophy of some gill filaments and the inner area of the branchiostegite. By histology, hypertrophied areas at the level of the gill filaments or branchiostegite had an identical structure corresponding to a large cyst filled with a fluid containing a few free haemocytes, limited on one side by the cuticle and on the other by the subcuticular epithelial layer. Analysis of the diseased prawns did not reveal any pathogenic agent leading us to conclude that the disease is idiopathic, probably due to suboptimal water quality conditions.

Response of crayfish, Procambarus clarkii, haemocytes infected by white spot syndrome virus.

White spot syndrome virus (WSSV) is a serious pathogen of aquatic crustaceans. Little is known about its transmission in vivo and the immune reaction of its hosts. In this study, the circulating haemocytes of crayfish, Procambarus clarkii, infected by WSSV, and primary haemocyte cultures inoculated with WSSV, were collected and observed by transmission electron microscopy and light microscopy following in situ hybridization. In ultra-thin sections of infected haemocytes, the enveloped virions were seen to be phagocytosed in the cytoplasm and no viral particles were observed in the nuclei. In situ hybridization with WSSV-specific probes also demonstrated that there were no specific positive signals present in the haemocytes. Conversely, strong specific positive signals showed that WSSV replicated in the nuclei of gill cells. As a control, the lymphoid organ of shrimp, Penaeus monodon, infected by WSSV was examined by in situ hybridization which showed that WSSV did not replicate within the tubules of the lymphoid organ. In contrast to previous studies, it is concluded that neither shrimp nor crayfish haemocytes support WSSV replication.

Simultaneous detection of Macrobrachium rosenbergii nodavirus and extra small virus by a single tube, one-step multiplex RT-PCR assay.

Post-larvae of Macrobrachium rosenbergii infected with white tail disease were collected from hatcheries and nursery ponds in India. The causative organisms have been identified as Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV). A one-step multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to detect these viruses simultaneously in naturally and experimentally infected prawns. Several parameters were assayed in order to optimize the protocol for simultaneous detection. Naturally and experimentally infected prawns showed two prominent bands of 681 and 500 bp for MrNV and XSV, respectively, as in separate RT-PCR assays. Experimentally infected adult prawns showed two bands for these two viruses in all the organs, except hepatopancreas and eyestalk, as seen in normal RT-PCR. The sensitivity test carried out on the primer sets of MrNV and XSV revealed that these primers could simultaneously detect the two viruses at a level of 25 fg of total RNA prepared from infected samples using this multiplex RT-PCR protocol.

White tail disease of the giant freshwater prawn, Macrobrachium rosenbergii: separation of the associated virions and characterization of MrNV as a new type of nodavirus.

White tail disease of the farmed freshwater prawn, Macrobrachium rosenbergii, is the cause of mortalities in the French West Indies, China and India. Two different sized particles, both developing in the cytoplasm of target cells, are found associated with diseased animals. These two viruses were separated, purified and subsequently characterized. The larger one, called MrNV, is icosahedral in shape and 27 nm in diameter. Its genome is composed of two fragments of linear single-stranded RNA (ss-RNA), of 2.9 and 1.3 kb, respectively and its capsids exhibited a single polypeptide of 43 kDa. These characteristics and the partial sequence of a cloned fragment of RNA-1 suggest this agent is a member of the family Nodaviridae, but with differences from both the genera Alphanodavirus and Betanodavirus. The smaller virus, named XSV, is icosahedral in shape, 15 nm in diameter, possesses a linear ss-RNA genome of about 0.9 kb, and its capsid exhibits two polypeptides of 16 and 17 kDa, respectively. The relationships between these two viruses remain unknown.

Purification and characterization of a new reovirus from the Chinese mitten crab, Eriocheir sinensis.

A new reovirus was recently isolated from a freshwater crab, the Chinese mitten crab, Eriocheir sinensis, in China. The complete viral particles are 55 nm in diameter, icosahedral, non-enveloped and have a mean buoyant density of 1.39 g cm(-3) in CsCl gradient. The viral genome is composed of 12 pieces of dsRNA with an electrophoretic pattern of 3/4/2/3. This virus infects connective tissue of the gills, gut and hepatopancreas. Partial cDNA cloning and sequence analysis showed that the RNA-dependent RNA polymerase is located in the first RNA segment. From its biochemical, ultrastructural and physicochemical properties, this virus is quite different from the genus Aquareovirus (Reoviridae). It may represent a new genus of Reoviridae, different from the other crab reoviruses, P and W(2).

A synthetic antibacterial peptide from Mytilus galloprovincialis reduces mortality due to white spot syndrome virus in palaemonid shrimp.

White spot syndrome virus (WSSV) isolated from Penaeus monodon was found to be highly infective for the western Mediterranean shrimp, Palaemon sp. Using polymerase chain reaction (PCR), it was demonstrated that such shrimp are not naturally carriers of WSSV. Following challenge with virus, mortality reached 100% 3.5-4 days after injection at 22 degrees C. Incubation of infected shrimp at 10 degrees C totally suppressed the mortality which rapidly developed when shrimp were returned to 18 or 22 degrees C. Preincubation of WSSV with mature synthetic mytilin significantly reduced shrimp mortality with a 50% efficient dose of about 5 microM. Survival of shrimp was not due to the development of an active mechanism of defence as re-injection of WSSV produced the same mortality pattern. Mortality was probably due to WSSV replication as dot blot failed to detect viral DNA in the injection sample but was positive 1 day post-injection. Protection by mytilin was by interaction at the virus level, preventing replication as no WSSV nucleic acid was detected by PCR even after 7 days in shrimp injected with WSSV preincubated with 10 or 50 microM mytilin.

Experimental transmission and tissue tropism of Macrobrachium rosenbergii nodavirus (MrNV) and its associated extra small virus (XSV).

White tail disease (WTD) was found to be a serious problem in hatcheries and nursery ponds of Macrobrachium rosenbergii in India. The causative organisms have been identified as M. rosenbergii nodavirus (MrNV) and its associated extra small virus (XSV). Experimentally transmitted to healthy animals, they caused 100% mortality in post-larvae but failed to cause mortality in adult prawns. The RT-PCR assay revealed the presence of both viruses in moribund post-larvae and in gill tissue, head muscle, stomach, intestine, heart, hemolymph, pleopods, ovaries and tail muscle, but not in eyestalks or the hepatopancreas of experimentally infected adult prawns. The presence of these viruses in ovarian tissue indicates the possibility of vertical transmission. Pleopods have been found to be a suitable organ for detecting these viruses in brooders using the RT-PCR technique.

Genome-based detection methods of Macrobrachium rosenbergii nodavirus, a pathogen of the giant freshwater prawn, Macrobrachium rosenbergii dot-blot, in situ hybridization and RT-PCR.

The availability of specific and rapid detection methods is essential for monitoring the health status of farmed species, particularly in viral diseases as in this case early diagnosis is a critical factor in containing disease outbreaks. Three complementary genome-based methods were developed for the detection of Macrobrachium rosenbergii nodavirus (MrNV), i.e. dot-blot hybridization, in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR). Detection limits were established for dot-blot hybridization and RT-PCR and are c. 7 fg and 8 pg of viral RNA, respectively. In situ hybridization indicated that infection was confined to the striated muscle tissue. As a result of its sensitivity, RT-PCR can be used for in-depth investigations to examine the extent of the viral infection and establish the onset of infection in hatcheries. The application of RT-PCR on samples collected from prawn farms in China showed the possible use of this method in routine health monitoring.

Extra small virus-like particles (XSV) and nodavirus associated with whitish muscle disease in the giant freshwater prawn, Macrobrachium rosenbergii.

A disease of Macrobrachium rosenbergii, the giant freshwater prawn, farmed in China was recently recorded in Zhejiang, Jiangsu, Shanghai, Guangxi and Guangdong provinces. The clinical sign of the disease, which develops in post-larvae (PL), is a whitish appearance of the muscles, particularly noticeable in the abdomen. Mortalities may reach 100% in some hatcheries. Investigations by transmission electron microscopy after negative staining of diseased PL homogenates showed the presence of two types of viral particles: one, unenveloped, icosahedral in shape, 26-27 nm in diameter, the second, much smaller, about 14-16 nm in diameter, designated extra small virus particle (XSV). The large virus has a genome with two pieces of ssRNA (RNA-1 and RNA-2), of 3 and 1.2 kb, respectively. Hybridization tests confirmed that this large virus is closely related to M. rosenbergii nodavirus (MrNV) which was isolated from diseased prawns in a hatchery in the French West Indies. Its very small size and hypothesized biochemical and biological characteristics suggest XSV is a new type of crustacean virus. As XSV has always been found associated with the larger virus (nodavirus) and is located in muscle and connective cells of diseased animals, it could be an autonomous virus, a helper-type virus or a satellite-like virus.

A sandwich enzyme linked immunosorbent assay (S-ELISA) for detection of MrNV in the giant freshwater prawn, Macrobrachium rosenbergii (de Man).

A sandwich enzyme-linked immunosorbent assay (S-ELISA) was developed to improve diagnosis of white tail disease of the giant freshwater prawn, Macrobrachium rosenbergii, caused by the nodavirus, MrNV. Polyclonal antibodies were produced by immunization of Balb/C mice using a purified suspension of the virus and IgG anti-MrNV were purified from ascitic fluid. A sandwich method was successfully developed, coating first with unlabelled antibody and detecting trapped antigens with a second biotinylated antibody. Reaction was demonstrated using an avidin-peroxidase conjugate. Tissue extracts from M. rosenbergii infected with MrNV or purified viral extracts (control) were successfully identified in an individual ELISA, thus confirming the validity of the method. This S-ELISA should be the technique of choice for epidemiological studies of this disease and is a rapid and inexpensive assay with high specificity and sensitivity.