Laem-Singh Virus Research Articles

Full genome characterization of Laem Singh virus (LSNV) in shrimp Penaeus monodon

Laem Singh virus (LSNV) was discovered in 2006 and proposed as a necessary but insufficient cause of retarded growth in the giant tiger shrimp Penaeus monodon. Its closest relatives were plant viruses including an unassigned Sobemovirus and viruses in the family Luteoviridae. During succeeding years, attempts to obtain the full LSNV genome sequence by genome walking failed. However, recent publication of the full sequence of Wenzhou shrimp virus 9 (WZSV 9) at GenBank revealed that LSNV sequences in our database shared 99% sequence identity with it. Thus, we hypothesized that LSNV and WZSV 9 were different isolates of the same virus species. Here we confirm that hypothesis by cloning and sequencing of the full genome of LSNV from P. monodon and by showing that it consists of two fragments each with 99% identity to the matching fragments of WZSV.

Major viral diseases in culturable penaeid shrimps: a review

The practice and development of the culture of commercially important penaeid shrimp results in the occurrence of infectious and non-infectious diseases worldwide. Most of the diseases are caused by the opportunistic microorganisms which are part of the microflora and fauna of the penaeid shrimp. Once the pathogens get a favourable condition, it will cause diseases to the host organisms. Most of the organisms are ubiquitous and are found and reported all over the cultured area in the world. The pathogens are the bacteria, viruses, parasites and protozoans. Viruses are the most important disease-causing agents in penaeid shrimps. The penaeid viruses are widely spread in geographical areas of shrimp culture. The shipment of broodstock and post larvae from one geographical region to another region often results in spreading of viral diseases. The penaeid viruses are the Whispovirus, white spot syndrome virus (WSSV), Penaeus monodon baculovirus (monodon baculovirus), infectious hypodermal and haematopoietic necrosis virus (IHHNV), infectious myonecrosis virus (IMNV), Taura syndrome virus (TSV), yellow head virus (YHV), white tail disease (WTD), covert mortality nodavirus (CMNV), Laem–Singh virus (LSNV) and shrimp haemocyte iridescent virus (SHIV). The review examines and highlights the aquaculture penaeid shrimp viral diseases in detail and concludes the need and importance of severe disease surveillance of the global countries through networking and coordination to safeguard the aquaculture practice for our future generation.

Feasibility of dsRNA treatment for post-clearing SPF shrimp stocks of newly discovered viral infections using Laem Singh virus (LSNV) as a model

Using post-larvae derived from specific pathogen free (SPF) stocks in penaeid shrimp farming has led to a dramatic increase in production. At the same time, new pathogens of farmed shrimp are continually being discovered. Sometimes these pathogens are carried by shrimp and other crustaceans as persistent infections without gross signs of disease. Thus it is that a 5-generation stock of Penaeus monodon SPF for several pathogens was found, post-stock-development, to be persistently-infected with newly-discovered Laem Singh virus (LSNV). In this situation, the stock developers were faced with destroying their existing stock (developed over a long period at considerable cost) and starting the whole stock development process anew in order to add LSNV to its SPF list. As an alternative, it was hypothesized that injection of complementary dsRNA into viral-infected broodstock prior to mating might inhibit replication of the target virus sufficiently to reduce or eliminate its transmission to their offspring. Subsequent selection of uninfected offspring would allow for post-clearing of LSNV from the existing stock and for conversion of the stock to LSNV-free status. Testing this hypothesis using the LSNV-infected stock described above, we found that transmission was substantially reduced in several treated broodstock compared to much higher transmission in buffer-injected broodstock. Based on these results, the model is proposed for post-clearing of SPF stocks using dsRNA treatment. The model may also be applicable to post-clearing of exceptional, individual performers from grow-out ponds for return to a nucleus breeding center.

Current status of viral diseases in Indian shrimp aquaculture.

The intensification of aquaculture has been unique in showing the overwhelming changes in global food production in the last 100 years. Presently, it is playing a vital role in the economies of several countries. Conversely, it is also to be noted that the progression of aquaculture has been the foundation of anthropogenic alteration of a gigantic hierarchy and hence not astonishingly, it resulted in spread and emergence of an increasing group of new unknown diseases. In India, Penaeus monodon, black tiger shrimp was previously the foremost-cultivated shrimp species. Subsequently in 2008, the American white leg shrimp Litopenaeus vannamei has effectively replaced it. The change in dominant species has affected disease concerns in India as well as in world shrimp aquaculture. White spot syndrome virus (WSSV) is the most deleterious for both species. Hepatopancreatic parvovirus (HPV), Monodon baculovirus (MBV) and Infectious hypodermal and hematopoietic necrosis virus (IHHNV) are the other significant infectious agents of P. monodon and L. vannamei. An emerging disease of loose shell syndrome (LSS) was already reported from India during late 1998. A more recent disease of L. vannamei in India is monodon slow growth syndrome (MSGS), a component of which seems to be Laem-Singh virus (LSNV). Thus, most of the information in this review relates to new emerging pathogens that threaten the cultivation shrimp industry in India.

Demonstration of a very inexpensive, turbidimetric, real-time, RT-LAMP detection platform using shrimp Laem-Singh virus (LSNV) as a model.

Rapid and accurate detection of pathogens under field laboratory conditions is necessary for effective control of veterinary pathogens. Here we describe a prototype, portable, pathogen detection device developed for single tube, real-time, reverse transcription, loop-mediated isothermal amplification (RT-LAMP) using Laem-Singh virus (LSNV) as a model. LSNV is an RNA virus and a component cause of growth retardation in black tiger shrimp. We chose its RNA-dependent RNA polymerase (RdRp) gene as the target for our tests. The basis for detection was measurement of turbidity arising from formation of a white, insoluble magnesium pyrophosphate precipitate byproduct upon amplification of the RdRp target sequence from 100 ng template RNA extracted from shrimp. The measurement device consisted of a heating block to maintain constant temperature in the RT-LAMP reaction for 8 Eppindorf sample tubes, a light-emitting diode (LED) light source providing red light emission at 650 nm wavelength to pass through sample tubes, a light dependent resistance (LDR) photo-detector and a software program to report turbidity events and could potentially be marketed for under US$3000. The device was connected to a computer to display real-time results in a variety of formats. The optimized protocol for LSNV detection consisted of incubation of the sample tubes at 65°C for 1 h during which turbidity was continuously measured, and quantitative results could be obtained by reaction time measurement. The sensitivity of detection was comparable to that of conventional nested RT-PCR and there was no cross reaction with other common shrimp viruses. The device was used for quantitative measurement of relative copy numbers of LSNV RdRp in 8 shrimp tissues and they were found to be highest in the gills followed in order by the lymphoid organ and hemolymph (p≤0.05). This platform can be easily adapted for detection of other pathogens under field laboratory settings.

Therapeutic effect of Artemia enriched with Escherichia coli expressing double-stranded RNA in the black tiger shrimp Penaeus monodon

We exploited Artemia as a double-stranded (ds)RNA-delivery system to combat viral diseases in shrimp. First, the transformed Escherichia coli (E. coli) expressing red fluorescent protein (RFP) was tested in the Artemia enrichment process. RFP signals detectable in the gut of Artemia under confocal microscope were evident for the successful encapsulation. Second, the Artemia enrichment process was performed using E. coli producing Laem-Singh virus (LSNV)-specific dsRNA, which has been previously shown to inhibit the viral infection in the black tiger shrimp Penaeus monodon by intramuscular injection and oral administration. The enriched Artemia nauplii were confirmed to contain dsRNA-LSNV by RT-PCR, and were subjected to the feeding test with P. monodon postlarvae. Quantitative RT-PCR indicated that a number of LSNV copies in most of the treated shrimp were, at least, 1000-fold lower than the untreated controls. During 11–17weeks after feeding, average body weight of the treated group was markedly increased relative to the control group. A smaller differential growth rate of the treated group as compared to the control was also noticed. These results suggested that feeding shrimp with the dsRNA-enriched Artemia can eliminate LSNV infection, which is the cause of retarded growth in P. monodon. The present study reveals for the first time the therapeutic effect of dsRNA-enriched Artemia for shrimp disease control.

Using double-stranded RNA for the control of Laem-Singh Virus (LSNV) in Thai P. monodon

Viral inhibition by double-stranded (ds)RNA is a potential therapeutic approach for controlling shrimp viral diseases. Here, we describe the successful oral application of dsRNA targeting Laem-Singh Virus (LSNV) to diminish monodon slow growth syndrome (MSGS) in Thai Penaeus monodon. Shrimp feed formulated with bacterially expressed LSNV-dsRNA was given to shrimp for 9 weeks. RT-PCR results revealed that all control shrimp were LSNV-positive at the end of experiment, while the shrimp that received dsRNA-feed exhibited 20–60% LSNV reduction. The average body weight of treated shrimp (number of shrimp=100) was significantly higher than that of the control group. Such increase is likely due to the elimination of MSGS caused by LSNV, as size variation of the treated group is much lower than that in the control group. This study demonstrates for the first time that feed with LSNV-specific dsRNA promotes the overall growth of P. monodon and relieves MSGS condition in LSNV-infected shrimp. The work reaffirms the potential of dsRNA application for controlling viral disease in shrimp farming.

Laem-Singh Virus: A Probable Etiological Agent Associated with Monodon Slow Growth Syndrome in Farmed Black Tiger Shrimp (Penaeus monodon)

Among the emerging diseases in shrimp aquaculture, monodon slow growth syndrome (MSGS) is a major concern in South and Southeast Asia. Shrimp farming in Thailand was severely affected during 2000-2002 due to MSGS, which caused an economic loss, of about US$ 300 million. MSGS is characterized by abnormally slow growth with coefficients of size variation of >35%, that has impacted P. monodon production in Thailand. A new shrimp virus, Laem-Singh virus (LSNV) was identified to be associated in MSGS affected shrimp. LSNV a RNA virus of about 25nm diameter is phylogenetically related to the insect-borne viruses in the families Barnaviridae, Tymoviridae and Sobemoviridae an important histopathological observation is exclusively noticed in growth-retarded shrimp. The LSNV infections have been confirmed in various organs of infected shrimp such as lymphoid organ, gills and nervous tissues by various diagnostic techniques such as reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization, quantitative real-time RT-PCR and reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick (RT-LAMP-LFD) and these tools are available for the diagnosis of LSNV. Recently, an integrase containing element has been identified in absolute association with LSNV in stunted growth shrimp. The transmission of LSNV through horizontal and vertical routes has been experimentally demonstrated. The known natural host-range of LSNV includes P. monodon and other penaeid shrimp. The putative RdRp gene involved in replication of LSNV was targeted for dsRNA-mediated gene silencing and appeared to be effective in a dose-dependent manner. Since the discovery of LSNV in 2006 in Thailand, it has been added to the list of viruses to be excluded from domesticated specific pathogen-free stocks of P. monodon and it has been recommended that shrimp farmers avoid stocking post larvae positive for LSNV to prevent MSGS in their farms.

Decreased level of crustacean hyperglycemic hormone (CHH) in black tiger shrimp Penaeus monodon suffering from Monodon Slow-Growth Syndrome (MSGS)

Monodon Slow-Growth Syndrome (MSGS), a pathological condition in the black tiger shrimp Penaeus monodon, is associated with Laem–Singh virus (LSNV) infection. Infected shrimp grow slowly when the virus invades the part of the retina called the zona fasciculata. Since molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH) are synthesized in the optic lobe and are related to molting activity and growth of the shrimp, the purpose of this study is therefore to determine the levels of these hormones and related parameters in MSGS shrimp. P. monodon juveniles were sampled from normal and MSGS ponds and were divided into small-negative, large-negative, small-positive and large-positive groups, depending on the size of the shrimp and whether they were LSNV-negative or LSNV-positive. Individual shrimp were measured for duration of each molt stage and molt interval. Levels of MIH1 and CHH1 transcripts were determined from the optic lobe, using real-time reverse transcriptase–polymerase chain reaction. The results revealed that the small-positive and the small-negative shrimp did not differ in durations of molt stages or molt intervals, as well as on the levels of MIH1 transcript in the optic lobe, MIH1 peptide in the optic lobe and hemolymph, or ecdysteroids in the hemolymph. Differences in molting activities and related transcript and hormones were observed between the small- and large-sized shrimp, but not in the status of LSNV infection. While levels of CHH1 transcript in the optic lobe of both small-negative and small-positive shrimp did not differ, levels of CHH1 peptide, as well glucose, in the hemolymph of the small-positive shrimp were significantly lower than those of the small-negative group. Levels of glycogen in the hepatopancreas of the small-positive shrimp were also significantly higher than that of the small-negative ones. The results suggest that growth retardation in MSGS shrimp is related to the suppression of the release of CHH1 peptide by LSNV invasion in the zona fasciculata, consequently causing decreased hepatopancreatic glycogenolysis and persistent hypoglycemia, resulting in growth stunting.

Natural host-range and experimental transmission of Laem-Singh virus (LSNV)

Slow growth caused by viral diseases has become a major constraint in shrimp aquaculture. Laem-Singh virus (LSNV), a positive-sense single-stranded RNA (ssRNA) virus, has been identified in Penaeus monodon showing slow growth syndrome. To examine the host-range and transmission modes of the virus, 6 species of penaeid shrimp of varying life stages, sourced from the wild and from farms, as well as juvenile mud crabs Scylla serrata, were screened using RT-nested PCR. LSNV was detected in P. monodon, Fenneropenaeus merguiensis, Metapenaeus dobsoni, and Litopenaeus vannamei, but not in E indicus, Marsupenaeus japonicus or S. serrata. LSNV was most prevalent in P. monodon followed by M. dobsoni, F. merguiensis, and L. vannamei, and real-time quantitative RT-PCR (qRT-PCR) showed that LSNV infection loads were highest in P. monodon, followed by L. vannamei, M. dobsoni, and E merguiensis. The nucleotide sequence of the LSNV RdRP gene fragment amplified by RT-nested PCR was highly conserved (99% identity) across these 4 penaeid species. LSNV was detected in both small and normal-sized P. monodon collected from the same pond. In experimental infections of both P. monodon and S. serrata, LSNV infection loads increased over time. The present study extends the known natural penaeid host-range and geographical distribution of LSNV and shows for the first time the potential susceptibility of S. serrata.

Rapid and sensitive detection of Laem-Singh virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick

Laem-Singh virus (LSNV) was discovered recently in Thailand in farmed Giant Tiger shrimp (Penaeus monodon) displaying signs of slow growth syndrome. Loop-mediated isothermal amplification (LAMP) allows DNA to be amplified rapidly at a constant temperature. Here a reverse transcription (RT)-LAMP method was combined with a chromatographic lateral-flow dipstick (LFD) to detect LSNV RNA rapidly and specifically. The reaction was optimized at 65°C for 30min and amplified DNA hybridized to an FITC-labeled oligonucleotide probe for 5min was detected at LFD test line 5min after application. Including 10min for rapid RNA extraction, test results could be generated within 1h and did not require electrophoresis. Compared to an existing RT-PCR method, the RT-LAMP-LFD was also ∼1000-fold more sensitive in detecting LSNV RNA.

A novel integrase-containing element may interact with Laem-Singh virus (LSNV) to cause slow growth in giant tiger shrimp

BackgroundFrom 2001-2003 monodon slow growth syndrome (MSGS) caused severe economic losses for Thai shrimp farmers who cultivated the native, giant tiger shrimp, and this led them to adopt exotic stocks of the domesticated whiteleg shrimp as the species of cultivation choice, despite the higher value of giant tiger shrimp. In 2008, newly discovered Laem-Singh virus (LSNV) was proposed as a necessary but insufficient cause of MSGS, and this stimulated the search for the additional component cause(s) of MSGS in the hope that discovery would lead to preventative measures that could revive cultivation of the higher value native shrimp species.ResultsUsing a universal shotgun cloning protocol, a novel RNA, integrase-containing element (ICE) was found in giant tiger shrimp from MSGS ponds (GenBank accession number FJ498866). In situ hybridization probes and RT-PCR tests revealed that ICE and Laem-Singh virus (LSNV) occurred together in lymphoid organs (LO) of shrimp from MSGS ponds but not in shrimp from normal ponds. Tissue homogenates of shrimp from MSGS ponds yielded a fraction that gave positive RT-PCR reactions for both ICE and LSNV and showed viral-like particles by transmission electron microscopy (TEM). Bioassays of this fraction with juvenile giant tiger shrimp resulted in retarded growth with gross signs of MSGS, and in situ hybridization assays revealed ICE and LSNV together in LO, eyes and gills. Viral-like particles similar to those seen in tissue extracts from natural infections were also seen by TEM.ConclusionsICE and LSNV were found together only in shrimp from MSGS ponds and only in shrimp showing gross signs of MSGS after injection with a preparation containing ICE and LSNV. ICE was never found in the absence of LSNV although LSNV was sometimes found in normal shrimp in the absence of ICE. The results suggest that ICE and LSNV may act together as component causes of MSGS, but this cannot be proven conclusively without single and combined bioassays using purified preparations of both ICE and LSNV. Despite this ambiguity, it is recommended in the interim that ICE be added to the agents such as LSNV already listed for exclusion from domesticated stocks of the black tiger shrimp.

Antiviral effect of PmRab7 knock-down on inhibition of Laem-Singh virus replication in black tiger shrimp

PmRab7 is a Penaeus monodon small GTPase protein possibly involved in replication of several shrimp viruses. In this study RNA interference (RNAi) using double-stranded RNA (dsRNA) targeting PmRab7 gene (dsRNA-PmRab7) was employed to silence the expression of PmRab7 to investigate the inhibitory effect on Laem-Singh virus (LSNV) replication. Injection of dsRNA-PmRab7 24 h before challenge with the virus resulted in a drastic decrease of PmRab7 mRNA and complete inhibition of LSNV replication at 3 days post-challenge. In a therapeutic mode, shrimp injected with dsRNA-PmRab7 1 day but not at 3 or 5 days post-LSNV challenge resulted in inhibition of LSNV replication. These results pave the way to use dsRNA-PmRab7 to prevent or cure LSNV infection in shrimp.

Detection of Laem-Singh virus in cultured Penaeus monodon shrimp from several sites in the Indo-Pacific region

Laem-Singh virus (LSNV) is a positive-sense single-stranded RNA (ssRNA) virus that was recently identified in Penaeus monodon shrimp in Thailand displaying signs of slow growth syndrome. A total of 326 shrimp collected between 1998 and 2007 from countries in the Indo-Pacific region were tested by RT-PCR for evidence of LSNV infection. The samples comprised batches of whole postlarvae, and lymphoid organ, gill, muscle or pleopod tissue of juvenile, subadult and adult shrimp. LSNV was not detected in 96 P. monodon, P. japonicus or P. merguiensis from Australia or 16 P. monodon from Fiji, Philippines, Sri Lanka and Mozambique. There was no evidence of LSNV infection in 73 healthy juvenile P. vannamei collected during 2006 from ponds at 9 locations in Thailand. However, LNSV was detected in each of 6 healthy P. monodon tested from Malaysia and Indonesia, 2 of 6 healthy P. monodon tested from Vietnam and 39 of 40 P. monodon collected from slow-growth ponds in Thailand. A survey of 81 P. monodon collected in 2007 from Andhra Pradesh, India, indicated 56.8% prevalence of LSNV infection but no clear association with disease or slow growth. Phylogenetic analysis of PCR amplicons obtained from samples from India, Vietnam, Malaysia and Thailand indicated that nucleotide sequence variation was very low (>98% identity) and there was no clustering of viruses according to site of isolation or the health status of the shrimp. The data suggests that LSNV exists as a single genetic lineage and occurs commonly in healthy P. monodon in parts of Asia.

Detection of viruses in Penaeus monodon from India showing signs of slow growth syndrome

The presence of viruses implicated in slow growth of Penaeus monodon, hepatopancreatic parvovirus (HPV), monodon baculovirus (MBV), infectious hypodermal and hematopoietic necrosis virus (IHHNV) and Laem–Singh virus (LSNV) was studied by polymerase chain reaction (PCR) in 72 P. monodon samples, which showed slow growth rate. The prevalence of IHHNV was highest 18/72 (25%) followed by HPV 5/72 (6.9%), LSNV 3/72 (4.2%) and MBV 3/72 (4.2%). Further, 20/72 (27.7%) samples were positive for WSSV and 18/72 (25%) had clinical conditions typical of monodon slow growth syndrome (MSGS) of which eight samples had IHHNV, two had LSNV and remaining eight were free from all known slow growth viruses. In a hatchery based study, 71 post larval samples were analysed and the incidence of IHHNV was highest 44/71 (61.9%) followed by MBV 7/71 (9.8%), HPV 6/71 (8.4%) and WSSV 2/71 (2.8%). Hatchery rearing of IHHNV infected P. monodon nauplii up to 50 days confirmed the virus to be responsible for slow growth. The study suggests the presence of IHHNV in India and indicates that this virus could be one of the causes of slow growth in cultured P. monodon.

Current Status of Viral Diseases in Asian Shrimp Aquaculture

The giant or black tiger shrimp, Penaeus monodon, was formerly the dom- inant cultured shrimp species in Asia. Since approximately 2002, it has been essentially replaced by the domesticated American whiteleg shrimp P. vannamei. The change in dominant species has affected disease concerns. For both species, white spot syndrome virus (WSSV) and yellow head virus (YHV) are the most lethal. For P. monodon, the next most important dis- eases are hepatopancreatic parvovirus (HPV) and monodon baculovirus (MBV). For P. vannamei, they are taura syndrome virus (TSV) and infec- tious hypodermal and hematopoietic necrosis virus (IHHNV). TSV was introduced to Asia in 1998 by careless importation of shrimp stocks for aquaculture but has not been reported to cause problems with local crus- tacean species. IHHNV, which is endemic in Asia, is harmless to P. mon- odon but poses a constant threat to IHHNV-free stocks of P. vannamei if they are hatched and reared in Asia under non-biosecure conditions. An emerging disease for P. monodon is monodon slow growth syndrome (MSGS), a component of which seems to be Laem-Singh virus (LSNV). Infectious myonecrosis virus (IMNV) is a P. vannamei disease, first report- ed from Brazil but now reported in Indonesia where it was probably intro- duced by careless importation of shrimp aquaculture stocks. So far, IMNV has not been reported in other Asian countries. Penaeus vannamei nodavirus (PvNV) is a new pathogen first reported from Belize in 2004 with gross and histological signs that are indistinguishable from those of IMNV. The disease has not yet affected Asian culture. A more recent disease of P. vannamei in Asia is abdominal segment deformity disease (ASDD), possi- bly caused by a yet unknown local virus.

Abdominal segment deformity disease (ASDD) of the whiteleg shrimp Penaeus vannamei reared in Thailand

From 2004–2006, shrimp farmers in Thailand, Malaysia and Indonesia have complained of cultured whiteleg shrimp Penaeus vannamei that grow and survive normally but have abdominal segment deformity disease (ASDD). This is characterized by abdominal segments that are enlarged or twisted laterally and/or dorso-ventrally, sometimes accompanied by opaque muscles. Histological analysis revealed abdominal muscle necrosis and degeneration, with haemocytic infiltration. To study the cause, affected shrimp were tested by polymerase chain reaction (PCR) methods for the presence of known viruses including infectious hypodermal and haematopoietic necrosis virus (IHHNV), infectious myonecrosis virus (IMNV), P. vannamei nodavirus ( PvNV), Macrobrachium rosenbergii nodavirus ( MrNV), Taura syndrome virus (TSV) and Laem Singh virus (LSNV). Except a few shrimp that were positive for IHHNV, TSV and LSNV, the diseased shrimp were negative for all of the viruses tested. Their negative status was confirmed by in situ hybridization specific for IHHNV, IMNV, MrNV, TSV and LSNV. By transmission electron microscopy (TEM), 20–22 nm, non-enveloped viral particles were observed in the cytoplasm of the muscle lesions, connective tissue and glial cells of neural tissues and gills. Affected neural tissues included the optic lobe, brain, thoracic ganglia, abdominal ganglia and ventral nerve cord. The findings suggest that ASDD in P. vannamei may be caused by a new virus that primarily invades neural tissue and results in neuromuscular dysfunction and abnormal morphology.

Retinopathy in stunted black tiger shrimp Penaeus monodon and possible association with Laem-Singh virus (LSNV)

Monodon slow-growth syndrome (MSGS) is a condition of Penaeus monodon cultivation ponds characterized by abnormally slow growth and coefficients of size variation greater than 35%. Shrimp from these ponds often show the presence Laem-Singh virus (LSNV) in the lymphoid organ, heart and other tissues. We compared various tissues of abnormally small and normal size (large) P. monodon from 3 MSGS ponds and from 2 reference ponds with normal growth (control ponds) both histologically and for the presence of LSNV. Small and large shrimp from the MSGS ponds were positive for LSNV by reverse transcriptase polymerase chain reaction (RT-PCR) tests while shrimp from one normal-growth pond were positive and from another negative. The LSNV infections were confirmed by transmission electron microscopy (TEM) and in situ hybridization (ISH). The only striking difference between the small and large LSNV-positive shrimp was retinopathy exclusively in the small shrimp from the MSGS ponds. Retinopathy comprised abnormally enlarged haemolymphatic vessels, haemocytic infiltration and rupture of the membrane that separated the fasciculated zone from the overlying row of retinular cells. By TEM and ISH, LSNV was detected in the fasciculated zone and in onion bodies of the organ of Bellonci in the small shrimp from the MSGS ponds, but not in those tissues and cells of the large shrimp from the MSGS pond or from the normal-growth ponds, whether LSNV-positive or not. The results suggested that retinopathy associated with LSNV may be linked causally to stunting of P. monodon in MSGS ponds.

Detection of Laem-Singh virus (LSNV) in cultured Penaeus monodon from India

Viral diseases have become a major constraint on the growth of shrimp aquaculture in India. During a study of new diseases in cultured tiger shrimp Penaeus monodon, diseased samples randomly collected from the southwestern and southeastern coasts of India were analyzed for white spot syndrome virus (WSSV), monodon baculovirus (MBV), and hepatopancreatic parvovirus (HPV) by nested PCR, and for Laem-Singh virus (LSNV) by reverse transcription PCR (RT-PCR). Of the 56 samples analyzed, 3 were positive for LSNV. These samples had signs of loose shell syndrome (LSS). Of the 3 samples that tested positive for LSNV, 2 were also positive for WSSV and MBV, and of these 2 samples, 1 was also positive for HPV. This is the first reported presence of LSNV in cultured shrimp in India.

A new RNA virus found in black tiger shrimp Penaeus monodon from Thailand

A new, apparently innocuous virus was found while investigating the cause of monodon slow growth syndrome (MSGS) in cultured black tiger shrimp ( Penaeus monodon). It was identified via plasmid vector clones of E. coli containing randomly amplified cDNA fragments produced from total nucleic acid extracts of hemolymph from MSGS shrimp. Of 421 clones, 30 that failed to give positive dot blot hybridization with a digoxigenin (DIG)-labeled shrimp DNA probe were sequenced and compared to GenBank records. Of these, 22 corresponded to known shrimp DNA records. Of eight that did not, one (20A) showed significant deduced amino acid sequence similarity to RNA-dependent RNA polymerases (RdRp) of the viruses in the family Luteoviridae and alignment revealed commonly conserved amino acids including a GDD motif believed to be at the enzyme active site. However, phylogenetic analysis showed that the virus sequence did not cluster with the Luteoviridae or other known RNA virus sequences. Thus, in accordance with frequent practice, it was named according to the area where it was first collected as Laem-Singh virus (LSNV). In situ hybridization with a DIG-labeled 20A insert revealed strong cytoplasmic staining confined to the lymphoid organ (LO), the heart and hepatopancreatic connective tissue in both normal and MSGS shrimp. RT-PCR assays based on the 20A clone sequence also gave positive results with both normal and MSGS shrimp. Transmission electron microscopy (TEM) of LO tissue revealed viral-like particles of approximately 27 nm diameter (within the Luteoviridae size range) in locations that matched those of positive in situ hybridization reactions in parallel samples. Although not directly associated with MSGS in Penaeus monodon, the presence or effect of this virus with other crustacean species is presently unknown.