Warachin Gangnonngiw

Impact of yellow head virus outbreaks in the whiteleg shrimp, Penaeus vannamei (Boone), in Thailand.

Abstract Yellow head virus (YHV) is known as a major pathogen in the black tiger shrimp, Penaeus (Penaeus) monodon. It can also cause serious mortality in farmed whiteleg shrimp, Penaeus (Litopenaeus) vannamei. However, there is no published information on the economic and/or production impact of the disease in P. vannamei. Shrimp with gross signs of YHV disease (faded body colour and 60–70% mortality) were observed in 20 study farms rearing P. vannamei in the central part of Thailand from the end of 2007 through early 2008. The estimated economic loss for these farms according to the Thai Animal Aquaculture Association was approximately US

False rumours of disease outbreaks caused by infectious myonecrosis virus (IMNV) in the whiteleg shrimp in Asia

3 million. Detailed sequence analysis of RT‐PCR amplicons from shrimp in all the study ponds revealed the presence of YHV Type 1b (YHV‐1b) alone (characterized by a 162‐bp deletion in the ORF3 region encoding the structural gene for gp116) and the absence of YHV Type 1a (YHV‐1a), the original YHV type reported from Thailand. Despite the large 162‐bp deletion (= 54 deduced amino acids) in the gp116 structural gene, histopathology of YHV‐1b infections was identical to that of YHV‐1a infections, and electron microscopy revealed that YHV‐1b virions were morphologically indistinguishable from those previously reported for YHV‐1a. In addition, an existing commercial RT‐PCR detection kit and an immunochromatographic test strip for the detection of YHV were proven to have been valid tests for both YHV‐1b and YHV‐1a. The source of the virus for these outbreaks was unlikely to have been the post‐larvae used to stock the ponds, as they were derived from domesticated specific pathogen‐free stocks free of YHV. Thus, it is possible that they originated from an unknown, natural reservoir.

Non-virulence of a recombinant shrimp nidovirus is associated with its non structural gene sequence and not a large structural gene deletion

BackgroundInfectious myonecrosis virus (IMNV) disease outbreaks in cultivated whiteleg shrimp Penaeus (Litopenaeus) vannamei are characterized by gross signs of whitened abdominal muscles and by slow mortality reaching up to 70%. In 2006 the first disease outbreaks caused by IMNV in Asia occurred in Indonesia. Since then rumours have periodically circulated about IMNV disease outbreaks in other Asian countries. Our findings indicate that these are false rumours.FindingsOur continual testing by nested RT-PCR of shrimp samples suspected of IMNV infection from various Asian countries since 2006 has yielded negative results, except for samples from Indonesia. Our results are supported by the lack of official reports of IMNV outbreaks since January 2007 in the Quarterly Report on Aquatic Animal Diseases (QAAD) from the Network of Aquaculture Centers in Asia Pacific (NACA). In most cases, our shrimp samples for which tissue sections were possible showed signs of muscle cramp syndrome that also commonly causes muscle whitening in stressed whiteleg shrimp. Thus, we suspect that most of the false rumours in Asia about IMNV outside of Indonesia have resulted because of muscle cramp syndrome.ConclusionsResults from continual testing of suspected IMNV outbreaks in Asian countries other than Indonesia since 2006 and the lack of official country reports of IMNV outbreaks since January 2007, indicate that rumours of IMNV outbreaks in Asian countries outside of Indonesia are false. We suspect that confusion has arisen because muscle cramp syndrome causes similar signs of whitened tail muscles in whiteleg shrimp.

Successful propagation of shrimp yellow head virus in immortal mosquito cells.

RT-PCR using a commercial kit for yellow head virus (YHV) detection in growth-retarded shrimp yielded an unusual 777 bp amplicon instead of expected amplicons of 277 bp for YHV type-1 (YHV-1) or 406 bp for YHV type-2 (YHV-2). Cloning and sequencing (GenBank EU170438) revealed approximately 80% identity to non-structural (NS) ORF1b sequences of both YHV-1 (GenBank AA083987) and YHV-2 (GenBank AF227196), indicating an atypical YHV type (A-YHV) phylogenetically equidistant from both types. An RT-PCR test specifically designed for A-YHV revealed that it was uncommon and that its occurrence in shrimp culture ponds did not correlate with growth retardation or mortality. By immunohistochemistry with YHV-specific monoclonal antibodies, the A-YHV gave positive reactions for envelope protein gp64 and capsid protein p20, but not for envelope protein gp116, even though gp116 and gp64 originate from a polyprotein of ORF3. Lack of gp116 immunoreactivity correlated with a large ORF3 deletion (GenBank EU123854) in the region of the protein targeted by an MAb against gp116. Transmission electron microscopy of A-YHV-infected shrimp revealed only unenveloped pre-virions. During manuscript revision, information received revealed that typing of YHV isolates based on sequences of ORF1b and ORF3 had yielded several geographical types, including one virulent type (YHV-1b) with an ORF3 deletion sequence that matched the sequence of A-YHV. Using these sequences and an additional A-YHV sequence (EU853170) from the ORF1b typing region, A-YHV potentially represents a recombinant between type 1b and type 5. SDS-PAGE and Western blot analysis revealed that type 1b produced a gp116 deletion protein that did not bind with the MAb or polyclonal Ab to normal gp116. Overall, the information suggested that lack of A-YHV virulence was associated with the NS gene sequence linked to ORF1b rather than the deletion in ORF3.

Monodon baculovirus (MBV) infects the freshwater prawn Macrobrachium rosenbergii cultivated in Thailand

Research on crustacean viruses is hampered by the lack of continuous cell lines susceptible to them. To overcome this problem, we previously challenged immortal mosquito and lepidopteran cell lines with shrimp yellow head virus (YHV), followed by serial, split-passage of whole cells, and showed that this produced cells that persistently expressed YHV antigens. To determine whether such insect cultures positive for YHV antigens could be used to infect shrimp Penaeus monodon with YHV, culture supernatants and whole-cell homogenates were used to challenge shrimp by injection. Shrimp injected with culture supernatants could not be infected. However, shrimp injection-challenged with whole-cell homogenates from Passage 5 (early-passage) of such cultures died with histological and clinical signs typical for yellow head disease (YHD), while homogenates of mock-passaged, YHV-challenged cells did not. By contrast, shrimp challenged with cell homogenates of late-passage cultures became infected with YHV, but survived, suggesting that YHV attenuation had occurred during its long-term serial passage in insect cells. Thus, YHV could be propagated successfully in C6/36 mosquito cells and used at low passage numbers as a source of inoculum to initiate lethal infections in shrimp. This partially solves the problem of lack of continuous shrimp cell lines for cultivation of YHV.

Parvo-like virus in the hepatopancreas of freshwater prawns Macrobrachium rosenbergii cultivated in Thailand

Field specimens of post-larvae of the giant freshwater prawn (Macrobrachium rosenbergii) from Thailand showed hepatopancreatic tubule epithelial cells that contained central, eosinophilic inclusions within enlarged nuclei and marginated chromatin. These inclusions resembled those produced by some baculoviruses prior to formation of occlusion bodies that enclose virions in a polyhedrin protein matrix. By electron microscopy, the intranuclear inclusions contained bacilliform, enveloped virions (approximately 327+/-29nmx87+/-12nm) with evenly dense, linear nucleocapsids surrounded by trilaminar envelopes with lateral pockets containing nucleoproteinic filaments. In some cases, these were accompanied by moderately electron dense, spherical particles of approximately 20nm diameter resembling polyhedrin subunits of occlusion bodies (OB) of a bacilliform virus of the black tiger shrimp Penaeus monodon, previously reported from Thailand and called monodon baculovirus (MBV). It is currently listed by the International Committee on Taxonomy of viruses as Penaeus monodon nucleopolyhedrovirus (PemoNPV). Two polymerase chain reaction (PCR) assays for MBV gave positive results with DNA extracts prepared from M. rosenbergii samples using the hot phenol technique. One of these assays targeted the polyhedrin gene of MBV to which the resulting amplicon showed 100% sequence identity. Presence of the Penaeus monodon virus polyhedrin gene was confirmed by in situ hybridization assays and by positive immunohistochemical reactions in one sample batch. The data revealed that MBV can be found but may rarely produce polyhedrin occlusion bodies in M. rosenbergii.

Experimental infections reveal that common Thai crustaceans are potential carriers for spread of exotic Taura syndrome virus.

A survey of cultivated giant freshwater prawns Macrobrachium rosenbergii from Thailand revealed the presence of unusual spherical to ovoid inclusions in nuclei of hepatopancreas tubule epithelial cells. These began as small eosinophilic inclusions that became more basophilic as they increased in size. They were present in both R-cells and E-cells but were largest and deeply basophilic only in the E-cells. Confocal laser microscopy revealed that stained nucleic acid fluorescence from the inclusions was lost by treatment with DNase I specific for double- and single-stranded DNA and also lost or reduced by treatment with mungbean nuclease specific for single-stranded nucleic acids. Transmission electron microscopy (TEM) revealed that the inclusions contained tightly packed, unenveloped, viral-like particles of approximately 25 to 30 nm diameter, resembling those produced by shrimp parvoviruses. However, PCR, in situ hybridization and immunohistochemical tests for shrimp parvoviruses previously reported from Thailand were all negative. These results suggested that the inclusions contained a parvo-like virus, not previously reported from M. rosenbergii in Thailand.

In vitro cultivation of shrimp Taura syndrome virus (TSV) in a C6/36 mosquito cell line

Taura syndrome virus (TSV) was first reported as a serious cause of shrimp mortality limited to reared Penaeus (Litopenaeus) vannamei in the Americas, where it spread principally through regional and international transfer of live post larvae (PL) and broodstock. Subsequently, through importation of infected broodstock, TSV outbreaks spread to Asia, first to Taiwan and China and then to Thailand, Indonesia and Korea. Since its introduction to Thailand, outbreaks have occasionally been reported from rearing ponds stocked with batches of specific pathogen free (SPF) P. vannamei PL that tested negative for TSV by nested RT-PCR assay. Since it was possible that the outbreaks may have occurred via horizontal transfer of TSV from wild carrier species, we tested 5 common native crustaceans that live in and around shrimp ponds (2 palaemonid shrimp species, Palaemon styliferus and Macrobrachium lanchesteri, and 3 species of crabs, Sesarma mederi, Scylla serrata and Uca vocans) for susceptibility to TSV in experimental challenges. We found that U. vocans, S. serrata and S. mederi did not die but, respectively, gave strong RT-PCR reactions indicating heavy viral load at 5, 10 and 15 d post-injection of TSV and 10, 15 and up to 50 d after feeding with TSV-infected P. vannamei carcasses. Also after feeding, P. styliferus did not die, but a high proportion gave strong RT-PCR reactions at 5 d post-challenge and no reactions at 15 d. Similarly after feeding, M. lanchesteri showed no mortality and gave only light RT-PCR reactions at 2 d, moderate reactions at 5 d and no reaction at 15 d. By contrast, transmission experiments from the TSV-infected crabs and palaemonid shrimp via water or feeding resulted in death of all the exposed P. vannamei from 8 to 12 d post-challenge and all were positive for heavy viral load by RT-PCR assay. Despite the results of these laboratory challenge tests, natural TSV infections were not detected by nested RT-PCR in samples of these species taken from the wild. These results indicated that transmission of TSV from infected crabs and palaemonid shrimp via water or feeding might pose a potential risk to shrimp aquaculture.

Laminin receptor protein is implicated in hemocyte homeostasis for the whiteleg shrimp Penaeus (Litopenaeus) vannamei.

Journal of Fish Diseases Volume 34, Issue 10, pages 805–810, October 2011 Additional Information How to Cite Arunrut, N., Phromjai, J., Gangnonngiw, W., Kanthong, N., Sriurairatana, S. and Kiatpathomchai, W. (2011), In vitro cultivation of shrimp Taura syndrome virus (TSV) in a C6/36 mosquito cell line. Journal of Fish Diseases, 34: 805– 810. doi: 10.1111/j.1365-2761.2011.01286.x Author Information 1 Page 1 of 4 In vitro cultivation of shrimp Taura syndrome virus (TSV) in a C6/36 mosquito cell line Arunrut 20...

Duplex PCR assay and in situ hybridization for detection of Francisella spp. and Francisella noatunensis subsp. orientalis in red tilapia.

Here we show that knockdown of laminin receptor (Lamr) with PvLamr dsRNA in the whiteleg shrimp Penaeus (Litopenaeus) vannamei (Pv) caused a dramatic reduction specifically in hyaline hemocytes prior to death. Since apoptosis was not detected in hemocytes or hematopoietic cells, other possible causes of hemocyte loss were investigated. Reports that suppression of crustacean hematopoietic factor (CHF)-like protein or hemocyte homeostasis-associated protein (HHAP) also reduced shrimp hemocyte counts led us to carry out yeast two-hybrid (Y2H) and co-immunoprecipitation (co-IP) assays to test for interactions between Lamr and Pv homologues to these proteins (PvCHF-like and PvHHAP). The assays revealed that Lamr bound to both these homologues, but that the homologues did not bind to each other. Subsequent RT-PCR assays confirmed that PvLamr dsRNA injection significantly reduced expression levels for both PvCHF-like and PvHHAP genes. Further work is needed to determine how interaction among these three proteins can regulate shrimp hemocyte homeostasis.