Up-regulation of ribophorin I after yellow head virus (YHV) challenge in black tiger shrimp Penaeus monodon

This work constitutes the second report from a continuing investigation of shrimp genes that may be involved in apoptosis associated death resulting from yellow head virus (YHV) infection. Here, we describe from the black tiger shrimp Penaeus monodon, a ribophorin I-like gene that is probably a subunit of the oligosaccharyltransferase complex (OST), a key enzyme in N-linked glycosylation that occurs in the endoplasmic reticulum. The OST complex also contains DAD1 (defender against apoptotic death 1) that has been reported to control apoptosis and that we have previously reported from P. monodon. The full length ribophorin I of P. monodon comprised 2157 bp with the ORF of 1806 bp corresponding to 601 deduced amino acids and three putative N-linked glycosylation sites. Analysis revealed hydrophobic properties implying that it could be a membrane protein. Tissue distribution analysis using real-time RT-PCR with SYBR Green revealed that ribophorin I was endogenously expressed in all examined tissues of normal shrimp. However, unlike DAD1 that was down-regulated after YHV challenge, ribophorin I expression was up-regulated and remained high until the moribund stage.     

A cDNA microarray approach for analyzing transcriptional changes in Penaeus monodon after infection by pathogens

A cDNA microarray comprised of 9990 different ESTs obtained from the Penaeus monodon EST project (http://pmonodon.biotec.or.th) was employed to identify viral (white spot and yellow head viruses) and bacterial (Vibrio harveyi) responsive genes in the hemocytes of P. monodon at 6, 24 and 48 h post-injection (hpi). The number of differentially expressed genes found was highest in shrimps infected with white spot virus (1954 genes) followed by yellow head virus (1136 genes) and V. harveyi (420 genes). Changes in shrimp gene expression were highest at the late infection stage for both viruses, whilst that for V. harveyi induced gene expression was mainly found at the early infection stage, but the repression of genes was mainly found in the mid stage of infection. Shrimp genes specifically upregulated by each particular pathogen are identified and are summarized.     

Suppression of PmRab7 by dsRNA Inhibits WSSV or YHV Infection in Shrimp

Viral entry into host cells requires endocytosis machineries of the host for viral replication. PmRab7, a Penaeus monodon small GTPase protein, was investigated for its function in vesicular transport during viral infection. The double-stranded RNA of Rab7 was injected into a juvenile shrimp before challenging with white spot syndrome virus (WSSV) or yellow head virus (YHV). PmRab7 mRNA was specifically decreased at 48 h after dsRNA-Rab7 injection. Silencing of PmRab7 dramatically inhibited WSSV-VP28 mRNA and protein expression. Unexpectedly, the silencing of PmRab7 also inhibited YHV replication in the YHV-infected shrimp. These results suggested that PmRab7 is a common cellular factor required for WSSV or YHV replication in shrimp. Because PmRab7 should function in the endosomal trafficking pathway, its silencing prevents successful viral trafficking necessary for replication. Silencing of PmRab7 could be a novel approach to prevent both DNA virus (WSSV) and RNA virus (YHV) infection of shrimp.     

Development of a monoclonal antibody specific to yellow head virus (YHV) from Penaeus monodon

A monoclonal antibody specific to yellow head virus (YHV) was produced from a mouse immunized with gill extracts prepared from laboratory-reared Penaeus monodon dually infected with YHV and white spot syndrome virus (WSSV). One clone designated V3-2B specifically bound to native and SDS-treated viral specific antigens. Immunocytochemical studies of infected gills revealed viral specific immunoreactivities in the cytoplasm of gill tissue and in haemocytes. No antibody binding was observed in gills from non-infected shrimp. In addition, immunocytochemical examination of tissues from shrimp experimentally infected with YHV gave a positive reaction, while tissues from uninfected control shrimp or shrimp experimentally infected with WSSV did not. Western blot analysis indicated that the antibody reacted with a protein of approximately 135 kD that was present only in shrimp infected with YHV. In dot-blot indirect immunoperoxidase assays, the antibody was able to detect viral associated antigen in diluted haemolymph up to 1:50 dilution and in an ammonium sulfate precipitate of haemolymph up to 1:1000 dilution. The results suggested that this antibody might be useful for development of effective diagnostic techniques for both heavy and mild YHV infections in shrimp.     

Differences in susceptibility of palaemonid shrimp species to yellow head virus (YHV) infection

Five species of palaemonid shrimp, Macrobrachium rosenbergii, M. lanchesteri, M. sintangense, Palaemon styliferus and P. serrifer, were collected from Penaeus monodon farming areas in Thailand. Some of each species were artificially infected with yellow head virus (YHV) by injection and then monitored by RT-PCR and by immunohistochemistry using monoclonal antibodies specific to 116 kDa, 64 kDa, and 20 kDa proteins of YHV. Natural YHV infections were not detected in any of the shrimp examined. In YHV injection experiments, a high proportion of P. serrifer, P. styliferus and M. sintangense exhibited mild to moderate YHV infections at 3 d post-injection. The severity of infection was reduced in shrimp that survived to 10 and 30 d post-injection. Using immunohistochemistry and RT-PCR, a small proportion of M. lanchesteri showed very mild YHV infections at Day 3 but no infections at Days 10 and 30. No YHV infections resulted in M. rosenbergii. The evidence suggested that M. sintangense, P. styliferus and P. serrifer are susceptible to YHV and carry it for some time. In contrast, M, rosenbergii and M. lanchesteri appear to resist YHV infection and eliminate YHV efficiently. Because they display a range of responses to YHV, palaemonid shrimp may serve as a good model for studying YHV defense mechanisms in shrimp.     

Viral diseases of penaeid shrimp with particular reference to four viruses recently found in shrimp from Queensland

The culture of penaeid shrimp world-wide is primarily dependent on wild-caught broodstock which has an enormous potential to introduce new pathogens, particularly viruses, into culture systems. Of the 13 viruses described for cultured penaeid shrimp, seven have been described within the past 5 years; the most devastating viral epidemics on record for cultured penaeid shrimp have also occurred within the past 5years. During examination of local wild and cultured shrimp, four new viruses were found. Bennettae baculovirus was discovered in the digestive gland of wild Metapenaeus bennettae. It closely resembles monodon baculovirus (MBV) but has a more slender virion, does not cross-react with a DNA probe for MBV and is not infectious to Penaeus monodon. Two morphologically indistinguishable viruses, one pathogenic (gill-associated virus, GAV) and the other benign (lymphoid organ virus, LOV), were found in cultured P. monodon. LOV and GAV closely resemble yellow head virus (YHV) of Thailand. A parvo-like virus was found recently in dying post-larvae of P. japonicus. As the intensity of shrimp culture world-wide increases, researchers can expect to discover more penaeid viruses. The need to close the life cycle of P. monodon and other cultured species and develop rapid diagnostic methods for viral infections has become imperative.     

Historic emergence, impact and current status of shrimp pathogens in Asia

It is estimated that approximately 60% of disease losses in shrimp aquaculture have been caused by viral pathogens and 20% by bacterial pathogens. By comparison, losses to fungi and parasites have been relatively small. For bacterial pathogens, Vibrio species are the most important while for viral pathogens importance has changed since 2003 when domesticated and genetically selected stocks of the American whiteleg shrimp Penaeus (Litopenaeus) vannamei (Boone 1931) replaced the formerly dominant giant tiger or black tiger shrimp Penaeus (Penaeus) monodon (Fabricius 1798) as the dominant cultivated species. For both species, white spot syndrome virus (WSSV) and yellow head virus (YHV) are the most lethal. Next most important for P. vannamei is infectious myonecrosis virus (IMNV), originally reported from Brazil, but since 2006 from Indonesia where it was probably introduced by careless importation of shrimp aquaculture stocks. So far, IMNV has not been reported from other countries in Asia. Former impacts of Taura syndrome virus (TSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) on this species have dramatically declined due to the introduction of tolerant stocks and to implementation of good biosecurity practices. Another problem recently reported for P. vannamei in Asia is abdominal segment deformity disease (ASDD), possibly caused by a previously unknown retrovirus-like agent. Next most important after WSSV and YHV for P. monodon is monodon slow growth syndrome (MSGS) for which component causes appear to be Laem Singh virus (LSNV) and a cryptic integrase containing element (ICE). Hepatopancreatic parvovirus (HPV) and monodon baculovirus (MBV) may be problematic when captured P. monodon are used to produce larvae, but only in the absence of proper preventative measures. Since 2009 increasing losses with P. vannamei in China, Vietnam and now Thailand are associated with acute hepatopancreatic necrosis syndrome (AHPNS) of presently unknown cause. Despite these problems, total production of cultivated penaeid shrimp from Asia will probably continue to rise as transient disease problems are solved and use of post larvae originating from domesticated SPF shrimp stocks in more biosecure settings expands.     

Development of simple sequence repeat markers from expressed sequence tags of the black tiger shrimp (Penaeus monodon)

In this study, we describe the development of expressed sequence tag-simple sequence repeat markers from expressed sequence tags of the black tiger shrimp (Penaeus monodon) deposited in public sequence databases. A total of 46 primer pairs were designed and screened on 26 individuals of P. monodon from a natural population. Of these, 16 primer pairs showed polymorphic profiles with between two and five alleles per locus. The average unbiased and direct count heterozygosities were 0.4662 and 0.3516, respectively. Cross-amplification was tested with five individuals of Penaeus vannamei and polymorphic products were detected at five loci.     

YHV-protease dsRNA inhibits YHV replication in Penaeus monodon and prevents mortality

Yellow head virus infects cultured shrimps and causes severe mortality resulting in a great economic loss. Haemolymph injection of dsRNA(pro) corresponding to the protease motif of YHV genome resulted in a complete inhibition of YHV replication. The effect of dsRNA lasted for at least 5 days. Injecting sequence-unrelated dsRNA(gfp) or dsRNA(TSV-pol) also resulted in an inhibition of YHV replication but at a comparatively much less extent. Shrimp mortality was monitored for 10 days when more than 90% shrimps receiving no dsRNA died within 8 dpi. However, those receiving dsRNA(pro) showed no mortality. A partial mortality was observed among the shrimps receiving dsRNA(gfp) or dsRNA(TSV-pol). Thus, Penaeus monodon possesses the sequence-specific protection to YHV infection, most likely through the RNAi pathway, in addition to sequence-independent protection. It gives a new notion that dsRNA induction of antiviral immunity in shrimp goes through two pathways, sequence-independent and sequence-dependent.