RT-nested PCR detection of Mourilyan virus in Australian Penaeus monodon and its tissue distribution in healthy and moribund prawns

Mourilyan virus (MoV) is a newly identified virus of Penaeus monodon prawns that is genetically related to the Uukuniemi virus and other phleboviruses of the Bunyaviridae. This paper describes an RT-nested PCR test that can reliably detect between 2 and 6 copies of a synthetic MoV RNA. Total RNA isolated from the lymphoid organ, gills and haemocytes of P. monodon with moderate infections gave comparable amplicon yields in the RT-PCR step of the test. However, in prawns with extremely low-level infections, haemocytes and gill tissue proved slightly more reliable in detecting MoV RNA following nested PCR. The distribution of MoV in tissues of healthy and moribund P. monodon was examined by in situ hybridisation (ISH) using a digoxigenin-labelled DNA probe to a approximately 0.8 kb M RNA segment cDNA insert in clone pMoV4.1. The DNA probe targeted a region in the MoV M RNA segment containing a coding sequence with homology to the C-terminus of the G2 glycoprotein of phleboviruses. In healthy prawns harbouring an unapparent MoV infection, ISH signal primarily occurred in the lymphoid organ, where it was more prominent in hypertrophied cells of 'spheroids' than within cells of normal tubules. ISH signal was also sometimes detected in cells of cuticular epithelium, segmental nerve ganglion and the antennal and tegmental glands. MoV was distributed widely throughout these and other cephalothoracic tissues of mesodermal and ectodermal origin in moribund P. monodon following experimental infection or collected from farm pond edges during disease episodes. Transmission electron microscopy of gill of moribund, captive-reared P. monodon identified spherical (approximately 85 nm diameter) to ovoid MoV particles (approximately 85 x 100 nm) in and around highly necrotic cells in which the nucleus and other organelles had disintegrated. MoV virions co-existed with rod-shaped virions of gill-associated virus and were often seen clustered within cytoplasmic vacuoles or associated with the outer rim of concentric ring-shaped structures comprised of endoplasmic membranes likely to represent degenerated Golgi.     

Detection of gill-associated virus (GAV) by in situ hybridization during acute and chronic infections of Penaeus monodon and P. esculentus

Chronic and acute gill-associated virus (GAV) infections were examined by in situ hybridization (ISH) using a DNA probe targeting a 779 nucleotide region of the ORF1b-gene. Chronic GAV infections were observed in healthy Penaeus monodon collected from farms and healthy P. esculentus surviving experimental infection. During chronic-phase infections in both species, GAV was detected only in partitioned foci of cells with hypertrophied nuclei (spheroids) within the lymphoid organ. Acute-phase infections were observed in moribund P. monodon and P. esculentus infected experimentally with a high dose of GAV, and in moribund P. monodon collected from farms during outbreaks of disease. During acute experimental infections in P. monodon, ISH detected GAV throughout the lymphoid organ, in gills and in connective tissues throughout the cephalothorax. In moribund P. monodon collected from natural outbreaks of disease, GAV was also detected in the gills and in connective tissues of the cephalothorax, but the distribution of virus within the lymphoid organ varied. In acutely infected P. esculentus, GAV was detected in connective tissues, but was restricted to the inner stromal matrix cells and endothelial cells of intact lymphoid organ tubules. The tissue distribution of GAV identified by ISH suggests that shrimp are able to control and maintain chronic asymptomatic infection by a process involving lymphoid organ spheroids. Acute phase infections and the development of disease appear to be dose-related and involve the systemic distribution of virus in connective tissues throughout the cephalothorax.     

The gene encoding the nucleocapsid protein of Gill-associated nidovirus of Penaeus monodon prawns is located upstream of the glycoprotein gene

The ORF2 gene of Gill-associated virus (GAV) of Penaeus monodon prawns resides 93 nucleotides downstream of the ORF1a-ORF1b gene and encodes a 144-amino-acid hydrophilic polypeptide (15,998 Da; pI, 9.75) containing 20 basic (14%) and 13 acidic (9%) residues and 19 prolines (13%). Antiserum to a synthetic ORF2 peptide or an Escherichia coli-expressed glutathione S-transferase-ORF2 fusion protein detected a 20-kDa protein in infected lymphoid organ and gill tissues in Western blots. The GAV ORF2 fusion protein antiserum also cross-reacted with the p20 nucleoprotein in virions of the closely related Yellow head virus. By immuno-gold electron microscopy, it was observed that the ORF2 peptide antibody localized to tubular GAV nucleocapsids, often at the ends or at lateral cross sections. As GAV appears to contain only two structural protein genes (ORF2 and ORF3), these data indicate that GAV differs from vertebrate nidoviruses in that the gene encoding the nucleocapsid protein is located upstream of the gene encoding the virion glycoproteins.     

Penaeus monodon is protected against gill-associated virus by muscle injection but not oral delivery of bacterially expressed dsRNAs

Gill-associated virus (GAV) is a nidovirus that commonly infects Penaeus monodon (black tiger shrimp) in eastern Australia, causing morbidity and mortalities in the acute stage of disease. Here we explored the possibility of inhibiting GAV replication and disease using double-stranded (ds)RNAs expressed in bacteria and delivered either orally or by muscle injection. To enhance potential RNA interference (RNAi) responses, 5 long dsRNAs were used that targeted open reading frame 1a/1b (ORF1a/b) gene regions and thus only the genomic length RNA. To examine oral delivery, P. monodon were fed pellets incorporating a pool of formalin-fixed bacteria containing the 5 GAV-specific dsRNAs before being injected with a minimal lethal GAV dose. Feeding with the pellets continued post-challenge but did not reduce mortality accumulation and elevation in GAV loads. In contrast, muscle injection of the dsRNAs purified from bacteria was highly effective at slowing GAV replication and protecting shrimp against acute disease and mortalities. In synergy with these data, dsRNA targeted to P. monodon beta-actin mRNA caused 100% mortality following injection, whilst its oral delivery caused no mortality. Findings confirm that injected dsRNA can mount effective RNAi responses in P. monodon to endogenous shrimp mRNA and exogenous viral RNAs, but when delivered orally in bacteria as a feed component, the same dsRNAs are ineffective. The efficacy of the RNAi response against GAV provided by injection of dsRNAs targeted to multiple genome sites suggests that this strategy might have general applicability in enhancing protection against other shrimp single-stranded (ss)RNA viruses, particularly in hatcheries or breeding programs where injection-based delivery systems are practical.     

The haemocytic origin of lymphoid organ spheroid cells in the penaeid prawn Penaeus monodon

Studies on lymphoid organ spheroid (LOS) cells of Penaeus monodon were undertaken. Phenoloxidase and peroxidase assays showed that LOS cells have characteristics similar to semi-granular and, in particular, large granular haemocytes. The mean percentage of LOS cells positive for phenoloxidase and peroxidase was 85 +/- 23 and 82 +/- 23%, respectively. There was no significant difference between the sites of phenoloxidase and peroxidase activity in LOS cells (t = 1.617, df = 29, p > 0.05). The relative sectional area occupied by LOS cells relative to that of the stromal matrix cells from both laboratory-held and farmed prawns was not correlated to increasing weight or total length of the prawns (p > 0.05). An apoptosis detection assay showed that LOS cells were often apoptotic whilst stromal matrix cells were not. There was a significant difference (t = -5.533, df = 58, p < 0.05) in the mean percentage of apoptotic spheroid cells between laboratory-held prawns (52 +/- 24%) and farmed prawns with midcrop mortality syndrome (MCMS) (80 +/- 12%). In conclusion, LOS cells have the characteristics of exocytosed, granular haemocytes that have phagocytosed foreign material, particularly viruses, and probably constitute a major mechanism for penaeid antiviral defense.     

RNA-binding domain in the nucleocapsid protein of gill-associated nidovirus of penaeid shrimp

Gill-associated virus (GAV) infects Penaeus monodon shrimp and is the type species okavirus in the Roniviridae, the only invertebrate nidoviruses known currently. Electrophoretic mobility shift assays (EMSAs) using His(6)-tagged full-length and truncated proteins were employed to examine the nucleic acid binding properties of the GAV nucleocapsid (N) protein in vitro. The EMSAs showed full-length N protein to bind to all synthetic single-stranded (ss)RNAs tested independent of their sequence. The ssRNAs included (+) and (-) sense regions of the GAV genome as well as a (+) sense region of the M RNA segment of Mourilyan virus, a crustacean bunya-like virus. GAV N protein also bound to double-stranded (ds)RNAs prepared to GAV ORF1b gene regions and to bacteriophage M13 genomic ssDNA. EMSAs using the five N protein constructs with variable-length N-terminal and/or C-terminal truncations localized the RNA binding domain to a 50 amino acid (aa) N-terminal sequence spanning Met(11) to Arg(60). Similarly to other RNA binding proteins, the first 16 aa portion of this sequence was proline/arginine rich. To examine this domain in more detail, the 18 aa peptide (M(11)PVRRPLPPQPPRNARLI(29)) encompassing this sequence was synthesized and found to bind nucleic acids similarly to the full-length N protein in EMSAs. The data indicate a fundamental role for the GAV N protein proline/arginine-rich domain in nucleating genomic ssRNA to form nucleocapsids. Moreover, as the synthetic peptide formed higher-order complexes in the presence of RNA, the domain might also play some role in protein/protein interactions stabilizing the helical structure of GAV nucleocapsids.     

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.     

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.     

Fatal,virus-associated peripheral neuropathy and retinopathy in farmed Penaeus monodon in eastern Australia. I. Pathology

Lesions are described in farmed Penaeus monodon affected with a previously unreported, fatal disease, 'peripheral neuropathy and retinopathy' (PNR). Outbreaks, associated with minor to heavy mortalities, occurred in 22 of 25 ponds on a farm in eastern Australia during the mid to late 1998/99 growout period. Moribund prawns, 5 to 26 g mean body weight, gathered at pond edges and were typically reddish in colour, lethargic, with mild to moderate epibiotic fouling and 1 or more partially amputated appendages. Histologically, there was mild to severe, focal to diffuse degeneration and necrosis of axons and their sheaths, together with associated glial cell apoptosis, in peripheral nerve fibres. Of the 3 appendage types examined systematically, these pathognomonic lesions were most common and severe in proximal antennal nerves and less common and severe in distal antennal nerves, antennular nerves and pereiopod nerves. Mild to severe, acute to chronic retinitis, associated with degeneration and necrosis of retinular cells and their axons, was also present in most clinically affected prawns. Transmission electron microscopy revealed moderate to large numbers of intracytoplasmic rod-shaped, helical nucleocapsids and enveloped virions, morphologically consistent with a yellow head-like virus, in putative glial cells in the antennal nerve, in the fasciculated zone of the eye and in putative sensory nerve cells of antennules. Immunohistochemical examination revealed lesions, but not histologically normal tissues, in peripheral nerves, eyes, lymphoid organ and vas deferens that consistently stained positively for a yellow head-related virus. The findings strongly suggest that a yellow head-related virus such as the Australian gill-associated virus (GAV) is causally associated with PNR. It is likely that PNR was not recognised during earlier investigations of mid-crop mortalities of farmed P. monodon in eastern Australia because appropriate peripheral nerves and eyes were not routinely examined histologically.     

Differences in the susceptibility of some penaeid prawn species to gill-associated virus (GAV) infection

Four species of penaeid prawn cultured in Australia (Penaeus monodon, Penaeus esculentus, Marsupenaeus japonicus and Fenneropenaeus merguiensis) were injected with a virulent preparation of gill-associated virus (GAV). P. monodon (average weight = 8.9, 13.9 and 19.2 g), P. esculentus (average weight = 19.5 g), F. merguiensis (average weight = 10.5 g), and small (average weight = 5.8 g) M. japonicus displayed overt signs of disease and mortalities which reached 82 to 100% within 23 d post-injection. Cumulative mortalities in P. esculentus and F. merguiensis were significantly lower than for P. monodon of the same size class. Medium (average weight = 13.0 g) M. japonicus also developed overt signs of disease but cumulative mortalities were not significantly higher than uninfected controls. Large (average weight = 20.3 g) M. japoncius did not display symptoms of disease and there were no significant mortalities up to 23 d post-injection.     

Isolation and characterization of Vibrio alginolyticus isolated from diseased kuruma prawn, Penaeus japonicus

K.-K. LEE, S.-R. YU, T.-I. YANG, P.-C. LIU AND F.-R. CHEN. 1996. Outbreaks of serious mortality among cultured kuruma prawns ( Penaeus japonicus ) with white spotted syndrome in the carapace occurred in the summer of 1993 in I-Lan, Taiwan. A swarming bacterium, strain Swy, was isolated from the hepatopancreas of the moribund prawns using tryptic soy agar supplemented with 1% NaCl and/or thiosulphate citrate bile salt sucrose agar. This strain was characterized and identified as Vibrio alginolyticus on the basis of a number of biochemical tests. The Swy strain was virulent to both kuruma prawns ( P. japonicus ) and tiger prawns ( P. monodon ) with LD₅₀ values of 4.43 times 10⁴ and 1.57 times 10⁵ cfu g body weight^-1, respectively.     

The 3C-like proteinase of an invertebrate nidovirus links coronavirus and potyvirus homologs

Gill-associated virus (GAV), a positive-stranded RNA virus of prawns, is the prototype of newly recognized taxa (genus Okavirus, family Roniviridae) within the order NIDOVIRALES: In this study, a putative GAV cysteine proteinase (3C-like proteinase [3CL(pro)]), which is predicted to be the key enzyme involved in processing of the GAV replicase polyprotein precursors, pp1a and pp1ab, was characterized. Comparative sequence analysis indicated that, like its coronavirus homologs, 3CL(pro) has a three-domain organization and is flanked by hydrophobic domains. The putative 3CL(pro) domain including flanking regions (pp1a residues 2793 to 3143) was fused to the Escherichia coli maltose-binding protein (MBP) and, when expressed in E. coli, was found to possess N-terminal autoprocessing activity that was not dependent on the presence of the 3CL(pro) C-terminal domain. N-terminal sequence analysis of the processed protein revealed that cleavage occurred at the location (2827)LVTHE downward arrow VRTGN(2836). The trans-processing activity of the purified recombinant 3CL(pro) (pp1a residues 2832 to 3126) was used to identify another cleavage site, (6441)KVNHE downward arrow LYHVA(6450), in the C-terminal pp1ab region. Taken together, the data tentatively identify VxHE downward arrow (L,V) as the substrate consensus sequence for the GAV 3CL(pro). The study revealed that the GAV and potyvirus 3CL(pro)s possess similar substrate specificities which correlate with structural similarities in their respective substrate-binding sites, identified in sequence comparisons. Analysis of the proteolytic activities of MBP-3CL(pro) fusion proteins carrying replacements of putative active-site residues provided evidence that, in contrast to most other 3C/3CL(pro)s but in common with coronavirus 3CL(pro)s, the GAV 3CL(pro) employs a Cys(2968)-His(2879) catalytic dyad. The properties of the GAV 3CL(pro) define a novel RNA virus proteinase variant that bridges the gap between the distantly related chymotrypsin-like cysteine proteinases of coronaviruses and potyviruses.     

Diseases of the eye of farmed shrimp Penaeus monodon

Lesions were found in the eyes of cultured shrimp Penaeus monodon that displayed non-specific signs of disease, including lethargy, dark pigmentation, brown gills, empty midgut, anorexia, white tail muscle, necrosis of uropods and fouled cuticle. Eye lesions were associated with sexual development in moribund shrimp in at least 1 disease event. Suppurative inflammation, granuloma and malacia were observed in histological examination of the eye and the causative agents of lesions appear to be Vibrio spp. and a rod-shaped virus (similar to Lymphoid Organ Virus, Gill-Associated Virus [GAV] and Yellow-Head Virus). Suppurative inflammation was characterised by edema, infiltration of haemocytes and local sites of abscesses. Eyes with granuloma usually appeared white in pond-side examinations, and histology showed that fibrous tissue replaced ommatidia, ganglia and internal structures of the eye. Malacia of the eye was characterised by necrosis of nervous tissue, vacuolation and vascular proliferation in the medulla ganglia. Levels of presumptive Vibrionaceace were high in moribund specimens and Gram-negative rods were observed in some specimens as free particles in the interstitial fluid and haemolymph in the eye. Transmission electron microscopy showed that nerve cells in the fasciculated zone (near the basement membrane) contained cytoplasmic vesicles (1 to 3 microm in diameter) with particles (15 to 26 nm in diameter) and rod-shaped nucleocapsids. The rods were similar to those of GAV and were 130 to 260 nm long, 10 to 16 nm in diameter and had helical symmetry with a screw-like thread (2.4 to 3.5 nm pitch). Also, unidentified enveloped virions, averaging 74 nm in diameter, were observed in cytoplasmic vesicles in the fasciculated zone. In conclusion, it is suggested that bacterial and viral infections of the eye could result in impaired neuroendocrine functions, which may cause a range of clinical signs of disease.     

Diagnosis of Penaeus monodon-type baculovirus by PCR and by ELISA of occlusion bodies

The black tiger prawn Penaeus monodon is a valuable aquaculture product in Taiwan. Two specific diagnostic methods were established for P. monodon-type baculovirus, one using polymerase chain reaction (PCR) technology and the other enzyme-linked immunosorbent assay (ELISA) technology. Monodon-type baculovirus (MBV) was purified by sucrose gradient centrifugation from occlusion bodies of MBV-infected postlarvae of P. monodon. MBV DNA was subsequently purified from the occlusion bodies and its presence was confirmed by PCR using primers of the polyhedrin gene. Based on conserved sequences of the DNA polymerase genes of Autographa californica nuclear polyhedrosis virus (AcMNPV) and Lymantria dispar nuclear polyhedrosis virus (LdMNPV), primers were designed and synthesized to yield a 714 bp PCR fragment from MBV. However, the sequence of this fragment revealed low homology with that of LdMNPV and AcMNPV. From the DNA sequence of this fragment, a second set of primers was designed, and using these primers, a 511 bp DNA fragment was amplified only when MBV DNA was the template. DNA templates from AcMNPV, white spot syndrome diseased shrimp, or PMO cells (a cell line derived from the Oka organ of Penaeus monodon) did not give any amplified DNA fragment. Therefore, this primer pair was specific for the diagnosis of MBV. By using intraspleenic immunization of rabbits with purified MBV occlusion bodies, a polyclonal rabbit antiserum against MBV was obtained. This antiserum could detect nanogram levels of MBV, but did not cross react with white spot syndrome virus (WSSV), homogenates of PMO cells, postlarvae, hepatopancreatic tissue or intestinal tissue of black tiger prawns by competitive ELISA. This sensitive method could detect MBV even in tissue homogenates.