Hepatopancreatic Parvovirus Research Articles

Different reactions obtained using the same DNA detection reagents for Thai and Korean hepatopancreatic parvovirus of penaeid shrimp.

Hepatopancreatic parvovirus (HPV) can cause stunted growth and death in penaeid shrimp including Penaeus monodon. We used PCR primers and a commercial DNA probe designed from HPV of Penaeus chinensis (HPVchin) to examine HPV-infected Thai P. monodon (HPVmon). We found that the PCR primers produced a 732 bp DNA amplicon rather than the 350 bp amplicon obtained with HPVchin template and that the DNA probe gave weak to variable in situ DNA hybridization results. In addition, hybridization to PCR products from HPVmon was weak compared with hybridization with PCR products from HPVchin. By contrast, the 732 bp amplicon hybridized strongly with HPVmon-infected cells by in situ hybridization but not with uninfected shrimp tissue or other shrimp viruses, thus confirming its origin from HPVmon. Cloning, sequencing and analysis of the 732 bp amplicon showed that 696 bp (excluding the primer sequences) contained 47% GC content and had only 78% homology to 701 aligned bases from a 3350 bp DNA fragment of HPVchin from GenBank. These results explain why the reagents based on HPVchin gave a different PCR product and weak hybridization results with HPVmon, and they show that multiple primers or degenerate primers may be necessary for general detection of HPV varieties. Together with previously published information on the estimated total genome sizes for HPVchin (approximately 4 kb) and HPVmon (approximately 6 kb), these data support the contention that HPVchin and HPVmon are different varieties or species, in spite of their similar histopathology.

Detection and quantification of infectious hypodermal and hematopoietic necrosis virus in penaeid shrimp by real-time PCR

A real-time PCR method using a fluorogenic 5' nuclease assay and a PE Applied Biosystems GeneAmp 5700 sequence detector was developed to detect infectious hypodermal and hematopoietic necrosis virus (IHHNV) in penaeid shrimp. A pair of PCR primers to amplify an 81 bp DNA fragment and a fluorogenic probe (TaqMan probe) were selected from ORF1 (open reading frame 1) of the IHHNV genome. The primers and TaqMan probe used in this assay were shown to be specific for IHHNV and did not react with either hepatopancreatic parvovirus (HPV), white-spot syndrome virus (WSSV), or shrimp DNA. A plasmid, pIHHNV-P4, containing the target IHHNV sequence was constructed and used as a positive control. The concentration of pIHHNV-P4 was determined through spectrophotometric analysis and the plasmid was used for quantitative studies. This real-time PCR assay had a detection limit of 10 copies and a log-linear range up to 5 x 10(7) copies of IHHNV DNA. The assay was then used to quantify IHHNV in infected shrimp collected from 5 locations: Hawaii, Panama, Mexico, Guam, and the Philippines. The quantitative analysis showed that wild-caught, large juvenile Penaeus stylirostris collected from the Gulf of California (Mexico) in 1996 were naturally infected with IHHNV and contained up to 10(9) copies of IHHNV microg(-1) of DNA. Similar quantities of IHHNV were detected in hatchery-raised, small juvenile P. stylirostris collected from Guam in 1995 and in farm-raised, post-larval P. monodon from the Philippines in 1996. Laboratory-infected P. stylirostris contained approximately 10(8) copies of IHHNV 31 d after being fed with IHHNV-infected shrimp tissue. In contrast, individuals of Super Shrimp, a line of P. stylirostris selected for IHHNV resistance, showed no signs of infection 32 d after ingesting IHHNV-infected shrimp tissue. Laboratory-infected P. vannamei also contained approximately 10(8) copies of IHHNV 30 d after being fed infected shrimp tissue. A time-course study of IHHNV replication in juvenile P. vannamei showed that the doubling time in the exponential growth phase was approximately 22 h.

A non-destructive method based on the polymerase chain reaction for detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp.

Current methods to detect hepatopancreatic parvovirus (HPV) infection of penaeid shrimp depend on invasive techniques that require dissecting the organs infected by this virus. However, sacrificing valuable stocks in order to determine their HPV status can be a drawback in the case of breeding programs. A method was developed for HPV detection by applying a polymerase chain reaction (PCR) assay to fecal samples collected from live HPV-infected shrimp Penaeus chinensis. A pair of PCR primers, 1120F/1120R, which amplify a 592 base pair (bp) region from the virus genome, was designed from previously known HPV sequence information (HPV clone HPV8). PCR amplification with these primers generated a product of the expected size directly from the crude feces of HPV-infected shrimp but not from the feces of specific pathogen-free (SPF) shrimp. The HPV origin of the amplified product was validated by means of an in situ hybridization assay where the product of the amplification, labeled with digoxigenin (DIG)-11-dUTP, showed an intense reaction within hepatopancreatic cells displaying characteristic HPV lesions on HPV-infected shrimp. No reaction to this probe was observed when reacted in situ with sections of the hepatopancreas of SPF specimens or to sections of shrimp infected by the infectious hypodermal and hematopoietic necrosis virus (IHHNV), another parvovirus of penaeid shrimp. These primers were tested for specificity against homologous and nonhomologous viruses and no product was amplified. A fragment of the expected size was obtained only when purified HPV or purified HPV8 plasmid was used as template DNA. Under optimized conditions, these primers detected as little as 1 fg of purified HPV8 plasmid DNA, equivalent to approximately 300 HPV particles. Analysis of fecal samples by PCR may prove useful for non-lethal screening of valuable shrimp of unknown HPV status. This same strategy also might be used for detection of other enteric viruses that infect penaeid shrimp.

Statistical correlation between severity of hepatopancreatic parvovirus infection and stunting of farmed black tiger shrimp ( Penaeus monodon)

In response to complaints from shrimp farmers, samples were taken to determine whether stunting in cultured Penaeus monodon was related to the presence of known infectious agents. Shrimp were arbitrarily defined as normal at >8 cm length and stunted at <8 cm (base of the rostrum to base of telson) and 10 of each type were collected at harvest from each of four production ponds. Mean length for the normal shrimp was 10 cm (range 8.5–12.3) while that of stunted shrimp was 6 cm (range 2.8–7.7 cm). The shrimp were examined histologically for infections by hepatopancreatic parvovirus (HPV), infectious hypodermal and haematopoietic necrosis virus (IHHNV), monodon baculovirus (MBV), white-spot syndrome virus (WSSV), yellow-head virus (YHV) and the microsporidian, Agmasoma penaei. Infections were found for only HPV, IHHNV and MBV. For all 80 specimens, prevalences were HPV 49% (39), MBV 50% (40) and IHHNV 4% (3). Mean prevalences for HPV in the normal and stunted groups (28±8% and 70±15%, respectively) were not significantly different ( P=0.057). Nor were the mean prevalences of MBV infection (45±9% and 58±15%, respectively; P=0.496). For HPV and MBV, severity of infection was estimated by an ocular grid severity index. Pearson product moment correlation tests on a subgroup of 71 pooled samples showed a negative correlation between HPV severity index and length ( P<0.01) and a positive correlation ( P<0.01) between severity of HPV and severity of MBV infections. In addition, an ANOVA comparison was made for four sub-groups: uninfected (mean length 9.3 cm=17.2 g), MBV infected (mean length 8.3 cm=13.8 g), HPV infected (mean length 6 cm=6.3 g) and dually MBV/HPV infected (mean length 6.5 cm=7.6 g). Results showed that mean lengths of only the HPV and dually infected groups were significantly shorter than mean length of the uninfected group ( P<0.05). The results suggest either that stunted shrimp are more susceptible to HPV infection or that HPV infection contributes to stunting. In our opinion, the similar size distribution for HPV infected and dually HPV/MBV infected shrimp weighs in favour of the latter relationship.

Characterization and PCR detection of hepatopancreatic parvovirus (HPV) from Penaeus monodon in Thailand.

Hepatopancreatic parvovirus (HPV) causes disease in several species of penaeid shrimp. Heavy infections may result in poor growth and reduced production for shrimp farmers. From one southern Thai shrimp pond with a high prevalence of HPV infection, 790 shrimp were sampled randomly and the hepatopancreas (HP) removed. Most HP were preserved in liquid nitrogen. However, every 10th HP (79 total) was divided into 2 parts appropriately fixed for examination by transmission electron microscopy (TEM) and light microscopy. Based on light microscopy, the prevalence of HPV infection in the pond was approximately 30% and its presence was confirmed by TEM of parallel samples. The virus was subsequently purified from hepatopancreatic homogenates of the samples preserved in liquid nitrogen. Negative staining of the purified viral preparation revealed unenveloped, icosahedral viral particles 22 to 24 nm in diameter. Agarose gel electrophoresis of nucleic acid extracts revealed the presence of 2 fragments, one very intense (5.8 kb) and the other weak (4.2 kb). The larger fragment was degraded by DNase I and S1 nuclease, indicating single-stranded DNA (ssDNA) characteristic of the viral family Parvoviridae. The smaller fragment was degraded by DNase I but not by S1 nuclease, indicating that it comprised double-stranded DNA. A genomic DNA library of the 5.8 kb ssDNA was constructed in pUC18 and a clone containing a 659 bp fragment specific and sensitive for HPV was selected for sequencing. Based on this sequence, an HPV-specific primer set was designed to yield a 156 bp amplicon by polymerase chain reaction (PCR) amplification. The expected 156 bp amplicon was obtained only in the presence of HPV DNA template (at as little as 1 fg purified DNA) and not with nucleic acid templates extracted from healthy shrimp tissue or other shrimp pathogens. It is hoped that this PCR assay will be useful to shrimp aquaculturists for early detection and screening of shrimp larvae, parental broodstock or other possible carriers of HPV in the shrimp cultivation system.

Taura syndrome of penaeid shrimp: cloning of viral genome fragments and development of specific gene probes.

The ssRNA genome extracted from purified Taura Syndrome Virus (TSV) was transcribed into double-stranded, blunt-ended cDNA and was used to construct cDNA libraries either in pUC 18 or in pBluescript II KS-vectors. Twelve recombinant plasmids chosen after screening of the libraries were subjected to restriction enzyme digestions for determination of size inserts and restriction maps. Two of them, pP15 and pQ1, were selected for probe construction. The inserts, 1500 and 1300 base pairs (bp) respectively, were DIG-11dUTP-labelled and the corresponding probes were named P15 and Q1. On northern blots and dot blots, using different denaturation methods, the 2 probes hybridized specifically with extracted RNA-TSV genome, TSV and infected TS shrimp homogenates. No positive hybridization was obtained with other shrimp viruses tested [Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and Hepatopancreatic Parvovirus (HPV)]. The specificity of the 2 probes was confirmed by in situ hybridization on histological sections of TS diseased shrimps.

Partial cloning of the genome of an unusual shrimp parvovirus (HPV):use of gene probes in disease diagnosis

The DNA genome of the hepatopancreatic parvovirus (HPV) of penaeid shnmp was partially cloned The largest insert obtained, from the clone OF44, was 2 3 kbp In size and characterized by mapping w ~ t h restnction enzymes It was used to construct 2 different DIG-11 dUTP labelled probes, which appeared specific to the virus, without any cross-reactivity w ~ t h uninfected tissues or with a second shnmp parvovirus, infectious hypodermal and haematopoietlc necrosis virus (IHHNV) In sltu hybridization on paraffin sections of the hepatopancreas of infected and uninfected shrimp showed the probes react with Infected tlssue and not with umnfected tissue In positive hepatopancreas, the probes produced a strong labelling of infected nuclei, especially of the nucleolus and the karyoplasm peripheric to the intranuclear inclusion body Intranuclear inclusion bodies, which are the principal diagnostic feature of HPV infections by histology, also reacted with the probes, but less intensely In heavily infected hepatopancreas, cell d e b r ~ s reacted strongly wlth the probes, as did the microvilli on the surface of eplthelial cells

Development and application of a simple and rapid diagnostic method to studies on hepatopancreatic parvovirus of penaeid shrimp

Hepatopancreatic parvovirus (HPV) is one of about a dozen viruses known to infect penaeid shrimps. HPV is apparently cosmopolitan in distribution, having been reported from wild and/or cultured penaeid shrimp in Asia, Africa, Australia and North and South America. However, the importance of the virus as a pathogen remains poorly understood, possibly due in part to the relative difficulty of diagnosis of HPV infections and to the possibility that HPV infections may be masked in multiple infections with other pathogens. Until now diagnosis of HPV has been dependent upon the histological demonstration of pathognostic Feulgen positive, basophilic intranuclear inclusion bodies in hepatopancreatic epithelial cells, which has limited diagnostic capability for HPV to suitably equipped laboratories. A rapid field diagnostic method for HPV infections was developed that uses Giemsa-stained impression smears of the hepatopancreas. The impression smear method was shown to compare favorably in diagnostic sensitivity to the standard histopathological method for HPV when applied to the diagnosis of HPV infections in postlarval and juvenile Penaeus chinensis (Osbeck).

Lymphoidal parvovirus-like particles in Australian penaeid prawns

Prawns in northern Queensland exhib~ted mult~nucleated g ~ a n t cell formation in hypertrophied lymphoid organs. Mostly farmed Penaeus monodon, P, merguiensls and P esculentus were involved The transformation involved mild nuclear hypertrophy, marglnated chromatin and basophilic da rken~ng of increased cytoplasm In a multinucleate giant cell. Areas of transformation were d~scre te , often bounded by elongated f~brocytes and sometimes fully encapsulated Basophilic inclusions occurred con~monly and eos~nophlhc lntranuclear inclus~ons rarely. These inclus~ons talned for DNA with acndlne orange fluorescence. Associated \nth lympho~dal changes was the presence of rounded F l~ke cells with basophilic, pyknolc nucleoli. Under the electron microscope, transformed lympho~dal cells d~splayed virus-hke parlcles of 18 to 20 nm diameter associated with inclusion b o d ~ e s in the nucleus These particles were similar to a parvovlrus and named lymphoidal parvovirus-like (LP\/). Their similarities and relationship to hepatopancreatic parvovirus and infectious hypodermal and haematopoletic necrosis virus are discussed. INTRODUCTION MATERIALS AND METHODS The lymphoid organ (or Oka organ) is a relatively newly discovered organ in penaeid prawns (Oka 1969) and is becoming increasingly important in disease studies. Lightner et al. (1987) described Oka organ hyperplastic spheroids and metastatic foci in ectopic localities in both Penaeus monodon Fabricus and P. penicillatus from Taiwan. Prlmary cell cultures from the lymphoid organ of P. n~onodon were capable of supporting in vitro replication of Monodon baculovirus when inoculated into the cultures (Chen & Kou 1989). Lymphoid t~ssue necrosis and haenlocytic infiltration lead to encapsulation, nodule formation and melanization and are often the earliest changes associated with bacterial infections (Nash 1990). In Australia, the lymphoidal spheroids in Penaeus nlerguiensis de Man have been associated with moribund wild spawners (Owens & Hall-Mendelin 1990). Suggestions were made that there were similarities between tissues showing lymphoid metastasis with the lymphoidal spheroids and tissues attacked by infectious haematopoietic and hypodermal necrosis virus (IHHNV). This paper reports on further investigations of lymphoidal changes in prawns and the probable causahve agent. Banana prawns Penaeus merguiensis were collected by a cast net in the Burdekin Rver , northern Queensland. Tiger prawns (P. monodon and P. esculentus Haswell) were collected from prawn farms in the local area. Prawns were split longitudinally, and fixed in Davidson's fixative. Histological sections were cut and stained using standard techniques (Culling et al. 1985). For electron microscopy, the diced lymphoid organ was fixed in 2 .5 % glutaldehyde/2 O/O paraforn~aldehyde in cacodylate buffer and postfixed in 1 O/O osmium tetroxide. The tissue was dehydrated and mounted in Spurr (Tm) resin. Sections were cut on a LKB Ultratome at 50 nm stained with uranyl acetate/ 70 O/O methanol and lead citrate and viewed at 80 kV on a Joel 2000FX transmission electron microscope