DNA fragmentation, an indicator of apoptosis, in cultured black tiger shrimp Penaeus monodon infected with white spot syndrome virus (WSSV)

Fifty black tiger shrimp Penaeus monodon from commercial cultivation ponds in Malaysia were examined by Tdt-mediated dUTP nick-end labeling (TUNEL) fluorescence assay and agarose gel electrophoresis of DNA extracts for evidence of DNA fragmentation as an indicator of apoptosis. From these specimens, 30 were grossly normal and 20 showed gross signs of white spot syndrome virus (WSSV) infection. Of the 30 grossly normal shrimp, 5 specimens were found to be positive for WSSV infection by normal histology and by nested polymerase chain reaction (PCR) analysis. All of the specimens showing gross signs of WSSV infection were positive for WSSV by normal histology, while 5 were positive by nested PCR only (indicating light infections) and 15 were positive by 1-step PCR (indicating heavy infections). Typical histological signs of WSSV infection included nuclear hypertrophy, chromatin condensation and margination. None of the 25 grossly normal shrimp negative for WSSV by 1-step PCR showed any signs of DNA fragmentation by TUNEL assay or agarose gel electrophoresis of DNA extracts. The 10 specimens that gave PCR-positive results for WSSV by nested PCR only (i.e., 5 grossly normal shrimp and 5 grossly positive for WSSV) gave mean counts of 16 +/- 8% TUNEL-positive cells, while the 25 specimens PCR positive by 1-step PCR gave mean counts of 40 +/- 7% TUNEL-positive cells. Thus, the number of TUNEL positive cells present in tissues increased with increasing severity of infection, as determined by gross signs of white spots on the cuticle, the number of intranuclear inclusions in histological sections, and results from single and nested PCR assays. DNA extracts of PCR-positive specimens tested by agarose gel electrophoresis showed indications of DNA fragmentation either as smears or as 200 bp ladders. Given that DNA fragmentation is generally considered to be a hallmark of apoptosis, the results suggested that apoptosis might be implicated in shrimp death caused by WSSV.     

WSSV和IHHNV二重实时荧光PCR检测方法的建立

根据基因库中对虾白斑综合征病毒WSSV(AF369029)和传染性皮下及造血器官坏死病毒IHHNV(AF218226)基因序列,设计了WSSV和IHHNV的两对特异性引物和两条用不同荧光基团标记的TaqMan探针。对反应条件和试剂浓度进行优化,建立了能够同时检测WSSV和IHHNV的二重实时荧光PCR方法。该方法特异性好,对WSSV和IHHNV的检测敏感性分别达到2和20个模板拷贝数;此外抗干扰能力强,对WSSV和IHHNV不同模板浓度进行组合,仍可有效地同时检测这二个病毒。对保存的30份经常规PCR检测仅为WSSV或IHHNV阳性的样品进行二重实时荧光PCR检测,结果都为阳性,其中1份为WSSV和IHHNV混合感染。本研究建立的二重实时荧光PCR方法用于WSSV和IHHNV的检测具有特异、敏感、快速、定量等优点。     

Usefulness of dead shrimp specimens in studying the epidemiology of white spot syndrome virus (WSSV) and chronic bacterial infection

This paper describes the utility of dead shrimp samples in epidemiological investigations of the white spot syndrome virus (WSSV) and chronic bacterial infections. A longitudinal observational study was undertaken in shrimp farms in Kundapur, Karnataka, India, from September 1999 to April 2000 to identify risk factors associated with outbreaks of white spot disease (WSD) in cultured Penaeus monodon. As a part of the larger study, farmers were trained to collect and preserve dead and moribund shrimp (when observed) during the production cycle. At the end of the production cycle, 73 samples from 50 ponds had been collected for histopathology and 55 samples from 44 ponds for PCR. Intranuclear viral inclusion bodies diagnostic of WSSV infection were detected in dead samples from 32 ponds (64 %). Samples of dead shrimp from 18 ponds (36%) showed no histopathological evidence of WSSV infection. However, of these, samples from 13 ponds (26%) showed clear evidence of shell, oral, enteric and systemic chronic inflammatory lesions (CIL) in the form of haemocytic nodules, typical of bacterial infection. Samples from 5 ponds (10%) were negative for both WSSV and CIL. Samples from 8 ponds had dual WSSV and CIL, although both WSSV and CIL were only observed in the same shrimp from 1 pond. Useful information was obtained from these shrimp despite the presence of post-mortem changes. Samples from 19 ponds (43%) tested positive for WSSV by 1-step PCR and samples from an additional 10 ponds (22.7%) were positive by 2-step nested PCR. Samples from 15 ponds (34.1%) were negative for WSSV by 2-step nested PCR. There was moderate to substantial agreement between PCR and histopathology in the diagnosis of WSSV infection in dead shrimp. WSSV infection in dead shrimp was significantly associated with crop failures as defined by a shorter length of the production cycle (<90 d) and lower average weight at harvest (<22 g). WSSV infection was also associated with lower survival (<50%), but this was not significant. Ponds with CIL did not experience any crop failures, and the presence of CIL was significantly associated with successful crops. The study demonstrates that samples of dead shrimp can provide useful information for disease surveillance and epidemiological investigations of WSSV and chronic bacterial infections.     

Development of a real-time PCR assay for detection of monodon baculovirus (MBV) in penaeid shrimp

A real-time PCR method was developed to detect monodon baculovirus (MBV) in penaeid shrimp. A pair of MBV primers to amplify a 135 bp DNA fragment and a TaqMan probe were developed. The primers and TaqMan probe were specific for MBV and did not cross react with Hepatopancreatic parvovirus (HPV), White spot syndrome virus (WSSV), Infectious hypodermal and haematopoietic virus (IHHNV) and specific pathogen free (SPF) shrimp DNA. A plasmid (pMBV) containing the target MBV sequence was constructed and used for determination of the sensitivity of the real-time PCR. This real-time PCR assay had a detection limit of one plasmid MBV DNA copy. Most significantly, this real-time PCR method can detect MBV positive samples from different geographic locations in the University of Arizona collection, including Thailand and Indonesia collected over a 13-year period.     

Development of a Rapid Method for Identifying Carryover Contamination of Positive Control DNA,Using a Chimeric Positive Control and Restriction Enzyme for the Diagnosis of White Spot Syndrome Virus by Nested PCR

Chimeric positive plasmids have been developed to minimize false-positive reactions caused by polymerase chain reaction (PCR) contamination. Here, we developed a rapid method for identifying false-positive results while detecting white spot syndrome virus (WSSV) by nested PCR, using chimeric positive plasmids. The results of PCRs using WSSV diagnostic primer sets showed PCR products of a similar size (WSSV 1st PCR product, 1,447 bp; WSSV 2nd PCR product, 941 bp) using WSSV chimeric plasmids or DNA from shrimp infected with WSSV. The PCR products were digested with DraI for 1 h at 37 °C. The digested chimeric DNA separated into two DNA bands; however, the WSSV-infected shrimp DNA did not separate. Thus, chimeric plasmid DNA may be used as positive control DNA instead of DNA from WSSV-infected shrimp, in order to prevent PCR contamination. Thus, the use of restriction enzyme digestion allowed us to rapidly distinguish between WSSV DNA and WSSV chimeric plasmid DNA.     

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.     

Conservation of the DNA sequences encoding the major structural viral proteins of WSSV

A cDNA library was constructed from white spot syndrome virus (WSSV)-infected penaeid shrimp tissue. cDNA clones with WSSV inserts were isolated and sequenced. By comparison with DNA sequences in GenBank, cDNA clones containing sequence identical to those of the WSSV envelope protein VP28 and nucleoprotein VP15 were identified. Poly(A) sites in the mRNAs of VP28 and VP15 were identified. Genes encoding the major viral structural proteins VP28, VP26, VP24, VP19 and VP15 of 5 WSSV isolates collected from different shrimp species and/or geographical areas were sequenced and compared with those of 4 other WSSV isolate sequences in GenBank. For each of the viral structural protein genes compared, the nucleotide sequences were 100 to 99% identical among the 9 isolates. Gene probes or PCR primers based on the gene sequences of the WSSV structural proteins can be used for diagnoses and/or detection of WSSV infection.     

White spot syndrome virus isolates of tiger shrimp Penaeus monodon (Fabricious) in India are similar to exotic isolates as revealed by polymerase chain reaction and electron microscopy

Microbiological analysis of samples collected from cases of white spot disease outbreaks in cultured shrimp in different farms located in three regions along East Coast of India viz. Chidambram (Tamil Nadu), Nellore (Andhra Pradesh) and Balasore (Orissa), revealed presence of Vibrio alginolyticus, Vibrio parahaemolyticus, and Aeromonas spp. but experimental infection trials in Penaeus monodon with these isolates did not induce any acute mortality or formation of white spots on carapace. Infection trials using filtered tissue extracts by oral and injection method induced mortality in healthy P. monodon with all samples and 100% mortality was noted by the end of 7 day post-inoculation. Histopathological analysis demonstrated degenerated cells characterized by hypertrophied nuclei in gills, hepatopancreas and lymphoid organ with presence of intranuclear basophilic or eosino-basophilic bodies in tubular cells and intercellular spaces. Analysis of samples using 3 different primer sets as used by other for detection of white spot syndrome virus (WSSV) generated 643, 1447 and 520bp amplified DNA products in all samples except in one instance. Variable size virions with mean size in the range of 110 x 320 +/- 20 nm were observed under electron microscope. It could be concluded that the viral isolates in India involved with white spot syndrome in cultured shrimp are similar to RV-PJ and SEMBV in Japan, WSBV in Taiwan and WSSV in Malaysia, Indonesia, Thailand, China and Japan.     

High variation in repetitive DNA fragment length for white spot syndrome virus (WSSV) isolates in Thailand

White spot syndrome virus (WSSV) presently causes the most serious losses to shrimp farmers worldwide. Earlier reports of high DNA sequence homology among isolates from widely separated geographical regions suggested that a single virus was the cause. However, we have found surprisingly high variation in the number of 54 bp DNA repeats in ORF94 (GenBank AF369029) from 55 shrimp ponds (65 shrimp samples) experiencing WSSV outbreaks in Thailand in 2000 and 2002. These were detected by PCR amplification using primers ORF94-F and ORF94-R flanking the repeat region. Altogether, 12 different repeat groups were found (from 6 to 20 repeats) with 8 repeats being most frequent (about 32%). Extracts prepared from individual shrimp in the same outbreak pond belonged to the same repeat group while those collected at the same time from separate WSSV outbreak ponds, or from the same ponds at different times, usually belonged to different repeat groups. This suggested that different outbreaks were caused by different WSSV isolates. In contrast to the highly variable numbers of repeats, sequence variation within the repeat region was confined to either T or G at Position 36. These variations may be useful for epidemiological studies on the local and global movement of WSSV, since there is high variation in the number of repeats (good for local studies) but little sequence change (good for global studies).     

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.     

White spot syndrome virus (WSSV) in cultured Penaeus monodon in the Philippines

The prevalence and geographic distribution of white spot syndrome virus (WSSV) infection among cultured penaeid shrimp in the Philippines was determined from January to May, 1999, using PCR (polymerase chain reaction) protocol and Western blot assays. A total of 71 samples consisting of 18 post-larvae (PL) and 53 juvenile/adult shrimp samples (56 to 150 days-of-culture, DOC) were screened for WSSV. Of the 71 samples tested, 51 (72%) were found positive for WSSV by PCR: 61% (31/51) after 1-step PCR and 39% (20/51) after 2-step, non-nested PCR. Of the PL and juvenile/adult shrimp samples tested, 50 and 79% were positive for WSSV, respectively. By Western blot, only 6 of the 51 (12%) PCR-positive samples tested positive for WSSV. Of the 20 samples negative for WSSV by PCR, all tested negative for WSSV by Western blot assay. This is the first report of the occurrence of WSSV in the Philippines.     

The role of Pm-fortilin in protecting shrimp from white spot syndrome virus (WSSV) infection

Crustacean fortilin or the product of the translationally controlled tumor protein (TCTP) gene isolated from Penaeus monodon, is well conserved and has a Ca(++) binding domain. Pm-fortilin has anti-apoptotic properties and is present at high levels during the onset of viral infections in P. monodon. The possibility of using rFortilin to protect against white spot syndrome virus (WSSV) infection was tested. Injection of shrimp with rFortilin, after infection with WSSV, resulted in 80-100% survival and detection of very low levels of WSSV by PCR, whereas in moribund samples WSSV levels were very high. This result implies that injection of recombinant rFortilin decreases viral infection by an unknown mechanism, but probably by inhibiting viral replication. Using a yeast two-hybrid screen for cellular protein partners to rFortilin we identified an unknown protein that bound to fortilin. This is a novel polypeptide of 93 amino acids with a number of XPPX signature sequences that are often reported to have a function in antiviral peptides.     

多重RT-PCR同时检测鉴别三种对虾病毒的研究与应用

根据基因库中对虾桃拉综合征病毒（TSV）、白斑综合征病毒（WSSV）、传染性皮下和造血器官坏死病毒（IHHNV）的基因序列,分别设计了三对特异性引物,通过对多重RT—PCR扩增条件的优化,研究建立了可同时检测鉴别TSV、WSSV和IHHNV的多重RT—PCR。该技术对同一样品中的TSV RNA、WSSV DNA和IHHNV DNA模板进行扩增,结果均同时得到3条大小与实验设计相符的231bp（TSV）、593bp（WSSV）和356bp（IHHNV）的特异性多重RT—PCR扩增带,对其它对虾病原核酸的扩增结果为阴性。敏感性试验结果表明,该技术最低能检测到10pgTSV RNA、100pg WSSV DNA和100pg IHHNV DNA。临床检测试验结果表明,该技术对TSV、WSSV和I—HHNV的检出率明显高于传统的临床症状观察和组织病理学检查,提示该技术适用于这三种病毒的临床快速检测和鉴别诊断。     

Controlled bioassay systems for determination of lethal infective doses of tissue homogenates containing Taura syndrome or white spot syndrome virus

In vivo bioassay is the predominant method for evaluating the infectivity of materials potentially harboring viable shrimp pathogens and determining the relative susceptibility of shrimp species to viral infections. A controlled bioassay system for white spot syndrome virus (WSSV) and Taura syndrome virus (TSV) was developed utilizing 260 ml tissue culture flasks modified with an air exchange vent. Individual shrimp (1.00 +/- 0.25 g) were placed in separate flasks containing artificial seawater (100 to 150 ml) and held in an incubator at 27 degrees C. After a 48 h acclimation period, shrimp were either injected intramuscularly with viral inoculum or exposed to virus-laden water. Water was exchanged and shrimp were fed a commercial food pellet daily except 24 h post-infection (p.i.). Bioassays were performed with serial dilutions of stock viral preparations and shrimp mortality was recorded for 7 d p.i. Mortality rates of test animals permitted the estimation of the lethal infective doses, LD50 and LD90. The LD50 of the TSV injection preparation was estimated at viral dilutions of 1:7.692 x 10(7) (Trial 1) and 1:6.667 x 10(7) (Trial 2). The LD50s of 2 different WSSV injection preparations were estimated at 1:4.444 x 10(6) and 1:4.505 x 10(6). The LD50 for the TSV waterborne challenge was 1:9916 (Trial 1) and 1:15 710 (Trial 2) at 20 degrees C and 1:1272 at 27 degrees C. A second waterborne TSV inoculum challenge at 27 degrees C produced an LD50 of 1:2857. WSSV doses used in the waterborne challenge only reached 39% mortality, which did not allow for the estimation of effective lethal doses. Bioassay by injection proved to be a more reliable method of estimating viral infectivity compared to waterborne method. The dose-response curves developed can serve as a basis for controlled comparisons of relative levels of viral infectivity of specific tissue preparations and for controlled comparisons of relative susceptibility of shrimp species or stocks to viral pathogens.     

Single-tube multiplex PCR for rapid and sensitive diagnosis of subgenus B and other subgenera adenoviruses in clinical samples

We developed a new diagnostic method of subgenus (Sub) B adenovirus (Ad) in clinical samples using non-nested polymerase chain reaction (PCR). Sequences of the conserved hexon-coding region of representative strains of eight serotypes (3, 7, 11, 14, 16, 21, 34 and 35) of Sub B Ad were heterogeneous. In order to distinguish Ad serotype 3 (Ad 3) and Ad 7 from the other serotypes of Sub B Ad, and to differentiate Ad 3 and 7 from each other, 3 different downstream primers were designed based on the sequence heterogeneity. By a single-tube PCR method using a combination of 6 primers including the 3 new primers, Ads demonstrated to amplify 188, 206, 284, and 301 bp DNA fragments for Ad 3, Ad 7, other Sub B Ads, and non-Sub B Ads, respectively. A total of 114 clinical samples were selected to evaluate the direct applicability of our PCR. The results were compared with previous culture results. Sixty-seven out of 71 (94%) Sub B Ad culture-positive samples, and 15 out of 19 (79%) Sub C or E-positive samples amplified products of the expected size. Two of 20 (10%) culture-negative samples from pharyngoconjunctival fever patients were identified as Ad 3 by the PCR. Four samples, from which non-Ad viruses were isolated, were negative by the PCR. The present study might provide a rapid and sensitive diagnosis method for infections caused by Sub B Ads.     

Detection of hepatopancreatic parvovirus in Thai shrimp Penaeus monodon by in situ hybridization,dot blot hybridization and PCR amplification

Hepatopancreatic parvovirus (HPV) infects the hepatopancreas in penaeid shrimp and retards their growth. The DNA sequence of HPV from Thai shrimp Penaeus monodon (HPVmon) differs from HPV of Penaeus chinensis (HPVchin) by approximately 30%. In spite of this difference, commercial PCR primers (DiagXotics) developed from HPVchin to yield a 350 bp PCR product do give a 732 bp product with HPVmon DNA template. On the other hand, the sensitivity of HPVmon detection with these primers and with hybridization probes designed for HPVchin is significantly lower than it is with HPVchin. To improve sensitivity for HPVmon detection, we used the sequence of the 732 bp HPVmon PCR amplicon described above to develop specific PCR primers (H441F and H441R) and hybridization probe. The primers could detect as little as 1 fg of purified HPVmon DNA while the 441 bp digoxygenin-labeled PCR product gave strong, specific reactions with in situ hybridization and with hybridization blots. In contrast, negative results were obtained using DNA from all other pathogens tested and from DNA of P. monodon. Supernatant solution from boiled, fresh shrimp fecal and postlarval samples homogenized in 0.025% NaOH/0.0125% SDS could be used to detect as little as 0.1 pg HPVmon DNA by the PCR reaction. By dot blot hybridization, a visible signal was obtained with purified HPVmon DNA at 0.01 pg, but detection in spiked feces and postlarval samples was only 1 and 0.1 pg, respectively.