Infectious Hypodermal And Hematopoietic Necrosis Research Articles

Molecular cloning, characterization and gene expression of an antioxidant enzyme catalase (MrCat) from Macrobrachium rosenbergii

In this study, we reported a full length of catalase gene (designated as MrCat), identified from the transcriptome database of freshwater prawn Macrobrachium rosenbergii. The complete gene sequence of the MrCat is 2504 base pairs in length, and encodes 516 amino acids. The MrCat protein contains three domains such as catalase 1 (catalase proximal heme-ligand signature) at 350-358, catalase 2 (catalase proximal active site signature) at 60-76 and catalase 3 (catalase family profile) at 20-499. The mRNA expressions of MrCat in healthy and the infectious hypodermal and hematopoietic necrosis virus (IHHNV) challenged M. rosenbergii were examined using quantitative real time polymerase chain reaction (qRT-PCR). The MrCat is highly expressed in digestive tract and all the other tissues (walking leg, gills, muscle, hemocyte, hepatopancreas, pleopods, brain and eye stalk) of M. rosenbergii taken for analysis. The expression is strongly up-regulated in digestive tract after IHHNV challenge. To understand its biological activity, the recombinant MrCat gene was constructed and expressed in Escherichia coli BL21 (DE3). The recombinant MrCat existed in high thermal stability and broad spectrum of pH, which showed over 95% enzyme activity between pH 5 and 10.5, and was stable from 40 °C to 70 °C, and exhibited 85–100% enzyme activity from 30 °C to 40 °C.

Effect of infectious hypodermal and haematopoietic necrosis virus (IHHNV) infection on caspase 3c expression and activity in freshwater prawn Macrobrachium rosenbergii

Caspase 3c ( MrCasp3c) was sequenced from the freshwater giant prawn Macrobrachium rosenbergii using Illumina Solexa Genome Analyzer Technique. MrCasp3c consisted of 2080 bp nucleotide encoded 521 polypeptide with an estimated molecular mass of 59 kDa. MrCasp3c sequence contains caspase family p20 domain profile and caspase family p10 domain profile at 236–367 and 378–468 respectively. The quantitative real time PCR analysis revealed a broad expression of MrCasp3c with the highest expression in haemocyte and the lowest in stomach. The expression of MrCasp3c after challenge with the infectious hypodermal and haematopoietic necrosis virus (IHHNV) was tested in haemocyte. In addition, MrCasp3c was expressed in Escherichia coli by prokaryotic expression plasmid pMAL-c2x. The enzyme activity of MrCasp3c was also found to be up-regulated by IHHNV in haemocyte and hepatopancreas tissues. This study suggested that MrCasp3c may be an effector caspase associated with the induction of apoptosis which is potentially involved in the immune defence of M. rosenbergii.

Genomic sequence of infectious hypodermal and hematopoietic necrosis virus (IHHNV) KLV-2010-01 originating from the first Korean outbreak in cultured Litopenaeus vannamei

Due to the need to track and monitor genetic diversity, the genome of the infectious hypodermal and hematopoietic necrosis virus (IHHNV) strain KLV-2010-01 in cultured Litopenaeus vannamei shrimp that originated from the first Korean outbreak in 2010 was sequenced and analyzed. The genome, with a length of 3914 nucleotides, was sequenced from the Korean IHHNV. The genome encoded three large and overlapping open reading frames: ORF1 (NS-1) of 2001 bp, ORF2 (NS-2) of 1092 bp and ORF3 (capsid protein) of 990 bp. The overall organization, size and predicted amino acid sequence of the three ORFs in Korean IHHNV were highly similar to those of members of the infectious IHHNV group, and the most closely related strains were IHHNVs described from Ecuador and Hawaii. Additionally, phylogenetic analysis showed that the Korean IHHNV was clustered with lineage III in the infectious IHHNV group and was most similar to IHHNV isolates from Ecuador, China and Taiwan.

Detection and identification of white spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) of Litopenaus vannamei from Bushehr and Sistan and Baloochestan provinces (Iran), during 2009-2010.

The first time the white spot disease (WSD) and IHHNV were reported in Iran was in 2004 and 2008, respectively in Bushehr Province. In Sistan and Balochestan province only the WSD was reported in 2008.The aim of this study was detecting these two viral diseases in these provinces, during December 2009 to April 2010. A total of 364 samples were collected according to suspected gross signs from hatcheries and shrimp farms in Bushehr and Sistan and Blochestan provinces (I.R. Iran) respectively, including larvae (72, 43 samples), post larvae (48, 37 samples), juveniles (57, 32 samples), sub adults (39, 22 samples) and broodstock (29, 13 samples) of Litopenaus vannamei . WSD was detected from juveniles (23 samples), sub adults (14 samples), and broodstock (14 samples), and IHHNV was also detected from juveniles (16 samples), sub adults (9 samples) and broodstock (5 samples) based on gross signs, PCR and histopathological changes from Bushehr province but from Sistan and Blochestan province only WSD was detected from juveniles (26 samples), sub adults (18 samples) and broodstock (7 samples). Histopathological observations of WSSV showed basophilic Cowdry type A inclusion bodies in all tissues such as gills, haematopoietic tissue, cuticle epithelium, lymphoid organ and connective tissue. However histologically, the hepatopancreas tissue showed vacuolization of B cells, without inclusion bodies, but histopatholgical changes caused by IHHNV including eosinophilic Cowdry type A inclusion bodies which were ectodermal, mesodermal and rarely endodermal. Keyword: WSSV, IHHNV, L. vannamei, Bushehr province, Sistan and Blochestan province,

Bioinformatic characterization and gene expression pattern of apoptosis inhibitor from Macrobrachium rosenbergii challenged with infectious hypodermal and hematopoietic necrosis virus

Apoptosis is genetically programmed cellular killing processes that execute unnecessary or infected cells. It plays an important role in embryogenesis, homeostasis, insect metamorphosis and immunity. Apoptosis inhibitor ( MrIAP) was sequenced from the freshwater giant prawn Macrobrachium rosenbergii using Illumina Solexa Genome Analyzer Technique. MrIAP consisted of 1753 base pair nucleotides encoded 535 polypeptide with an estimated molecular mass of 60 kDa. MrIAP amino acid sequence contains IAP superfamily domain between 5 and 490. The deduced amino acid sequences of the MrIAP were aligned with the other IAP family members. The highest sequence similarity was observed in IAP-5 from ant Camponotus floridanus (67%) followed by IAP from body louse Pediculus humanus corporis (66%) and the lowest (62%) in IAP-5 isoform-5 from common chimpanzee Pan troglodytes and IAP-5 from Aedes aegypti. The IAP phylogenetic tree showed that MrIAP closely related to other arthropod blacklegged tick Ixodes scapularis, formed a sister group with IAP from a hemichordate acorn worm Saccoglossus kowalevskii and finally clustered together with IAPs from fish groups. The quantitative real time PCR analysis revealed that significantly ( P < 0.05) highest expression was noticed in hepatopancreas and significantly ( P < 0.05) lowest expression in pleopods. Based on the results of gene expression analysis, MrIAP mRNA transcription in M. rosenbergii challenged to infectious hypodermal and hematopoietic necrosis virus (IHHNV) was highly induced in hepatopancreas. The collective results of this study indicate that the MrIAP is an essential immune gene and influences the immune response against IHHNV infection in M. rosenbergii.

The Infectious Hypodermal and Haematopoietic Necrosis Virus: A Brief Review of What We Do and Do Not Know

Given its high prevalence, its wide distribution and its remarkable capacity to cause severe mortality in shrimp, the infectious hypodermal and haematopoietic necrosis virus (IHHNV) may deserve far more attention than it has received, as it remains considered as one of the most serious problems plaguing the global shrimp farming industry. Furthermore, its real measurable impact over wild shrimp populations remains unknown. Undeniably, the progress that we have reached today on the knowledge of its geographical distribution, clinical signs, genetic diversity, transmission and virulence may help to identify and understand important aspects of its biology and pathogenesis. However, the information regarding the molecular events that occur during the infection process is scarce. Thus, it may not be surprising to find that there are no therapeutic options available for the prophylaxis or treatments to reduce the deleterious impact of this viral pathogen to date. The aim of this review is to integrate and discuss the current state of knowledge concerning several aspects of the biology of IHHNV and to highlight potential future directions for this area of research.

Detection and genetic profiling of infectious hypodermal and haematopoietic necrosis virus (IHHNV) infections in wild berried freshwater prawn, Macrobrachium rosenbergii collected for hatchery production

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) has been detected widely in penaeid culture facilities in Asia and the Americas. IHHNV infection on sub-adult and postlarvae of the giant freshwater prawn, Macrobrachium rosenbergii which had caused up to 80% mortalities was first reported in Southeast Taiwan in 2006. In Malaysia, although, there has been no report on IHHNV infections in M. rosenbergii, preliminary work suggests that there is an urgent need to setup a screening protocol for IHHNV for both wild and cultured populations. In this study, polymerase chain reaction based screening was carried out on 30 randomly sampled berried wild M. rosenbergii before and after spawning. All samples did not showed any sign of IHHNV infection. However, the results showed that 20% of the samples were IHHNV positive. Sequence analysis of the amplified band using NCBI-BLAST showed that the putative IHHNV sequence had 98% nucleotide sequence (388bp) identity with the IHHNV isolate AC-05-005 non-structural protein 1 gene and seven other IHHNV strains in the data bank further affirming the suggestion on the presence of IHHNV in wild freshwater prawn populations in Malaysia.

Selection of shrimp breeders free of white spot syndrome and infectious hypodermal and hematopoietic necrosis

The objective of this work was to select surviving breeders of Litopenaeus vannamei from white spot syndrome virus (WSSV) outbreak, adapted to local climatic conditions and negatively diagnosed for WSSV and infectious hypodermal and hematopoietic necrosis virus (IHHNV), and to evaluate if this strategy is a viable alternative for production in Santa Catarina, Brazil. A total of 800 males and 800 females were phenotypically selected in a farm pond. Nested-PCR analyses of 487 sexually mature females and 231 sexually mature males showed that 63% of the females and 55% of the males were infected with IHHNV. Animals free of IHHNV were tested for WSSV, and those considered double negative were used for breeding. The post-larvae produced were stocked in nine nursery tanks for analysis. From the 45 samples, with 50 post-larvae each, only two were positive for IHHNV and none for WSSV. Batches of larvae diagnosed free of virus by nested-PCR were sent to six farms. A comparative analysis was carried out in growth ponds, between local post-larvae and post-larvae from Northeast Brazil. Crabs (Chasmagnathus granulata), blue crabs (Callinectes sapidus), and sea hares (Aplysia brasiliana), which are possible vectors of these viruses, were also evaluated. The mean survival was 55% for local post-larvae against 23.4% for post-larvae from the Northeast. Sea hares showed prevalence of 50% and crabs of 67% of WSSV.

Gene profiling and characterization of arginine kinase-1 (MrAK-1) from freshwater giant prawn ( Macrobrachium rosenbergii)

Arginine kinase-1 (MrAK-1) was sequenced from the freshwater prawn Macrobrachium rosenbergii using Illumina Solexa Genome Analyzer Technique. MrAK-1 consisted of 1068 bp nucleotide encoded 355 polypeptide with an estimated molecular mass of 40 kDa. MrAK-1 sequence contains a potential ATP:guanido phosphotransferases active domain site. The deduced amino acid sequence of MrAK-1 was compared with other 7 homologous arginine kinase (AK) and showed the highest identity (96%) with AK-1 from cherry shrimp Neocaridina denticulate. The qRT-PCR analysis revealed a broad expression of MrAK-1 with the highest expression in the muscle and the lowest in the eyestalk. The expression of MrAK-1 after challenge with the infectious hypodermal and hematopoietic necrosis virus (IHHNV) was tested in muscle. In addition, MrAK-1 was expressed in Escherichia coli by prokaryotic expression plasmid pMAL-c2x. The optimum temperature (30 °C) and pH (8.5) was determined for the enzyme activity assay. MrAK-1 showed significant ( P < 0.05) activity towards 10–50 mM ATP concentration. The enzyme activity was inhibited by α-ketoglutarate, glucose and ATP at the concentration of 10, 50 and 100 mM respectively. Conclusively, the findings of this study indicated that MrAK-1 might play an important role in the coupling of energy production and utilization and the immune response in shrimps.

Complete nucleic acid sequence of Penaeus stylirostris densovirus ( PstDNV) from India

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) of shrimp, recently been classified as Penaeus stylirostris densovirus ( PstDNV). The complete nucleic acid sequence of PstDNV from India was obtained by cloning and sequencing of different DNA fragment of the virus. The genome organisation of PstDNV revealed that there were three major coding domains: a left ORF (NS1) of 2001 bp, a mid ORF (NS2) of 1092 bp and a right ORF (VP) of 990 bp. The complete genome and amino acid sequences of three proteins viz., NS1, NS2 and VP were compared with the genomes of the virus reported from Hawaii, China and Mexico and with partial sequence available from isolates from different regions. The phylogenetic analysis of shrimp, insect and vertebrate parvovirus sequences showed that the Indian PstDNV isolate is phylogenetically more closely related to one of the three isolates from Taiwan ( AY355307), and two isolates ( AY362547 and AY102034) from Thailand.

Prevalence of three shrimp viruses in Zhejiang Province in 2008

White spot syndrome virus (WSSV), Taura syndrome virus (TSV) and Infectious hypodermal and haematopoietic necrosis virus (IHHNV) are three shrimp viruses responsible for major pandemics affecting the shrimp farming industry. Shrimps samples were collected from 12 farms in Zhejiang province, China, in 2008 and analyzed by PCR to determine the prevalence of these viruses. From the 12 sampling locations, 8 farms were positive for WSSV, 8 for IHHNV and 6 for both WSSV and IHHNV. An average percentage of 57.4% of shrimp individuals were infected with WSSV, while 49.2% were infected with IHHNV. A high prevalence of co-infection with WSSV and IHHNV among samples was detected from the following samples: Bingjiang (93.3%), liuao (66.7%), Jianshan (46.7%) and Xianxiang (46.7%). No samples exhibited evidence of infection with TSV in collected samples. This study provides comprehensive information of the prevalence of three shrimp viruses in Zhejiang and may be helpful for disease prevention control in this region.

Detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Litopenaeus vannamei shrimp cultured in South Korea

In October 2010, cuticular deformities with abnormal growth suppression were detected among cultured Litopenaeus vannamei shrimp in one farm located on the Taean peninsula along the western coast of Korea. We detected infectious hypodermal and haematopoietic necrosis virus (IHHNV) from the moribund L. vannamei shrimp and its pond water via PCR assays. The IHHNV strain isolated from the shrimps was designated K1 and the strain isolated from the pond water was designated K2, and the capsid genes were sequenced for genetic comparison and phylogenetic analysis with others obtained from different geographical regions. The results demonstrated that the Korean strains were similar to infectious IHHNV and were closely related to the Type 1 strains. This is the first report, to the best of our knowledge, of the detection of IHHNV from L. vannamei in Korean cultured shrimp.

Natural co-infection with infectious hypodermal and hematopoietic necrosis virus (IHHNV) and infectious myonecrosis virus (IMNV) in Litopenaeus vannamei in Brazil

Cultivation of whiteleg shrimp ( Litopenaeus vannamei) constitutes a growing aquaculture industry in northeast Brazil. Similar to other animals that are intensively farmed, shrimp experience disease outbreaks, which are a constant threat and cause eventually significant economical losses. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) and infectious myonecrosis virus (IMNV) are prevalent epizootic viral agents in Brazil. In a routine monitoring program for the diagnosis of IHHNV and IMNV, using molecular techniques like conventional PCR, reverse transcription coupled with PCR (RT-PCR) and absolute quantitative PCR (qPCR), we found that most positive samples of shrimp were simultaneously co-infected with both viruses. This survey is the first to show the occurrence of a natural co-infection that is caused by IHHNV and IMNV in penaeid shrimp attacked by viral disease that were cultivated in Northeast Brazil.

Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of white spot syndrome virus and infectious hypodermal and hematopoietic necrosis virus in penaeid shrimp

A multiplex LAMP (mLAMP) assay was developed for the simultaneous detection of two penaeid shrimp viruses, namely, white spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV), which are the major viral pathogens of penaeid shrimp worldwide. Two sets of the LAMP primers were designed for VP28 gene of WSSV and non-structural protein 1 (NS1) gene of IHHNV, in which a restriction enzyme cleavage site was inserted into two pairs of species-specific primers, to construct the mLAMP assay by combining these two sets totaling eight primers. The mLAMP method was distinguishable between WSSV and IHHNV due to the subsequent restriction enzyme analysis. The sensitivities of the mLAMP method were 102 and 103 times higher on the detection limits for WSSV and IHHNV, respectively, than those of the nested-PCR (nPCR) and classical PCR methods. No positive results were displayed when nucleic acids from other shrimp pathogen DNAs were used as mLAMP templates. Uracil-N-glycosylase (UNG) was used to prevent carry-over contamination of mLAMP products. In addition to its high specificity and sensitivity, the developed multiplex LAMP assay offers an efficient, convenient and rapid tool for screening penaeid shrimp viruses since both WSSV and IHHNV can be diagnosed in a single reaction, and will be useful for the control of these viruses in shrimp.

Rapid and sensitive detection of infectious hypodermal and hematopoietic necrosis virus by loop-mediated isothermal amplification combined with a lateral flow dipstick

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is an important shrimp pathogen that causes mortality in Penaeus stylirostris and stunting (called runt deformity syndrome or RDS) in Penaeus vannamei. Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acids under isothermal conditions. It can be combined with a chromatographic lateral flow dipstick (LFD) for highly specific, rapid and simple visual detection of IHHNV-specific amplicons. Using this protocol, a 30-min amplification followed by 5 min hybridization with an FITC-labeled DNA probe and 5 min LFD resulted in visualization of DNA amplicons trapped at the LFD test line. Thus, 10 min for rapid DNA extraction followed by LAMP combined with LFD detection resulted in a total assay time of approximately 50 min. Detection sensitivity was comparable to other methods used commonly for nested PCR detection of IHHNV but had the additional advantages of reduced assay time, confirmation of amplicon identity by hybridization and elimination of electrophoresis with carcinogenic ethidium bromide.

COMPARATIVE STUDY OF SPECIFICITY AND SENSITIVITY FOR IHHNV DETECTION BETWEEN REAL-TIME PCR AND LAMP METHODS

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the most important pathogens infecting penaeid shrimp and causes huge economic losses in the shrimp culture industry worldwide. Because no commercial vaccine is yet available, the most effective way of containing the disease is by the routine screening of juveniles and adults for the presence or absence of the virus. Therefore, our goal is to establish a simple and rapid examination system for infectious hypodermal and hematopoietic necrosis virus in places such as shrimp ponds. Two detection methods, real-time PCR and loop-mediated isothermal amplification (LAMP), were developed for IHHNV diagnosis, and then their specificity and sensitivity were compared in the study. Using the real-time PCR method, the assay had a detection limit of six copies of DNA template of IHHNV, and had a correlation coefficient of 0.99521 between template concentration and threshold cycle value at the template concentration of 6.038×104 to 6.038×109cps/mL. Furthermore, the approach had no signal response to genomic DNA of white spot syndrome virus and shrimp. The result revealed that the detection method had high specificity and sensitivity for IHHNV detection. However, the costly real-time thermal cycler and technically demanding were deemed to be not appropriate for IHHNV detection in field conditions. LAMP was a novel, sensitive and rapid detection technique and could be applied for disease diagnosis in aquaculture. Here, a set of four primers was designed using PrimerExplorer V4 software by targeting the IHHNV genome DNA, and used to develop the LAMP method for IHHNV detection. Using the detection system, target DNA was amplified and visualized on agarose gel within 60min under isothermal condition at 64℃. Also, the LAMP amplicon was observed directly in the reaction tube by addition of SYBR Green I for a naked-eye inspection. Sensitivity assay showed that the method also had a detection limit of six copies of DNA template of IHHNV. Moreover, genomic DNA of white spot syndrome virus and shrimp were unable to be detected within 60min using the LAMP method. Overall, these data revealed that the LAMP method had an equivalent to the real-time PCR method in specificity and sensitivity for IHHNV detection. Considering that the LAMP method had great advantage in its performance and low cost, this technique was more suitable for IHHNV detection in field conditions. Therefore, the LAMP method could be applied routinely to check the shrimp in hatcheries and growout ponds, so that virus carrying shrimp could be found during the early infection stages and counter measures could be devised before the infection becomes epizootic.

Prevalence of shrimp viruses in wild Penaeus monodon from Brunei Darussalam

Over a period of 30 months, wild black tiger shrimp ( Penaeus monodon) were collected from the South China Sea in South-east Asia, in an effort to establish specific pathogen free (SPF) stocks of P. monodon in Brunei Darussalam. The viral status of the broodstock was assessed by real-time and conventional polymerase chain reaction (PCR) using DNA and reverse transcribed RNA extracted from the gill, hepatopancreas and pleopods. Histopathology was performed to confirm PCR results. Over 270 P. monodon were collected, screened and spawned. Of the nine viruses assessed, infectious hypodermal and hematopoietic necrosis virus (IHHNV) was most commonly detected (19.6%), followed by monodon baculovirus (MBV) (7.4%), hepatopancreatic parvovirus (HPV) (3.8%), and Mourilyan virus (MoV) (0.9%). The only multiple viral infections found were a combination of IHHNV and MBV (2.2%). Interestingly, the two most virulent viruses for P. monodon, white spot syndrome virus (WSSV) and yellowhead virus (YHV) were not detected in any shrimp. It is concluded that wild P. monodon from Brunei waters have a low prevalence of viral pathogens, which has permitted the development of SPF stocks as a foundation population for selective breeding.

Genetic Signature of Rapid IHHNV (Infectious Hypodermal and Hematopoietic Necrosis Virus) Expansion in Wild Penaeus Shrimp Populations

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a widely distributed single-stranded DNA parvovirus that has been responsible for major losses in wild and farmed penaeid shrimp populations on the northwestern Pacific coast of Mexico since the early 1990's. IHHNV has been considered a slow-evolving, stable virus because shrimp populations in this region have recovered to pre-epizootic levels, and limited nucleotide variation has been found in a small number of IHHNV isolates studied from this region. To gain insight into IHHNV evolutionary and population dynamics, we analyzed IHHNV capsid protein gene sequences from 89 Penaeus shrimp, along with 14 previously published sequences. Using Bayesian coalescent approaches, we calculated a mean rate of nucleotide substitution for IHHNV that was unexpectedly high (1.39×10−4 substitutions/site/year) and comparable to that reported for RNA viruses. We found more genetic diversity than previously reported for IHHNV isolates and highly significant subdivision among the viral populations in Mexican waters. Past changes in effective number of infections that we infer from Bayesian skyline plots closely correspond to IHHNV epizootiological historical records. Given the high evolutionary rate and the observed regional isolation of IHHNV in shrimp populations in the Gulf of California, we suggest regular monitoring of wild and farmed shrimp and restriction of shrimp movement as preventative measures for future viral outbreaks.

Fast short-fragment PCR for rapid and sensitive detection of shrimp viruses

This article describes a fast short-fragment PCR method for the detection of white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and monodon baculovirus (MBV). Fast two-temperature (95 °C denaturation and 60 °C annealing/extension) PCRs were performed in 5–10 μl volume samples in miniaturized microplates using a fast Peltier thermal cycler. 40 cycles were completed in 25–30 min. Rapid high-resolution agarose gel electrophoresis of 70–150 bp PCR fragments was performed in 10 min. High sensitivity of PCR product detection (50–100 pg) was obtained using ultra sensitive dyes such as GelStar ® and a gel documentation system equipped with a blue-light transilluminator. This novel method is faster and more sensitive than its TaqMan ® real-time PCR counterparts.

Differential diagnosis of active hypodermal and hematopoietic necrosis virus based on gene choice and reverse transcription coupled with PCR

The Pacific whiteleg shrimp Litopenaeus vannamei (Penaeidae) is one of the most important cultivated species in world aquaculture. In Brazil, the northeastern states are home to the main shrimp producers. As shrimp aquaculture has expanded and intensified, diseases have progressively become one of the most serious threats to this industry. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is an enzootic viral agent in Brazilian shrimp farms. Its is usually diagnosed by histological methods. However, to detect sub-clinical or acute IHHNV infection, more refined methods based on molecular techniques have been utilized. We found that by using "universal" primers and a single-step PCR diagnostic test, it was difficult to distinguish between non-infective forms of the virus and active IHHNV. Detection of IHHNV was more accurate when we used two alternative molecular strategies, namely 1) single-step PCR amplification based on gene choice and 2) reverse transcription coupled with PCR.