Infectious Hypodermal And Hematopoietic Necrosis Research Articles

Development and Evaluation of a Shrimp Virus (IHHNV)-Mediated Gene Transfer and Expression System for Shrimps.

An efficient gene transfer and expression tool is lacking for shrimps and shrimp cells. To solve this, this study has developed a shrimp DNA virus-mediated gene transfer and expression system, consisting of insect Sf9 cells for viral packaging, the shrimp viral vector of pUC19-IHHNV-PH-GUS and the baculoviral vector of Bacmid or Bacmid-VP28 encoding the shrimp WSSV envelope protein VP28. The pUC19-IHHNV-PH-GUS vector was constructed by assembling the genomic DNA of shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), which has shortened inverted terminal repeats, into a pUC19 backbone, and then an expression cassette of baculoviral polyhedron (PH) promoter-driven GUS (β-glucuronidase) reporter gene was inserted immediately downstream of IHHNV for proof-of-concept. It was found that the viral vector of pUC19-IHHNV-PH-GUS could be successfully packaged into IHHNV-like infective virions in the Sf9 cells, and the gene transfer efficiency of this system was evaluated and verified in three systems of Sf9 cells, shrimp hemolymph cells and tissues of infected shrimps, but the GUS expression could only be detected in cases where the viral vector was co-transfected or co-infected with a baculovirus of Bacmid or Bacmid-VP28 due to the Bacmid-dependence of the PH promoter. Moreover, the packaging and infection efficiencies could be significantly improved when Bacmid-VP28 was used instead of Bacmid.

A novel dual CRISPR-Cas assay for detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in penaeid shrimp without false positives from its endogenous viral elements (EVEs)

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a single-stranded DNA virus posing significant economic challenges in shrimp aquaculture. Depending on the shrimp species, IHHNV infections can lead to mass mortalities, deformities, or growth retardation, all of which impact production output. Timely identification of IHHNV, particularly using nucleic acid detection methods, is paramount for effective prevention. However, PCR and methods relying on nucleic acid probes face challenges due to the presence of chromosome-integrated, endogenous viral elements (EVEs) of IHHNV sharing identical sequences to their infectious counterparts. While long-range PCR assays, such as LongAmp PCR (LA-PCR), have shown promise in minimizing false positive results, their applicability in the field is limited by their long turnaround times and reliance on costly equipment. To address these limitations, we have developed a rapid and potentially field-deployable platform termed Cas9-RPA-Cas12a (“CRPAC”) capable of differentiating the infectious form of IHHNV from EVEs, capitalizing on the fact that the genome of IHHNV is single-stranded, while EVEs are double-stranded DNA. This method employs Cas9 endonuclease to deplete EVEs, thereby enriching for single-stranded genomic DNA. This enriched DNA is then subjected to recombinase polymerase amplification (RPA) coupled with the Cas12a diagnostic assay. Here, we show that CRPAC gives positive results exclusively for infectious IHHNV, appearing as strong fluorescence or a distinct pattern on lateral flow strips that facilitates naked-eye evaluation. In field sample testing, CRPAC demonstrated excellent agreement with LA-PCR, identifying 8 out of 16 samples containing infectious IHHNV, while the WOAH-recommended PCR assays gave false positive results to all samples containing EVEs. Collectively, our findings underscore the utility of CRPAC as a robust and field-adaptable tool for distinguishing between infectious IHHNV and EVEs, irrespective of their nucleotide sequence similarities.

Parvoviruses of Aquatic Animals.

Family Parvoviridae consists of small, non-enveloped viruses with linear, single-stranded DNA genomes of approximately 4-6 kilobases, subdivided into three subfamilies, Parvovirinae, Densovirinae, and Hamaparvovirinae, and unassigned genus Metalloincertoparvovirus. Parvoviruses of aquatic animals infect crustaceans, mollusks, and finfish. This review describes these parvoviruses, which are highly host-specific and associated with mass morbidity and mortality in both farmed and wild aquatic animals. They include Cherax quadricarinatus densovirus (CqDV) in freshwater crayfish in Queensland, Australia; sea star-associated densovirus (SSaDV) in sunflower sea star on the Northeastern Pacific Coast; Clinch densovirus 1 in freshwater mussels in the Clinch River, Virginia, and Tennessee, USA, in subfamily Densovirinae; hepatopancreatic parvovirus (HPV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) in farmed shrimp worldwide; Syngnathid ichthamaparvovirus 1 in gulf pipefish in the Gulf of Mexico and parts of South America; tilapia parvovirus (TiPV) in farmed tilapia in China, Thailand, and India, in the subfamily Hamaparvovirinae; and Penaeus monodon metallodensovirus (PmMDV) in Vietnamese P. monodon, in unassigned genus Metalloincertoparvovirus. Also included in the family Parvoviridae are novel parvoviruses detected in both diseased and healthy animals using metagenomic sequencing, such as zander parvovirus from zander in Hungary and salmon parvovirus from sockeye salmon smolts in British Columbia, Canada.

Real-time triplex loop-mediated isothermal amplification (LAMP) using a turbidimeter for detection of shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV).

The World Organization for Animal Health still regulates the infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimp. The existing disease identification approach is time consuming, necessitates expensive equipment, and requires specialized expertise, thereby limiting the accessibility of shrimp disease screening on farms. Loop-mediated isothermal amplification (LAMP) is recognized for its ability to detect inhibitory substances with high sensitivity and specificity. We developed a real-time triplex LAMP assay that combines the simplicity of point-of-care testing with the accuracy of a turbidimeter. Using a set of three LAMP primers, our technology enables rapid DNA amplification in a single reaction within 45 min and with a low detection limit (10 copies/reaction). We tested 192 shrimp samples from different sources and demonstrated the clinical utility of our method, achieving 100% specificity (95% confidence interval = 93.40-100.00%), 100% sensitivity (97.36-100.00%), and 100% accuracy (98.10-100.00%) in detecting IHHNV DNA, with a high Cohen's kappa value (1) compared to the standard quantitative polymerase chain reaction assay. The high technology readiness level of our method makes it a versatile platform for any real-time LAMP assay, and its low cost and simplicity make it well suited for fast deployment and use in shrimp farming.

Development of recombinase amplification assays for the rapid detection of infectious myonecrosis virus

Infectious myonecrosis virus (IMNV) has affected shrimp farming in many countries, such as northeastern Brazil and southeast Asia, and poses a serious threat to the global shrimp industry. Reverse transcription enzymatic recombinant amplification technology (RT-ERA) is a rapid DNA amplification assay with high specificity in isothermal conditions and has been widely applied to the pathogen’s detection. In this study, two novel ERA assays of IMNV, real-time RT-ERA and an RT-ERA combined with lateral flow dipsticks assay (RT-ERA-LFD), were developed and evaluated. The real-time RT-ERA assay could be carried out at 38–42 °C and had the highest end-point fluorescence value and the smallest Ct value at 41 °C. The brightness and width of the detection line were at a maximum at 39 °C and 30 min, and these conditions were selected in RT-ERA-LFD. Both real-time RT-ERA and RT-ERA-LFD produced positive results with IMNV standard plasmids only and showed no cross-reaction with Vibrio parahaemolyticus, which causes acute hepatopancreatic necrosis disease (VpAHPND); white spot syndrome virus (WSSV); infectious hypodermal and hematopoietic necrosis virus (IHHNV); or Ecytonucleospora hepatopenaei (EHP). Meanwhile, we compared the sensitivities of nested RT-PCR, real-time RT-PCR, real-time RT-ERA, and RT-ERA-LFD. The sensitivities of real-time RT-ERA and RT-ERA-LFD were both 101 copies/μL. The detection sensitivities of nested RT-PCR and real-time RT-PCR were 100 and 102 copies/μL, respectively. As a result, two ERA assays were determined to be specific, sensitive, and economical methods for the on-site diagnosis of IMNV infection, showing great potential for the control of IMNV infections.

Development of a Melting Curve-Based Triple Eva Green Real-Time PCR Assay for Simultaneous Detection of Three Shrimp Pathogens.

Infections with Enterocytozoon hepatopenaei (EHP), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and Decapod iridescent virus 1 (DIV1) pose significant challenges to the shrimp industry. Here, a melting curve-based triple real-time PCR assay based on the fluorescent dye Eva Green was established for the simultaneous detection of EHP, IHHNV, and DIV1. The assay showed high specificity, sensitivity, and reproducibility. A total of 190 clinical samples from Shandong, Jiangsu, Sichuan, Guangdong, and Hainan provinces in China were evaluated by the triple Eva Green real-time PCR assay. The positive rates of EHP, IHHNV, and DIV1 were 10.5%, 18.9%, and 44.2%, respectively. The samples were also evaluated by TaqMan qPCR assays for EHP, DIV1, and IHHNV, and the concordance rate was 100%. This illustrated that the newly developed triple Eva Green real-time PCR assay can provide an accurate method for the simultaneous detection of three shrimp pathogens.

Propagation of IHHNV (Infectious hypodermal and haematopoietic necrosis virus)/PstDVI and MBV (Monodon baculovirus)/PmNV in shrimp primary haemocyte culture

In vitro propagation of shrimp viruses IHHNV (Infectious hematopoietic hypodermal necrosis virus)/PstDV1 and MBV (Monodon baculovirus)/PmNV in the primary haemocyte culture was successfully be accomplished in the present study. The haemocytes were maintained in double-strength L15 medium with growth supplements and antibiotics. Cytopathic changes observed in the cells were detachment, slendering of cells, vacuolation, and rounding of the cells. Replication of viruses in the culture was confirmed through the expression of DNA polymerase, polyhedrin and lef 4 of MBV and capsid gene of IHHNV. Ultrathin sections of haemocytes observed through electron microscopy revealed scattered chromatin, virogenic stroma and presence of viruses and occlusion bodies (MBV). The study revealed that primary haemocyte culture could be used as a platform for the propagation of a variety of shrimp viruses.

Development of a multiplex real-time enzymatic recombinase amplification assay for differentiation of yellow head virus genotype 1 and 2 in Penaeus vannamei

At present, eight genotypes of yellow head virus (YHV) have been identified, and the main pathogenic genotypes are yellow head virus 1 (YHV1) and gill-associated virus (GAV), which have caused mass death and serious economic losses of shrimp industry. The prevention of YHV complex infection is highly dependent on early and rapid diagnostic, however, the available molecular technologies require complex procedures or expensive equipment. In this study, a multiplex rapid detection method of YHV1 and GAV based on real-time enzymatic recombinase amplification (YHV1/GAV-ERA) was established. Primers and probes were designed to detect the conserved ORF1b gene of YHV1 and GAV. Best primers and probes were selected to effectively amplify the target gene, and the sensitivity was reached 102 copies/μL within 6.17 ± 0.49 min. While multi-nested PCR could reach the detection limit of 1.9 copies/μL and 2.1 copies/μL for YHV1 and GAV by consuming higher time and instrument cost. The YHV1/GAV-ERA can effectively amplify the target gene at 32–45 °C within 30 min, and the optimum reaction temperature is 42 °C. No cross reaction with white spot syndrome virus (WSSV), Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VpAHPND), Enterocytozoon hepatopenaei (EHP), infectious hypodermal and hematopoietic necrosis virus (IHHNV) and healthy shrimp DNA were observed. Additionally, the test results of YHV1/GAV-ERA were 100% consistent with the industrial standard multi-nested PCR in shrimp samples. This method can distinguish the two genotypes of YHV and get rid of the dependence on the thermal cycle instrument, and provides a feasible rapid detection method for on-site screening of pathogens.

Stock Health of Penaeid Shrimps in Sri Lanka: a Comprehensive Molecular-Based Study on Seven Diseases Found in the Asian Region

Purpose: Although shrimp aquaculture in Sri Lanka has mainly been plagued by recurrent outbreaks of White Spot Syndrome Virus (WSSV), recent symptomatic and analytical field data of shrimps were incoherent with typical WSSV infections. As transboundary infection of new diseases is not uncommon in world shrimp aquaculture, the possibility of new diseases established in the country surfaced. Hence, the present study was aimed at evaluating the stock health of shrimps in Sri Lanka for additional six diseases commonly available in the Asian region, namely Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV), Infectious Myonecrosis Virus (IMNV), Taura Syndrome Virus (TSV), Yellow Head Virus (YHV), Acute Hepatopancreatic Necrosis (AHPND) and Necrotizing Hepatopancreatitis Bacterium (NHPB). Research method: Sampling was performed using broodstocks, postlarvae, cultured Penaeus monodon, wild P. semisulcatus, P. indicus and P. monodon from different locations and broodstock collecting areas in the country. Other crustacean species were also collected as possible carrier species. The presence of pathogenic organisms was identified by Polymerase Chain Reaction using respective IQ 2000TM test kits and a total of 2060 PCR tests were conducted. Findings: Of all the samples tested, no samples were positive for IMNV, TSV, YHV, AHPND and NHPB. However, confirmatory evidence was found in Sri Lanka for the presence of IHHNV with the prevalence of 31.3% in broodstock samples, 24.07% in hatchery-produced postlarvae and 18.03% in cultured shrimps. WSSV was also recorded with a prevalence of 10.34% in broodstock samples and 55.56% in cultured shrimp samples collected. None of the wild collected samples was positive for any of the tested diseases. Value: Reinstating the presence of WSSV in the Sri Lankan shrimp industry, provides confirmatory evidence of the presence of IHHNV in the country, highlighting the possibility of the emergence of new diseases in shrimp farming in Sri Lanka. Also, commonly suggested disease carriers seem to be not the major cause of disease spread during the sampling time. This study, therefore, provides much-needed information about the stock health of shrimps in Sri Lanka.

First detection of white spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) from wild-caught giant tiger prawn, Penaeus monodon Fabricius, 1798 (Penaeoidea: Penaeidae) from the Gulf of Mexico and Northwestern Atlantic Ocean

First detection of white spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) from wild-caught giant tiger prawn, Penaeus monodon Fabricius, 1798 (Penaeoidea: Penaeidae) from the Gulf of Mexico and Northwestern Atlantic Ocean

The immune response of Caspase-3 gene in infectious hypodermal and hematopoietic necrosis virus (IHHNV) infection of Litopenaeus vannamei

The pathogenicity of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in litopenaeus vannamei (L. vannamei) is closely related to the age and size of the host, with juveniles being more susceptible to IHHNV infection and exhibiting higher pathogenicity. In our previous research, transcriptome sequencing results revealed a significant downregulation of the Caspase-3 (LvCAP-3) gene in juveniles compared to adults. Based on these findings, we hypothesize that the LvCAP-3 gene plays an immunological role in the defense against IHHNV in L. vannamei. To validate this hypothesis, we analyzed the differential expression of the LvCAP-3 gene in various tissues (pleopods, muscles, gills, intestine, hepatopancreas) of L. vannamei using Real-time quantitative PCR (qPCR), and investigated the impact of LvCAP-3 gene interference using RNA interference (RNAi) technology on IHHNV viral load. The research findings indicated that the LvCAP-3 gene was expressed in all 5 tissues mentioned above, with the highest expression in hepatopancreas and the lowest expression in pleopods. L. vannamei injected with IHHNV showed an increasing viral load from 24 to 120 h, accompanied by an upregulation of LvCAP-3 gene expression. Furthermore, the interference of the LvCAP-3 gene resulted in a higher IHHNV viral load in L. vannamei injected with IHHNV compared to the control group. In conclusion, interference with the LvCAP-3 gene makes L. vannamei more susceptible to IHHNV infection. It can be deduced that the higher susceptibility of juvenile shrimp to IHHNV associated with lower expression levels of the LvCAP-3 gene.

Comparison of Polymerase Chain Reaction (PCR) assay performance in detecting Decapod penstylhamaparvovirus 1 in penaeid shrimp

Decapod Penstylhamaparvovirus 1, commonly known as infectious hypodermal and hematopoietic necrosis virus (IHHNV), remains an economically important viral pathogen for penaeid shrimp aquaculture due to its effects on growth performance. The World Organization for Animal Health (WOAH, Paris, France) recommended methods for the detection of IHHNV include both conventional and real-time PCR. However, published reports and anecdotal evidence suggest the occurrence of non-specific amplifications when testing for IHHNV using the WOAH protocols. Studies were designed to develop a sensitive, robust TaqMan PCR method for detection of IHHNV in the three commercially important penaeid shrimp: Penaeus vannamei, P. monodon and P. stylirostris. We compared the performance of the WOAH-recommended real-time PCR method to several published as well as in-house designed primer/probe sets spanning the entire genome of IHHNV. Our results show that (1) more than one primer/ probe set is needed when testing for the infectious form of IHHNV in all three species of shrimp and (2) primer pairs qIH-Fw/qIH-Rv and 3144F/ 3232R have diagnostic characteristics that would enable IHHNV detection in all three shrimp species. These findings are valuable for a large-scale screening of shrimp using a TaqMan real-time PCR assay.

Development of a duplex PCR for the simultaneous detection of EHP and IHHNV and analysis of the correlation between these two pathogens

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the linearly single-stranded DNA viruses. Ecytonucleospora hepatopenaei (EHP) is an intracellular parasitic microsporidian. IHHNV and EHP are pathogens that have been widely prevalent in shrimp farming. Both of them are associated with growth retardation of the penaeid shrimp, which causes serious economic losses to shrimp farming. Shrimp can be co-infected with IHHNV and EHP. In this study, a rapid duplex polymerase chain reaction (PCR) was developed and optimized for the simultaneous detection of EHP and IHHNV. The detection limit of the duplex PCR could reach 1.5 × 102 copies for EHP and IHHNV. A total of 578 Litopenaeus vannamei samples were detected by the established duplex PCR detection method. The results suggested that 398 samples were infected with EHP, 362 samples were infected with IHHNV, and 265 samples were co-infected with EHP and IHHNV. The case-control analysis of the detected shrimp samples showed a certain synergistic effect between EHP and IHHNV.

Development of a real-time enzymatic recombinase amplification assay for rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimp Penaeus vannamei

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is classified as a reportable crustacean disease by the World Organisation for Animal Health (WOAH), which causes poor growth in Penaeus vannamei. According to genome sequence alignment analysis, enzymatic recombinase amplification (ERA) primers and probe were designed based on the ORF1 region of IHHNV, and a real-time ERA assay for IHHNV detection (IHHNV-ERA) was established. The experimental results show that IHHNV-F2/IHHNV-R2 and IHHNV-Probe can effectively amplify the target gene, and the sensitivity is 1.4 × 101 copies/μL within 14.97 ± 0.19 min, while the qPCR using primers 309F/309R could reach the detection limit of 1.4 × 101 copies/μL within 21.76 ± 0.63 min, and the sensitivity results of one-step PCR could be as low as 1.4 copies/μL with expense of time and false positives. The IHHNV-ERA system can effectively amplify the target gene at 42 ℃ within 20 min, and has no cross-reaction with white spot syndrome virus (WSSV), Ecytonucleospora hepatopenaei (EHP), Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VpAHPND), and healthy shrimp genomic DNA. Test results of practical samples showed that the detection rate of IHHNV-ERA (18/20) was better than the industrial standard qPCR assay (17/20). Compared with the existing technology, the useful results of this detection assay are: (1) get rid of the dependence on the thermal cycle instrument in the PCR process; (2) the experimental procedure is simple, time-consuming and fast; (3) the detection sensitivity is high. This study provides an ERA based detection assay for IHHNV, which can be used not only for the rapid detection of IHHNV infection, but also for the field screening of pathogens. This assay can also be applied to clinical inspection, customs detection, enterprise quality inspection and other fields, and has obvious practical application value.

Establishment of two visual interpretation methods of DIV1 LAMP amplification products

Viral diseases have become a significant impediment to the sustainable development of the global shrimp aquaculture industry. Decapod iridescent virus 1 (DIV1) is an emerging shrimp virus that has affected shrimp in China recent years. Rapid detection of DIV1 could improve enhance the effectiveness of prevention, control and treatment in the absence of good prevention and control measures. This study established loop-mediated isothermal amplification (LAMP) along with two visual interpretation methods, LAMP-dye and LAMP-LFD, to detect DIV1. The newly developed method would not cause cross-reactions with other shrimp pathogens such as white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), Enterocytozoon hepatopenaei (EHP), and Vibrio parahaemolyticus acute hepatopancreatic necrosis disease (VpAHPND). The detection limit of DIV1 LAMP was as low as 103 copies of DIV1 per reaction, with a reaction time of less than 40 min. The diagnostic sensitivity and diagnostic specificity of this method were determined to be 88% and 100%, respectively, when compared with the conventional PCR. Both of the LAMP-dye and LAMP-LFD methods are cost-effective and do not require expensive amplification equipment. They can be combined with LAMP and other temperature amplification methods for rapid on-site detection, effectively prevent aerosol contamination, and which are convenient and suitable for field testing or preliminary infection rish prediction experiments to predict the risk of infection.

A real-time fluorogenic recombinase polymerase amplification microfluidic chip (on-chip RPA) for multiple detection of pathogenic microorganisms of penaeid shrimp

Pathogenic microorganisms pose a significant threat to global shrimp production, often resulting in widespread reductions in production across continents. In this study, we developed a centrifugal microfluidic chip integrating real-time fluorogenic recombinase polymerase amplification (on-chip RPA) for the parallel detection of five important pathogenic microorganisms: acute hepatopancreatic necrosis disease-causing Vibrio spp. (VAHPND), white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), shrimp hemocyte iridescent virus (SHIV), and Enterocytozoon hepatopenaei (EHP). This method enabled the parallel analysis of six genetic markers from a single sample through a single pipetting operation for sample injection. It also allowed for the highly automated detection of up to four samples simultaneously using a chip format. The on-chip RPA reaction was performed in a reduced system with a volume as low as 5 μL at a temperature of 39 °C for 20 min. This assay achieved a limit of detection (LOD) of 10 copies/μL of the recombinant plasmid of the target genes. The coefficient of variation (CV) for the time to positive (Tp) value was less than 0.10, even using concentrations at the LOD for multiple pathogens. This indicates a robust reproducibility among replicates. The clinical sensitivity and specificity for detecting field shrimp samples were 96.4% and 100%, respectively, compared with polymerase chain reaction (PCR) combined with DNA sequencing. On-chip RPA assays allow for low reagent consumption, ease of use, accelerated analysis, the ability to analyze multiple samples and genetic targets, and applicability for on-site detection and routine monitoring of multiple pathogens in shrimp aquaculture.

Prevalence of Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) in Farmed Procambarus clarkii of the Middle and Lower Reaches of the Yangtze River in China.

Procambarus clarkii is an important economic aquaculture species worldwide. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) infects numerous crustacean hosts, including P. clarkii. However, there have been few reports on the prevalence of IHHNV in P. clarkii. In this study, 200 farmed P. clarkii were collected from Anhui, Jiangsu, Zhejiang, Hunan, Hubei, and Sichuan provinces in China. PCR detection was employed per the protocol by the World Organization for Animal Health (WOAH) to identify and detect the presence of IHHNV. The positive rate of IHHNV in different provinces ranged from 16.7 to 56.7%, and the overall IHHNV-positive rate was 38.5%. IHHNV strains isolated in this study related closely to infectious IHHNV and split into two major distinct branches. Besides, the IHHNV strains shared a high homology (93.4-99.4%). These findings suggest that a high prevalence of IHHNV was established in farmed P. clarkii in the middle and lower reaches of the Yangtze River.

Abolishment of spawner-isolated mortality virus and where the remaining science leads.

In the late 1980s, there was histological and electron microscopy evidence for a parvovirus-like virus in Australian prawns. The data were consistent with infectious hypodermal and haematopoietic necrosis virus (IHHNV). However, these cases did not fit the then current paradigms of the known viruses and sequencing did not find any meaningful sequence homology. The virus was named spawner-isolated mortality virus (SMV; GenBank AF499102.1) in order to allow publication of the information about its occurrence to inform the scientific and aquacultural communities. This virus was present in the early years of mid-crop mortality syndrome (1993-1995). However, as time passed, nucleotide and protein databases have expanded and sequence investigation tools have become more cost effective. The sequence of the entity known as SMV is now shown to be of Carnobacterium divergens (CP016843.1). Therefore, the publications with regard to SMV have been assessed and a recommendation to abolish the name with the still valid science transferred to IHHNV and C. divergens.

SIMULTANEOUS PATHOGEN DETECTION OF SHRIMP VIRUSES ON CULTURED TIGER SHRIMPS (Penaeus monodon) IN INDONESIA

The multiple-pathogen infection causes severe economic impact to shrimp industry in Indonesia and worldwide due to mass mortality and multiple abnormalities of the survived infected shrimps. However, multiple-pathogen detection tools in shrimp diseases have not yet widely used. The purpose in this study was to develop and applied simultaneous detection system using multiplex polymerase chain reaction (PCR) assay from natural infections caused by white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV) and monodon baculovirus (MBV) in Black tiger shrimp culture. To analyze multiple-pathogen infections in the shrimp, the study designed and used three pairs of specific primers targeting DNA virus from the shrimp diseases. All amplifications used a specific master mix for multiplex PCR assay and standardized extracted nucleic acid from the samples. This mPCR assay successfully amplified the DNA of three viruses in a single tube-run by multiplex PCR for each virus. Based on the results, the study confirms that multiple-pathogen infection contributes the highest mass mortality rather than from single infection by either WSSV, IHHNV or MBV. This study also confirms that the mPCR assay is a faster, cheaper, and efficient method to detect and subsequently prevent the spreading of multi-pathogen shrimp diseases.

Study of infectious hypodermal and hematopoietic necrosis virus (IHHNV) infection in different organs of Penaeus vannamei

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) is a major viral pathogen in cultured shrimp. It is generally believed that the target organs of IHHNV in shrimp include tissues of ectodermal and mesodermal origin, but do not normally include organ systems of endodermal origin, such as hepatopancreas. In this study, the feeding challenge of IHHNV in different organs (pleopods, muscles, gills, and hepatopancreas) of Penaeus vannamei was studied. The PCR results showed that hepatopancreas of P. vannamei had the strongest IHHNV positivity (100% positive, 19.4 copies/mg) in the feeding challenge experiment. Gills and pleopods had similar infectivity to IHHNV (86.7% positive, 10.6 and 10.5 copies/mg). Among the four organs tested in this study, the IHHNV positivity of muscles was the weakest (33.3% positive, 4.7 copies/mg). The IHHNV infection to hepatopancreas of P. vannamei was also histological confirmed. Our current data indicated that the shrimp tissues derived from the endoderm such as hepatopancreas could also be infected by IHHNV.