Detection Of Infectious Myonecrosis Virus Research Articles

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.

Detection of infectious myonecrosis virus in Penaeus vannamei using the multiplexed PCR platform shrimp MultiPath™

Infectious myonecrosis virus (IMNV) causes 40–80% cumulative mortality in Penaeid shrimp aquaculture, with severity being dependant on pathogen variant and culture conditions. With an increasing presence of IMNV in shrimp aquaculture and devastating impacts in recent years on commercial production, there is increased demand for cost effective surveillance, early detection, and early mitigation strategies for this pathogen in combination with other devastating pathogens including White Spot Syndrome virus (WSSV), virulent strains of Vibrio parahaemolyticus (which cause acute hepatopancreatic necrosis disease (AHPND)) and Enterocytozoon hepatopenaei (EHP) that are also highly prevalent around the world. This study undertook assay validation of the IMNV test within the commercially available Shrimp MultiPath™ (SMP) technology in comparison to an existing quantitative PCR recommended by the World Organisation for Animal Health (WOAH). A total of 254 samples from three Penaeus vannamei populations from farms in the Indo-Pacific region that had reduced feed intake were used for this study. Key performance metrics for analytical and diagnostic performance of the IMNV assay in Shrimp MultiPath™ were assessed in addition to reproducibility and repeatability following the WOAH validation pathway. Establishing these metrics is important to provide the global industry with a cost-effective reliable tool that can be used to look at complete pathogen profiles to mitigate disease risk in shrimp farming. Limit of detection was determined to be 6.6 copies per microlitre of total nucleic acid sample. The diagnostic sensitivity of the IMNV SMP was 99.2% and diagnostic specificity 98.0%, whereas the diagnostic sensitivity and specificity of qPCR were estimated to be 86.9% and 98.2%, respectively. Importantly, the SMP IMNV assay detects both Indo-Pacific and Eastern Latin American variants of IMNV. Overall, the IMNV SMP assay outperforms currently published assays for early detection, and early risk mitigation of this pathogen in shrimp culture, whilst having the added benefit of a full pathogen profile due to its multiplexed nature.

Modification of PCR Program for Detection of Infectious Myonecrosis Virus

Modification of PCR Program for Detection of Infectious Myonecrosis Virus

Sensitivity improvement of immunochromatographic strip test for infectious myonecrosis virus detection

An immunochromatographic strip test with enhanced sensitivity for the detection of infectious myonecrosis virus (IMNV) was developed using three monoclonal antibodies specific to N- and C-termini fragments of IMNV capsid protein. This strip test can detect IMNV with 20 times higher sensitivity than the previous test strip and approximately 50 times lower than that of one-step RT-PCR. The shrimp sample can be heat-treated to augment the release of antigen from the muscular tissue and ensure the sterilization of the sample. Due to its high specificity, field friendly and rapid result, this test strip is suitable for surveillance of IMNV outbreaks and confirmation of IMNV infection in shrimp farming. Statement of relevanceThe IMNV strip test will help farmers to monitor IMNV infection during shrimp culture. The test kit has sensitivity of approximately 50 times lower than that of one-step RT-PCR.

A real-time PCR for the detection of infectious myonecrosis virus in penaeid shrimp

Infectious myonecrosis virus (IMNV) is a recently observed shrimp virus, which threats the cultured Litopenaeus vannamei and can cause huge economic loss in shrimp farming industry. The specific aim of this study was to develop a new sensitive real-time PCR method for the specific detection of shrimp IMNV. A real-time PCR assay with a pair of primers to specifically amplify a 101bp IMNV cDNA fragment and a corresponding TaqMan probe was developed, which shown to be specific for IMNV without cross reaction with DNA samples prepared from four other shrimp viruses including white spot syndrome virus (WSSV), hepatopancreatic parvovirus (HPV), monodon baculovirus (MBV), and infectious hypodermal and haematopoietic virus (IHHNV). The method could detect as low as one single copy of IMNV plasmid cDNA.

Simple and rapid detection of infectious myonecrosis virus using an immunochromatographic strip test

A strip test was developed for detection of infectious myonecrosis virus (IMNV) using a pair of monoclonal antibodies (MAbs), called IMN7 and IMC6, that are specific for the N and C fragments, respectively, of the IMNV capsid protein. The test strips were placed in plastic cassettes and stored desiccated in sealed plastic bags. In detection assays using the test-strip cassettes, 100-μl samples of application buffer containing homogenates from muscles or pleopods of normal or IMNV-infected shrimp were applied to the cassette sample chamber. Subsequent flow through the glass-fiber pad and the nitrocellulose membrane strip led to the development of visible antibody-protein complexes within 15min. In samples containing IMNV, viral capsid protein bound to gold-labeled IMN7 in the glass-fiber pad and the complex was subsequently captured by MAb IMC6 at the T line to form a reddish-purple band. Any unbound gold-labeled IMN7 migrated past the T line to be captured by the GAM antibody to form a band at the C line. Samples without IMNV or containing it below the test detection limit gave reddish-purple bands only at the C line. The sensitivity of the test was comparable to that of dot blot tests using single MAbs but was ~300-fold less sensitive than a one-step RT-PCR test for IMNV. Despite this lower sensitivity, the strip test has advantages of low cost, speed and simplicity (i.e., no sophisticated equipment or specialized skills required), and it is appropriate for use by farmers for pathogen confirmation when IMNV is suspected in diseased shrimp.

Detection of infectious myonecrosis virus using monoclonal antibody specific to N and C fragments of the capsid protein expressed heterologously

The gene encoding the capsid protein in ORF1 of the genome of infectious myonecrosis virus (IMNV) (GenBank AY570982) was amplified into three parts named CP-N (nucleotides 2248–3045), CP-I (nucleotides 3046–3954) and CP-C (nucleotides 3955–4953). The CP-N fragment was inserted into expression vector pTYB1 while CP-I and CP-C were each inserted into expression vector pGEX-6P-1 for transformation of BL21 E. coli strain. After induction, intein-CP-N (84 kDa), glutathione-S-transferase (GST)-CP-I (60 kDa) and GST-CP-C (62 kDa) fusion proteins were produced. They were separated by SDS-PAGE and electroeluted before immunization of Swiss mice for monoclonal antibody (MAb) production. Two MAbs specific to CP-N and one MAb specific to CP-C were selected for use for detection of natural IMNV infections in Penaeus vannamei by dot blotting, Western blotting and immunohistochemistry. There was no cross-reaction with shrimp tissues or common shrimp viruses including white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), Penaeus monodon nucleopolyhedrovirus (PemoNPV), Penaeus stylirostris densovirus (PstDNV) and Penaeus monodon densovirus (PmDNV). The detection sensitivities of the MAbs were approximately 6 fmol/spot of purified recombinant intein-CP-N protein and 8 fmol/spot of GST-CP-C as determined by dot blotting. A combination of all three MAbs resulted in a twofold increase in sensitivity over use of any single MAb. However, this sensitivity was approximately 10 times lower than that of one-step RT-PCR using the same sample. Immunohistochemical analysis using MAbs specific to CP-N and CP-C in IMNV-infected shrimp revealed intense staining patterns in muscles, the lymphoid organ, gills, the heart, hemocytes and connective tissue.

Detection of infectious myonecrosis virus in penaeid shrimps using immunoassays: usefulness of monoclonal antibodies directed to the viral major capsid protein

Despite the economic impact of the infectious myonecrosis virus (IMNV) on shrimp farms in several countries, no method for immunological detection is currently available. With the aim of developing immunodiagnostic methods for IMNV detection in infected shrimps, a recombinant fragment of the IMNV major capsid protein gene encoding amino acids 105-297 (rIMNV₁₀₅₋₂₉₇ was heterologously expressed in Escherichia coli and used to immunize Balb/c mice, generating monoclonal antibodies (MAbs). Six hybridomas were obtained, and four of these recognized the presence of IMNV in tissue homogenates from naturally infected shrimps by immunodot blot assay. Among these MAbs, three were able to detect a ~100-kDa protein, which corresponds to the predicted mass of the IMNV major capsid protein, as well as viral inclusion bodies in muscle fibroses by western blot and immunohistochemistry. Two MAbs showed high specificity and sensitivity, showing no cross-reaction with healthy shrimp tissues in any assays, indicating their usefulness for IMNV detection.

Development of a method for the detection of infectious myonecrosis virus by reverse‐transcription loop‐mediated isothermal amplification and nucleic acid lateral flow hybrid assay

We report the development of a reverse-transcription loop-mediated isothermal amplification and nucleic acid lateral flow method (RT-LAMP-NALF) for detection of infectious myonecrosis virus (IMNV). The RT-LAMP-NALF method combines simplified nucleic acid extraction, a reverse-transcription loop-mediated isothermal amplification platform, and one-step visual colorimetric confirmation of the IMNV amplified sequences using a generic NALF qualitative detection test strip. The sensitivity of RT-LAMP (using two and three primer pairs) and nested RT-LAMP (using three primer pairs) was compared by real-time reverse-transcription-polymerase chain reaction (RT-PCR) using TaqMan probe. The detection of RT-LAMP (three primer pairs) products was accomplished by using a NALF-test strip. The RT-LAMP-NALF showed equivalent sensitivity to RT-LAMP (using three primer pairs), and it was found to be 100 and 10 times more sensitive than one-step RT-PCR and RT-LAMP (two primer pairs), respectively. On the other hand, the RT-LAMP-NALF was 10 and 100 times less sensitive than nested RT-PCR and real-time RT-PCR, respectively. The simplified RNA extraction method ranged from 4.4 x 10(6) to 2.2 x 10(8) IMNV copy numbers microL(-1) RNA, and it was similar with the standard RNA extraction (from 1.2 x 10(6) to 6.3 x 10(7) IMNV copy numbers microL(-1) RNA). These results clearly demonstrate that the RT-LAMP-NALF method is specific, sensitive, can shorten the time for analysis, and has potential application for IMNV diagnosis in resource-poor diagnostic settings.

Detection of infectious myonecrosis virus (IMNV) of penaeid shrimp by reverse-transcriptase polymerase chain reaction (RT-PCR)

Infectious myonecrosis virus (IMNV) infecting cultured Litopenaeus vannamei in Brazil is a double-stranded RNA virus that causes a slowly progressive disease with cumulative mortalities of up to 70%. The disease is currently diagnosed using a combination of gross signs (primarily skeletal tail muscle necrosis with white opaque discoloration), histopathology, and in situ hybridization with a digoxigenin-labeled gene probe. A rapid and sensitive method for definitive diagnosis of the disease was developed using reverse-transcriptase polymerase chain reaction (RT-PCR). Two primer sets were used to detect 328 and 139 bp amplicons in a nested RT-PCR assay. Using RNA extracted from purified virions, the first step reaction detected 100 copies of the IMNV viral genome whereas the nested step detected 10 copies. The primers were shown to be specific for IMNV and no amplicons were detected using RNA extracted from shrimp infected with other penaeid shrimp viruses (Taura syndrome virus [TSV], yellowhead virus [YHV], infectious hypodermal hematopoietic necrosis virus [IHHNV] and white spot syndrome virus [WSSV]).