Infectious Myonecrosis Research Articles

Association of dual viral infection with mortality of Pacific white shrimp (Litopenaeus vannamei) in culture ponds in India

Pacific white shrimp, Litopenaeus vannamei has been introduced recently for culture practice in India. Though SPF stocks are imported for larval production and thereafter culture practice, these are prone to infection with the existing viruses in the environment. Here we report mortality of L.vannamei in several farms in India with minimum biosecurity. The shrimp were harvested early within 50-72days of culture due to the onset of disease and consequent mortality. As per the analysis carried out, the shrimp were infected with two virus, white spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV). About 80% of the samples collected had either or both of the viruses. A majority of these samples (60%) had dual infection with WSSV and IHHNV. Infection of shrimp with WSSV and IHHNV could be detected both by PCR and histopathology. Some of the samples had either exclusively WSSV infection or IHHNV infection and were also harvested before the completion of the required culture period. All the samples analyzed were negative for taura syndrome virus, yellow head virus and infectious myonecrosis virus. While it is difficult to point out the exact etiological agent as the cause of mortality, strict biosecurity measures are advisable for the continuity of L. vannamei culture in India.

Non-Specific Immune Response of Vanname Shrimp (Litopeaneus vanname) Induced by Chitosan as an Immunomodulator against IMNV (Infectious Myonecrosis Virus) Exposure

Chitosan is one of polysaccharide derivatives commonly known as poly D-glucosamin-2 amino-2 dexoy-D-glucose, and glucosamine has a nearly similar chemical structure with β-glucan. It is capable of activating Prophenoloxidase (ProPO) to enhance immune system of shrimp. Chitosan is added to the food with treatment dose of 3 ml, 6 ml, 9 ml, and 12 ml. Then one negative control (not infected with IMNV) and one positive control (infected with IMNV). Shrimps are infected with IMNV through injection and THC, DHC, phagocytosis activity, super anionoxide and phenoloxidase (PO) are carefully observed. The most optimum result was obtained in the treatment of 3 ml Chitosan/kg food and the lowest result was found in the positive control.

Rapid and sensitive detection of shrimp infectious myonecrosis virus using a reverse transcription loop-mediated isothermal amplification and visual colorogenic nanogold hybridization probe assay

This study reports a novel strategy for the detection of reverse transcription loop-mediated isothermal amplification (RT-LAMP) products derived from infectious myonecrosis virus (IMNV), causes a serious myonecrosis in Penaeus (Litopenaeus) vannamei, by using a ssDNA-labeled with gold nanoparticle (AuNP) probe. This technique relies on a self-aggregation method, when the AuNP aggregation is induced by an increasing of salt concentrations with visual detection. The presence of IMNV-LAMP target prevented an AuNP aggregation and a solution remained as pink color of AuNP, while non-complementary targets cannot prevent AuNP aggregation, resulting in a visible color change to purple color after addition of salt. By using the combination of LAMP and AuNP probe system, the total assay interval required approximately 50min (exclude RNA preparation). Detection limit was 10copies of IMNV RNA in vitro transcript that comparable to that of LAMP followed by LFD and nested RT-PCR, but it was 100-times more sensitive than RT-PCR methods. This assay can be adapted easily for rapid detection of other shrimp infectious diseases agents at low-cost with robust reagents and using a simple colorimetric detection method.

Investigation of possible presence of infectious myonecrosis virus in shrimp in China

Investigation of possible presence of infectious myonecrosis virus in shrimp in <scp>C</scp>hina

Sequence-optimized and targeted double-stranded RNA as a therapeutic antiviral treatment against infectious myonecrosis virus in Litopenaeus vannamei

Infectious myonecrosis virus (IMNV) is a significant and emerging pathogen that has a tremendous impact on the culture of the Pacific white shrimp Litopenaeus vannamei. IMNV first emerged in Brazil in 2002 and subsequently spread to Indonesia, causing large economic losses in both countries. No existing therapeutic treatments or effective interventions currently exist for IMNV. RNA interference (RNAi) is an effective technique for preventing viral disease in shrimp. Here, we describe the efficacy of a double-stranded RNA (dsRNA) applied as an antiviral therapeutic following virus challenge. The antiviral molecule is an optimized dsRNA construct that targets an IMNV sequence at the 5' end of the genome and that showed outstanding antiviral protection previously when administered prior to infection. At least 50% survival is observed with a low dose of dsRNA administered 48 h post-infection with a lethal dose of IMNV; this degree of protection was not observed when dsRNA was administered 72 h post-infection. Additionally, administration of the dsRNA antiviral resulted in a significant reduction of the viral load in the muscle of shrimp that died from disease or survived until termination of the present study, as assessed by quantitative RT-PCR. These data indicate that this optimized RNAi antiviral molecule holds promise for use as an antiviral therapeutic against IMNV.

Administration of Hot Water Extract Diatomae Caetoceros Ceratosporum via Injection Enhances the Immune Resistance of White Shrimp Litopenaeus vannamei against Infectious Myonecrosis Virus

Immune resistance of white shrimp against disease can be enhanced through the administration of immunostimulant. Some of sea microalgae extracts has been proved trough examination to be able to stimulate immune system of shrimp. The objective of this study is to know immune response of Litopenaeus vannamei injected with hot water extract Chaetoceros ceratosporum (HWEC) doses of 5, 10, 15 and 20 µg·g -1 body weight (BW) against Infectious Myonecrosis (IMNV) virus infection. The findings show that C . ceratosphorum administration can enhance immune response of L. vannamei infected by Infectious Myonecrosis virus, indicated by the increasing number of hemocytes (THC), total protein plasma (TPP), phenolokxidase (PO) activity, and respiratory burst (RB) activity. Highest immune response is achieved by injection dose of 15 µg·g -1 shrimp body weight. The increase of shrimp immune response leads to the increase of shrimp immune system against IMNV infection, so the survival rate of L. vannamei infected by IMNV is higher than positive control and similar to those of negative control.

Profiling infectious myonecrosis virus infection in Litopenaeus vannamei using nextgen sequencing reveals novel IMNV sequence

Profiling infectious myonecrosis virus infection in Litopenaeus vannamei using nextgen sequencing reveals novel IMNV sequence

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.

Distribusi Penyakit Infectious Myo Necrosis Virus (IMNV) Pada Udang Vannamei (Litopenaeus vannamei) Di Pantai Utara Jawa Timur &lt;br&gt;&lt;i&gt;[ Distribution Of Infectious Myo Necrosis Virus (IMNV) In White Shrimp Vannamei (Litopenaeus vannamei) In North Coast, East Java]&lt;i&gt;

Abstract White shrimp vannamei (Litopenaeus vannamei) is the main commodities among shrimp farmers because it has a rapid growth, can be cultivated with high density and world market demand is large enough .Since being introduced in Indonesia, vannamei shrimp has spread to the north of East Java pesisisr like Probolinggo, Pasuruan, Lamongan and Tuban. Shrimp growth factors are must be faced on vannamei shrimp culture, including the problems of disease. One type of virus disease is an Infectious Myo Necrosis Virus (IMNV). IMNV has affecting to the 70% of shrimp mortality (Tang et al., 2008). This study aims to determine the distribution of IMNV on white shrimp vannamei from the northern coast of East Java. This research uses descriptive method by taking samples from five different location which 75 samples totaly. In this research also take the water quality parameter including pH, DO, salinity and temperature. Whole of the samples performed the PCR test to determine how many samples which is indicating of IMNV. Based on measurements of water quality at five different locations, the plots I, II and III obtained the temperature range between 29.3-30.2 °C, salinity 90-30 ppt, pH 7.5-7.9, and DO 3.8-5.7 ppm. Temperature, pH and DO parameters show normally on white shrimp vannamei, while salinity shows different valve from normal conditions. Abnormallity happened in Situbondo with the salinity betwen 32-33 ppt and Pasuruan betwen 9-10 ppt. Results showed that the number of the lowest infection is Lamongan (53%) following Probolinggo (60%), Tuban (67%), Pasuruan (73%) and the highest is from Situbondo (100%)

Infectious myonecrosis virus and white spot syndrome virus co-infection in Pacific white shrimp (Litopenaeus vannamei) farmed in Brazil

Over the past few years, infectious myonecrosis virus (IMNV) and white spot syndrome virus (WSSV) have caused substantial economic losses to Brazilian shrimp farmers. Initially reported from different geographical locations within the country, these pathogens are becoming increasingly prevalent throughout Brazil. In the present study, 30 diseased shrimp collected from a farm in Northeastern Brazil were screened for viral infection. The shrimp were submitted to histological analysis and quantitative real-time PCR (qPCR). New probes and primers were designed for the qPCR assays, and IMNV detection was based on a modified protocol. Clinical signs of both white spot syndrome and infectious myonecrosis were observed in histological preparations from four of the sampled shrimp. The presence of both IMNV and WSSV was confirmed by qPCR in twelve specimens. Viral co-infection has previously been reported in farmed shrimp. However, this is the first Brazilian report of co-infection with IMNV and WSSV in farmed Litopenaeus vannamei.

Dual infections of IMNV and MrNV in cultivated Penaeus vannamei from Indonesia

Whitening of muscle tissue in farmed whiteleg shrimp Penaeus (Litopenaeus) vannamei can result from stress-induced muscle cramps and from viral infections caused by infectious myonecrosis virus (IMNV) or Penaeus vannamei nodavirus (PvNV). A similar viral-induced whitening of muscles can be caused in the river prawn Macrobrachium rosenbergii and in larvae of the penaeid shrimp Penaeus (Penaeus) monodon and Penaeus (Fenneropenaeus) indicus by a mixed infection with Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV). Here we describe mixed infections of IMNV and MrNV in juvenile cultivated P. vannamei from Indonesia, detected by nested RT-PCR and immunohistochemical analysis. Muscle lesions in the dually-infected shrimp gave positive immunohistochemical reactions for both IMNV and MrNV, while connective tissue in the same samples gave positive reactions for MrNV only, indicating some differences in tissue specificity between the two viruses. Although it is not known whether the dual infections are more lethal to shrimp than single IMNV infections, this is possible since earlier work has shown that MrNV alone can increase mortality of P. vannamei under stress.

Differential induction of HSP-70 expression in response to IHHNV in white shrimp Litopenaeus vannamei naturally co-infected with IHHNV and IMNV

Abstract Brazil is becoming one of the main global producers of the shrimp Litopenaeus vannamei. Worldwide outbreaks of viral disease place this aquaculture industry at risk, causing episodic economical loss. The primary viruses for L. vannamei, particularly in northeastern Brazil, are the infectious hypodermal and hematopoietic necrosis virus (IHHNV) and the infectious myonecrosis virus (IMNV). After a period of unusual rainfall, we detected that farmed shrimp developing IMN or IHHN disease were co-infected with both viruses, and the disease outcome resulted from reciprocal IHHNV and IMNV proliferation. To comprehend how the key molecules of innate immunity respond to this double infection, the levels of HSP-70, crustin, penaeidin-3a, and C-type lectin-br1 were assessed by quantitative PCR. HSP-70 expression was expressively up-regulated by IHHNV infection in the gills of double-infected shrimp but not by IMNV infection; the other transcripts were not significantly altered. These findings implicate the HSP-70 as a differential modulator of viral co-infection in shrimp.

Development of TaqMan Probe-Based Insulated Isothermal PCR (iiPCR) for Sensitive and Specific On-Site Pathogen Detection

Insulated isothermal PCR (iiPCR), established on the basis of Ralyeigh-Bénard convection, is a rapid and low-cost platform for nucleic acid amplification. However, the method used for signal detection, namely gel electrophoresis, has limited the application of iiPCR. In this study, TaqMan probe-based iiPCR system was developed to obviate the need of post-amplification processing. This system includes an optical detection module, which was designed and integrated into the iiPCR device to detect fluorescent signals generated by the probe. TaqMan probe-iiPCR assays targeting white spot syndrome virus (WSSV) and infectious myonecrosis virus were developed for preliminary evaluation of this system. Significant elevation of fluorescent signals was detected consistently among positive iiPCR reactions in both assays, correlating with amplicon detection by gel electrophoresis analysis. After condition optimization, a threshold value of S/N (fluorescent intensityafter/fluorescent intensitybefore) for positive reactions was defined for WSSV TaqMan probe-iiPCR on the basis of 20 blank reactions. WSSV TaqMan probe-iiPCR generated positive S/Ns from as low as 101 copies of standard DNA and lightly infected Litopenaeus vannamei. Compared with an OIE-certified nested PCR, WSSV TaqMan probe-iiPCR showed a sensitivity of 100% and a specificity of 96.67% in 120 WSSV-free or lightly infected shrimp samples. Generating positive signals specifically and sensitively, TaqMan probe-iiPCR system has a potential as a low-cost and rapid on-site diagnostics method.

A Nonluminescent and Highly Virulent Vibrio harveyi Strain Is Associated with “Bacterial White Tail Disease” of Litopenaeus vannamei Shrimp

Recurrent outbreaks of a disease in pond-cultured juvenile and subadult Litopenaeus vannamei shrimp in several districts in China remain an important problem in recent years. The disease was characterized by “white tail” and generally accompanied by mass mortalities. Based on data from the microscopical analyses, PCR detection and 16S rRNA sequencing, a new Vibrio harveyi strain (designated as strain HLB0905) was identified as the etiologic pathogen. The bacterial isolation and challenge tests demonstrated that the HLB0905 strain was nonluminescent but highly virulent. It could cause mass mortality in affected shrimp during a short time period with a low dose of infection. Meanwhile, the histopathological and electron microscopical analysis both showed that the HLB0905 strain could cause severe fiber cell damages and striated muscle necrosis by accumulating in the tail muscle of L. vannamei shrimp, which led the affected shrimp to exhibit white or opaque lesions in the tail. The typical sign was closely similar to that caused by infectious myonecrosis (IMN), white tail disease (WTD) or penaeid white tail disease (PWTD). To differentiate from such diseases as with a sign of “white tail” but of non-bacterial origin, the present disease was named as “bacterial white tail disease (BWTD)”. Present study revealed that, just like IMN and WTD, BWTD could also cause mass mortalities in pond-cultured shrimp. These results suggested that some bacterial strains are changing themselves from secondary to primary pathogens by enhancing their virulence in current shrimp aquaculture system.

Infections of MrNV (Macrobrachium rosenbergii nodavirus) in cultivated whiteleg shrimp Penaeus vannamei in Asia

Macrobrachium rosenbergii nodavirus (MrNV) is well-known as a major pathogen that causes whitened muscles and high mortality in the freshwater prawn Macrobrachium rosenbergii. Recently, it has also been reported to cause white muscles and high mortality in postlarvae of the marine shrimp Penaeus (Penaeus) monodon and Penaeus (Fenneropenaeus) indicus in India. The latter report stimulated us to re-examine specimens from Asian shrimp farms that had experienced high mortality in Penaeus (Litopenaeus) vannamei with white muscles but tested negative for infectious myonecrosis virus (IMNV) and Penaeus vannamei nodavirus (PvNV) by RT-PCR analysis. Some of these specimens were indeed positive for MrNV by RT-PCR. Sequences of the two single-stranded RNA fragments in the MrNV genome were amplified from these P. vannamei specimens and found to share ~97% nucleic acid sequence identity with the MrNV sequences deposited at GenBank (NC_005094 and NC_005095NC_005094NC_005095). Extra small virus (XSV) usually associated with MrNV, was detected in some but not all of the samples. Infectivity tests were performed by feeding P. vannamei with minced tissues from MrNV-infected M. rosenbergii. The assays were preformed at low salinity of 2ppt and at two different water temperatures of approximately 22°C and 28°C. It was revealed that shrimp exhibited a higher infectivity and mortality at the lower temperature. Our findings suggested that P. vannamei is an additional species that is susceptible to MrNV and that low temperature together with low salinity of rearing water may increase the severity of infections leading to significant mortality.

DsRNA provides sequence-dependent protection against infectious myonecrosis virus in Litopenaeus vannamei

Viral diseases are significant impediments to the sustainability of shrimp aquaculture. In addition to endemic disease, new viral diseases continue to emerge and cause significant impact on the shrimp industry. Disease caused by infectious myonecrosis virus (IMNV) has caused tremendous losses in farmed Pacific white shrimp (Litopenaeus vannamei) since it emerged in Brazil and translocated to Indonesia. There are no existing antiviral interventions, outside of pathogen exclusion, to mitigate disease in commercial shrimp operations. Here, we describe an iterative process of panning the genome of IMNV to discover RNA interference trigger sequences that initiate a robust and long-lasting protective response against IMNV in L. vannamei. Using this process, a single, low dose (0.02 µg) of an 81 or 153 bp fragment, with sequence corresponding to putative cleavage protein 1 in ORF1, protected 100 % of animals from disease and mortality caused by IMNV. Furthermore, animals that were treated with highly efficacious dsRNA survived an initial infection and were resistant to subsequent infections over 50 days later with a 100-fold greater dose of virus. This protection is probably sequence dependent, because targeting the coding regions for the polymerase or structural genes of IMNV conferred lesser or no protection. Interestingly, non-sequence specific dsRNA did not provide any degree of protection to animals as had been described for other shrimp viruses. Our data indicate that the targeted region for dsRNA is a crucial factor in maximizing the degree of protection and lowering the dose required to induce a protective effect against IMNV infection in shrimp.

Quantitation of infectious myonecrosis virus in different tissues of naturally infected Pacific white shrimp, Litopenaeus vannamei, using real-time PCR with SYBR Green chemistry

The Pacific white shrimp, Litopenaeus vannamei, is the most important shrimp species in volume in world aquaculture. However, in recent decades, outbreaks of diseases, especially viral diseases, have led to significant economic losses, threatening the sustainability of shrimp farming worldwide. In 2004, Brazilian shrimp farming was seriously affected by a new disease caused by the Infectious myonecrosis virus (IMNV). Thus, disease control based on rapid and sensitive pathogen detection methods has become a priority. In this study, a specific quantitation method for IMNV was developed using real-time PCR with SYBR Green chemistry and viral load of the principal target tissues of chronically infected animals was quantified. The quantitative analysis revealed that mean viral load ranged from 5.08 × 10 8 to 1.33 × 10 6 copies/μg of total RNA in the hemolymph, 5.096 × 10 5 to 1.26 × 10 3 copies/μg in the pleopods, 6.85 × 10 8 to 3.09 × 10 4 copies/μg in muscle and 8.15 × 10 6 to 3.90 × 10 3 copies/μg in gills. Different viral loads of IMNV were found with greater values in the hemolymph and muscle, followed by the pleopods and gills.

False rumours of disease outbreaks caused by infectious myonecrosis virus (IMNV) in the whiteleg shrimp in Asia

BackgroundInfectious myonecrosis virus (IMNV) disease outbreaks in cultivated whiteleg shrimp Penaeus (Litopenaeus) vannamei are characterized by gross signs of whitened abdominal muscles and by slow mortality reaching up to 70%. In 2006 the first disease outbreaks caused by IMNV in Asia occurred in Indonesia. Since then rumours have periodically circulated about IMNV disease outbreaks in other Asian countries. Our findings indicate that these are false rumours.FindingsOur continual testing by nested RT-PCR of shrimp samples suspected of IMNV infection from various Asian countries since 2006 has yielded negative results, except for samples from Indonesia. Our results are supported by the lack of official reports of IMNV outbreaks since January 2007 in the Quarterly Report on Aquatic Animal Diseases (QAAD) from the Network of Aquaculture Centers in Asia Pacific (NACA). In most cases, our shrimp samples for which tissue sections were possible showed signs of muscle cramp syndrome that also commonly causes muscle whitening in stressed whiteleg shrimp. Thus, we suspect that most of the false rumours in Asia about IMNV outside of Indonesia have resulted because of muscle cramp syndrome.ConclusionsResults from continual testing of suspected IMNV outbreaks in Asian countries other than Indonesia since 2006 and the lack of official country reports of IMNV outbreaks since January 2007, indicate that rumours of IMNV outbreaks in Asian countries outside of Indonesia are false. We suspect that confusion has arisen because muscle cramp syndrome causes similar signs of whitened tail muscles in whiteleg shrimp.

Purification of infectious myonecrosis virus (IMNV) in species of marine shrimp Litopenaeus vannamei in the State of Ceará

In Brazil, shrimp farming has been developed most intensely in the Northeast Region. Recently, however, exporters have become concerned over the appearance of Infectious Myonecrosis (IMN), the etiological agent of which is a virus called Infectious Myonecrosis Virus (IMNV). Although IMNV has been characterized extensively, purification methods are complicated to reproduce and very expensive. The objective of this study was to purify the IMNV virus using an easy reproductive method and to produce anti-IMNV antibodies to be used in diagnostic methods. Shrimp samples showing symptoms of IMN obtained from two aquaculture farms in Ceará were used for this purpose.IMNV-positive shrimps were macerated in phosphate buffer, pH 7.5, enriched with antioxidants, clarified with chloroform and the supernatant was submitted to differential centrifugation, precipitated using PEG and NaCl and finally loaded on a discontinuous gradient of sucrose. Purified IMNV was submitted to RT-PCR and electrophoresis either in agarose gel or SDS-PAGE, which revealed RNA and protein bands, characteristic of IMNV. IMNV induced humoral immune response in Swiss mice when administered subcutaneously. Anti-IMNV antibodies were identified by ELISA (enzyme-linked immunosorbent assay) and Western blotting methods and produced a response against purified IMNV and the crude extract obtained from the infected shrimp. However, antibodies specific to the crude extract obtained from uninfected shrimp were not detected. This is the first report of IMNV having been purified in Brazil and the first time that specific antibodies against IMNV proteins have been produced. These results suggest that easy methods can be developed to produce specific antiserum for viral diagnosis on a large scale.