Infectious Myonecrosis Virus (IMNV) – An alarming viral pathogen to Penaeid shrimps

Accepted Manuscript Infectious Myonecrosis Virus (IMNV) – An alarming viral pathogen to Penaeid shrimps

Kurcheti Pani Prasad, KU Shyam, Husne Banu, K Jeena, Rahul Krishnan PII: DOI: Reference:

S0044-8486(16)30653-6 doi: 10.1016/j.aquaculture.2016.12.021 AQUA 632460

To appear in:

aquaculture

Received date: Revised date: Accepted date:

5 October 2016 19 December 2016 20 December 2016

Please cite this article as: Kurcheti Pani Prasad, KU Shyam, Husne Banu, K Jeena, Rahul Krishnan , Infectious Myonecrosis Virus (IMNV) – An alarming viral pathogen to Penaeid shrimps. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Aqua(2016), doi: 10.1016/j.aquaculture.2016.12.021

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ACCEPTED MANUSCRIPT Infectious Myonecrosis Virus (IMNV) – An alarming viral pathogen to Penaeid shrimps

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Kurcheti Pani Prasad*, K U Shyam, HusneBanu, K Jeena, Rahul Krishnan Aquatic Environment and Health Management division, Indian Council of Agricultural Research-Central Institute of Fisheries Education, Mumbai, India- 400 061

Abstract

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*Corresponding author: Kurcheti Pani Prasad, [email protected], Phone no: +919867241101

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Intensification and diversification of the aquaculture practices made an opening to the emergence of new viral diseases daunting the farmers to achieve a sustained production. Viruses are

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obligatory parasites abundant in the aquatic environments and are being introduced in the sector directly or indirectly. Infectious myonecrosis virus (IMNV) is an emerging shrimp RNA virus

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causing the disease, infectious myonecrosis (IMN). The disease was reported first from Brazil and currently the geographical locations of infection span in Brazil and Indonesia. Research are

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centered on the viral pathogenesis, viral entry, disease prevention and epidemiology, diagnostics and molecular pathology. The recent developments in the synthetic and molecular biology

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techniques have paved way to explore IMNV at its molecular levels, yet further research has to be conducted to fully understand the virus as well as diagnostics of the disease with cause. This

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review covers all the aspects of the virus, IMNV and the disease IMN, research developments and emphasizes on the current progress and the future prospects of the research in control and

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prevention strategies.

Keywords: Infectious myonecrosis, IMNV, Penaeid shrimp, double-stranded RNA virus

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ACCEPTED MANUSCRIPT Introduction The farming of Litopenaeus vannamei, white leg shrimp has positively influenced the global shrimp production and export scenario. Global production of farmed shrimp has increased from 3.4 million tons in 2013 to 3.6 million tons in 2014 (Aquaculture Culture Asia-Pacific Magazine). The farmers of Vietnam, Indonesia, and India are shifting from Black tiger shrimp

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farming to L. vannamei culture. As a result,the production of L. vannamei in Asia increased from

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2.12 million tons in 2013 to 2.37 million tons in 2014 (FAO, Glob fish, shrimp May-2015). The

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culture of monodon was a true sufferer of many havocs like White spot Disease (WSD), Loos Shell Syndrome (LSS) and Monodon Slow Growth Syndrome (MSGS).Today world has moved

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on to L. vannamei due to its high productivity, low-cost involvement and availability of SPF seed. The huge production rate of L. vannamei has boosted the export of India in 2013–2014

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fiscal year by 1,34,372 tons (USD 1.47 billion) compared with 69 565 tons (USD 540.8 million) during the previous fiscal year (Shrimp, 2014). But now L. vannamei is no more resistant to diseases like TSV, YHV Type-1, IHHNV, and EMS. The availability of L. vannamei SPF stocks

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for WSSV has reduced the outbreaks of WSSV.But still, it is suffering from new emerging

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diseases like EMS, IMNV and abdominal segment deformity disease (ASDD) (Sakaew et al., 2008). In this paper, we are reviewing the current research status for IMNV which is an

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emerging viral disease of L. vannamei.

Infectious myonecrosis virus is an emerging novel virus which has affected the Vannamei

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culture of countries like north-eastern Brazil (Andrade et al., 2007; Lightner et al., 2004, Nunes et al., 2004; Poulos et al., 2006) and in the East Java Island (Senapin et al., 2007) as well as in

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other South-East Asian countries. The disease is not threatening as other viral diseases viz., WSSV, YHV etc, but there may be high mortality due to adverse environmental conditions. As per the bioassay results there were no mortality until 9 to 13 days after exposure to IMNV (Tang et al., 2005), while significant mortality can occur within 1 to 3 days in bioassays with TSV, YHV, or WSSV (Lu et al., 1994; Overstreet et al., 1997; Tang and Lightner, 2000). Although disease progression is slower in comparison to other viral diseases.There can be a significant economic loss due to the persistent mortality and increased feed conversion efficiency (Lightner, 2004). Cumulative mortalities up to 80% has been reported in Brazil (Poulose et al., 2006). There is a reduction in the market value of survivors with necrotic muscle.The disease was first 2

ACCEPTED MANUSCRIPT reported in shrimp farming areas of Brazil in 2004 and by 2006, it was spread to Indonesia, which is due to the improper transportation of live animals.It was responsible for economic losses that valued at approximately US$20 million (OIE, World Animal Health Organization, 2009) in 2003. Hence to develop the awareness and necessary control measures for the emergence of IMNV, it was listed in OIE (International Organisation of Epizootics) report

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during 2005.

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Infectious Myonecrosis Virus (IMNV)

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Infectious Myonecrosis Virus (IMNV) is an emerging potential shrimp virus reported to cause a considerable economic loss in shrimp aquaculture (Nunes et al., 2004). The disease was

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first reported in the State of Piaui, North-East Brazil, 2002 in the Pacific whiteleg shrimp, Litopenaeus vannamei and initially was named as Idiopathic Myonecrosis (Lightner et al.,

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2004a, b). Finally, the disease was renamed as Infectious myonecrosis (IMN) and the etiological agent was identified as a virus, named Infectious Myonecrosis Virus (IMNV) (Poulos et al.,

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2006; Tang et al., 2008). Apart from naturally susceptible L. vannamei, the other species reported to be experimentally susceptible are Farfantepenaeus subtilis, Penaeusmonodon, and

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Litopenaeus stylirostris. (Lightner et al., 2004a, b; Tang et al., 2005; Coelho et al., 2009). The virus infects all the life stages of shrimp including post larvae, juveniles, and adult, but the

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mortality was observed in the juveniles and adult with a cooked appearance (Nunes et al., 2004). The reported survival range during infection is35 to 55% in 12 g shrimp with a stocking density

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of 60/m2and the economic loss was estimated to be US$20 million in 2003 (Nunes et al., 2004). Till date, the disease was restricted only to Brazil in South America and Indonesia in Asia.

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(Lightner et al., 2004a, b; Senapin et al., 2007). Losses caused by the disease in 2003 alone were estimated to be 20 million dollars (OIE, 2006). Brazilian shrimp farmers suffered an economic loss of 440 million USD as a result of IMNV outbreak at the end of 2005 (Andrade et al., 2007) and by the end of 2011, an estimate has been reported from both Brazil and Indonesia that the financial loss was increased to more than 1 billion US$ (Lightner et al., 2012). Studies reported that the infectious myonecrosis virus can appear as a co-infection with Macrobrachium rosenbergii Noda virus (MrNV) and white spot syndrome virus (WSSV) in Litopenaeus vannamei (Senapin et al., 2013; Feijo et al., 2013) as well as with Vibrio harveyi (Oktaviana et al., 2014). 3

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Geographical distribution of the virus The virus was first reported from the North- East Brazil in South America in 2002. Up to 2007, the disease was restricted only to Brazil. But in 2007, the virus crossed its border and was

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reported in Indonesia for the first time from an Asian country where it was seen in farms of west

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Java, Sumatra, Bangka, west Borneo, south Sulawesi, Bali, Lombok and Sumbawa in SouthEast Asia (Sutanto, 2011). Thailand also claimed the infection with IMN virus but further

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investigation revealed that other than Indonesia, no other Asian countries were infected. (Senapin et al., 2011). Since it was first reported from Brazil, this virus is thought to be South American in

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origin. It is restricted in geographical distribution. The spread of the disease to new locations like Indonesia is believed to be due to the illegal transboundary movement of the infected or carrier

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broodstock and postlarvae for aquaculture (Flegel, 2006; Lightner, 2012; Senapin et al., 2007; Walker and Mohan, 2009; Walker and Winton, 2010). Rumors are already existing regarding the

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presence of virus in Asian countries other than Indonesia say, India, China, Malaysia, Thailand, and Vietnam. But the report by Senapin et al in 2011, states them false as contamination or

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muscle cramp syndrome (whitening of the muscle).

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Fig. Geographical distribution of Infectious Myonecrosis Virus (IMNV) in worldwide.

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Host range and strains of IMNV IMN virus exclusively infects the Penaeid shrimps. Till date, there is no report of any

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infection among the wild populations (Tang et al., 2005). The virus naturally infects Pacific whiteleg shrimp, L. vannamei and Southern brown shrimp, Farfantepenaeus subtilis and has been experimentally proving to infect, Penaeus monodon, and L. stylirostris (Lightner et al., 2004a, b; Tang et al., 2005; Coelho et al., 2009). There have been no reports of death in P. monodon due to the infection, but it can be a potential carrier of the virus (Tang et al., 2005). Since the main target tissue of IMNV infection is skeletal muscles and which is not a vital organ, the infection is not that much fatal in comparison with other virulent shrimp viruses like WSSV, YHV, and TSV. Also, the damage initiated to the muscle tissues can be repaired at the early 4

ACCEPTED MANUSCRIPT stages of the infection (Tang et al., 2005). Studies have been undertaken for the identification of potential vectors or carriers of the IMNV and recently da Silva et al., 2015 reported that Artemia franciscana, aquaculture live feed organism, as a vector for the IMNV infection. But mass mortality of the shrimp L. vannamei fed with infected A. franciscana were not observed. This is because maybe it act as a source of IMNV in growing out culture ponds (da Silva et al., 2015). Bivalves and polychaete worms are also reported positive to IMNV infection from the infected

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pond without any reliability to confirm that they are true vectors or carriers. The presence of the

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virus may be either due to the ingestion of the contaminated tissues or water from the infected

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area (Andrade, 2009).

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(Table 1. Host range of IMNV)

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To date, four strains of IMNV have been described and nine strains partially sequenced. The first strain (IMNV_Brazil_2006) was reported from Brazil in 2006 (Poulose et al., 2006)

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followed by the second (IMNV_Indonesia_2006) from Indonesia in 2007 (Senapin et al., 2007). The partial and complete genome of the isolates of these strains is available in Genebank. The

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following table shows updated information about the genome sequence available in the database.

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(Table 2. Identified strains of IMNV)

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The viral genome sequence of IMNV isolated from Indonesian shrimp farm shows 99.6% similarity with Brazilian sequence (Senapin et al., 2007). Multiple alignments of Brazilian

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IMNV viral capsid protein coding sequence of 372 bp shows a high degree of similarity with sequences of Brazil and Indonesia (Melo et al., 2014). This analysis suggested that the Brazilian and Indonesia strains are genetically identical and the Indonesian strain may be originated from Brazil. Subsequently, Naim et al. (2014) have determined and deposited two other Indonesian IMNV strains collected from Lampung province in 2011 and East Java province in 2012. Along with the complete sequencing, 6 other partial sequences were also attempted. But they reported a clear evidence of genetic diversification between the Brazilian and Indonesian strains as well as within the partially sequenced Indonesian IMNV strains. Comparison done among the four whole genome sequences available, only a total of 9 aa substitution were found (Naim et al., 5

ACCEPTED MANUSCRIPT 2014). In compliance with Naim et al. (2014) reports, Coelho-Melo et al. (2014) also confirmed that the Indonesian strain shared a high identity with the Brazilian strain isolated and characterized from Ceara state, Brazil. The characterization of major capsid protein (MCP) of the virus revealed that there is a genetic conservation among the viral isolates of Ceara state, Brazil (Coelho-Melo et al., 2014). The latest phylogenetic analysis confirmed that the Brazilian isolates are more variable than Indonesian isolate (Dantas et al., 2015). These conclusions were

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reconfirmed by Kibenge and Godoy (2016) by constructing an unrooted phylogenetic tree based

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on available maximum length of each strain (8 strains). The analytical reports were consistent

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and confer the monophyletic branch of each geographical group (Naim et al., 2014; Dantas et al., 2015; Kibenge and Godoy, 2016).

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Based on the RNA-dependent RNA polymerase (RdRp) sequence phylogeny it is evident that arthropod Totivirus are close to Giardiavirus clade (Oliveira et al., 2014). However recently

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Dantas et al (2016) have reported enough evidence of Arthropod Totiviruses to be grouped separately under“Artivirus”genus within Totiviridae family, as previously proposed by Zhai et

Viral transmission

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al. (2010).

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The exact mode of transmission of IMN virus has not been fully understood. The cannibalistic behaviour of the infected shrimp can be the horizontal way of disease transmission

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(Lightner, 2010; Poulos et al., 2006). The probability of survival of the infectious non-enveloped viral particles is high in the gastrointestinal tract of the organisms which feed on the infected

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animals, therefore the propagation of the virus via ingestion is likely to happen (OIE, 2012). Transmission through the potential carriers such as A. fransiscana, aquaculture live feed organism is cannot be eliminated. Even though there are deficiencies in specific data on the IMNV vectors (OIE, 2012; da silva et al., 2015). The probability of transmission via water and vertical transmission from broodstock to progeny cannot be disregarded. Recently experimental evidence for vertical transmission has been reported in L. vannamei through real-time PCR analysis of naturally and experimentally infected broodstock (Silva et al., 2016). Based on the 100% positive detection of IMNV in ovaries and lower viability of sperm cells in naturally infected males, the source of vertical transmission is suggested to be the maternal origin. The 6

ACCEPTED MANUSCRIPT virus which belongs to the family Totiviridae is getting transmitted to the uninfected cells only during cell division, sporogenesis in the case of fungi infecting viruses and cell fusion (Poulose et al., 2006). But IMN virus and the Piscine myocarditis virus (PMCV) which uniquely infecting Atlantic salmon (Salmo salar L.) showed extracellular transmission strategy during the infection (Haugland et al., 2011). The studies generalizing that the virus can be transmitted to susceptible animals through horizontal mode via vectors, co-habitation through water, cannibalism and

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maternally derived vertical route (Lightner, 2011; Poulose et al., 2006; OIE, 2012; da Silva et al.,

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2015; da Silva et al., 2016).

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Viral morphology and genome characteristics

Characterization of IMNV infecting the Brazilian L. vannamei in details was carried out

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by Poulos et al., (2006). The transmission electron microscopy reveals the single layered isometric viral particles as icosahedral, non-enveloped with T=1, 120 subunits and 5 fiber complexes as surface projections of size 80 Å extended beyond at the fivefold axes. The virion

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capsid structure more likely resembles as that of Totiviridae prototype virus, Saccharomyces

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cerevisiae virus L-A (ScV-L-A) (Tang et al., 2008). Ultrastructurally, a single protrusion composed of an outer knob, a middle stalk and an inner capsid (pentagon) anchored fiber foot.

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These subunits were composed of 60 copies each of two identical subunits namely MCP-A and B which is arranged in a T=2 fashion. 12 Pentameric fivefold (5f) axes were formed by the A

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subunits likewise 20 threefold (3f) axes were surrounded by B subunits in the form of a trimer. A tightly compacted decamer structure was formed at each 5f axis in which 5 number of B subunits were arranged in staggered fashion around each A pentamer. As a result, the virion outer capsid

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constituted with 12 pentagons delimited by broad canyons (Tang et al., 2008). The size of the particles was determined to be 40 nm in diameter. It has a density of 1.366 g ml-1 in CsCl. The genome consists of single non-segmented dsRNA of 8226 -8230bp. The packaged dsRNA genome characteristically witnessed as 5 to 6 concentric rings inside the capsid coating (Tang et al., 2008). Similar to that of other Orthoreoviruses, the rings were spaced at least of 26 Å (Reinisch et al., 2000) distance but less than that of ScV-L-A which is of 30 Å (Tang et al., 2008). The internally projecting fusion protein (MCP-RdRp) is the responsible factor for the

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ACCEPTED MANUSCRIPT circular arrangement of the genome which anchoring the one end of dsRNA and directing the other end. The full-length sequencing of available strains revealed that the IMNV genome has at least 8226 bp length (8226-8230 bp) (GeneBank Accession No. AY570982.3 and KF836757.1). The genetic material made up of two overlapping Open Reading frames (ORFs) namely ORF 1

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and ORF 2 (Poulos et al., 2006; Dantas et al., 2015; Loy et al., 2012). The ORF 1 starts at nt 470

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and runs a total of 5127 nt, which translates a 1708 aa long polyprotein of 106 kDa. Which hew

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to give a putative RNA binding protein (RNP) of 196 aa, two small proteins (SP1 of 284 aa and SP2 of 327 aa) and a 901 aa containing major capsid protein (MCP). dsRNA binding motif (dsRBM or RNP) plays a vital role in viral replication or modulation of host’s immune response

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(Poulos et al, 2006) whereas the function of small proteins is still unclear suggested that which may be involved in assembly, cell entry and extracellular transmission of the virus. Dantas et al.

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(2016) have done a more detail study on ORF1 and based on the presence of

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polypeptide cleavage sites, the singularity of Arthropod Totiviruses is proved. They have

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identified a total of 220 polymorphic sites in 7561 bp genome with a highest in the hypervariable region which translates the small proteins SP1 and SP2 (Dantas et al., 2015). ORF 2 covers 2739

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nt from nt 5395 to nt 8133 encodes for a 912 aa virus-specific RNA-dependent RNA polymerase (RdRp) of 85 kDa (Poulos et al., 2006; Mello et al., 2011; Dantas et al., 2015, 2016). Apart from

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this ORF 2 translated sequence shows a high similarity to RNA-dependent RNA polymerase (RdRp) of Totiviridae family. Also underlined that the ORF 2 coding strategies of IMNV are

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similar to that of GLV and other family members in the Totiviridae, suggested the RdRp conserved domain (Nibert, 2007). The genome is flanked by untranslated region (UTR) of 469 nt

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at 5’ extreme and 93 nt at 3’ extreme. Two stem loops (H1-544 to 574 nt, H2-584 to 646 nt) and two pseudoknots (327-356 nt& 705-732 nt) are present in the 5’ terminal. 3’-end possesses a putative stem-loop and a slippery motif (GGGUUUU-nt 8185 to 8191). A pseudoknot (nt 81848213) present in the 3’ extremity acts as the ribosomal stopping point and adjacent to that a hairpin (nt 8151-8165) was also found. Thus, these regions involved in the initiation and termination of the translation process of the viral mRNA (Dantas et al., 2016). The export of transcribed viral mRNAs and/or import of infectious viral nucleic acid to the cell cytoplasm is not well understood. Since the IMNV viral capsid lacks the capsid pores 8

ACCEPTED MANUSCRIPT through which the export/import occurs, there are two probable explanations for the support. One is that at the time of initial cell entry, the protrusions were disoriented and those potential pores will be available for the release of transcripts and transport of nucleic acid. The second possibility is that the fusion protein incorporated site at 5f axis is not sufficient to anchor the protrusions, thus those pores will be made available for the translocation (Tang et al., 2008).

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Fig. Schematic diagram of genome of IMNV showing the ORF positions from 5’ to 3’ direction

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Disease diagnosis

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Clinical signs

The graveness of the disease is more prevalent in juveniles and adult stages and the

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cumulative mortality in subadult and adult stages range from 40% to 70% (Poulose et al., 2006). Considerable reduction in market value was reported due to more than 40% mortality during the

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late production cycle and total mortality escalates and reaches up to 70% because of natural infection in aquaculture farms (Nunes et al., 2004; Tang et al., 2005). Severely affected shrimps

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become moribund and show non-specific behavioural signs such as lethargy during or soon after stressful events such as netting, feeding, sudden changes in water temperature, sudden reductions

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in water salinity. These increase in stress level escalates the mortality rate (Lightner, 2011; Lightner et al., 2004a, b; Nunes et al., 2004; Poulos et al., 2006). Temperature plays a crucial

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role in the occurrence of disease and it can onset in both the seasons. The increase in temperature can lead to excessive feeding and thereby production of ammonia above the normal level. These

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condition augmenting the stress factors to shrimps and results in elevated mortalities. Crop loss usually reported from the farms when the culture period crosses 40 days or more (Flegel et al., 2008; Taukhid et al., 2009). The infected shrimps were shown reduced feed intake and it was reported that the feed conversion ratio (FCR) was rose from 1.5 to 4.4 in culture ponds as result the survival rate has fallen to 21% (Tang et al., 2005; Coelho et al., 2009; Loy, 2014). Clinical manifestation is prominent in the acute phase of IMNV infection. Skeletal muscle is the primary site of infection but gills and lymphoid organ can also be affected (Lightner et al., 2004a, b). There are focal to extensive white necrotic areas in striated (skeletal) muscles, mostly in the distal abdominal segments and tail fan. In the chronic stage, lesions are accompanied by 9

ACCEPTED MANUSCRIPT liquefying of the necrotic muscles. The muscles and appendices exhibit a reddish coloration, giving the appearance of cooked shrimps (Nunes et al., 2004). The acute phase of infected animal exhibits coagulative muscle necrosis which progresses to liquefactive necrosis as disease progress to the chronic stage (Lightner, 2004). The first gross sign of muscle necrosis was experimentally observed after 3 days of injection with purified IMNV and complete skeletal

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muscle lesions observed on the 5th day of injection (Poulose et al., 2006).

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Histology

Conventional diagnostic methods like histopathology of the affected tissues are one of the best ways to diagnose IMNV infection in shrimps (Lightner et al., 1996). The striated muscles

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(skeletal and less commonly cardiac muscles), connective tissues, haemocytes and the lymphoid organ tubule parenchyma cells are major targeted organs in the acute phase whereas the infection

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is restricted only to lymphoid organ in the chronic phase (OIE, 2012; Lightner et al., 2004a, b; Poulose et al., 2006). The IMNV infection is characterized by the presence of Lymphoid organ

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spheroid which is 3 to 4 times larger than the normal lymphoid organ tubules, virus-specific darkly basophilic inclusion bodies in the myocyte, haemocyte, and connective tissues (Lightner

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et al., 2004a, b; Poulose et al., 2006). Other manifestations can be observed in the H&E stained sections are coagulative striated muscle necrosis, oedema, haemocyte infiltration, fibrosis

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(Poulose et al., 2006). IMNV can be easily differentiated from Muscle Cramp Syndrome of L. vannamei by the presence of infiltrated haemocytes in the coagulated muscle, which is absent in

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the latter. (Senapin et al., 2011). IMNV will not replicates in the enteric tissues rendered the

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tissues unsuitable for the diagnosis (OIE, 2012).

Nucleic acid-based diagnosis In-situ hybridization along with 993 bp digoxigenin-labeled probe gives a strong signal with inclusion bodies present in the cytoplasm of infected skeletal muscle cells (Tang et al., 2005). To detect dsRNA through ISH the tissue sections were denatured at 84°C for 10 min. Andrade et al., (2008) has modified ISH protocol described by Lightner (1996) for detection of IMNV in infected tissue. He has also evaluated the impact of Davidson’s fixative on ISH. A rapid and sensitive method for definitive diagnosis of the disease was developed using nested 10

ACCEPTED MANUSCRIPT reverse-transcriptase polymerase chain reaction (RT-PCR) by Poulos and Lightner (2006) which has a detection limit of 10 viral genome copies.In order to improve the sensitivity of IMNV detection Senapin et.al., (2007) has developed an alternative, nested RT-PCR detection method with specific primers to target the viral RdRp region instead of the capsid gene targeted by the commercial kit. The two sets of primers were designed to amplify an internal fragment of 282 bp from the 600 bp amplicons. Andrade (2007) has developed a real-time PCR for detection of

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IMNV with primers and probes against an ORF-1 region of viral genome which is more sensitive

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in comparison to nested PCR. Melo et. al. (2014) has developed a diagnostic technique through

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RT-PCR of 372bp Nucleotide sequence corresponding to the viral capsid protein.Puthawibool et. al. (2008) has reported a combined system of RT-LAMP and LFD for detection of IMNV. The

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combined system takes less than 75 min with a sensitivity of detection as comparable to nested RT-PCR detection of IMNV. Lateral flow device reduces amplicon detection time and use of

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carcinogenic ethidium bromide when compared to electrophoresis. Andrade and Lightner (2009) has also developed RT-LAMP-NALF method which is showing equivalent sensitivity to RT-

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LAMP (using three primer pairs) and it is 100 and 10 times more sensitive than one-step RT-

Antibody-based diagnosis

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PCR and RT-LAMP (two primer pairs), respectively.

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In order to develop an an immunodiagnostic method which is faster and cheaper in comparison to molecular methods Polyclonal antibody has developed against IMNV (Melo et.

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al., 2011). The virus purification was done through sucrose gradient centrifugation method which is different from CsCl ultracentrifugation done by Poulos et al. (2006). The specific antibody is

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able to detect IMNV through western blotting. Development of monoclonal antibody is necessary for immunodiagnosis of a disease. There are two mAbs developed by Kunanopparat et. al. (2010) against capsid protein of IMNV which are detecting natural IMNV infections in Litopenaeusvannamei by dot blotting, Western blotting, and immunohistochemistry. The detection sensitivities of the MAbs were approximately 6 – 8fmol/spot of purified recombinant protein. The combination of all three MAbs sensitivity was approximately 10 times lower than that of one-step RT-PCR using the same sample but these are specific to detect IMNV and do not cross-react with other viruses and shrimp tissues. In the same year, Seibert et al. (2010) have developed fourMAbs which are specific to IMNV100 kDa major capsid protein. More than 11

ACCEPTED MANUSCRIPT MAbs, rapid diagnosis immunochromatographic test strips has been developed for IMNV detection (Chaivisuthangkura et al., 2013; Wangman et al., 2016). The sensitivity of immunodiagnosis has been increased by conjugating MAbs with the colloidal gold particle.

Host immune response

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Anti-viral activity of crustacean immune response is a new field of interest. Many types

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of research need to be carried out for understanding the crustacean immune activity in details. Till now what we understand that crustacean immune system means the innate defense activity

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of the animal. It consists of both humoral and cellular immune system. There are a diverse group of pattern recognition receptors in crustaceans. The cellular immune response consists of three

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classes of haemocyte including hyaline cells (HCs), semi-granular haemocytes (SGHs) and granular haemocytes (GHs) Lin and Söderhäll (2011). There are a number of antimicrobial

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peptides present in crustaceans such as penaeidins (Destoumieux et. al., 1997), antilipopolysaccharide factors (ALFs) (Supungul et. al., 2002) and crustins (Bartlett et. al., 2002).

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A study of Vannamei immune system in response to IMNV infection has been done by

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Costa (2009). There is 30% reduction in circulating haemocyte numbers in heavily infected shrimp. The cause of such reduction is not yet clear as there is no evidence of haemopoietic

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tissue infection by the virus. The percentage of apoptotic haemocytes are also very less and this information suggests that haemocytes are not the primary target of the virus. Heavy Infected

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animals have significantly (7%) less granular haemocytes which may be a cause of the reduction in pro-phenol oxidase activity. However, there is an increase in reactive oxygen intermediates. The hemolymph of infected vannamei shows anti-microbial activity against Micrococcus luteus

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and Escherichia coli but not against the marine Vibrio harveyi. Study of immune parameters through conventional methods is not enough and it needs molecular level investigation by realtime PCR. The result of immune response investigation through quantitative RT-PCR of L. vannamei co-infected with IHHNV and IMNV has shown an increased level of HSP-70 expression by IHHNV infection in the gills of double-infected shrimp but not by IMNV infection (Girão et al., 2012). The increased level of HSP in response to viral infection proves it act as an immune modulator and helps in inducing innate immunity in the case of IMNV infection. The expression levels of crustin, penaeidin, and C-type lectin are not influenced by such type of co12

ACCEPTED MANUSCRIPT infection. Significant mortalities were also observed in the co-infected shrimps with IMNV and Vibrio harveyi (Sukenda et al., 2015). The administration of the probiotics SKT-b and the prebiotics oligosaccharide from sweet potatoes (Ipomea batatas L) were upregulated the immune response of the whiteleg shrimp against the IMNV infection (Sepatiani, 2011). The similar results were proved by Oktaviana et al. (2014) by administrating the similar symbiotic, Vibrio alginolyticus SKT-bR and prebiotics from sweet potatoes (I. batatas L) oligosaccharide apart

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from the higher growth performance (Oktaviana et al., 2014).

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Disease control and prevention

Unlike fish, in crustaceans, common vaccination does not work because they have any

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memory in their immune system. Research in the field of vaccination against crustacean viral diseases is mostly RNAi oriented. Immuno-stimulation with sequence specific dsRNA gives

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protection against target viral diseases. There are few research for IMNV protection because most of the researches were carried out for characterization of IMNV. However for the first time

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Loy et al., (2012) has reported that a single, low dose (0.02 mg) of an 81 or 153 bp fragment (dsRNA95-475), with sequence corresponding to putative cleavage protein 1 in ORF1gives

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100% protection against IMNV and were resistant to subsequent infections over 50 days later with a 100-fold greater dose of virus. He has also reported that the dsRNA targeted to structural

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protein and RdRp (dsRNA3764–4805 and dsRNA5518–6391 respectively) is not able to give satisfactory protection unlike WSSV (Robalino et. al., 2004) and YHV (Tirasophon et al., 2005;

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Yodmuang et al., 2006). The mode of vaccination is very important. The common method of shrimp vaccination is through intramuscular injection but Sriyotee Loy (2014) has reported that

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vaccination with dsRNA95-475 (Loy et al., 2012) through reverse gavage can elicit statistically significant (p<0.05) protection against IMNV. It is well known that bacteria and fungi have LPS and β -1,3-glucan on their surface which is recognized as nonself by host immune response. In another way they use to trigger host immune response and provide non-specific immunity. In insects and crustaceans LPS and β-1,3glucans, upon binding to LPS and glucan binding protein, activate proPO, the coagulation cascade, and the genes for antibacterial proteins (Hoffmann et al., 1996; Soderhall et al., 1996; Iwanaga et al., 1994). Neto and Nunes (2015) has reported that addition of 1,000 mg/kg of β13

ACCEPTED MANUSCRIPT 1,3/1,6-glucan which is a polysaccharide extracted and purified from the cellular wall of the baker’s yeast Saccharomyces cerevisiae in a diet for L. vannamei enhances shrimp survival when orally exposed to IMNV without any side effects after a long term exposer. Use of Chitosan which is a glucosamine having chemical structure nearly similar to β-1,3glucan in the feed with treatment dose of 3 ml/kg feed activates Prophenoloxidase (ProPO) of L. vannamei (Nindarwi et al, 2013). Protein interaction analysis using yeast two-hybrid assay revealed that Laminin

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receptor (Lamr) specifically interacted with capsid/envelope proteins of RNA viruses IMNV and

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YHV and immunization with the recombinant-Lamr protein expressed in yeast elicited

immunostimulant against IMNV (Busayarat et al., 2011).

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protection against YHV in lab condition. So there is a future prospect of using rLmar as an

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The basic viral diseases control and preventive strategies like use of Specific Pathogen Free (SPF) and Specific Pathogen Resistant (SPR) animals, biosecurity measures and genetic

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selection process for disease resistant varieties (Moss et al., 2012). The University of Arizona has developed a lab challenge protocol for identification of IMNV resistant L. vannamei family

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lines and subsequent breeding of resistant brood stocks. They have challenged 21 L. vannamei family lines for a minimum of 20 days and the results were shown significant improvement in

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survival rate from 3.2% in the F1 generation to 55.3% in F7 generation (White-Noble et al., 2010). The selective breeding process is currently undertaking so these results have not been

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published in any peer-reviewed publications (Lima et al., 2013). There is 20% mortality in case of experimental infection of L. vannamei but no mortalities were observed in either L. stylirostris

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or P. monodon (Tang et al., 2005). Hence, restocking of ponds with IMNV resistant variety such as P. monodon and P. stylirostris can possibly reduce loss due to mortality. It is believed that

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IMNV supposed to transfer vertically and in such condition disinfection of egg and larvae is necessary. Other general husbandry practices like use of SPF broodstock, biosecurity measures, and proper quarantine condition should be properly implemented.

Conclusion L. vannamei production has a great future for shrimp farmers but the outbreak of infectious diseases may hamper countries export as well as the socio-economic status of farmers and other stakeholders. The present situation of Black tiger shrimp is not responsible for the 14

ACCEPTED MANUSCRIPT single virus but it is due to multiple viral diseases. L. vannamei production in few countries like Thailand is already affected by EMS. So it is time to think about all emerging viral disease whether it is more or less infectious. Well, developed diagnostic procedures and upcoming growth in research on prophylactics constituted strongness to the IMNV research whereas the fundamental studies in virus biology, epidemiology and therapeutics still in infant stages. There should be more research about IMNV to conform its mode of transmission, possible vector, host-

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pathogen interactions and development of preventive measure.

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Plates

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Plate 1. Geographical distribution of Infectious Myonecrosis Virus (IMNV) in worldwide.

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Plate 2. Schematic diagram of genome of IMNV showing the ORF positions from 5’ to 3’ direction

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Tables

1

2

Age of

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Susceptible Species

susceptabilty

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No

Reported

Litopenaeus vannamei

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Table 1. Host range of IMNV

Farfantepenaeus subtilis

Subadult

Study

Other variable

Region

(seasonal/temperature)

State of

Lightneret al.,

Piaui, NE

2004

Brazil 30 ups Salinity, 27-29°C Juveniles

Brazil

temperature, pH 7.8 – 7.9, DO 6.4 – 6.6 ppm

3

Litopenaeus stylirostris

Juveniles

4

Penaeus monodon

Juveniles

Reference

Coelho et al., 2009

Arizona,

Tang et al.,

USA

2005

Arizona,

Tang et al.,

USA

2005

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organism -

(as a vector)

Brazil

temperature, stocking density 20 nauplii/mL

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nauplii

Sl no

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Table 2. Identified strains of IMNV

Strain Name BZ-03

2

GeneBank Accession No

type

AY570982.2

Full

ID-EJ-06

EF061744

Full

3

BZ-04-ZS2011001

KC200075

Partial

4

BZ-07-1

HM030799

Partial

BZ-07-2

HM357803

Partial

ID-LP-11

KJ636782

Full

ID-EJ-12-1

KJ636783

Full

8

ID-EJ-12-2

KJ636784

Partial

9

ID-LP-12-1

KJ636785

Partial

10

ID-EJ-12-3

KJ636786

Partial

11

ID-LP-12-2

KJ636787

Partial

12

ID-BB-12

KJ636788

Partial

6 7

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5

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1

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5

Live feed

Artemiafranciscana

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da Silva et al., 2015

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KJ636789

Partial

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ACCEPTED MANUSCRIPT Highlights  Infectious myonecrosis virus (IMNV) is an emerging penaeid shrimp virus belongs to the family Totiviridae  The virus is endemic to Brazil and Indonesia with a potential chance to

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spread transboundary.

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 World Animal Health Organization (OIE) is listed the IMNV as one of

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major viral pathogen to crustaceans and initiated the active surveillance  The infection is characterized by whitish muscles along the abdomen and tail

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region

 Good Management Practices can prevent the virus and diagnostics are under

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research phase

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