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The complete sequence of the Australia recognizate of Macrobrachium rosenbergiinodavirus which causes white tail disease
Orachun Hayakijkosol, Graham Burgess, Kathy La Fauce, Leigh Owens ⁎School of Veterinary and Biomedical Sciences, James Cook University, Queensland 4811, Australia
⁎ Corresponding author at: School of Veterinary and BUniversity, Townsville, Queensland 4811, Australia.
Article history:Received 28 August 2012Accepted 28 August 2012Available online 4 September 2012
Keywords:White tail disease (WTD)Macrobrachium rosenbergii nodavirus (MrNV)The Australian recognizate of MrNVProtein B2Complete sequence
White tail disease (WTD) caused by Macrobrachium rosenbergii nodavirus (MrNV) has been found in the giantfreshwater prawn (Macrobrachium rosenbergii) and has recently been the cause of high mortalities in manycountries such as India, China, Taiwan and Thailand. In mid 2004, the index case of WTD in Australia presentedin adult broodstockM. rosenbergii from Flinders River in western Queensland. In order to understand the phylo-genetic relationship of the Australian recognizate of MrNV to other MrNV recognizates, the complete sequencesof the AustralianMrNV (RNA1 and RNA2) were determined. Nucleotide and phylogenetic analysis revealed thatthe Australian strain of MrNV (RNA1) was between 94% and 97% identical to Malaysian, the FrenchWest Indies,Chinese and the Thai recognizates. Also, the nucleotide sequence of the Australian MrNV (RNA2) was 92% iden-tical to the French West Indies, Chinese and the Thai recognizates. The nucleotide comparison of protein B2showed a different relationship between MrNV and fish nodaviruses. However, the phylogenetic tree of proteinB2 determined that some fish nodaviruses are closely related to insect nodavirus. These results can be used todevelop effective diagnostic tests and design specific RNA interference (RNAi) against protein B2 to controlother nodaviruses in the future.
The culture of the giant freshwater prawn (Macrobrachiumrosenbergii) began in the early 1960s by Shao Wen Ling (Daisy,2007). M. rosenbergii require brackish water to breed and he suc-cessfully used this information to commercialise the culture ofM. rosenbergii in 1965 (New, 2002).
White tail disease (WTD) or white muscle disease (WMD) is associ-atedwithMacrobrachium rosenbergiinodavirus (MrNV) and extra smallvirus (XSV) (Sahul Hameed et al., 2004). WTD was first reported ina hatchery in Guadeloupe in 1997 (Arcier et al., 1999) and was foundlater in many countries around the world such as Martinique, theFrench West Indies (Arcier et al., 1999), People's Republic of China(Qian et al., 2003), India (Sahul Hameed et al., 2004), Thailand(Yoganandhan et al., 2006), Taiwan (Tung et al., 1999; Wang et al.,2008) and Australia (Owens et al., 2009).
MrNV caused high mortalities and massive economic losses inhatcheries and nursery ponds in many countries including Taiwan(Wang et al., 2008). Clinical signs of white tail disease include whitemuscle in the abdominal area associated with a decline in feeding andswimming behaviour. Mortalities occurred after the first clinical signsand all the population was often lost in less than 1 week (Bonami andSri Widada, 2011). Similarly, Ravi et al. (2009) reported that mortality
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reached 100% after the appearance of whitish muscle. Also, 2 weeksafter postlarvae were transferred to the growing pond, mortality be-tween 5% and 70% was reported by Bonami and Sri Widada (2011).Moreover, clinical signs and mortality patterns are similar in differentcountries and the movement of prawn populations might be onecause of the worldwide distribution of WTD.
MrNV has been placed in the family of Nodaviridae (Alphanodavirus)based on its characteristics and genome sequences (Bonami and SriWidada, 2011; Sahul Hameed et al., 2004). MrNV is a small icosahedralnon-enveloped virus, 26 to 27 nm in diameter. The genome of MrNVis formed by two pieces of positive sense, single stranded RNA(ssRNA): RNA1 (3.2 kb) andRNA2 (1.25 kb), having a single polypeptideof 43 kDa in the capsid (Bonami and Sri Widada, 2011; Romestand andBonami, 2003). MrNV RNA1 contains the coding sequences of twoproteins, protein A and protein B required for MrNV replication anddevelopment in the host cells. Specifically, protein B2 is important forthe intracellular accumulation of viral RNA because it is a sequence-nonspecific binding protein preventing host antiviral immunity (Fenneret al., 2006) via RNA interference.
In Australia, phylogenetic analysis of representatives from 18populations of M. rosenbergii has been reported by De Bruyn et al.(2004). Significant DNA divergence between eastern and westernM. rosenbergii determined thatM. rosenbergiimay actually representtwo distinct phylogenetic species in Australia. The index case ofMrNV described by Owens et al. (2009) was in a lineage II Easternform (De Bruyn et al. 2004) of M. rosenbergii. No sequence data ofMrNV from the Eastern form has been published.
8 RNA2 396R Reverse TGT GCC ATC TAT AACGCT CCC AAA ATTGCG ATA GAC CA
358–396 396
RNA2 954F Forward CCA ACT TTA ACC CCATTG TCG
141–162 954
9 RNA2 954R Reverse CAC CCT GAT AATCGG TCA CT
1075–1095 954
RNA2 535F Forward AAC AAC TAT TCC ATTGAT TG
578–598 535
10 RNA2 535R Reverse AAC AAC ACC CTGATA ATC
1095–1113 535
RNA2 325F Forward CAA GCA AAC TTATAC TCA AGA TAT TACTGG TTT GAA GCC AA
850–891 325
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A number of diagnostic techniques for MrNV have been developedfor MrNV including a sandwich enzyme-linked immunosorbent assay(S-ELISA) (Romestand and Bonami, 2003), dot blot hybridization, insitu hybridization and reverse transcription polymerase chain reaction(RT-PCR) (Hsieh et al., 2006; Yoganandhan et al., 2006). Genome-baseddetection methods such as RT-PCR and real-time RT-PCR have beenused to detect MrNV and were considered a sensitive diagnostic methodin routine health monitoring for white tail disease (Hayakijkosol et al.,2011; Sri Widada et al., 2003). MrNV sequences have been analysed inorder to develop effective diagnostic techniques for the detection ofMrNV infection. However, few complete viral genomes of MrNV havebeen published. In order to analyse the nucleotide sequence similaritiesand amino acid changes of the Australian recognizate of MrNV comparedto other recognizates, the complete sequences ofMrNV (RNA1andRNA2)were analysed in this study. This study aims to determine the firstcomplete sequence of the Australian MrNV (RNA1 and RNA2) includingprotein B2 from the Eastern form of the giant freshwater prawn(M. rosenbergii) in Australia.
2. Materials and methods
2.1. Samples of MrNV
The giant freshwater prawn (M. rosenbergii) was collected fromthe Flinders River system near the Gulf of Carpentaria, Queensland,Australia in 2004.M. rosenbergii infected MrNV as confirmed by histo-pathology and RT-PCR (Owens et al., 2009) became the source mate-rial of the Australian recognizate of MrNV.
2.2. RNA extraction and RT-PCR assay
35 mg of M. rosenbergii muscle (tail) was used for RNA extractionusing SV total RNA isolation system (Promega, Australia) according tothe manufacturer's instructions. RNA samples were used for RT-PCRaccording to Owens et al. (2009). cDNA was produced using ImProm-IIReverse Transcriptase System (Promega, Australia) following themanufacturer's protocol before DNA amplification (Immomix, BiolineAustralia). Specific primers were designed from sequence data in the Na-tional Center for Biotechnology Information (NCBI), GenBankof theMrNV(Table 1). The cycle parameters consisted of incubation at 95 °C for 4 min,followed by 35 cycles at 94 °C for 30 s and 55 °C for 30 s and 72 °C for1 min and 5 min at 72 °C. PCR products were electrophoresed on 0.8%agarose gels and stained with 0.5 μg/ml ethidium bromide. Bands werecut from gels and purified for use in cloning and sequencing.
2.3. Oligonucleotide primers
Oligonucleotide primers were designed by the Oligo programmeversion 7 using all published sequences of MrNV obtained throughthe NCBI (Table 1). Published MrNV sequences have been depositedat GenBank under accession number AY222839, AY231436, FJ379531,DQ146969, DQ459203, DQ459204, DQ459205, DQ459206, DQ459207,DQ459208, DQ521574, JN187416 and FJ751226 for RNA1, andAY222840, AY231437, DQ521575, EU150126, EU150127, EU150128,EU150129, HM565741, GU300102, NC_005095 and FJ751225 for RNA2.The programme selected primer sets with appropriate melting tempera-ture for PCR.
2.4. Cloning and sequencing
PCR products were purified from agarose gels using Wizard® SV Geland PCR Clean-UP system (Promega, Australia) and directly transformedinto Escherichia coli JM109High Efficiency cells using pGEM-T®Easy Vec-tor System (Promega, Australia). 100 μl of transformation reaction wasspread plated onto duplicated Luria Bertani (LB) agar plates containing80 μg/ml 5-bromo-4-chloro-3-indoylgalactoside (X-Gal) and 0.5 mM
isopropyl-D-thiogalactopyranoside (IPTG), 100 μg/ml ampicillin and incu-bated at 37 °C overnight. Three white colonies, putatively containingMrNV insert, were inoculated to universal vials with 15 ml LB brothsupplemented with 100 mg/ml ampicillin and incubated at 37 °Covernight shaking at 150 rpm. Bacteria were pelleted at 4000 rpmfor 5 min using Eppendorf 5804R centrifuge (Eppendorf, Germany) andrecombinant plasmids were extracted from pelleted bacteria usingWizard® Plus SV Minipreps DNA Purification System (Promega,Australia), according the manufacturer's instructions. Four replicates ofplasmids with DNA inserts of MrNV were sent to Macrogen Inc. (Seoul,Korea) for sequencing.
2.5. Sequence analysis
Three forward and three reverse sequences fromMacrogen Inc. wereproduced for each clone and analysed using Sequencher™ software(Gene Codes Corporation, USA). Sequences were aligned and comparedto available sequences using Basic Local Alignment Search Tool (BLAST),through the National Centre for Biotechnology Information (NCBI). Align-ment of nucleotide sequences was performed using GeneDoc softwareversion 2.6.002 developed by Nicholas, Karl B. and Nicholas, Hugh B. in
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1997. Nucleic acids of protein B2 at positions 2725 to 3126 bp of theAustralianMrNV(RNA1)were compared to other protein B2 recognizatesincluding those from MrNV and Penaeus vannamei nodavirus in crusta-ceans; Nodamura virus, Boolarra virus, Flock House Virus (FHV), Blackbeetle virus and Drosophila melanogaster American nodavirus in in-sects; Striped Jack Nervous Necrosis Virus (SJNNV) and Atlantic hali-but nodavirus in fish (Table 2). Phylogenetic trees of nucleotidesequenceswere constructedusingmolecular evolutionary genetics analy-sis (MEGA) software version 5. The evolutionary history was inferredusing the Minimum Evolution (ME) method. The tree is drawn to scale,with branch lengths in the same units as those of the evolutionary dis-tances used to infer the phylogenetic tree. The evolutionary distanceswere computed using the Maximum Composite Likelihood method andare in the units of the number of base substitutions per site. The MEtreewas searched using the Close-Neighbor-Interchange (CNI) algorithmat a search level of 0. The Neighbor-joining algorithm was used togenerate the initial tree.
3. Results
3.1. Complete sequences of the Australian recognizate of MrNV (RNA1and RNA2)
Complete sequences of the Australian recognizate of MrNV (RNA1(3203 bp) and RNA2 (1173 bp))were successfully sequenced (Genbankaccession numbers JN619369 (RNA1) and JN619370 (RNA2)).
3.2. Potential open reading frames (ORF)
Potential ORFs in the Australian recognizate of MrNV were deter-mined by NCBI ORF finder (www.ncbi.nlm.nih.gov/projects/gorf). MrNV
Table 2Comparison of protein B2 (402 bp) of crustacean, insect and fish nodaviruses.
Fromaccessionnumber
Recognizate/year
Description Similarity(%)
1 JN619369 Australia2012
B2 protein (Macrobrachiumrosenbergii nodavirus)
100
2 FJ751226 China22012
B2 protein (M. rosenbergiinodavirus)
98
3 AY222839 French WestIndies 2005
B2 protein (M. rosenbergiinodavirus)
98
4 AY313773 France 2005 B2 protein (M. rosenbergiinodavirus)
98
5 GU300103 India 2011 B2 protein (M. rosenbergiinodavirus)
97
6 AY231436 China1 2006 B2 protein (M. rosenbergiinodavirus)
97
7 JN187416 Malaysia2011
B2 protein (M. rosenbergiinodavirus)
95
8 NC_005094 France 2009 B2 protein (M. rosenbergiinodavirus)
NA
9 NC_014978 Belize 2011 B2 protein (Penaeus vannameinodavirus)
NA
10 NC_003448 Japan 2008 B2 protein (Striped jack nervousnecrosis virus)
NA
11 AB025018 Japan 2003 B2 protein (Striped jack nervousnecrosis virus)
NA
12 AB056571 Japan 2002 B2 protein (Striped jack nervousnecrosis virus)
14 GQ342965 USA 2010 B2 protein (Drosophila melanogasterAmerican nodavirus)
NA
15 NC_002690 USA 2008 B2 protein (Nodamura virus) NA16 M33065 USA 1993 B2 protein (Black beetle virus) NA17 NC_004146 USA 2008 B2 protein (Flock house virus) NA18 JF461541 France 2011 B2 protein (Flock house virus) NA19 NC_004142 Australia 2009 B2 protein (Boolarra virus) NA
(RNA1) sequence contained two overlapping ORFs. The first ORF wasthe largest of the ORFs indentified for MrNV (RNA1). It started at nucleo-tide 24with ATG codon and terminated with TAA codon at position 3140containing 1038 amino acids (3117 bp). This protein is speculated to en-code protein A or an RNA-dependent RNA polymerase. In particular fromamino acid 525 Alanine (A) to 769 Serine (S), the sequence is similar to areverse transcriptase mobile element containing RNA‐dependent poly-merase. The second ORF started at nucleotide 2725 with ATG codon andterminated with TAA codon at position 3126 containing 133 aminoacids (402 bp). This protein is speculated to encode protein B2 whichhas an RNA-binding domain. All small ORFs inMrNV (RNA1) did not con-tain significant sequence homology to available sequences using BLAST.MrNV (RNA2) had one major ORF which started at nucleotide 38 withATG codon and terminated with TAG codon at position 1075 containing345 amino acids (1038 bp)which is speculated to encode the capsid pro-tein. All small ORFs in MrNV (RNA2) had no significant sequence similar-ity to other nodavirus recognizates using BLAST.
3.3. Nucleotide sequence analysis
Nucleotide sequence analysis showed that the identities of theAustralian MrNV (RNA1) were 94%, 95%, 95% and 97% similar toMalaysian (JN187416), the French West Indies (AY222839), Chinese1(AY231436) and Chinese2 (FJ751226) recognizates, respectively. Nu-cleotide sequence analysis showed that the identities of the Australianrecognizate of MrNV (RNA2) had 92% similarity when compared withthe French West Indies (AY222840), Chinese1 (AY231437), Chinese2(FJ751225) and the Thai (EU150126, EU150127, EU150128 andEU150129) recognizates. Nucleotide sequences of the completedAustralian MrNV (RNA1 and RNA2) recognizate were translated intoamino acids to determine amino acid changes against other MrNVrecognizates (Figs. 1 and 2).Many protein changes in the amino acid se-quence of the Australian recognizate of MrNV (RNA1) were observed(Fig. 1), whilst the protein changes in amino acid sequence of MrNV(RNA2) were less extensive (Fig. 2).
3.4. Phylogenetic comparison
TheAustralian recognizate ofMrNV (RNA1) is phylogenetically close-ly related to Chinese2 and the French West Indies recognizates and aregrouped into a separate clade from the Chinese1 and Malaysian MrNV(RNA1) recognizates (Fig. 3). The Australian, Chinese1 and 2 MrNV(RNA2) recognizates are not phylogenetically closely related to otherMrNV recognizates and form separate clades whereas the French WestIndies and Thai (EU150126, EU150127, EU150128 and EU150129)recognizates of MrNV (RNA2) are in the same cluster (Fig. 4).
Phylogenetic analysis protein B2 nodaviruses (317 bp) showed simi-larity between the insect, crustacean and fish nodaviruses and recognisedtwo groups of nodavirus; MrNV/insect/fish nodaviruses and fish/insect/P.vannamei nodaviruses (Fig. 5). Protein B2 of the MrNV group and blackbeetle virus are phylogenetically closely related and are different to fishnodaviruses. However, the incomplete sequence of P. vannameinodavirusfrom Belize is closely related to fish nodavirus at this stage.
4. Discussion
Many countries in Asia have studied and monitored MrNV infectionin the past 10 years. Only one paper from Australia has been publishedon MrNV (Owens et al., 2009) which reported that the Australianrecognizate ismost closely related to the Chinese1 recognizate. However,in this study using the whole of RNA1 revealed that the Australianrecognizate is phylogenetically closely related to Chinese2 and FrenchWest Indies recognizates.
In invertebrates such as insects and crustacea, RNA interference(RNAi) is the primary response against viral infection, whereasvertebrates such as fish use combinations of host immunity such as
Fig. 1. Comparison of the amino acids from the complete sequences (3126 bp) of Macrobrachium rosenbergii nodavirus (RNA1) from white tail disease recognizates. The sequence name obtained from GenBank is given on the left and thenumbering of the deduced amino acid is on the right. FJ751226 from China2 (2011), AY222839 from The French West Indies (2005), AY231436 from China1 (2006) and JN187416 from Malaysia (2011).
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Fig. 2. Comparison of the amino acids from the complete sequences (1170 bp) of Macrobrachium rosenbergii nodavirus (RNA2) from white tail disease recognizates. The sequence name obtained from GenBank is given on the left and thenumbering of the deduced amino acid is on the right. FJ751225 from China2 (2011), AY222840 from the French West Indies (2005), AY231437 from China1 (2006), EU150126 from Thailand (2007), EU150127 from Thailand (2007),EU150128 from Thailand (2007) and EU150129 from Thailand (2007).
Fig. 4. Phylogenetic tree deduced from analysis of 1170 bp nucleotide sequences of complete MrNV (RNA2) of the Australian recognizate of Macrobrachium rosenbergii nodaviruscompared with other nodavirus recognizates. The evolutionary history was inferred using the Minimum Evolution (ME) method. The optimal tree with the sum of branch length=0.15061489 is shown. The analysis involved 8 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing datawere eliminated. There were a total of 1170 positions in the final dataset. Evolutionary analyses were conducted in MEGA programme version 5.
Fig. 3. Phylogenetic tree deduced from analysis of 3126 bp nucleotide sequences of complete MrNV (RNA1) of the Australian recognizate of Macrobrachium rosenbergii nodaviruscompared with other nodavirus recognizates. The evolutionary history was inferred using the Minimum Evolution (ME) method. The optimal tree with the sum of branch length=0.07071728 is shown. The analysis involved 4 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing datawere eliminated. There were a total of 3126 positions in the final dataset. Evolutionary analyses were conducted in MEGA programme version 5.
Fig. 5. Phylogenetic tree of nucleotide (317 bp) of protein B2 of the Australian MrNV compared with fish, insect and other crustacean nodaviruses. The evolutionary history wasinferred using the Minimum Evolution method. The optimal tree with the sum of branch length=14.24306802 is shown. The analysis involved 19 nucleotide sequences. Codonpositions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 317 positions in the final dataset. Evo-lutionary analyses were conducted in MEGA programme version 5.
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cell-mediated immunity and interferon to defend against viral infec-tions (Fenner et al., 2007; Qi et al., 2011). Protein B2 of Alphanodavirusand Betanodavirus is important for the intracellular accumulation ofviral RNA in the cells. Protein B2 is able to block the RNA silencing
mechanism of the viral defence mechanism and enables completion ofviral replication in the cells (Fenner et al., 2006). This study demonstrat-ed different nucleic acids of protein B2 in fish, insect and crustaceannodavirus (Fig. 5). This suggests that sequences of protein B2 from
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different nodaviruses are not similar and hence, RNAi targeting proteinB2 has to be specific for each virus to inhibit the synthesis of proteini.e. a universal RNAi for all nodaviruses might not be achievable.Hayakijkosol and Owens (2012) studied specific RNAi againstprotein B2 of the Australian MrNV using a redclaw crayfish (Cheraxquadricarinatus) model. The results showed that mortalities and histo-pathological lesions decreased significantly. This current study suggeststhat specific RNAi against protein B2 needs to be reanalysed for fish andinsect nodavirus if the technology is to be used to prevent and controlthe diseases.
Based on incomplete data, the sequence of protein B2 from P.vannameinodavirus appears to be an anomaly at this stage. It is really im-perative that this virus be fully sequenced with particular attention tothe area of protein B2. If this preliminary data is correct, then the impli-cation is that the sharing of nodaviruses between insects, fish and crusta-ceans may have happened more than once.
In conclusion, this is the first study involving the complete sequencesof the AustralianMrNV (RNA1 and RNA2) and the phylogenetic relation-ship between existingMrNV recognizates that can be used to form a bet-ter understanding of the true phylogenetic relationship of MrNV. Thisinformation will be used to develop effective diagnostic tools to detectthe Australian recognizate of MrNV. This study suggests that sequence-specific RNAi against protein B2 can be designed and used to controlother nodavirus infections in the future.
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