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omplete nucleic acid sequence of Penaeus stylirostris densovirus (PstDNV)rom India
raveen Rai, Muhammed P. Safeena, Iddya Karunasagar, Indrani Karunasagar ∗
epartment of Fishery Microbiology, Karnataka Veterinary, Animal and Fisheries Sciences University, College of Fisheries, Mangalore 575 002, India
r t i c l e i n f o
rticle history:eceived 16 November 2010eceived in revised form 26 February 2011ccepted 7 March 2011
a b s t r a c t
Infectious hypodermal and hematopoietic necrosis virus (IHHNV) of shrimp, recently been classified asPenaeus stylirostris densovirus (PstDNV). The complete nucleic acid sequence of PstDNV from India wasobtained by cloning and sequencing of different DNA fragment of the virus. The genome organisation ofPstDNV revealed that there were three major coding domains: a left ORF (NS1) of 2001 bp, a mid ORF
(NS2) of 1092 bp and a right ORF (VP) of 990 bp. The complete genome and amino acid sequences of threeproteins viz., NS1, NS2 and VP were compared with the genomes of the virus reported from Hawaii, Chinaand Mexico and with partial sequence available from isolates from different regions. The phylogeneticanalysis of shrimp, insect and vertebrate parvovirus sequences showed that the Indian PstDNV isolateis phylogenetically more closely related to one of the three isolates from Taiwan (AY355307), and two
Y102
enomehylogenetic analysis
isolates (AY362547 and A
. Introduction
Penaeus stylirostris densovirus (PstDNV) (Tattersall et al.,005)/Infectious hypodermal and hematopoietic necrosis virusIHHNV) is one of the major viral pathogens of penaeid shrimpsorldwide (Lightner and Redman, 1998). PstDNV was first reported
n blue shrimp, Penaeus (Litopenaeus) stylirostris cultured in Hawaiin the early 1980s (Lightner et al., 1983a, 1983b). It causes aisease condition called runt deformity syndrome (RDS), charac-erized by reduced growth, deformities of cuticle and rostrum intocks of P. (Litopenaeus) vannamei and Penaeus monodon (Bell andightner, 1984; Kalagayan et al., 1991; Primavera and Quinitio,000). PstDNV has been reported to be present in P. monodon fromaiwan, Thailand and Philippines (Flegel, 2006). In India, we haveecently presented molecular evidence of the presence of PstDNVssociated with slow growth in P. monodon (Rai et al., 2009a).
PstDNV is a small typical Densovirus (Family: Parvoviridae,ub-family: Densovirinae), with nonenveloped, icosahedral, virionsveraging 22–23 nm in diameter and containing linear single-tranded DNA of 4.1 kb in length. Genome of PstDNV is comprised
f 3 large open reading frames, ORF1, 2 and 3 (Bonami et al., 1990;ari et al., 1993; Shike et al., 2000).To date only two complete sequence of genomes of PstDNV
re available: from Hawaii (GenBank Accession No. AF218266;
3909 bp) and China (GenBank Accession No. EF633688; 3833 bp). Inaddition to these partial sequences are also available from Mexico(GenBank Accession No. AF273215; 3873 bp), Taiwan (GenBankAccession Nos. AY355307, AY355306, and AY355308), Thailand(GenBank Accession Nos. AY362547, and AY102034), Ecuador(GenBank Accession No. AY362548) and Australia (GenBank Acces-sion No. GQ475529). In addition to infectious PstDNV, virus-relatedsequences (non-infectious PstDNV) are integrated into the genomeof P. monodon in some parts of the world and has been reportedso far from Africa, Australia, Madagascar and India (Tang et al.,2003; Krabsetsve et al., 2004; Tang and Lightner, 2006; Rai et al.,2009b).
Complete genome information would be useful for assessing thespecificity of probes/primers to viruses from different geographi-cal areas. Comparisons of complete genome sequences will help usto gain insights into point mutations that can affect virulence ofthe virus. We sequenced the entire genome of the PstDNV fromP. monodon in India. The genome organization of Indian isolateof PstDNV was compared with that of other parvoviral sequences,shrimp parvoviruses in particular.
2. Materials and methods
2.1. Sample collection
Samples of PstDNV-infected P. monodon were collected fromhatcheries and culture systems from west coast of India and keptfrozen at −80 ◦C in the Fishery Microbiology Lab at College of Fish-eries, Mangalore. The PstDNV sequenced in the present study was
rom a single individual shrimp collected from west coast of Indiahich had a severe infection.
.2. Extraction of DNA
Approximately 50 mg of pleopods were taken separately fromach sample and viral DNA was extracted according to the protocolescribed by Otta et al. (2003). The precipitated DNA was dried ande-suspended in 1× TE buffer and stored at −20 ◦C till further use.
.3. Diagnostic PCR for PstDNV
The samples were initially screened for PstDNV infection byested PCR using IHHNV648F/R as the first step primers (Rai et al.,009a) and IHHNV309F/R as second step primers (Tang et al., 2007).CR was carried out in a 30 �l reaction mixture containing 1×ssay buffer with 1.5 mM MgCl2, 200 �M of each of the 4 dNTPs,U of Taq polymerase (Bangalore GeNei, Bangalore) and 10 pmolf each primers. PCR was performed using a DNA Engine DYADMJ Research, USA) thermal cycler. The amplified PCR productsesolved by agarose gel electrophoresis on 2% agarose gel, stainedith ethidium bromide (0.5 �g/ml) and photographed using a gelocumentation system (HeroLab). These primers yielded a productf 648 bp and 309 bp, respectively.
In the present study we designed different primer sets tomplify nonstructural protein 1 (NS-1), nonstructural protein
(NS-2) and structural protein (VP) genes of PstDNV and theon coding regions, based on the complete genome sequencef PstDNV from Hawaii (GenBank Accession No. AF218266).he primers were designed by using primer3 online soft-are (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3 www.cgi)
nd custom synthesized by Bioserve Biotechnologies (Hyderabad,ndia). The primer sequences and other details are given in theable 1.
.4. Cloning and sequencing of Indian PstDNV
The amplicons of genes and non coding regions were purifiedith a QIAquick PCR purification kit (Qiagen, Germany), and cloned
nto a pSC-A cloning vector using a stratacloneTM PCR cloning kitStratagene, USA) according to manufacturer’s instructions. Thehite colonies were further confirmed by PCR and for each PCR
mplicon at least three clones having the desired DNA fragments
ere sequenced with an automated ABI 3100 Genetic analyzersing fluorescent label dye terminators (M/s Bangalore Genei, Ban-alore).
The sequences analysis of PstDNV obtained was analyzedy using different computer based programmes. The Indian
h 158 (2011) 37–45
PstDNV sequence was compared with shrimp and insect parvovi-ral sequences available in the GenBank database. Comparativeanalysis of Indian strain of PstDNV sequences was performedusing Basic Local Alignment Search Tool (BLAST) (Altschul et al.,1997) of the National Center for Biotechnology Information(NCBI). The presence of open reading frame (ORF) was determinedby ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html).The amino acids deduced from the DNA sequences wereobtained through a web based java program, Molecular ToolKit (www.vivo.colostate.edu/molkit). Multiple sequence align-ment of PstDNV sequences was done by the ClustalW softwarepackage (version 1.8) (Thompson et al., 1994).
A phylogenetic tree was constructed by using aligned entiregenomes and partial genome sequences of shrimp and insect par-voviruses (Saitou and Nei, 1987). The tree was constructed by theneighbor-joining algorithm based on calculations from pair wisenucleic acid sequence distances by using Molecular EvolutionaryGenetics Analysis (MEGA) program (Version 4.1 Beta 2) (Kumaret al., 2008). The horizontal branch lengths are proportional tothe genetic distance, and numbers shown at branch point indicate1000 bootstrap values which determined the confidence indiceswithin the tree. Sequences of shrimp and insect parvovirusesfor comparisons were obtained from the GenBank database:Indian PstDNV (GQ411199), Hawiian PstDNV (AF218266), Mex-ican PstDNV (AF273215), Taiwanese PstDNV (AY355307), ThaiPstDNV (AY362547), Thai PstDNV (AY102034), Ecuadorian PstDNV(AY362548), Chinese PstDNV (EF633688), Taiwanese PstDNV(AY355306), Taiwanese PstDNV (AY355308), Australian PstDNV(GQ475529), Australian PstDNV (EU675312), Madagascan TypeA (DQ228358), East African Type B (AY124937), Indian PmDNV(Safeena et al., 2010; FJ410797), PmergDNV (La Fauce et al.,2007; DQ458781), AalDNV (X74945), AaeDNV (M37899), SMV(AF499102).
3. Result
3.1. Amplification and Nucleotide sequence analysis of PstDNV
The complete sequence of PstDNV was assembled from thesequences of overlapping amplicons. The total genome of PstDNVwas composed of 3908 nts (Fig. 1) and had a base compositionof 36.4% A, 20.6% T, 23.5% C and 19.4% G. Thus, the G + C contentand A + T content of the genome is 42.9% and 57.1%, respectively.The genome sequence has been deposited in GenBank with theaccession no. GQ411199. The Indian PstDNV sequence from P. mon-odon shared highest similarity of 94% to the complete genomesequence of Hawaiian PstDNV (3909 bp) (GenBank Accession No.AF218266) whereas it showed 93.8% with the Chinese isolate (Gen-Bank Accession No. EF633688). Nucleic acid similarity of IndianPstDNV sequence was compared with other partial sequencesreported so far are given in Table 2. Comparison of Indian PstDNVisolate with non-infectious PstDNV gave 86.0% similarity withType A sequence (GenBank Accession No. DQ228358) and 91.7%with Type B sequence from East Africa (GenBank Accession No.AY124937).
3.2. Coding domains
The overall organization and size of the coding sequences ofthe Indian PstDNV genome are very similar to those describedfrom Hawaii (GenBank Accession No. AF218266) and Mexico (Gen-
Bank Accession No. AF273215). Nucleotide sequence analysis ofthe Indian PstDNV genome revealed that there were three majorcoding domains: a left ORF (NS1) of 2001 bp, a mid ORF (NS2) of1092 bp and a right ORF (VP) of 990 bp (Fig. 2). The right and mid-dle ORFs are in the same reading frame where as the left ORF (NS1)
P. Rai et al. / Virus Research 158 (2011) 37–45 39
Fig. 1. Nucleotide sequence of the Indian Penaeus stylirostris densovirus (PstDNV) genome. Amino acid sequence of putative polypeptides corresponding to major ORFsis shown above the sequence. The putative transcription start ATG codons, stop codons and polyadenylation signals (AATAAA) are underlined, the putative initiation oftranscription (Inr, C/A) is indicated by an ‘→’. The nucleotide sequence of P2 and P61 promoter regions of PstDNV indicating the putative TATA-box, activator element, A-richregion, and downstream promoter element (DPE). Hairpin sequences are underlined with arrowhead.
40 P. Rai et al. / Virus Research 158 (2011) 37–45
(Conti
ioat
Fig. 1.
s in a different reading frame (Fig. 2). Similar to other two strainsf PstDNV, Indian PstDNV genome also contained non coding 5′
nd 3′ terminal sequences of 591 and 329 nt in length, respec-ively.
nued ).
3.3. Open reading frame 1 (ORF1)
The left ORF comprising of about 50% of the genome codesfor non-structural protein 1 (NS1) of PstDNV starts at nt
P. Rai et al. / Virus Research 158 (2011) 37–45 41
(Conti
6emi
Fig. 1.
48 and terminates with a TAA codon at 2648 nt. This ORFncodes a protein of 666 amino acids, corresponding to aolecular weight of 75.77 kDa. The nucleotide sequence sim-
larity of ORF1 (NS1) of PstDNV from India with all other
nued ).
sequences of ORF1 available in the GenBank are given inTable 2.
The ORF1 protein sequence contained highly conserved repli-cation initiator motifs (rolling-circle replication (RCR) motifs) and
42 P. Rai et al. / Virus Research 158 (2011) 37–45
Table 2Comparison of nucleotide and predicted amino acid sequences of PstDNV isolated from India with other PstDNV (The similarity was estimated based on the full lengthsequences of respective ORFs or partial sequences available).
Country/isolate Host species GenBank Acc. nos. Complete genome(% identity)
Hawaiia P. vannamei AF218266 94.0 96.20 97.06 94.14 91.89 95.86 96.35Mexico P. stylirostris AF273215 93.7 96.30 97.16 93.93 91.89 95.86 96.93Chinaa P. monodon EF633688 93.8 96.05 96.88 94.14 91.59 95.59 96.93Taiwan B P. monodon AY355307 94.8 98.50 99.54 93.43 94.89 98.89 96.65Thailand P. monodon AY102034 96.3 98.50 99.54 93.53 94.89 98.89 96.65Thailand P. monodon AY362547 94.2 97.75 98.90 93.63 93.99 98.35 96.65Taiwan A P. monodon AY355306 94.0 96.25 96.97 93 91.74 95.59 97.93Taiwan C P. monodon AY355308 93.8 96.25 96.97 94.24 91.74 95.59 96.65Australia P. monodon GQ475529 92.7 94.60 – 94.24 91.74 – 96.96Madagascar P. monodon DQ228358 86.0 87.75 – 86.56 79.57 – 92.40Tanzania P. monodon AY124937 91.7
aComplete genomes of PstDNV.
Ftt
Np4Bspn
owathatf2To(
3
nteTonio
ig. 2. Genomic organization of coding sequences on the plus and minus strands ofhe PstDNV genome. ORF2 (Mid ORF), ORF1 (Left ORF) and ORF3 (Right ORF) are inhe plus strand.
TP-binding and helicase domains (ATPase motifs) common to allarvoviruses which is located between aa 257 and 315 and aa80 and 579 (Fig. 3A and B), respectively (Afanasiev et al., 1991;oublik et al., 1994; Shike et al., 2000). The deduced amino acidequence identities of NS1 of PstDNV obtained from India was com-ared with all other PstDNV amino acid sequences for the putativeon-structural protein 1 (NS1).
As shown in Table 2, the non-structural protein 1 (NS1) sequencef the Indian PstDNV isolate showed 34 amino acid substitutionshen compared to the more closely related Taiwan (AY355307)
nd Thailand (AY102034) isolates. Though the replication initia-or motifs and the NTP-binding/helicase domains in NS1 protein isighly conserved among all PstDNV isolates, high degree of aminocid variation was observed in the NS1 protein when compared tohe other proteins. In case of Indian PstDNV NS1 gene, we observedrame-shift because insertion of an Adinine nucleotide at position491 and subsequent deletion of the Adinine at position 2573.his alone resulted in 25 amino acid substitutions in this regionf NS1 protein compared to the Taiwan (AY355307) and ThailandAY102034) isolates.
.4. Open reading frame 2 (ORF2)
The mid ORF of Indian strain of PstDNV consisted of 1092 bpucleotide sequences. ORF2 starts with ATG codon at nt 591 anderminates with a TAG codon at 1681 (Fig. 1). This ORF wouldncode a protein of 363 aa with a molecular mass of 42.11 kDa.
he nucleotide sequence and deduced amino acid sequence of NS2f Indian PstDNV obtained was compared with all other PstDNVon-structural protein 2 (NS2) available in the GenBank are shown
n Table 2. The multiple alignment of amino acid sequences of ORF2f Indian PstDNV isolate showed 3 amino acid substitutions com-
92.55 – 90.30 86.93 – 95.74
pared to the closest relatives from Thailand (AY102034) and Taiwan(AY355307) at 36th, 107th and 227th positions.
3.5. Open reading frame 3 (ORF3)
The ORF3 is the smallest (990 bp) among the three ORFs inthe Indian PstDNV genome which starts with ATG at nt 2590,and terminates with the TAA codon at nt 3577 (Fig. 1). This ORFencodes a protein of 329 aa with a molecular mass of 37.48 kDa. Thenucleotide and deduced amino acid sequence of structural proteinof Indian PstDNV isolate were compared with the other sequencesof structural protein of PstDNV available in the GenBank are shownin Table 2. The multiple alignment of amino acid sequences of ORF3of the Indian PstDNV isolate showed 11 amino acid substitutionscompared to its closest relatives (Taiwan; AY355307 and Thailand;AY102034).
3.6. Phylogenetic analysis
The phylogenetic tree was constructed based on the completeand partial genome sequences of shrimp and insect parvoviralsequences (Fig. 4). The tree generated was segregated into 3 mainlineages with the spawner isolated mortality virus (SMV) (Gen-Bank M37899) as an out-group. Lineage 1 included all strains ofP. stylirostris densovirus (PstDNV), where as lineage 2 includedIndian PmDNV, PmergDNV and the insect parvovirus Aedes aegyptidensovirus (AaeDNV) and Aedes albopictus densovirus (AalDNV).The Indian PstDNV isolate is phylogenetically more closely relatedto one of the three isolates of Taiwan (AY355307) and two Thailand(AY362547, and AY102034) isolates while the isolates from theAmericas (AY362548, AF218266, and AF273215), China (EF633688)and two isolates from Taiwan (AY355308, AY355306) clusteredtogether. The tree also showed that the infectious PstDNV strainreported from Australia (GQ475529) clustered with non infectiousPstDNV i.e. Type A from Australia (EU675312) and Madagascar(DQ228358) and Type B from East Africa (AY124937).
4. Discussion
Though PstDNV is widely distributed in different parts of theworld, complete nucleotide sequences is available only for the virus
from Hawaii (3909 bp) and China (3833 bp). Only partial nucleotidesequence is available for viruses from other regions. Here we reportthe complete genome of Indian strain of PstDNV from P. monodon.The genome is 3908 nt long and it is the first strain of PstDNV fromSouth Asia to be sequenced completely.
P. Rai et al. / Virus Research 158 (2011) 37–45 43
Fig. 3. (A) The most-conserved rolling-circle replication (RCR) motifs/replication initiator motifs I and II in the non structural protein 1 of vertebrate and invertebrateparvoviruses between members of the subfamilies Parvovirinae and Densovirinae. Consensus sequences are indicated in red colour. Numbers in parentheses indicate thep n nump familit icatedv ts.
Is(ABncAoistt(A
osition of sequences for each viral protein shown in the figure. GenBank accessiorotein 1 of vertebrate and invertebrate parvoviruses between members of the subhe NTP-binding and helicase domains/ATPase motifs. Consensus sequences are indiral protein shown in the figure. GenBank accession numbers are shown in bracke
The sequence analysis of PstDNV from India revealed that thendian PstDNV sequence shared 93.3–96.3% similarity with theequence of infectious PstDNV isolates from Asia, the AmericasGenBank Accession Nos. AF218266, AF273215, and AY362548),ustralia (GenBank Accession No. GQ475529) and China (Gen-ank Accession No. EF633688) but only 86.0–87.0% with theon infectious Type A PstDNV sequence reported from Madgas-ar (GenBank Accession No. DQ228358) and Australia (GenBankccession No. EU675312). The nucleotide sequence comparisonf Indian PstDNV showed higher identity of 91.7% to the nonnfectious Type B sequence from East Africa (GenBank Acces-
ion No. AY124937). The phylogenetic analysis suggests thathe Indian PstDNV isolate is more closely related to one of thehree isolates of Taiwan-AY355307 and two Thailand isolatesGenBank Accession Nos. AY362547, and AY102034) than themericas.
bers are shown in brackets. (B) The most-conserved motifs in the nonstructurales Parvovirinae and Densovirinae. Conserved sequence motifs in viral proteins arein red colour. Numbers in parentheses indicate the position of sequences for each
Despite minor differences, the genomes of several vertebrateand invertebrate parvoviruses exhibit similar organization. PstDNVappears to have low genetic diversity than other shrimp viruses.Geographic isolation has allowed the virus to evolve into two dis-tinct groups in Taiwan (Hsia et al., 2003). Though several PCRprimers have been described for detection of PstDNV (Nunan et al.,2000; Tang et al., 2000, 2003, 2007; Krabsetsve et al., 2004) virusesin regions are not detected by the available primers because ofthe presence multiple geographic variants of PstDNV (Tang andLightner, 2002; Tang et al., 2003). Recently in India the simulta-neous occurrence of both infectious PstDNV and integrated Type
A virus-related sequences was reported in 22.8% PL and 10.5%adults of P. monodon (Rai et al., 2009b). Hence understanding offull genome sequence of PstDNV from India is necessary to developnew control strategies for prevention of viral diseases in cultivatedshrimp.
44 P. Rai et al. / Virus Researc
Fig. 4. Phylogenetic tree generated from aligned shrimp and insect parvovirussequences available in GenBank. Aligned sequences consisted of both entiregnoi
PgstTtTcinstbMimhkraSVatotcetN
nmsc1a
enomes and fragments of sequenced genomes. The phylogeny was estimated byeighbor-joining method using MEGA program. Numbers indicate the percentagesf bootstrap support from 1000 replicates. Note: Country names (official short namesn English) as given by International Organization for Standardization (ISO).
The over all organisation and size of the coding sequences ofstDNV from India is very similar to those described from othereographical isolates. Like all other reports of PstDNV the presenttudy also shows that gene encoding for the nonstructural pro-ein 1 of Indian PstDNV is the largest among the three major ORFs.he nucleotide sequence of NS1 of Indian PstDNV isolate sharedhe highest nucleotide similarity (98.45%) with the Taiwan andhailand isolates but it showed 34 amino acid substitutions. Inase of Indian PstDNV NS1 gene, we observed frame-shift becausensertion of Adinine at 2491 and subsequent deletion of an Adi-ine at 2573rd position which alone resulted in 25 amino acidubstitutions in the C terminal region of the protein comparedo other part of NS1. There is no report on apparent associationetween clinical outcomes and particular amino acid substitutions.oreover this frame shift mutation does not affect any change
n the two highly conserved motifs, i.e. the replication initiatorotif/rolling-circle replication (RCR) motif and NTP-binding and
elicase (ATPase) domains at the N-terminal region which arenown to be involved in initiation and termination of rolling circleeplication of all parvoviruses (Iversen and Rhode, 1990; Kooninnd Ilyina, 1993; Nuesch et al., 1995; Bergoin and Tijssen, 1998;hike et al., 2000). Mutations in the NTP-binding motif of Minuteirus of Mice (MVM) NS-1 protein showed that it uncouples ATPasend DNA helicase activities (Jindal et al., 1994). In addition tohe putative NTP-binding domain, the N-terminus NS1 protein ofther parvoviruses contain an amino acid sequence with a dis-ribution of cysteine and histidine residues which resembles theonsensus sequence for metal-binding domains (Berg, 1986; Tsonist al., 1988). However, we have not observed such sequence dis-ribution represents a functional metal-binding domain in PstDNVS-1.
The mid ORF of Indian strain of PstDNV consisted of 1092 bpucleotide sequences and would encode a protein of 363 aa with aolecular mass of 42.11 kDa. The multiple alignment of amino acid
equences of ORF2 of Indian PstDNV isolate showed that the highlyonserved amino acids valine, tryptophan and aspartic acid at 36th,07th and 227th positions were substituted by alanine, argininend asparagine, respectively. Like all other PstDNV sequences, the
h 158 (2011) 37–45
Indian NS2 protein sequence has also got glutamic acid at 185thposition where as it is replaced by lysine in case of its closest rela-tive from Thailand. In addition to this Indian and Thailand has gotglutamic acid at position 190 instead of aspartic acid compared toall other strains of PstDNV. Predicted gene product of this proteinhas not shown any similarity with entries in the protein databaseshence the function of this protein is unknown.
The ORF3 is the smallest among the three ORFs in the IndianPstDNV genome with a nucleic acid sequence of 990 bp whichencode a protein of 329 aa with a molecular mass of 37.48 kDa.Among the three ORFs the highest nucleotide variation was foundin the region of right ORF, which encodes the major capsid pro-tein. By the analysis of PstDNV capsid protein gene sequences from89 penaeid shrimp, along with 14 previously published sequencesshowed a calculated mean mutation rate of 1.39 × 10−4 substitu-tions/site/year for PstDNV (Robles-Sikisaka et al., 2010).
Even though there are a lot of diagnostic techniques for thedetection of PstDNV, molecular technique like PCR was failed todetect different geographical specimens of PstDNV (Tang et al.,2003). The DNA probe developed from Hawaiian PstDNV did notreact with PstDNV like lesions described in shrimp from Australia(Owens et al., 1992; Owens, 1997). It has been already reportedthat the samples positive for PstDNV from Australia and Africa wereactually contained integrated sequences (Tang and Lightner, 2006).From India integrated Type A virus-related sequences was reportedin PL and adults of P. monodon (Rai et al., 2009b). Though the com-plete genome of PstDNV has been sequenced from different partof the world there was no information of genome organisationPstDNV from India. Complete genome sequence, allows compar-isons between genomes to identify any insertions or deletions thatare undoubtedly important in the different phenotypes of strains.A complete analysis of different genome sequence of PstDNV maylead to the development of new control strategies for preventionof viral diseases in cultivated shrimp.
Acknowledgements
The authors are kindly acknowledging the fund provided byDepartment of Biotechnology, Govt. of India, through COE in “Pro-gramme Support for Aquaculture and Marine Biotechnology”.
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