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Virus Research
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Laboratrio de Biologia Molecular Aplicada LAPLIC, Departamento de Bioqumica, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil
Laboratrio de Glicobiologia Molecular, Departamento de Bioqumica, Universidade Federal do Rio Grande do Norte, Natal, Brazil
c
Escola Agrcola de Jundia, Universidade Federal do Rio Grande do Norte, Brazil
d
Programa de Ps-Graduaco em Bioqumica, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil
b
a r t i c l e
i n f o
Article history:
Received 15 January 2014
Received in revised form 7 May 2014
Accepted 11 May 2014
Available online 24 May 2014
Keywords:
Shrimp nanism
White Spot Syndrome
Penaeus stitlirostris densovirus
Brevidensovirus
a b s t r a c t
A 3739 nucleotide fragment of Infectious hypodermal and hematopoietic necrosis virus (IHHNV) from
Brazil was amplied and sequenced. This fragment contains the entire coding sequences of viral proteins, the full 3 untranslated region (3 UTR) and a partial sequence of 5 untranslated region (5 UTR). The
genome organization of IHHNV revealed the three typical major coding domains: a left ORF1 of 2001 bp
that codes NS1, a left ORF2 (NS2) of 1091 bp that codes NS2 and a right ORF3 of 990 bp that codes VP.
Nucleotide and amino acid sequences of the three viral proteins were compared with putative amino
acid sequences of viruses reported from different regions. Comparisons among genomes from different geographic locations reveal 31 nucleotide regions that are 100% similar, distributed throughout the
genome. An analysis of secondary structure of UTR regions, revealed regions with high probability to
form hairpins, that may be involved in mechanisms of viral replication. Additionally, a maximum likelihood analysis indicates that Brazilian IHHNV belongs to lineage III, in the infectious IHHNV group, and is
clustered with IHHNV isolates from Hawaii, China, Taiwan, Vietnam and South Korea. A new nested PCR
targeting conserved nucleotide regions is proposed to detect IHHNV.
2014 Elsevier B.V. All rights reserved.
1. Introduction
Infectious hypodermal and hematopoietic necrosis virus
(IHHNV) is one of the viral pathogens of penaeid shrimps most
recurrent in the world (Lightner, 2011; Vega-Heredia et al., 2012).
The disease IHHN and its causative agent, IHHNV, were rst
described in the early 1980s, as the cause of acute epizootics and
mass mortality in blue shrimp (Litopenaeus stylirostris) farmed in
super intensive raceway systems in Hawaii, causing acute epizootics and mass mortalities (Brock et al., 1983; Lightner, 1983,
1988). After its discovery, IHHNV was found to be widely distributed in cultured shrimp in North, South, and Central America,
Caribbean and the Indo-Pacic (Lightner, 2011; Vega-Heredia et al.,
137
Table 1
Primers designed in this study to IHHNV genome amplication.
Primer name
Amplicon size
Tm ( C)
PF1
PR1
PF2
PR2
PF3
PR3
PF4
PR4
PF5
PR5
PF6
PR6
PUTF1
PUTR1
PUTF2
PUTR2
ATGTCAACGGACAGTGTCAACACTG
AATAAAGTCGGGTTGTGTTCCGCTA
GAAATAAAAGAACCAGAAACTCCA
TCATATATGGACATGGTCCGTCTA
ATATCAAGAGATGGATACTCTATCT
TAAGAGATTTTCCTGTTCCTGT
CCAACAACGACATCCGTGTACCAG
TCGTCTTCATTATGTGCATCCCTCC
ATCACCAGCGACGACTTCCTAGG
CCAAATTGTTGGATTGGGTCTTCAT
AAGGATACTACTGGACTACATAATC
GGAAGAAGTCGGCTTGTACTCT
GACGAGTGAAGAGGCTATTCCAAGT
CTTGGAGAAATTCCCTGGCTGGAGT
TTGGAAACCTAGTCTACCCAGC
CAGAAACCGTTAACTTAATATGTGA
662 pb
63.63
62.76
55.55
58.42
53.95
54.73
63.88
62.70
63.90
60.28
55.23
59.77
62.35
64.56
59.43
55.68
606 pb
588 pb
638 pb
663 pb
415 pb
594 pb
574 pb
designed for this study are described in Table 1. The PCR was carried
out in a 20 L reaction, containing 1 g of total extracted DNA,
5 pmol of each primer, 4 mM MgCl2 , 0.5 U of Tth DNA pol (Biotools),
and 200 M of each dNTP. Amplication was performed using a
Life Touch Thermal Cycler TC-96 (BIOER) and the following cycling
parameters: initial denaturation at 94 C for 3 min, followed by 40
cycles of 94 C for 1 min, 55 C for 1 min, and 72 C for 2 min, and
a nal extension at 72 C for 5 min. After amplication, aliquots of
the PCR products were analyzed in a 1.0 or 1.5% agarose gels stained
with ethidium bromide and then photographed.
The genome sequencing was performed from the amplicons
generated using primers described in Table 1 and the following cycle parameters: at 94 C for 3 min, followed by 40 cycles
of 94 C for 1 min, 59 C for 1 min, and 72 C for 2 min, and a
nal extension at 72 C for 5 min. The amplicons of coding and
noncoding regions were puried and submitted to sequencing
using the Applied Biosystems 3500 Genetic Analyzer platform, according to manufacturers specications. All amplicons
were sequenced at least twice (forward and reverse), assembled using basecall quality values and validated using pre-dened
parameters of Geneious software (Geneious version 7.0 Biomatters. Available from http://www.geneious.com/) and careful visual
inspection.
138
EU675312). Additional nucleic acid similarity values of Brazilian IHHNV sequence with other partial and complete genome
sequences reported so far are detailed in Table 2. The sequence
of Brazilian IHHNV is highly similar (>99.2%) to sequences that
were reported as members of an infectious group, including isolates
from Taiwan (AY355306), Ecuador AY362548, China (EF633688
and JX258653), Mxico (AF273215) and South Korea (JN377975)
(Lightner, 2011; Kim et al., 2011). A comparison of Brazilian IHHNV
isolate with non-infectious IHHNV gave 86.0% similarity with
Type A sequence (GenBank Accession No. DQ228358) and 91.3%
with Type B sequence from East Africa (GenBank Accession No.
AY124937).
The prediction of coding regions of Brazilian IHHNV reveals the
three typical major coding domains identied in other IHHNV isolates: the middle ORF1 that codes the non structural protein 1 (NS1)
of 2001 nt, a left ORF2 that codes the nonstructural protein 2 (NS2)
of 1092 nt and a right ORF3 that codes de capsid protein of IHHNV
(VP) of 990 nt. ORFs and its predicted amino acid sequences are
represented in Fig. 1. The ORF2 and ORF3 are in the same reading
frame and the ORF1 starts 57 nt after the rst nucleotide of ORF2, in
a different reading frame. The organization of the Brazilian IHHNV
genome is schematized in Fig. 2, which shows the 5 and 3 UTR
regions and the coding regions composed by the three typical ORFs.
The 162 nt fragment belonging to 5 UTR which was not sequenced
in this study is represented by a square with dotted line.
A search of nucleotide conserved regions in IHHNV genome was
performed in order to identify possible sites of genomic stability,
which are often associated with viral infection and replication processes in different viruses (Wang et al., 2009; Kraft et al., 2013;
Pollom et al., 2013). These regions are also ideal to PCR targeting especially in cases such as IHHNV which has a large number
of variants. An alignment of the distinct IHHNV sequences available in GenBank (Table 2), including Brazilian isolate, reveals 31
regions of at least 14 nt in length, that are 100% conserved in all
viruses (Fig. 1). The rst conserved region from the 5 end of IHHNV
genome was named as conserved region 1 (CR1) and the subsequent regions identied were numbered accordingly (Fig. 1). Only
3 of the 31 identied conserved regions are outside coding regions.
It is interesting to note that some conserved regions correspond to
conserved domains of viral proteins and regulatory regions in the
genome, while others are in regions that do not encode structured
protein domains. These points are discussed in the following topics.
3.3. ORF1 and non structural protein 1 (NS1)
The ORF1 starts at nucleotide 488 and terminates at nucleotide
2488 at a TAA codon. It encodes NS1 that contains 666 amino acids
and a molecular weight of 75,765 kDa. The nucleotide sequence
similarity of ORF1 of IHHNV from Brazil comparing it with ORF1
sequences available in the GenBank are given in Table 2. As previously observed in virus from other geographic locations, the
Brazilian IHHNV NS1 contains the replication initiator motif common to all parvoviruses located between aa 258 and 315, and a
stretch of conserved aa sequence characteristic of the NTP-binding
and helicase domains, shared by all parvoviruses located between
aa 480 and 579 (Afanasiev et al., 1991; Boublik et al., 1994; Shike
et al., 2000; Rai et al., 2011). The replication initiator motifs and the
NTP-binding/helicase domains in Brazilian IHHNV NS1 show 100%
identity with all IHHNV isolates.
The estimated NS1 amino acid sequence was compared with
putative amino acid sequences predicted from sequences available in Genbank and the similarities are listed in Table 2. The NS1
amino acid sequence of the Brazilian IHHNV shows high similarity with Hawaii (AF218266.2) and Taiwan (AY355306) isolates. The
predicted amino acid sequence of ORF1 showed only two substitutions in comparison with Hawaii isolate: a change from aspartate to
139
Fig. 1. Nucleotide sequence of partial Brazilian IHHNV genome. Putative aminoacid sequences of ORF1, ORF2 and ORF3 are shown above the sequence. The putative start
and stop codons correspondent to each ORF are in gray. The putative transcription initiation signals (CA) and polyadenilation signals (ATAAA) are underlined. The region of
P61 promoter is in bold. Conserved regions (CRs) are highlighted in black. CR3 and CR26 include the start codons of ORF1 and ORF3 respectively, and CR30 includes ORF3
stop codon. Conserved domains of proteins coded by ORF1 and ORF3 are highlighted in gray, in their respective aminoacid sequences.
140
Fig. 1. (Continued )
the CR26 begins at the start codon of capsid protein. The CR16 and
CR18 are inside the coding region of the rolling-circle replication
motifs I and II and CR23 and CR24 corresponds to a part of coding
sequence of NTP-biding and helicase domains. The CR25 comprises
part of one IHHNV promoter region, described by Dhar et al. (2001).
3.4. ORF2 and non-structural protein 2 (NS2)
The ORF2 starts at nucleotide 432 and terminates at nucleotide
1523 at a TAG codon, thus, only the 56 rst nucleotides of ORF2
does not overlap with ORF1. ORF2 encodes a protein containing
141
Fig. 1. (Continued )
Hawaii isolate. The same substitution at site 240 and other from
threonine to proline is observed when Brazilian IHHNV ORF2 is
compared with NS2 of Taiwan isolate. ORFs 1 and 2 share eighteen
conserved regions (CR3CR20). Until now, there is no information
about the NS2 function and about the possible functional domains
of this protein.
Table 2
Comparison of nucleotide and predicted amino acid sequences of Brazilian IHHNV and other IHHNV sequences available in GenBank.
Country/isolate
GenBank no
ORF2
ORF3
ORF1
ORF2
ORF3
Hawaiia
Taiwan A
Taiwan C
Ecuador
Chinaa
Mxico
Chinaa
South Koreaa
Chinaa
Vietnama
Vietnam
Thailand
Taiwan B
Thailand
Vietnam
Vietnama
Austrlia
ndiaa
East Africa
Madagascar
Austrlia
AF218266
AY355306
AY355308
AY362548
EF633688
AF273215
JX258653
JN377975
KF214742
JX840067
KC513422
AY362547
AY355307
AY102034
JN616415
KF031144
GQ475529
GQ411199
AY124937
DQ228358
EU675312
99.7
99.6
99.6
99.6
99.5
99.4
99.1
99.3
99.3
98.6
95.8
95.6
95.6
95.7
95.6
95.6
95.3
93.5
91.3
86.0
85.7
99.7
99.8
99.8
99.8
99.6
99.6
99.5
99.4
99.3
99.4
96.7
97
96.7
96.6
94.6
96.3
95.1
96.2
92.5
87.0
87.5
99.7
99.6
99.6
99.7
99.5
99.6
99.5
99.4
99.1
99.1
97.3
97.6
97.4
97.3
96.8
96.8
97.5
97
89.3
99.7
99.7
99.6
99.4
99.7
99.3
99.5
99.3
99.1
99.2
95.5
94.9
95.2
95.3
97.2
95.2
95.5
93.5
91.1
87.3
87.2
99.6
99.4
99.4
99.4
99.3
99.1
99.1
99
98.4
98.4
96.3
96.7
96.6
96.3
84.2
95.7
97
87.6
91.8
84.3
69.5
99.5
99.2
99.2
99.5
99.2
99.5
98.9
98.9
97.8
98.6
95.9
97
96.4
95.9
88.6
95.3
96.2
95.3
86
99.4
99.7
99.4
98.8
99.7
98.5
99.1
98.5
98.5
98.2
96.7
96.7
97
97
98.8
96.7
97.3
95.8
96.7
93.3
93
Complete genomes.
142
5UTR
|
1
ORF2
| |
432 488
ORF3
|
1.523
| |
2.430 2.488
3UTR
|
3.419
|
3.739
ORF1
Fig. 2. Genomic organization of Brazilian IHHNV genome. The Plus strand of the 3739 nt sequence that was sequenced in this study is represented by the rectangle. Regions
of ORF1, ORF2 and ORF3 are represented by keys and rst and last nucleotides of each ORF are represented by numbers. Untranslated regions (UTRs) are shown in gray. The
dotted square corresponds to the 5 UTR fragment that possibly exists in Brazilian IHHNV genome but was not sequenced in this study.
Fig. 3. Sequences of UTR regions, secondary structure bracket representation and minimum free energy (MFE) models of 5 UTR hairpin. 5 and 3 UTR sequences of Hawaii
IHHNV isolate (GenBank Accession No. AF218266.2) were analyzed using the software RNAfold. (A) The parentheses below the nucleotide sequences indicate base pairing and
dots indicate unpaired regions in DNA secondary structure. Hairpins with the highest probability of occurrence are highlighted in gray and conserved regions are highlighted
in black. The 77 nt sequence that is repeated in the 5 and 3 ends are underlined. (B) MFE model calculated using the rst 154 nucleotides that correspond to 5 end of the plus
strand of Hawaii isolate. (C) MFE model calculated using the correspondent 154 nucleotide sequence from minus strand of Hawaii isolate. The structures B and C are colored
according to base-pairing probabilities. Red color denotes the high probability and purple denotes low probability of a given base is paired or not. For unpaired regions the
color denotes the probability of being unpaired. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of the article.)
143
Brazilian_IHHNV
JX258653.1_China
35
76
39
KF214742.1_China
JN377975.1_South Corea
56
AY362548.1_Ecuador
59
LineageIII
JX840067.1_Vietnam
AF218266.2_Hawaii
100
55
AY355308.1_Taiwan
EF633688.1_China
AF273215.1_Mexico
100
GQ411199.1_India
AY362547.1_Thailand
99
JN616415.1_Vietnam
85
100
KC513422.1_Vietnam
48
77
80
GQ475529.1_Australia
LineageII
AY102034.1_Thailand
AY355307.1_Taiwan B
KF031144.1_Vietnam
LineageI
AY124937.1_East Africa
EU675312.1_Australia
100
DQ228358.1_Madagascar
0.02
Fig. 4. Pylogenetic tree of IHHNV strains based on partial genomes comprising the ORF1, ORF2 and ORF3 coding regions. The phylogenetic relationship was estimated by
maximum likelihood method using MEGA program. The infectious IHHNV strains were divided into three lineages, and the Brazilian IHHNV is clustered in lineage III (black
arrow). Numbers indicate the percentages of bootstrap support from 1000 replicates.
in both extremities (Fig. 3A underlined region). Interestingly, analyzing this repeated sequence fused to the next 77 nucleotides of
the 5 UTR region, it was possible to detect a hairpin with high
probability or occurrence that is represented in Fig. 3B. This hairpin is more probable when the corresponding sequence of minus
strand is used to generate the MFE model (Fig. 3C). Parvoviruses
that present 5 UTR and 3 UTR different ends, like minute virus of
mice, selectively encapsidates strands that are minus sense with
respect to transcription (Tattersall and Ward, 1976; Cotmore and
Tattersall, 2006). This predicted hairpin can be the starting point
for better understanding the mechanisms of IHHNV replication.
Three CRs were identied within UTRs: CR1 and CR2 are located
in the 5 UTR region and CR31 are located in the 3 UTR, as shown
in Figs. 1 and 3A. CR31 are inside the repeated sequence and is one
of the largest conserved regions identied, with 30 nt.
3.7. Phylogenetic analysis
A phylogenetic tree was constructed based on the alignment
of nucleotide coding IHHNV sequences available in GenBank,
including Brazilian IHHNV (Fig. 4). The maximum likelihood tree
suggest that the IHHNVs can be divided into two major groups,
one group corresponds to a group of isolates that produce the
infection and other non-virulent group of IHHNV, according to
previously described by (Kim et al., 2012). Likewise, our results
support the division of IHHNV infectious group in three distinct lineages. The lineage III comprising viruses JX258653.1, KF214742.1,
JN377975.1, AY362548.1, JX840067.1, AY355308.1, EF633688.1,
AF273215.1 and the Brazilian IHHNV isolate. The lineage II
is composed by strains GQ411199.1, AY362547.1, JN616415.1,
KC513422.1, AY102034.1, AY355307.1, KF031144.1, and the lineage I is composed uniquely by strain GQ475529.1 from Australia.
This subdivision was supported by very high bootstrap values, and
indicate potential genetic differences between lineages. The noninfectious group is composed of isolates from Australia (EU675312.1)
and Madagascar (DQ228358.1).
The three identied lineages are not limited to dened geographical areas, and more than one lineage may occur in the
same area. This observation corroborates with several studies that
144
Fig. 5. Semi-nested PCR to IHHNV detection. Primers PF3/PR3 were used in the rst PCR and PF3/PR2 in the nested PCR. (A) First PCR step, using the DNA templates submitted
to a serial dilution by a factor of 5. Lane 1, reaction with undiluted template; lanes 27, reactions using templates submitted to a progressive serial dilution by a factor of 5
(lane 2 = lowest dilution; lane 7 = highest dilution). The expected amplicon of rst step has 588 bp. (B) Nested reactions using rst step reaction products as templates. Lane
1, nested PCR using the product of reaction 1 of rst step as template; lanes 27, nested PCRs using the products of reactions 27 of rst step as templates, respectively. The
153 bp fragment indicates the positive result. M, DNA ladder; N, negative control; P, positive control. (C) DNA quality control using decapod specic primers. DP, DNA shrimp
sample used as positive control; DN, DNA shrimp sample used as negative control.
that are used in two subsequent PCR reactions. In the rst reaction the primers PF3 and PR3 are used and an amplicon of 588 bp is
generated. In the second reaction primer PF3 is used with primer
PR2, which aligns to an internal region of the amplicon generated in the rst reaction. The nested reaction generates a 153 bp
amplicon. This strategy allowed a signicant increase in test sensitivity, according to demonstrated by a serial dilution in Fig. 5.
The nested PCR increased the sensitivity of the test at least 5 times
and allowed a clear visualization of the positive result in reactions that were performed using 5 ng of total DNA as template
(Fig. 5B).
The development of molecular markers to detect IHHNV is
complex. The mean rate of nucleotide substitution for IHHNV is
high and comparable to that reported for RNA viruses (RoblesSikisaka et al., 2010). Moreover, as observed here and in previous
studies, IHHNV lineages are not restricted to specic areas (Tang
Acknowledgements
Primer name
Primer sequence 5 - 3 a
References
IHHNV392F
IHHNV392R
77012F
77353R
389F
389R
IHHNV309F
IHHNV309R
IHHNV648F
IHHNV648R
IHHNVF
IHHNVR1
IHHNV721F
IHHNV2860R
IHHN3065F
IHHN3065R
77012F
2553R
I2814F
I3516R
GGGCGAACCAGAATCACTTA
ATCCGGAGGAATCTGATGTG
ATCGGTGCACTACTCGGA
TCGTACTGGCTGTTCATC
CGGAACACAACCCGACTTTA
GGCCAAGACCAAAATACGAA
TCCAACACTTAGTCAAAACCAA
TGTCTGCTACGATGATTATCCA
GAACGGCTTTCGTATTTTGG
AGCGTAGGACTTGCCGATTA
ATGTGCGCCGATTCAACAAG
CTAAGTGACGGCGGACAATA
CTACTGCCTCTGCAACGAG
GTGGGTCTGGTCCACTTGAT
GACGACGAAGAATGGACAGA
TGCCTGGGTAGCTGGTATGTATA
ATCGGTGCACTACTGGGA
CGGACAATATCCCTGACT
TAATGAAGACGAAGAACACGCCGAAGG
TGGGTAGACTAGGTTTCCAAGGGATGGTT
145
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OIE (2003)
Tang et al. (2007)
Rai et al. (2009)
Tang and Lightner
(2002)
Tang and Lightner
(2002)
Tang et al. (2003)
Nunan et al. (2000)
Yang et al. (2007)
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