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Virus Research 189 (2014) 136146

Contents lists available at ScienceDirect

Virus Research
journal homepage: www.elsevier.com/locate/virusres

Infectious hypodermal and hematopoietic necrosis virus from Brazil:


Sequencing, comparative analysis and PCR detection
Douglas C.D. Silva a , Allan R.D. Nunes a , Drlio I.A. Teixeira c , Joo Paulo M.S. Lima b,d ,
Daniel C.F. Lanza a,
a

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.,

Corresponding author at: Laboratrio de Biologia Molecular Aplicada LAPLIC,


Departamento de Bioqumica, Centro de Biocincias, Universidade Federal do Rio
Grande do Norte, CEP: 59072-970 Natal, RN, Brazil. Tel.: +55 84 3215 3416;
fax: +55 84 3215 3415.
E-mail addresses: danielclanza@gmail.com,
daniel.lanza@cb.ufrn.br (D.C.F. Lanza).
http://dx.doi.org/10.1016/j.virusres.2014.05.008
0168-1702/ 2014 Elsevier B.V. All rights reserved.

2012). In Brazil, the rst ofcial report of IHHN occurrence was in


2009, at the Bahia state and subsequent studies have conrmed
the IHHNV presence in other states from Brazilian Northeast region
(Trindade et al., 2009; Braz et al., 2009; Teixeira-Lopes et al., 2011).
The prevalence of IHHNV has been also reported in other regions of
the world, such as China, India and Australia. Phylogenetic analysis
suggests that American IHHNV was introduced from the Philippines
(Tang et al., 2003; Krabsetsve et al., 2004; Tang and Lightner, 2006)
and that Indian IHHNV is originated from South-East Asia (Rai et al.,
2009).
The typical manifestation of IHHNV-induced disease in Litopenaeus vannamei includes growth retardation and runt deformity
syndrome, which is characterized by cuticular deformities as bent
rostrum, blistered cuticle, abdominal anomalies and curly antennae (Kalagayan et al., 1991; Browdy et al., 1993). IHHNV caused
high mortality rates in L. stylirostris some years after its emergency,
but this species has acquired tolerance to infection (MoralesCovarrubias et al., 1999; Tang and Lightner, 2002). Although L.
vannamei and Penaeus monodon do not show signicant mortality rates caused by IHHNV infection, the economic and production
losses are noteworthy, mainly due to the reduced and irregular

D.C.D. Silva et al. / Virus Research 189 (2014) 136146

growth which results in small sized shrimps at harvest (Bell and


Lightner, 1984; Kalagayan et al., 1991; Primavera and Quinitio,
2000).
IHHNV was classied as Penaeus stylirostris densovirus (PstDNV)
in the family Parvoviridae, sub-family Densovirinae (Fauquet et al.,
2005). It is the smallest penaeid shrimp virus, with nonenveloped
and icosaedral virions averaging 2223 nm in diameter and containing linear single stranded DNA of 3.9 kb in length (Shike
et al., 2000; Rai et al., 2011; Kim et al., 2012). IHHNV genome
is comprised of 3 large open reading frames, the left ORFs 1 and
2 that codies non structural proteins and the right OFR 3 that
codies the viral capsid protein (Mari et al., 1993; Shike et al.,
2000). To date, eight complete genome sequences of IHHNV are
available in GenBank. Two sequences correspond to isolates from
American continent, one complete genome from Hawaii (Accession No. AF218266; 3909 bp), and a complete coding sequence
(CDS) of a Mexican isolate (GenBank Accession No. AF273215;
3873 bp). Genomes and complete coding sequences from other
parts of the globe such as China, India, Korea, Vietnam, Taiwan,
Thailand, Philippines and Indonesia are also available. Two noninfectious related IHHNV sequences designed as Type A and Type B
were detected in P. monodon. The Type A was found in samples from
Madagascar and Australia and the Type B was detected in Tanzania
(Tang et al., 2003; Krabsetsve et al., 2004; Tang and Lightner, 2006).
Both virus-related A and B types contain the three ORFs and typical regulatory elements of IHHNV and show signicant divergence
when compared with the infectious species (Tang et al., 2003; Tang
& Lightner, 2006).
IHHNV related sequences may be integrated into the genome of
P. monodon as previously reported in shrimp samples from Africa,
Australia and India (Tang and Lightner, 2006; Rai et al., 2009). Due
to these ndings new targets for PCR detection has been selected in
IHHNV genome, to avoid the viral regions integrated into the host
genome, or in order to detect an integrated part of the virus genome
into the host genome (Tang et al., 2007; Rai et al., 2009).
In the present study we sequenced a 3739 nucleotide fragment of a Brazilian IHHNV detected in L. vannamei samples, and
compared this sequence with genomes and CDSs available in GenBank. Thereafter, conserved nucleotide sequences and putative
DNA secondary structures in IHHNV genome were observed and
phylogenetic relationships between IHHNV isolates from different
geographic regions were inferred. In addition, a new nested PCR to
IHHNV detection is proposed.
2. Materials and methods
2.1. Sample isolation and DNA extraction
Sub-adult shrimps L. vannamei (Boone, 1931) presenting viral
disease symptoms were sampled from one shrimp farm located in
the state of Rio Grande do Norte, Brazil. The samples were cleaned
to avoid any external contaminations and kept under refrigeration
(20 C), until DNA extraction.
DNA was extracted from 50 mg of muscular tissue extracted
from the third abdominal segment of frozen shrimp. Tissues samples were cut and immediately transferred to a buffer containing
proteinase K, according to specications of NucleoSpin Tissue kit.
After purication, DNA was eluted using EB buffer (5 mM Tris/HCl,
pH 8.5) and quantied using a NanoDrop 2000 Spectrophotometer
(Thermo Fisher Scientic).
2.2. Detection and sequencing of Brazilian IHHNV
First, IHHNV was detected using primers PF3 and PR3 specifically designed for the present study. All sequences of primers

137

Table 1
Primers designed in this study to IHHNV genome amplication.
Primer name

Primer sequence (5 3 )

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.

2.3. Sequence analysis and alignment


The characterization of Brazilian IHHNV and the rst comparative analysis with other sequences available in GenBank
was performed using Basic Local Alignment Search Tool (BLAST)
(Altschul et al., 1997) of the National Center for Biotechnology Information (NCBI). The open reading frames (ORF) were
determined using the ORF nder software (http://www.ncbi.nlm.
nih.gov/gorf/gorf.html). Amino acid sequence prediction and comparative analysis between nucleotide and aminoacid sequences
were performed using Geneious software (ver. 7.0, Biomatters). Multiple sequence alignment of coding IHHNV sequences
was carried out using Muscle algorithm (Edgar, 2004) with
default parameters in the Jalview software package Version 2.8
(Waterhouse et al., 2009). The nucleotide regions conserved in all
sequences were identied using the MEGA5 program Version 5.2
(Tamura et al., 2011) and conrmed visually.
The nucleotide secondary structure was predicted and the Minimum Free Energy models (MFE) were generated by RNAfold
(Gruber et al., 2008), using DNA parameters and rescaling the
energy parameters to a temperature of 28 C.

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D.C.D. Silva et al. / Virus Research 189 (2014) 136146

2.4. Phylogenetic analysis


A phylogenetic tree was constructed from alignment of partial
IHHNV sequences comprising ORF1, ORF2 and ORF3 coding regions,
using Molecular Evolutionary Genetics Analysis MEGA5 program
Version 5.2 (Tamura et al., 2011). The phylogenetic reconstruction
was performed using the maximum likelihood discrete method.
The model HKY (Hasegawa et al., 1985) was selected after a bestt nucleotide substitution model analysis, using estimated gamma
parameter (+G). Both model tests and phylogenetic tree reconstruction were carried out using complete deletion for gaps and very
strong branch swap lter. The horizontal branch lengths are proportional to nucleotide substitution rates, and numbers shown at
branch point indicate 1000 bootstrap values, which determined the
condence indices within the tree.
2.5. Nested PCR and serial dilution test
Total DNA extracted from a shrimp sample positive for IHHNV
was subjected to a serial dilution by a factor of 5. The rst PCR
reaction was performed using the PF3 and PR3 primers and 125 ng
of total DNA. After the rst reaction, each of seven reaction products were used as templates in the nested PCR, in which were
used primers PF3 and PR2. The following cycle parameters were
used in the two steps: 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 amplication products were analyzed on agarose gels ordered from lowest to highest dilution. A
positive control containing total DNA from a shrimp positive to
IHHNV and a negative control containing total DNA from a non
infected shrimp were inserted in the rst and second steps. An additional positive control using decapods specic primers preconized
by OIE, (143F 5 -TGCCTTATCAGCTNTCGATTGTAG-3 and 145R 5 TTCAGNTTTGCAACCATACTTCCC-3 ) yielding an 848 bp amplicon
was used to verify the quality of the extracted DNA.
3. Results and discussion
3.1. Viral genome amplication
Initially, 21 sequences available in GenBank (Table 2) corresponding to the complete genomes or coding sequences of IHHNV
were aligned. A set of primers that anneal in regions with low variability interspaced in IHHNV genome were designed to amplify
Brazilian IHHNV. Each primer used and its respective amplicon size
and melting temperature are detailed in Table 1. After amplication, the eight generated amplicons were sequenced and entire
sequences were assembled from the overlapping regions in each
amplicon.
3.2. Nucleic acid analysis and genomic organization of Brazilian
IHHNV
The sequence of Brazilian IHHNV is composed of 3739 nt and
comprise all coding regions, the major part of 5 UTR (lacking 160 nucleotides at 5 end that were not sequenced in this
study), and the entire 3 UTR region. The sequence are shown
in Fig. 1 and had a base composition of 36.7% A, 23.9% C, 18.8%
G and 20.6% T and the G + C and A + T content was calculated to
be 42.7% and 57.3%, respectively. The Brazilian IHHNV sequence
shared 99.7% similarity with the genome from Hawaii (GenBank Accession No. AF218266.2) and 99.6% similarity with IHHNV
genomes from Taiwan and Ecuador (GenBank Accession numbers AY355306 and AY362548, respectively). The lowest similarity,
(85.7%) was observed to the Australian isolate (GenBank Accession

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

D.C.D. Silva et al. / Virus Research 189 (2014) 136146

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

D.C.D. Silva et al. / Virus Research 189 (2014) 136146

Fig. 1. (Continued )

asparagine at positions 168 and 461. The comparison to the Taiwan


isolate reveals one substitution at the site 160, from asparagine to
threonine, and a substitution from aspartate to asparagine at site
168.
The nucleotide sequence of the ORF1 presents 24 conserved
regions (CR3CR26) and some of these regions comprise sites of
known importance in viral replication (Fig. 1). It is interesting,
though expected, to note that some conserved regions correspond
to segments where ORFs overlapping occurs, particularly the ones
next to start codons. The CR3 is located at the intersection point
between ORFs 1 and 2, covering the initiation codon of NS1 and

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

D.C.D. Silva et al. / Virus Research 189 (2014) 136146

141

Fig. 1. (Continued )

363 amino acid residues, and a molecular weight of 42,076 kDa. A


comparison of nucleotide and predicted amino acid sequence of
Brazilian IHHNV NS2 with all other predicted sequences of NS2
available in GenBank are shown in Table 2. The predicted amino
acid sequence of NS2 from Brazilian IHHNV had only one substitution, from arginine to lysine at site 240, when compared to the

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

Genome identity (%)

Nucleotide identity (%)


ORF1

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.

Amino acid identity (%)

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D.C.D. Silva et al. / Virus Research 189 (2014) 136146

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.

3.5. ORF3 and viral capsid protein (VP)


The ORF3 is in the same reading frame as the ORF2: starts
at nucletoide 2430 and terminates at nucleotide 3419 at a TAA
codon, and have a region of 59 nucleotides overlapping ORF1 end.
It encodes a viral capsid protein of 329 amino acids, corresponding
to a molecular weight of 37,429 kDa. The similarity degree between
nucleotide and predicted amino acid sequence of Brazilian IHHNV
VP and all other IHHNV correspondent sequences available in the
GenBank are shown in Table 2. The amino acid sequence of Brazilian IHHNV VP is identical to VP from China isolate and differs from

VPs from Hawaii and Taiwan isolates only by a substitution from


asparagine to glutamate at position 44.
Probably, VP is the unique protein composing the IHHNV capsid, and this feature is not observed in other parvoviruses. The
structure of VP protein was resolved by X-ray crystallography and
shows eight parvoviral conserved secondary elements that form
a jelly roll barrel motif, similar to that found in many icosahedral viruses, including other parvoviruses (Kaufmann et al., 2010).
These conserved secondary elements are highlighted in the amino
acid sequence shown in Fig. 1. It is curious to note that only CR27, of
the 5 CRs identied in ORF3, corresponds to a conserved secondary

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.)

D.C.D. Silva et al. / Virus Research 189 (2014) 136146

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.

structure in VP, considering the crystal structure elucidated by


Kaufmann et al., 2010. A more careful analysis shows that the most
part of CRs regions are inside the VP loops that are in most cases
unstructured or dynamic regions. These regions do not encode a
structured region of the protein, but are submitted to a selection
pressure equal to or greater than structured regions. Maybe, the
absence of a rigid protein structure promoting the conservation of
the nucleotide sequence in some manner.
3.6. UTR regions
The 5 UTR nucleotide sequence of Brazilian IHHNV, contains
431 nucleotides, 162 nucleotides less when compared to 5 UTR
from Hawaii isolate. The 3 UTR from Brazilian isolate shows 320
nucleotides, eight nucleotides absent in the 3 end when compared
with 3 UTR of Hawaii isolate. Disregarding the regions that are
absent in Brazilian IHHNV, the differences between UTR regions
from Brazil and Hawaii viruses are a transition from guanine to adenine at site 151 in 5 UTR and a transition from adenine to guanine
at site 27 in the 3 UTR. The 5 UTR and 3 UTR from Brazilian IHHNV
isolate presented here possibly are incomplete, since typical elements previously reported such as TATA box, a palindromic region
and other regulatory elements, observed in the rst nucleotides of
5 UTR were not detected (Shike et al., 2000; Rai et al., 2011).
Due to its high similarity with Brazilian IHHNV sequence, the
complete 5 UTR and 3 UTR sequences from Hawaii isolate were
used to prospect secondary structures, which are necessary to
rolling hairpin replication observed in parvoviruses (Tattersall and
Ward, 1976; Cotmore and Tattersall, 2006; Li et al., 2012). Analyzing the minimum free energy (MFE) models of plus strand, several
regions with potential to form hairpins in the UTR regions were
identied, as showed in bracket representation in Fig. 3A. The
highest base pair probability scores were observed to tree hairpins inside the 5 UTR sequence, the rst, from the 5 extremity,
which comprises the region from nucleotide 6296, the second the
region from nucleotide 293320, and the last comprises the region
from nucleotide 386412 (Fig. 3A). A more detailed examination
revealed a sequence that is repeated, located in the rst 77 nt of 5
UTR and in the last 77 nt of 3 UTR region, in the same orientation

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

D.C.D. Silva et al. / Virus Research 189 (2014) 136146

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.

demonstrate that the trade of breeding animals is one of the main


ways of virus dispersion (Lightner, 1988, 2011). Strains from East
Africa (AY124937.1) and Australia (GQ475529.1) can still preserve
characteristics of the primitive IHHNV. As indicated by the small
variation in the sizes of the branches inside each lineage, the major
events of differentiation may have occurred before the spread of
the virus across the globe.
3.8. Detection of IHHNV using nested PCR
A new semi nested PCR protocol to IHHNV detection with high
sensitivity and low cost has been developed, using primers corresponding to regions with little genetic variability that encodes
replication initiator motif and NTP-binding and helicase domains
in NS1. These new protocol comprises a set of tree primers PF3,
PR3 and PR2 (also used to genome sequencing, shown in Table 1)

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

D.C.D. Silva et al. / Virus Research 189 (2014) 136146


Table 3
Some primers used to IHHNV identication available so far.

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

Tang et al. (2000)

145

We would like to thank CAPES and FAPERN for nancial support


and the company Camanor for providing shrimp samples used in
this study.

OIE (2000)
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)

Underlined bases indicate the polymorphic sites.

and Lightner, 2002; Tang et al., 2003). Furthermore, fragments of


IHHNV are inserted in P. monodon genomes collected in Australia,
Africa and India (Krabsetsve et al., 2004; Tang and Lightner, 2006).
All these factors justify the need of continued development of
specic primers, in order to avoid false positive and false negative results. The primers recommended by the World Animal
Health Organization can amplify the IHHNV-related sequences
present in P. monodon, which has gotten integrated into the shrimp
genome, thus showing false positive results (Tang et al., 2006).
Several primers to IHHNV detection have been published, including primers that allow to detect the viral sequence integrated into
shrimp genome (Table 3). Many of these primes are effective to distinguish IHHNV variants, but most of them are located in regions
comprising polymorphic sites in the genome, as shown in Table 3.
Depending on its location (proximity to the 3 region), the mismatches may decrease the PCR efciency or causes the complete
inhibition of amplication. Considering all these points the system
presented here offers two major advantages. The rst is that the
primers were designed to align in regions that encoding conserved
domains of viral NS1 protein, which increases the chance to detect
the largest number of variants of the virus. The second is that the
use of tree primers and the increment of the annealing temperature
of the primer used in the nested reaction turned the method more
sensitive without loss the reaction specicity.
In summary, the IHHNV Brazilian isolate is very similar to
Hawaii isolate, and is closely related to a lineage which comprises
infectious variants of IHHNV. Some conserved nucleotide regions
identied in IHHNV genome correspond to putative regulatory
gene sites or regions that coding conserved protein domains. However, in some cases, it was not possible to correlate the conservation
of certain regions of its biological function. The UTR regions shows
segments with potential to form hairpins, and a repeated sequence
of 77 nt were observed in IHHNV genome extremities. Unexpectedly, a putative hairpin in the 5 UTR region of the positive IHHNV
strand was detected, and can be a candidate site of early viral replication. In addition, a new nested PCR protocol using tree primers
was efcient to detect IHHNV with high sensitivity. These results
will be important to direct further studies about genetic variability
of IHHNV and to guide the design of new markers to its variants
detection, as well as to the comprehensive understanding of the
gene regulation and replication mechanisms present in this virus
family.

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