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Virology 346 (20

Complete nucleotide sequence and genomic organization of

hepatopancreatic parvovirus (HPV) of Penaeus monodon

Wasana Sukhumsiricharta,*, Pongsopee Attasartb, Vichai Boonsaengc, Sakol Panyimb,c

aDepartment of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Sukhumvit 23, Bangkok 10110, ThailandbInstitute of Molecular Biology and Genetics, Mahidol University, Salaya, Nakorn Pathom 73170, Thailand

cDepartment of Biochemistry, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand

Received 22 March 2005; returned to author for revision 28 April 2005; accepted 17 June 2005

Available online 13 December 2005

Abstract

We have determined the genome of hepatopancreatic parvovirus (HPV), a minus, single-stranded DNA virus isolated from infected

Penaeus monodon in Thailand. Its genome consisted of 6321 nucleotides, representing three large open reading frames (ORFs) and two non-

coding termini. The left (ORF1), mid (ORF2), and right (ORF3) ORFs on the complementary (plus) strand may code for 428, 579, and 818

amino acids, equivalent to 50, 68, and 92 kDa, respectively. The 5V and 3V ends of viral genome contained hairpin-like structure length of

approximately 222 and 215 bp, respectively. No inverted terminal repeat (ITR) was detected. The ORF2 contained conserved replication

initiator motif, NTP-binding and helicase domain similar to NS-1 of other parvoviruses. Therefore, it most likely encoded the major

nonstructural protein (NS-1). The ORF1 encoded putative nonstructural protein-2 (NS-2) with unknown function. The ORF3 of the HPV

genome encoded a capsid protein (VP) of approximately 92 kDa. This may be later cleaved after arginine residue to produce a 57-kDa

structural protein. A phylogenetic tree based on conserved amino acid sequences (119 aa) revealed that it is closely related to

Brevidensoviruses, which are shrimp parvovirus (IHHNV) and mosquito densoviruses (AaeDNV and AalDNV). However, the overall

genomic organization and genome size of HPV were different from these parvoviruses, for instance, the non-overlapping of NS1 and NS2,

the larger VP gene, and the bigger genome size. This suggested that this HPV virus is a new type in Parvoviridae family. We therefore

propose to rename this virus P. monodon densovirus (PmDNV).

D 2005 Elsevier Inc. All rights reserved.

Keywords: Hepatopancreatic parvovirus; HPV genome; PmDNV genome; Genome organization; Genes; PmDNV

Introduction

Hepatopancreatic parvovirus (HPV) is a single-stranded

DNA virus that infect cultivated and wild penaeid shrimp

species including Penaeus monodon and Penaeus chinensis

(Lightner, 1996; Lightner et al., 1993). Heavy infections

resulted in poor growth, which reduces shrimp production

(Flegel et al., 1999).

Based on histological and cytological characteristics of

the virions and partial characterization of viral protein, HPV

belongs to the Parvoviridae family (Lightner and Redman,

0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.virol.2005.06.052

* Corresponding author. Fax: +66 2 2600125.

E-mail address: [email protected] (W. Sukhumsirichart).

1985; Bonami and Lightner, 1991; Francki et al., 1991).

However, the classification of HPV viruses is still uncertain,

because of their unusual capsid proteins and genome

structures (Bonami and Lightner, 1991).

The family Parvoviridae consisted of small icosaheral,

non-enveloped particle of 18–26 nm in diameter and a

single-stranded linear DNA ranging from 3.9 to 5.9 kb

(Berns et al., 1995). Parvoviridae can be divided into two

subfamilies: Parvovirinae and Densovirinae. Both of them

are able to infect vertebrates (Parvovirus, Erythrovirus, and

Dependovirus) and invertebrates (Densovirus). Densovi-

ruses (DNVs), which belong to Densovirinae subfamily,

have been isolated from a broad range of invertebrates, but

mainly from insects. They are highly pathogenic to their

hosts, causing severe diseases known as densonucleosis

06) 266 – 277

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http://dx.doi.org/10.1016/j.virol.2005.06.052

W. Sukhumsirichart et al. / Virology 346 (2006) 266–277 267

(Vago et al., 1964). DNVs are classified into three genera:

Densovirus, Iteravirus and Brevidensovirus. Densoviruses,

in which Junonia coenia DNV (JcDNV) and Galleria

mellonella DNV (GmDNV) are members, have 6-kb

genomes with very long inverted terminal repeats (ITRs);

their coding sequences are located on each strand (ambi-

sense genome). The second genus, Iteravirus, consisting of

Bombyx mori DNV-1 (BmDNV-1) and Casphalia extranea

densovirus (CeDNV), possess 5-kb genomes with coding

sequences located on one strand and contain inverted

terminal repeats of about 200 nts (Bando et al., 1987,

1990; Fediere et al., 2002). The third genus, Brevidensovi-

rus, includes two mosquito DNVs isolated from Aedes

aegypti (AaeDNV) and Aedes albopictus (AalDNV) and

one possible member, shrimp DNV isolated from Penaeus

stylirostris (PstDNV or Infectious Hypodermal and Hema-

topoietic Necrosis Virus; IHHNV).

The genomes of several vertebrate and invertebrate

parvoviruses have been partially or totally cloned and

sequenced (Fediere et al., 2002; Samulski et al., 1982;

Rhode and Paradiso, 1983; Srisvatava et al., 1983; Carlson

et al., 1985; Astell et al., 1986; Chen et al., 1986; Shade

et al., 1986; Reed et al., 1988; Bloom et al., 1988; Dumas

et al., 1992; Sakurai et al., 1989; Ranz et al., 1989;

Vasudevacharya et al., 1990; Deiss et al., 1990; Shike

et al., 2000; Afanasiev et al., 1991). Despite minor

differences, all of these genomes exhibit similar organ-

ization. The coding sequences on the complementary

strand consist of two major open reading frames (ORFs),

the left and right ORFs. The left ORF encodes non-

structural protein necessary for viral DNA replication

while the right ORF codes for one or three capsid

polypeptides. The 5V and 3V ends of the parvoviral genome

contain short palindromic sequences capable of forming

hairpin structure necessary for replication and encapsida-

tion of viral genome (Tattersall and Ward, 1976; Samulski

et al., 1983; Astell et al., 1985).

Recently, a partial nucleotide sequence and genomic

organization from another small parvovirus in shrimp,

infectious hypodermal and hepatopoietic necrosis virus

(IHHNV), have been reported (Shike et al., 2000). Its

genome (3873 nts) contains three open reading frames

(ORFs); the left, mid, and right ORFs on one strand.

Comparison of nucleotides, amino acid sequences, and

genome organization revealed that, IHHNV is more closely

related to densoviruses of the genus Brevidensovirus than to

any other groups of Parvoviridae.

IHHNV and HPV are shrimp parvoviruses with similar

structure such as virion size and shape but they infect

different target tissues. HPV infects hepatopancreatic

epithelial cells of shrimps whereas IHHNV infects all

multiple organs of ectodermal and mesodermal origin, but

not the midgut (Lightner et al., 1993). There may be because

of some differences in these viral genomes. Hence, it is

important to investigate HPV at the nucleotide level as was

done for IHHNV.

Here, we reported 6321 nucleotides of the HPV genome

that was isolated from P. monodon (HPVmon). The

nucleotide sequence, genome organization, genes, structural

protein, and phylogenetic tree were analyzed.

Results

Nucleotide sequence of HPV genome

The HPV genome of 6321 bp (Fig. 1) was obtained by

DNA sequencing using purified HPV genomic DNA and

recombinant plasmid (6 kb insert) as templates. The

nucleotide sequences at both ends were obtained by

modified methods (Materials and methods).

The base composition of the viral (minus) strand of the

HPVmon genome was 35.82% A, 17.17% C, 24.49% G,

and 22.53% T. The G + C content was 41.65%, and A + T

content was 58.35%. The viral strand of HPVmon has high

A + T content, which is similar to the shrimp parvovirus

(IHHNV 56.96%) (Shike et al., 2000), and the mosquito

brevidensoviruses; AaeDNV, 62.5% (Afanasiev et al.,

1991), and AalDNA, 61.8% (Boublike et al., 1994).

Potential coding domains

The open reading frames (ORFs) in the nucleotide

sequences were determined by computer analysis (Blast,

DNA Strider, Clone manager programs). The HPV genome

contained three major ORFs (Fig. 2A) and all of them are

found in the complementary strand of the viral genome. The

ORF1 (Left ORF) consisted of 1287 nucleotides while the

ORF2 (Mid ORF) contained 1740 nucleotides and ORF3

(Right ORF) consisted of 2457 nucleotides. The ORF1 and

ORF3 are in the different reading frame to that of ORF2.

The internal coding sequences were flanked by non-coding

5V and 3V terminal sequences of 222 and 215 nt in length,

respectively.

Structure of the ORFs

ORF1 (left ORF)

The ORF1 (Fig. 1) started at nucleotide 216 and

terminated with a TAA codon at nt 1500. The first in-frame

ATG codon at position 216 matched 6 out of 7 of the

consensus sequence (A/GCCAUGG) for initiation codon of

eukaryotic protein translation (Kozak, 1986) (Fig. 2B).

Assuming that this ATG codon is translated, the left ORF

would encode a product of 428 amino acids, corresponding

to a molecular weight of 50 kDa. However, no similarity

was found between the predicted gene product of the ORF1

and the entries in the protein databases. Thus, the function

of this protein is yet unknown. However, as was speculated

for the other densoviruses, the ORF1 of HPV genome may

encode a nonstructural protein, NS-2, whose function is

presently unknown.

W. Sukhumsirichart et al. / Virology 346 (2006) 266–277268

Fig. 1. Nucleotide sequence of the HPV genome. Amino acid sequence of putative polypeptides corresponding to major ORFs is shown under the sequence.

The putative translation initiation ATG codons and polyadenylation signals (AATAAA) are underlined. The three most likely functional p2, p22, and p48

promoters are indicated with TATA boxes. Inverted repeat sequences are underlines with arrowhead. DPE and Inr represent downstream promoter element and

transcriptional start signal, respectively.


ORF2 (mid ORF)

The ORF2 started at nt 1487 and terminated with a TAA

codon at nucleotide 3224. The ORF2 has a different reading

frame from the ORF1. Assuming that this ATG codon,

which matches 4 of the 7 nucleotides in the Kozak

consensus sequence, acts as a translation initiation codon,

then the coding sequence of the ORF2 would encode

a protein of 579 amino acids with a molecular mass of

68 kDa.

Fig. 2. (A) Organization of coding sequences on the plus and minus strands of the H

are in the plus strand. (B) Potential transcription/translation initiation sites of NS

The analysis of this predicted 579 amino acids gene

product revealed that it shared sequence homology to

nonstructural protein (NS-1) of other parvoviruses. Specifi-

cally, it showed 34%, 24%, and 23% identity with the NS-1

protein of IHHNV (Accession No. AF273215) (Shike et al.,

2000), AalDNV (Accession No. X74945) (Boublike et al.,

1994), and AaeDNV (Accession No. M37899) (Afanasiev

et al., 1991), respectively. Two regions within this sequence

contained highly conserved motifs commonly found in all

PV genome. Left ORF (ORF1), Mid ORF (ORF2), and Right ORF (ORF3)

1, NS2, and VP genes.


parvoviruses (Bergoin and Tijssen, 1998). The first one,

located between amino acids 87 to 134, contained the highly

conserved replication initiator motif (Fig. 3A). The other,

situated near the C-terminal end (position 390–490),

contained conserved sequence characteristic of NTP-bind-

ing and helicase domains of the NS-1 polypeptide shared by

all parvoviruses (Iversen and Rhode, 1990) (Fig. 3B).

Recently, the activity and crystal structure of the helicase

enzyme from adeno-associated virus type 2 (AAV-2) have

been determined (Hickman and Dyda, 2005).

A phylogenetic tree was constructed using PUAP

program version 4.0 based on the 119 amino acids

conserved region of NTP-binding and helicase domain

(Fig. 3C). It indicated that HPVmon is more closely related

to the shrimp virus, IHHNV or PstDNV and HPV from P.

chinensis (HPVchin) (Pantoja and Lightner, 2000; Phromjai

et al., 2001) as well as the two mosquito brevidensoviruses

(AaeDNV and AalDNV) rather than the other insect

parvoviruses (BmDNV, BgeDNV, CeDNV1, and JcDNV)

or other vertebrate parvoviruses (B19, MVM, AAV-2,

simian, pig-tailed macaque, mouse, and feline parvovi-

ruses). This indicated that HPVmon is a member of

Densovirinae subfamily. Moreover, the analysis revealed

32–35% amino acid identity between HPVmon and

IHHNV and the two mosquito brevidensoviruses (AalDNV

and AaeDNV) but only 29% identity between the mosquito

densoviruses and iteravirus (JcDNV and BmDNV).

ORF3 (right ORF)

The ORF3 started at nt 3642 and terminated with the

TAA codon at nt 6096 (Fig. 1). This is different from

mosquito brevidensoviruses in which the left and right

ORFs are overlapping. The first ATG matches 3 of the 7 nt

of the Kozak consensus sequence. If translation began there,

a protein of 818 amino acids with a molecular weight of 92

kDa is predicted. Based on the alignment of the protein

product (818 aa) with proteins from other parvoviruses, the

right ORF of HPVmon is encoded a capsid protein (VP).

The ORF3 has a highest sequence homology (78% identity)

to the capsid protein of HPV from P. chinensis (HPVchin).

Analysis of the conserved regions of HPVmon structural

polypeptide revealed a glycine-rich sequence downstream of

alanine (aa 274) of the right ORF (Fig. 4). These regions

were conserved among most parvoviruses. However, there

was no significant sequence homology between structural

protein of HPVmon with phospholipase A2 (PLA2)

signature sequence and other parvoviruses reported in

insects (CeDNV), vertebrates (PPV), and snake (Naja naja

snake venom) (Zdori et al., 2001) (Fig. 5).

Analysis of structural protein from HPVmon by SDS-

PAGE (Fig. 6) revealed a major band of 57 kDa and a minor

band of 54 kDa. A faint band was found at 97 kDa. This

result was unexpected since the deduced amino acid

sequence predicted from ORF3 is 92.17 kDa. However,

the result from N-terminal sequence of the 57-kDa protein

revealed the sequence of AAAGGGGSGGGGESA, which

implied that there may be a proteolytic cleavage of the larger

protein. Similar results have been reported in the rodent

parvoviruses in which a proteolytic site of their capsid

proteins (VP2) occurs at an arginine residue immediately

upstream of the glycine-rich stretch (Paradiso et al., 1984;

Rhode, 1985). In the HPV genome sequence, two arginine

residues are located upstream (aa 271) and downstream (aa

298) of the glycine-rich region, respectively. From Fig. 4, if

the cleavage occurred just after the first arginine residue, the

molecular weight of the resulting protein will be 57 kDa,

which will be in good agreement with the estimation for the

large protein seen on the gel (Fig. 6). If cleavage occurred at

second arginine residue just after the glycine-rich sequence,

it would generate a protein with molecular weight of about

54 kDa, agreeing with the estimation of the smaller protein

(54 kDa).

Genomic organization of HPV genome

The genome of HPV virus was organized as mono-

sense, non-overlapping between nonstructural (NS1, NS2)

and capsid protein (VP) genes. The sequence of the ORFs

which started from 5V end of the complementary strand

were ORF1 (NS2 gene), ORF2 (NS1 gene), and then

ORF3 (VP gene). This organization was different from

other parvoviruses in which the genes are organized as

Left ORF (NS1), Mid ORF (NS2) and Right ORF (VP)

(Shike et al., 2000, Boublike et al., 1994, Afanasiev et al.,

1991).

Potential promoters and transcriptional control signals

A computer search for putative transcription regulatory

sequences upstream and downstream from the ORF1,

ORF2, and ORF3 revealed several potential promoters

and polyadenylation signals. Upstream from the ORF1,

one TATA-like box appropriately spaced with respect to the

first ATG codon can be found at nt 144 (TATATAT) (Figs. 1

and 2B). An appropriately positioned upstream GC-rich

region (GGGGCGG, nt 121–127) could act as an activator

element. A canonical downstream AGAGTC (nt 174–179)

transcriptional start signal (Inr-box) and (GTGAAGTT), a

downstream promoter element (DPE) were also present.

Thus, this region is likely to be a functional promoter. This

promoter was named p2, according to its location at map

unit 2 (taking the length of HPVmon genome as 100 map

units).

A second set of putative transcriptional regulatory region

was found upstream from the ORF2. These included

TATAGTA, a TATA-box (nt 1380–1386); GGCACAG, a

GC-rich sequence (nt 1363–1369); TCACAG, an Inr-box

(nt 1419–1424) and GGAAAGATGT, a DPE sequence (nt

1443–1452). This putative promoter (p22) very likely

controls transcription of the ORF2.

A third set of putative transcription regulatory sequences

was found upstream from the right ORF. These included a

Fig. 3. (A) Replication initiator motif I and II in HPVmon and other parvoviruses, (B) Region of NS-1 with highly conserved 119-aa sequence. NTP-binding

and helicase domains A, B, and C are present in the HPVmon genome. Numbers in parentheses indicate the position of sequences for each viral protein shown

in this figure. GenBank accession numbers are shown in brackets. Consensus aa residues are shown in boldface type and are conserved either in all aligned

sequences (uppercase) or in only some aligned sequences (lowercase). An ampersand represents bulky hydrophobic residues (M, V, L, F, Y, I, V, W) and a dot

represents any residue. (C) Phylogenetic tree based on the 119-aa region shown in panel B. Alignment of insect and vertebrate parvoviruses showed that

HPVmon to be closely related to the shrimp and mosquito brevidensoviruses.


Fig. 4. Comparision of the amino acid sequences in the glycine-rich conserved region of the right ORF from MVM (Minute virus of mouse), H-1 (Parvovirus

H1), FPV (Feline panleukopenia virus), PPV (Porcine parvovirus), AaeDNV (Aedes densovirus), AalDNV (A. albopictus densovirus), BPV (Bovine

parvovirus), B19 (Erythrovirus B19), AAV2 (Adeno-associated virus 2), IHHNV (Hepatopoietic necrosis virus), HPVchin (HPV from P. chinensis), and

HPVmon (HPV from P. monodon). Arrow indicates proteolytic cleavage site.


TATA-box, TATAGCT (nt 3369–3375); a GC-rich activator

sequence, TGTCGGC (nt 3350–3356); an Inr-box,

TCATGGT (nt 3425–3431); and a DPE sequence, CAGA-

CACAGT (nt 3455–3464). This putative promoter (p48)

very likely controls transcription of the right ORF3.

Seven AATAAA sequences corresponding to potential

cleavage and poly-adenylation sites for messenger RNAs are

present in the HPVmon sequence. The first, at position 1,721

(AATAAA), is located 219 nt downstream from the ORF1

stop codon and is therefore likely to be functional. The

second (AATAAA), at position 3263, could be a functional

ORF2 transcription stop based on its location close to

canonical CAYTG sequence (CACTTG position 3140–

3145) and downstream G/T-rich sequence (GTTTG, nt

3211–3215). The third, at position 3705, could not be

functional because it was located far away from stop codon.

The other polyadenylated (AATAAA) sequences were found

at positions 4542, 4688, and 5189, which were unfavorably

located. The last site at position 6134–6139, i.e., 35 nt

downstream of the 3V end of the right ORF, is the only one

that is located close to both an almost canonical CAYTG

sequence (CAAATG at position 6112–6117) and located

downstream G/T-rich sequence (GTGGTTT, nt 6161–

6167). Consequently, this region appears to be the most

likely polyadenylation site for HPVmon messenger RNA.

Analysis of the 3V and 5V terminal non-coding sequences

The nucleotide sequences of 5V and 3V ends were

obtained by PCR amplification. Various sizes of amplified

Fig. 5. Sequence alignment of parvovirus PLA2 motifs and PLA2 representative. I

(Culex pipiens), and CeDNV (C. extranea). Vertebrate parvoviruses; PPV (Porcin

virus type 4). Phospholipase representative; PLA2 (N. naja snake venom).

PCR products, 200–500 bp, and 100–200 bp were

obtained from 5V and 3V ends, respectively. All of them

were cloned into pGEM-T Easy vector and transformed

into STBL2 at low temperature in order to stabilize inverted

repeat DNA, which commonly found in terminus of

parvovirus genomes. The results revealed that non-coding

region of the 3V extremity of the viral (minus) strand

contained 215 nucleotides. The non-coding sequence of the

5V extremity was 222 nt long. Both extremities contained

palindromic sequences capable of forming hairpin-like

structures by base pairing, a feature shared by all

parvoviruses previously studied (Astell, 1990). The nucleo-

tide sequence at 5V end (Fig. 7A) could be folded into a

hairpin-like structure but could not generate a typical Y- ,

T-, or J-form similar to other parvoviruses (Bergoin and

Tijssen, 2000). The stem can be formed by base pairing

between nucleotides 6152–6222 and nucleotides at 6251–

6321. There was an imperfect palindrome at nts 6223–

6250 that existed two orientations as reverse-complement;

flip and flop (Fig. 7A). The nucleotide sequence at 3Vterminal region, which obtained from seven independent

clones, also formed hairpin-like structure with 44 base pair

stem length but no flip and flop orientation as found at 5Vend (Fig. 7B). In addition, the analysis of the sequences at

the two termini failed to detect any significant sequence

complementary between the sequences of 5V and the 3Vextremities that can qualify as an inverted terminal repeat

(ITR). In this respect, the ends of HPV genome resembled

the genome of Brevidensovirus and most autonomous

vertebrate parvoviruses whose 5V and 3V terminal sequences

nsect parvoviruses; GmDNV (G. mellonella), JcDNV (J. coenia), CpDNV

e parvovirus), MVM (Minute virus of mice), and AAV-4 (Adeno-associated

Fig. 6. Identification of capsid protein by SDS-PAGE (12.5% gel). Lane M,

molecular weight markers. Lane 1, the viral structural protein of HPVmon.


are not complementary (Astell, 1990; Bergoin and Tijssen,

2000).

Discussion

This is the first report of complete nucleotide sequence

and genome organization of HPV virus from P. monodon.

The total genome length is approximately 6.3 kb, which

agreed with the result from alkaline gel and Southern blot

hybridization (Sukhumsirichart et al., 1999). This genome is

larger than HPV reported in P. chinensis (HPVchin) (4 kb)

(Bonami et al., 1995) or other parvoviruses. Moreover, since

only primers that are complementary to viral genome

provided nucleotide sequencing results but not primers in

the opposite strand, the majority of HPV DNA genome may

be in the minus strand.

The most time-consuming step in this experiment was the

cloning of the 5V and 3V termini of viral genome. However, we

were successful in obtaining the sequences of HPVends from

the modified method. By using a self-priming character of all

parvoviruses, a single primer was used for the PCR

Fig. 7. Analysis of the 5V and 3V terminal non-coding sequences of HPV genome. T

hairpin-like structure (B).

amplification. At 5V terminus, a specific primer was used to

synthesize a new complementary strand in the first round of

amplification. Then, the synthesized DNA should fold to

form hairpin-like structure at the 5V end and acted as the

primer for the second round of PCR step. The resultant DNA

fragment was then served as template for the next step of

amplification by using only one primer. Similar to the 5V end,the hairpin loop at 3V terminus served as primer for the first

round of amplification. The product was then the template for

the next cycle by the single primer. As a result of stem–loop

structure at both ends, which may lead to PCR amplification

failure by normal condition, therefore, two additives such as

DMSO (Chakrabarti and Schutt, 2001; Frackman et al., 1998)

and betaine (Henke et al., 1997) were added in the reaction

mixture to destabilize this structure. These two organic

solvents reportedly can enhance PCR amplification, partic-

ularly for hard-to-amplify high GC templates, which may

create secondary structure like hairpins. In addition, ramping

time of PCR condition was increased to facilitate the binding

between oligonucleotide primer and DNA template much

better than template self-priming (Bachmann et al., 2003).

High temperature (70 -C) for annealing step was also

performed in order to eliminate the forming of hampered

structure within DNA template. From these experimental

steps, it opens an opportunity for retrieving the sequence data

at the end of parvoviruses or a single-stranded virus genome

with folded termini.

Both flip and flop were found at 5V palindromic terminus

whereas only one orientation was detected from all seven 3Vend clones. Since only single-stranded virion DNAwas used

in this study, asymmetrical generation of these orientations or

encapsidation bias could not be ruled out. The feature of

terminal hairpin-like structure can act as the primer for viral

DNA replication, which has been proposed in two models.

According to replication model of parvovirus like AAV

(Cotmore and Tattersall, 1987), the hairpin transfer may

result in flip and flop orientations at both ends. Whereas, the

modified rolling hairpin model for autonomous parvovirus as

in MVM (Astell et al., 1985) was set to explain asymmetrical

he 5V end exists in flip and flop orientations (A). The 3V end forms only one


generation of flip and flop at both ends. Therefore, HPVmon

DNA may replicate by using the latter model.

The analysis of the genome sequence revealed that HPV

genome is the biggest among other parvoviruses so far

reported. The genome organization is also different from the

others in that the nonstructural protein genes, NS1 and NS2,

are non-overlapping. This characteristic may be resulted

from its larger genome size. The coding regions were found

only on the complementary strand but not on ambisense

strand. Interestingly, there were two possible start codons

for NS1 open reading frame that could generate two protein

products. These two alternative proteins may give survival

advantage to the virus.

Analysis of structural protein (VP) by SDS-PAGE

demonstrated a doublet protein band. A major band (57

kDa), which presumably functions as capsid protein and a

minor one (54 kDa) whose role is still unknown. These

proteins were smaller than the protein predicted from ORF3

(92.17 kDa). Three possible mechanisms may explain the

causes of protein shortening. One, the proteolytic cleavage of

92.17 kDa after arginine residue that resulted in 270 and 548

amino acids, which are equivalent to 30.42 kDa and 58.60

kDa, respectively. However, the smaller protein product could

not be detected in the SDS-PAGE. In addition, the result from

N-terminal amino acid sequencing of 57 kDa protein revealed

that it was a free end; therefore, this protein may be the

internal fragment, which had been cleaved by proteolytic

enzymes. Two, alternative splicing of largermRNA, however,

therewas no appropriate donor and acceptor sites as well as no

site for splicing regulatory (SR) binding protein was found.

Three, alternative translation of VP gene by using internal

start codon was another possibility. But DNA sequence

analysis data revealed that there was no internal ribosome

entry site (IRES) found in VP gene. Therefore, it seems to

speculate that VP protein underwent in vivo cleavage.

Phylogenetic tree analysis revealed that HPV is closely

related to mosquito densoviruses (AaeDNA and AalDNV)

and shrimp parvovirus (IHHNV); however, several charac-

teristics, such as genome organization and size, are

distinctively different from other parvoviruses. Therefore,

it may be concluded that HPVmon is a new member of

Densovirinae subfamily. According to the rules of the

Parvoviridae Study Group for the International Committee

on Taxonomy of Viruses, which establishes the nomencla-

ture for invertebrate parvoviruses (Berns et al., 1995), we

proposed to replace the name, HPVmon, with P. monodon

DNV (PmDNV), based on the initial recognition of HPV in

P. monodon (Flegel et al., 1992).

Materials and methods

Purification of HPV virions and DNA preparation

HPV virus was isolated from infected hepatopancrease of

P. monodon collected from ponds in southern and central

parts of Thailand by urografin ultracentifugation (Sukhum-

sirichart et al., 1999). For DNA extraction, the purified viral

particles were resuspended in NTE buffer (NaCl, Tris–HCl,

EDTA, pH 7.4) containing Proteinase K (50 Ag/ml final

concentration) and incubated at 37 -C for 1 h then sarcosyl

or SDS (2%) was added and further incubated at 37 -C for

2 h. The DNA was extracted directly from this mixture

using phenol and chloroform, and then the DNA was pre-

cipitated by 2 volumes of absolute ethanol in the presence

of 0.3 M sodium acetate. The DNA pellet was washed

with 70% ethanol and resuspended in sterile distilled water.

Identification of HPV DNA

The purified viral DNA was analyzed by agarose gel

electrophoresis and PCR amplification using primers that

specific only for HPV (Sukhumsirichart et al., 1999).

Nucleotide sequence of HPV

HPV genomic DNA libraries were constructed based on

the Thai isolate, HPVmon (Sukhumsirichart et al., 1999). A

plasmid clone, pMU659, containing 659 bp of viral insert

was initially sequenced. Then, the primer was designed and

used for further sequencing directly from purified HPV

genomic DNA. The other clones from genomic DNA library

and PCR products were also sequenced to verify the

sequence and to eliminate sequence artifacts.

The nucleotide sequences of the recombinant clones and

the PCR products from primer walking were performed by

Taq DyeDeoxy Teminator Cycle Sequencing using an

Automate DNA sequencer (PE Applied Biosystems; Model

377 version 3.0) and ABI100 DNA sequencing system

[Bioservice unit (BSU), NSTDA and Central Equipment

Laboratory, Mahidol University, Thailand].

In addition, the HPV genomic DNA was used as a

template for PCR amplification of the HPV genome by

using primers HPV12 (5V GTGAACTT TGTAAATAAC-

TTG 3V) and HPV6 (5V AAGGGTAAACCACG CACG 3V)and Proof-Reading Expand Long Template Taq DNA

polymerase (Roche). The PCR product of approximately 6

kb was then tailed with A nucleotide and ligated to pGEM-T

vector using pGEM-T Easy cloning kit (Promega). The

recombinant plasmids were screened and a clone HPV10

was selected. The nucleotide sequence of the clone HPV10

was determined on both strands and compared to the

original nucleotide sequencing data.

Cloning of the end termini

DNA sequences of the 5Vand 3Vends of HPV genomewere

obtained by cloning of PCR products. Modified PCR with

single primer was used to amplify both ends. High Tm

primers: SeqGC: 5V CCACCGCCGCAGCCGCAGTTG-

CCG 3V was used for the 5V end amplification and LeftGC:

5VGCCGACGCACCGCCCCTAGCTCC 3Vwas used for the


3V end. PCR amplification were performed using Taq DNA

polymerase (Promega) in the presence of 5%DMSO and 2M

betaine with increased ramping time. The PCR conditions

were 95 -C (2 min) and 40 cycles of 95 -C for 10 s (3 min

ramping from 95 -C to 70 -C), 70 -C for 30 s (2.5 min

ramping from 70 -C to 74 -C), 74 -C for 1 min and a cycle of

74 -C for 5 min. The amplified products were inserted into

pGEM-T Easy vector (Promega). Recombinant plasmids

were then transformed and maintained in Escherichia coli

STBL2 (Invitrogen) at low temperature (22–25 -C) and

sequenced using MegaBACE sequencer (Amersham).

Nucleotide sequences of viral DNA at the 3V and 5Vextremities containing stem–loop structures were per-

formed by using transcription sequencing (CUGA,WAGO

company).

Sequence analysis

All of the DNA sequence data were assembled and

analyzed using computer program. The restriction mapping

and open reading frame characterization were done using

BLAST (http://www.ncbi.nlm.nih.gov:80/gorf/orfig.cgi),

DNA strider, BCM Search Launcher (http://dot.imgen.

bcm.tmc.edu:9331/seq-util.html), DNAsis, Clone manager,

and GCG. The DNA and deduced amino acid sequences

were compared to the data in GenBank/EMBL, and

SWISSPORT database using FASTA and BLAST. The

multiple sequence alignments were done by using the

multiple alignment programs Clustal W (Thompson et al.,

1994) and Clustal X. Phylogenetic analysis based on NTP-

binding and helicase domain was performed using PUAP

program version 4.0.

The following selected NS-1 and VP proteins available in

GenBank were used in alignment and phylogenetic analysis;

HPVchin (AY008257), IHHNV (AF 273215), AalDNA

(X74945), AaeDNV (M37899), AAV2 (AF043303),

MVM (NC-001510), B19 (ABO30673), JcDNV (S47266),

BmDNV (M15123) and CeDNV (AF375296), BgeDNV

(NP_874381), Simain (AAC37927), Feline (BAA19023),

Mouse (NP_042345), and Pig-tailed macaque (AAF61213).

For Phospholipase A2 (PLA2) alignment; GmDNV

(AAA66966), JcDNV (Q90053), CeDNV (AF375296),

PPV (VCPVNA), MVM (VCPVIM), AAV4 (AAC58045),

PLA2 (I51017) and HPVmon (DQ002873).

Protein gel electrophoresis

Virus suspension was boiled for 10 min in cracking

buffer (50 mM Tris–HCl, pH 8.8, 10 mM EDTA, 2%

SDS, 1% h-mercaptoethanol) and electrophoresed on

12.5% polyacrylamide gels according to Laemmli

(1970). Gels were stained with Coomassie brilliant blue.

Standard molecular weight markers were phosphorylase b

(MW 97 kDa), bovine serum albumin (MW 66 kDa),

alcohol dehydrogenase (45 kDa), and carbonic anhydrase

(28 kDa).

Acknowledgments

The authors thank the Thailand Research Fund

(TRF) for financial support, Assist. Prof. Dr. Kusol

Pootanakit for his kind help in correcting the manuscript,

Prof. Dr. Boonsirm Withyachumnarnkul for shrimp

specimens and Dr. Miloslav Juricek for computer analysis

by GCG program. Dr. Pongsopee Attasart is a postdoc-

toral scholar of Faculty of Graduate Studies, Mahidol

University.

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