Laboratory and Epidemiology Communications

Identification of New Parvovirus DNA Sequencein Swine Sera from Myanmar
Minako Hijikata*, Kenji Abe), bin Maung Win2, Yohko K. Shimizu,
Naoto Keicho and Hiroshi Ybshikura3

Department ofRespiratoTy Diseases. Research Institute, International Medical Center ofJapan,
Toyama 1‑21‑1, Shinjuku‑ku, Tokyo 162‑8655,

IDepartmen i of Pathology, 3National Institute oflnfectious Diseases,

Toyama 1‑23‑1, Shinjuku‑ku, Too,0162‑8640 and2Department ofHepatology, Yangon General Hospital, Yangon. Myanmar
Communicated by Hiroshi Yoshikura(Accepted December 21, 2001)

Parvoviruses are among the smallest viruses of bothpreviously reported porcine parvovirus ( 1 ).

vertebrates and invertebrates. During an attempt to identify

Eighty‑six swine sera samples were collected in Myanmar.

the hepatitis E virus (HEV) genome, we.accidentally PCR‑
amplified a parvovirus DNA sequence ln swine sera from

Nucleic acids were extracted aom 50 LEI of sera with SMITEST
ExR&D (Genome Science Laboratories, Fukushima) andMyanmar. The sequence was only distantly related to thereverse‑transcribed using random hexamers with S uperscriptIIRNaseH‑ Reverse Transcriptase (Invitrogen, Carlsbad, Calif.,

USA). Then CDNA was subjected to a PCR with TaKaRa
ExTaq (TaKaRa, Shiga) and primers HE5‑I and HE5‑2 (2).

*Co汀eSpOnding author: Fax: +81‑3‑3207‑1038, E‑mail: minakoh@rijmcj.go.jp244Jpn. J. Infect. Dis., 54, 2001
No【‑codi【g region 186 【tGTP binding motif
Fig･ 1 I Overview of the ORFs and scqucnce alignment of the rcglOn conserved in cvcry vertebrate parvovirus nonstructural
protcin･ AAV I , adeno‑associatcdvirus 1 (GenBank accession n.o･ AFO63497); Muscovy, Muscovy duck parvoviruS (X75093);
BP3, bovine paⅣovims 3 (AF406967); Porcine, porcine paⅣovlruS (M38367); New, the sequence obtained in仙c prcscnt study.

Themal cycling condition was (95oC 1 min) + (95oC 15 sec,

The 5'and 3'non‑coding reg10nS including Inverted teminal

55oC 30 sec, 72oC 45 Eec) × 30 cycles + (72oC 7 min). PCR

repeats were not amplified in the present study.

products were electmphoresed in 2% agarose gels. The expected

Based on the homologleS aS described above, the sequencelength of the PCR product for the HEV sequence was 542 bp.

was provisionally named porcine parvovirus 2 sequence,However, none of the samples was positive fわr HEY; and athough the sequence was only distantly related to the previ‑

strong band of approximately 380 bp was observed in threeously reported porciTe.ParvOViruS ( 1 )･ Biochemical properties

samples･ These PCR products were purified with Wizard PCRof virion, Its infectlVlty and pathogenic nature should be

preps DNA Purification system (Promega, Madison, Wis.,

determined in the future.

USA) and directly sequenced using a BigDye teminator cycle
sequencing FS ready reaction kit (Applied Biosystems, FosterCity, Calif., USA). The obtained sequence was 339 nucleotides
inlength and was not identical to any sequences in the database,
based a BLAST search (DNA Data Bank orJapan [DDBJ],

Recently, two novel bovine parvoviruses were discoveredfrom bovine sera that had beenused as diluent while attemptlng
to丘nd hepatitis vimses (3). ln this study, we fわund a novel

DNA sequence related to parvovirus from swine sera while

attempting. to amplifyan HEV sequence･ These unexpectedShizuoka),and the most closely related sequence was a helicase

amplificatlOnS Of parvovirus DNA may indicate that thesequence of adeno‑associated virus 4 (GenBank accession
no. U89790).

unknown viral genomes.

advent of PCR teclmology accelerates discoveries of previouslyPrimers ql (5㌧GCG CAT TCG CCAAAC TAG CTC‑3')
and q2 (5′‑GTT TGC CCT TAA TGT CGA Tcc‑3′) Were

The nucleotide sequence reported in this paper will appear

designed based on the obtained sequence, and PCR wascarried out without reverse transcription. Nine of the 86

in DDBJ/GenBank/EMBL databases under the accession
number ABO76669.

(1 0･5%) samples were positive. When the extracted nucleic
acids were digested with Mung Beanでuclease (TaKaRa)

This work was supported by a grant f♭r htemational Healthaccording to the manufacturer's instuctlOnS, the sequence

Cooperation Research ( 1 2C‑4)from the Ministry of Labour,
Health and Welfare of Japan.

could not be ampli丘ed. These results suggested that the PCR

template was a slngle‑stranded DNA.
A sequence ofa total of5 kb was amplifiedfrom a sample
by three overlapplng PCR reactions with TaKaRa LA Taq

REFERENCES(TaKaRa), using primer set Ql and A2 (5'‑ATG TGG AATCGC AAT GCC AATl3'), primer set Q2 andAl (5'‑CGA GCT

I ･ Vasudevacharya, J･, Basak, S., Srinivas, R. V.and Compans,

GCA GTG GGC GTG GAC‑3'), and primer set (〕19 (5'‑GGT

infectious clone of porcine parvoviruS, strain NADL‑2.

R･ W･ (1990): The complete nucleotide sequence of an

ACG ACG AGC TCA TCA‑3') and (∋17 (5'一ccA GAr ArcⅥrology, 178, 611‑616.

GTC CTC GTAl3'). Two large open reading frames werefound in the 5 kb‑se9uence (Fig･ 1)･ ORF 1 coded 661 amlnO

2. Takahashi, K., Iwata, K., Watanabe, N., Hatahara, T.,Ohta, Y., Baba, K. and Mishiro, S. (2001): Full‑genome

acids, and the proteln Sequence Was most Closely related tonucleotide sequence of a hepatitis E vims strain that may

nonstmctural protein 1 ofMuscovy duck paⅣovirus (S52209)

be indigenous to Japan. Ⅵrology, 287, 9‑12.

by a FASTA search (DDBJ). ORF 2 coded 981 amino acids

3･ Allander, T･, Emerson, S. U., Engle, 良. E., Purcel, 良. H.

and the protein sequence was most closely related to capsid

and Bukh, J･ (200 1 ): A virus discovery method incoIPOrat‑protein of bovine parvovirus 3 (AF406967) by a FASmlng DNase treatment and its applicatlOn tO the identiflCa‑

search･ In the middle ofORF 1 , the helicase sequence, which istion of two bovine parvovims species. Proc. Natl. Acad.

most conserved in vertebrate parvoviruses, was found (Fig. 1 ).

Sci. USA, 98, 11609‑11614.

245