2008Phylogenetic Analysis and Isolation of CDV

198The Phylogenetic Analyses and Virus Isolation and of Canine Distemper Virus in Taiwan
INTRODUCTION
Canine distemper (CD) was first described in
Spain in 1791, and the agent (canine distemper virus,
CDV) was first isolated by Carre in 1905. The pathog-
enesis and clinical features of this infection have been
widely reported [1]. Over a period of 200 years after
the initial recognition of the disease, it has been the
most common viral infection in dogs, although CDV
can also infect wild carnivores and large cats [2,7,24].
CDV, which belongs to the genus Morbillivirus, fam-
ily Paramyxoviridae, is a non-segmented, single-
stranded negative RNA virus of approximately 15,690
nucleotides. Within the genome are six genes that en-
台灣獸醫誌 Taiwan Vet J 34 (4): 198-210, 2008
Phylogenetic Analysis and Isolation of Canine Distemper
Viruses in Taiwan
1,2
Chung-Tiang LIANG, 2
Ling-Ling CHUEH, 1
Kan-Hung LEE, 2
Hsuan-Shian HUANG,
3
Masashi UEMA, 3
Akira WATANABE, 3
Ryuichi MIURA, 3
Chieko KAI, 1
San-Chi LIANG,
1
Chun-Keung YU, *2
Chen-Hsuan LIU
1
2
3
（Received: August 25, 2008. Accepted: November 5, 2008）
[Liang CT, Chueh LL, Lee KH, Huang
HS, Uema M, Watanabe A, Miura R, Kai C, Liang SC, Yu CK, and *Liu CH. Phylogenetic Analysis and Iso-
lation of Canine Distemper Viruses in Taiwan. Taiwan Vet J 34 (4): 198-210, 2008. *Correspondence author
TEL/FAX: 886-02-23633289, E-mail: chhsuliu@ntu.edu.tw]

Chung-Tiang LIANG et al199
code for one envelope-associated protein (M), two
glycoproteins (the hemagglutinin/attachment protein
H and the fusion protein F), two transcriptase-associ-
ated proteins (the phosphoprotein P and the large pro-
tein L), and the nucleocapsid N that encapsulates the
viral RNA [28].
Dogs can be protected from the infection by im-
munisation with live attenuated vaccine. However,
due to the presence of maternal antibodies that inter-
fere with the vaccine virus, increasing incidences of
canine distemper in pups in suburban areas and urban
kennel shops have been noted in spite of vaccination
in Japan [14,15]. Antigenic changes have been specu-
lated as the cause for the currently prevalent CDV, ag-
ainst which the current vaccine fails to give complete
protection [14,17]. Although similar problems have
also been reported in Taiwan [22], the characteristics
of prevalent CDV in the field have not been investi-
gated due to difficulties in the isolation of wild type
CDV from field materials [12,16,25]. The most anti-
genic variation occurs in the H protein [3] and it has
been suggested that analyses of CDV genetic variation
should focus on this protein [9].
Many cell lines have been tested for their ability
to grow wild type CDV and maintain its virulence and
homogeneity. Among them, cells from the marmoset
B lymphoid line B95a have been reported to be highly
susceptible hosts [15,27]. The aim of this study, we
successfully obtained two CDV field isolates in Ta-
iwan by co-culturing peripheral blood leukocytes
(PBML) with B95a cells, four cloned H genes from
dogs infected with CDV, and totally six strains com-
pared their nucleic acid sequences of the H genes with
those of other reference isolates.
MATERIALS AND METHODS
Animals Seventeen non-vaccinated puppies, less
than 3-4 months old, from an animal shelter and 440
dogs clinically suspected of CDV infection from the
diagnostic services at National Taiwan University
were included in this study. In the 440 clinical cases, a
positive diagnosis of CDV infection was confirmed
for 166 (37.7 %) by P gene RT-PCR. From these posi-
tive cases, samples were selected quarterly for further
H gene analysis from October, 2003 to December,
2005. All 166 dogs showed at least one of the signs of
CDV infection, such as central nervous system signs
including seizure, ataxia, circling and myoclonus; di-
gestive signs including diarrhea, vomiting, depression
and anorexia; and respiratory signs including nasal
and ocular discharge, cough, dyspnea and sneezing.
Cell culture and virus isolation The B95a cel-
ls were propagated in RPMI medium 1640 (Gibco,
Grand Island, NY, USA) supplemented with 2 or 10%
heat-inactivated fetal bovine serum (FBS) (Gibco,
Grand Island, NY, USA) and 2 mM glutamine, 1.5 g/L
sodium bicarbonate, 4.5 g/L glucose, 10 mM Hepes,
and 1 mM sodium pyruvate under a 5% CO2 atmos-
phere. Virus isolation from peripheral blood leuko-
cytes (PBML) separated on Ficoll Hypaque solution
(Histopaque-1077, Sigma-Aldrich) was performed
using a modification of the method reported by Bli-
xenkrone-Moller [3]. Briefly, three to five mL hepa-
rin-stabilised whole blood of the 17 puppies with sus-
pected CDV infection were diluted 1:1 in RPMI 1640
supplemented with 2% heat-inactivated FBS. PBML
were isolated by centrifugation over a Ficoll density
gradient of 1.077 g/mL at 400 g for 45 min at room
temperature. The interface cells were harvested and
washed two times in RPMI 1640 with 2% FBS by cen-
trifugation at 400 g for 3 min. The 50 - 60% confluent
B95a cells cultured in 25 cm2
flasks were overlaid
with PBML for observation of cytopathic effect (CPE)
within 2-3 days. When B95a cells became 100% con-
fluent and no CPE was detected, a second blind pass-
age was carried out by removing the adherent B95a
cells with cell scrapers (Cell scraper 353085, Falcon,
Becton Dickinson Labware, Franklin Lakes, NJ, USA).
One mL of scraped adherent cells was suspended at
fresh 50% confluent B95a cells cultured in 25 cm2
fla-
sks (Corning, NY, USA) for the next passage and
examination.
CDV antigen test When the infected B95a cells
showed 40-50% CPE, the cells were scraped and four
drops of the suspension were dispensed into the Bioin-
dist International Technical (BIT) rapid color CDV
sample wells. The BIT rapid color CDV kit (Don-
ghwa-Ri, Bongdam-Eup, Hwaseong, KyungGi-Do,
Korea) was used to test for the presence of CDV anti-

gen. Briefly, whole blood (serum or plasma), ocular or
nasal discharge solution from suspected cases was dis-
pensed into the sample wells. The results were read by
naked eye after waiting for 5 to 10 min. One red or
purple band in the control line (C) with no apparent
band in the test line (T) indicated a negative result for
CDV infection. One red or purple band in the control
line (C) and one band in the test line (T) indicated a
positive result.
Indirect immunofluorescence assay (IFA)
When infected B95a cells in 25 cm2
flasks showed
40-50% CPE, the cells were harvested and spread on
10-well immunofluorescence assay (IFA) slides (Assi-
stant-Präzisions, Glaswarenfabrik, Karl Hecht KG,
Sondheim, Germany). The slides were air dried and
fixed by acetone/methanol (1:1) at 4℃ for 15 min. The
virus antigens in the fixed cells were examined micro-
scopically by IFA (Olympus BX51, Shinjku-Ku, To-
kyo, Japan). The primary antibody, a mouse anti-CDV
Ab(MCA 1893, Clone DV2-12; Serotec, Kidlington,
Oxford, UK) was diluted 1 in 150 in antibody diluent
(Ventana Medical System, Tucson, AZ, USA), and
then applied to the cells for 60 min at 37℃. The cells
were then immersed in phosphate buffered saline
(PBS; DakoCytomation, Carpinteria, CA, USA) for
10 min, followed by incubation with goat anti-mouse
IgG FITC-labelled secondary antibody (Chemicon
5008, Single Oak Drive Temecula, CA, USA) for 60 min
at 37℃. Then the cells were rinsed in PBS, counterstain-
ed with 0.02% Evans Blue dye and mounted with
mounting fluid (Chemicon 5013, CA, USA). When
the results were positive, the IFA test results were re-
confirmed by an eight-well Lab-Tek II Chamber slide
system (Nalge Nunc, Naperville, IL, USA). For this
assay, 4×104
fresh B95a cells in 0.5 mL RPMI me-
dium 1640 were seeded on each well of the slide and
incubated for one day, and then inoculated with 100
mL of field virus. Two to three days after inoculation,
once CPE was observed, the slides were fixed and
examined by IFA as described above.
Titration of CDV B95a cells infected with CDV
were frozen and thawed three times, and the titer of the
released virus was determined by endpoint dilution as-
says to calculate the tissue culture infective dose 50%
(TCID50). The assays were carried out in 96 well plates
(Costar 3599, Corning, NY, USA) by a limiting dilu-
tion method. In each well, 0.1 mL of 3×104
/mL B95a
cells was inoculated with 0.1 mL of sequential 10-fold
viral dilutions. During the next 7- 10 days, each well
was examined daily to detect the appearance of CPE,
and then viral titers were calculated.
RT-PCR of H gene Four positive results for the H
gene from the 166 CDV-infected clinical cases, NTU
1-2004, NTU 4-2003, NTU 3-2004, and NTU 2004,
and two CDV isolates from the virus-infected B95a
cells were analysed. Total RNA was extracted by
using the RNeasy Mini Kit (Qiagen). First-strand
cDNA synthesis was performed at 55 ℃ for one hour
with a specific reverse primer (CDVHR2, nucleotides
8928 to 8905, 5'-CAATTGARATGTGTATCATCATAC-3'),
by using SuperScriptTM
III reverse transcriptase (Invi-
trogen Inc., Carlsbad, CA, USA). H genes were amplif
ied by using Platinum®
Pfx DNA polymerase (Invitrog
en) with the primer set, CDHR2 and forward primer
(CDHF1, nucleotides 7063 to 7082, 5'-CTCAGGTAG-
TCCARCAATGC-3') yield an amplicon of 1,866 base
pairs, in the following conditions: 94℃ for 2 min, then
35 cycles of 94℃ for 15 sec, 53℃ for 30 sec and 68℃
for 2 min, with a DNA thermal cycler (Applied Biosys-
tems, CA, USA). The primers nucleotide position based
on the CDV strain 5804 (AY386315). The amplified
PCR products were purified from agarose gels with
the QIA quick PCR purification kit (Qiagen, Inc., CA,
USA).
Sequencing and accession numbers of H genes
from isolated CDVs Most purified PCR products
were directly sequencing using the primers used for
amplication. All direct sequencing reactions were
verified independently at least three times. Alternativ-
ely, some purified PCR products were 3'-end-adeny-
lated by incubation with dNTP and Platinum®
Taq
DNA polymerase, and then cloned into the pCR®
2.1-
TOPO®
TA cloning vector (Invitrogen, Inc., Carlsbad,
CA, USA). The direct and cloned H genes were se-
quenced using the ABI PRISM Big DyeTM
Terminator
Cycle Sequencing Ready reaction kit and an ABI 3730
XL DNA analyzer (Applied Biosystems, CA, USA).
Sequence analysis was performed with ABI PRISM

DNA sequencing analysis software V 5.2. The six
CDV strains identified in this study have been submit-
ted to GenBank. The code and GenBank accession
number for each of these strains are as follows: NTU
1-2004 (DQ191175), NTU 4-2003 (DQ191767), NTU
3-2004 (DQ191766), NTU 2004 (DQ191765), NTU
2005-1 (DQ887547), and NTU 2005-2 (DQ887548).
Phylogenetic analysis Phylogenetic analysis of
the nucleotide sequences of H genes was performed
with the LASERGENE Biocomputing (DNASTAR,
1998, Wisconsin, USA) software package. The tree
was inferred using the neighbor-joining (NJ) method
and the Kimura 2-parameter model, constructed with
the MEGA4 software package [29]. The codes, acces-
sion numbers and provenience for the sequences are as
follows (Table 1): Taiwan isolates, Dog/NTU 1-2004
(DQ191175, Taiwan), Dog/NTU 4-2003(DQ191767,
Taiwan), Dog/NTU 3-2004 (DQ191766, Taiwan), Dog/
NTU 2004 (DQ191765, Taiwan), Dog/ NTU 2005-1
(DQ887547, Taiwan), Dog/NTU 2005-2 (DQ887548,
Taiwan), and Dog/Taichung 2003 (AY378091, Taiwan);
China isolates, Dog/TN-China 2003 (AY390347, Chin-
a), Giant panda/China 1999 (AF178038, China), and
Lesser panda/China 1999 (AF178039, China); Japa-
nese isolates, Dog/KDK1 (AB025271, Japan), Dog/
Hamamatsu (D85754, Japan), Raccoon dog/Tanu 96
(AB016776, Japan), Dog/Ueno (D85753, Japan), Do
g/Yanaka (D85755, Japan), Dog/HM-3(AB040767,
Japan), Dog/HM-6(AB040768, Japan), Dog/26D
(AB040766, Japan), and Dog/98-002 (AB025270, Ja-
pan); European and USA isolates, Dog/404 (Z77671,
Germany), Dog/2544 (Z77672, Germany), Dog 324/03
(DQ494317, Italy 06), Mink/DK86 (Z47759, Den-
mark), Dog/DK91, B+C (Z47761, Denmark), Dog/
US89 (Z47762, USA), Black leopard/US91 (Z47763,
USA), Javelina/US89 (Z47764, USA), Raccoon/US89
(Z47765, USA); Old CDVs and vaccine isolates, On-
derstepoort (AF378705), Wyeth-Lederle (AF014953),
Convac vaccine (Z35493), Snyder Hill (AF259552,
USA), and A75-17(AF164967); the Arctic isolates,
Dog 179/04 (DQ226087, Italy 06) and Dog/GR88
(Z47760, Northern Greenland).
RESULTS
Isolation of field CDV strains Two CDV stra-
ins, NTU 2005-1 and NTU 2005-2, were successfully
isolated from 17 dogs with suspected CDV infection
between 2003 and 2005 in Taiwan. The appearance of
CPE was recognized in the fifth passage (12th day) of
B95a cells co-cultured with PBL of the NTU 2005-1
strain and in the second passage (6th day) of the NTU
2005-2 strain. The CPE was observed as a polygonal
to stellate or large round shape. Multinucleated giant
cells with more than 50 nuclei were observed (Fig.
1A). Mock controls of B95a cells showed normal ap-
pearances (Fig. 1B).
Indirect Immunofluorescence assay (IFA)
The IFA results for infected B95a cells showed posi-
tive labelling in the cytoplasm of altered cells. The
positive green-color labelling was more extensive and
distinct in the cytoplasm of syncytial cells with red-
color background (Fig. 2A). The same field counter-
staining with 0.02% Evans Blue also showed the posi-
tive FITC labeling cells in Fig. 2A was multinucleated
syncytial giant cells (Fig. 2B).
Titration of CDV The titers of the stock solution
of NTU 2005-1 and NTU 2005-2 strains were 3.1×103
TCID50/mL and 2.14×104
TCID50/mL, respectively.
Sequencing and phylogenetic analysis of H
gene The H gene of the six isolated CDV strains
was 1824 nucleotides long and the deduced amino
acid sequence was 607 amino acids long. Nucleic acid
sequences of the H genes of the NTU 2005-1 and NTU
2005-2 strains showed 98.6% identity, and their de-
duced amino acid sequences showed 98.8% identity
(Table 2). At the amino acid level, the NTU 2005-1
and NTU 2005-2 strains showed more than 98.2%
identity with Asia-1 isolates [10,13,26] (Hamamatsu,
KDK-1 and dog/TN-China 2003), 93.1% to 93.4%
identity with the Asia-2 isolate (98-002) [10,26], but
only 90.5% identity with the Onderstepoort vaccine
strain. At the nucleotide level, similar results were also
noted (Table 2). In the deduced H protein of the six
new isolates, nine asparagines that are potential sites
for N-linked glycosylation (N-X-S/T) were found at

amino acid positions 19-21, 149-151, 309-311,
391-393, 422-424, 456-458, 584-586, 587-589 and
603-605. Of these nine sites, the seventh (584-586)
that had been found in recent Japanese isolates
[10,11,13,18,19] was also identified in the Taiwan
strains. Within the H protein sequences of the six iso-
lates, 12 cysteine residues known to be important for
the secondary structure of the protein were conserved
Nucleotide sequence accession numbers of the H gene of the strains mentioned in this study
Strains Origin Accession Reference
NTU 1-2004 Dog, Taiwan DQ191175 Present study
NTU 2004 Dog, Taiwan DQ191765 Present study
Taichung 2003 Dog, Taiwan AY378091 Hsieh et al., 2003, unpublished
TN-China 2003 Dog, China AY390347 Meng et al.,2003, unpublished
China 1999 Giant panda, China AF178038 Li et al., 1999, unpublished
China 1999 Lesser panda, China AF178039 He et al., 1999, unpublished
KDK1 Dog, Japan AB025271 [26]
Hamamatsu Dog, Japan D85754 [13]
Tanu 96 Raccoon dog, Japan AB016776 Kai and Ohashi,1998, unpublished
Ueno Dog, Japan D85753 [13]
Yanaka Dog, Japan D85755 [13]
HM-3 Dog, Japan AB040767 [10]
HM-6 Dog, Japan AB040768 [10]
26D Dog, Japan AB040766 [10]
98-002 Dog, Japan AB025270 [26]
404 Dog, Germany Z77671 [9]
2544 Dog, Germany Z77672 [9]
324/03(Italy 2006) Dog, Italy DQ494317 [23]
DK86 Mink, Denmark Z47759 [4]
DK91, B+C Dog, Denmark Z47761 [4]
US89 Dog, USA Z47762 [4]
US91 Black leopard, USA Z47763 [4]
US89 Javelina, USA Z47764, [4]
US89 Raccoon, USA Z47765 [4]
GR88 Dog,Northern Greenland Z47760 [4]
Onderstepoort vaccine AF378705 [31]
Wyeth-Lederle vaccine AF014953 [28]
Convac vaccine Z35493 [17]
Snyder Hill vaccine AF259552 [9]
A75-17 vaccine AF164967 Wiederkehr et al.,1999 unpublished
179/04 (Italy 2006) Dog, Italy DQ226087 [23]

at positions 139, 154, 188, 283, 296, 377, 382, 390,
490, 566, 575 and 602 (Fig. 3). Phylogenetic analysis
of the H gene showed that all of the six CDV field stra-
ins isolated in this study belong to the Asia-1 group
that is distinct from the Asia-2 group (Fig. 4).
DISCUSSION
This study of CDV isolation was first carried out
in tissue homogenates of adult CDV-infected dogs di-
agnosed by RT-PCR or histopathology, but where this
failed to show CPE in either B95a or MDCK cell lines
as previously reported [9,21]. Thus, we also employed
a co-culture of PBML from 17 unvaccinated puppies
with suspected CDV infection with B95a cell lines.
The combination of IFA and CDV antigen tests suc-
cessfully identified the virus and found CPE in differ-
ent passages of these co-cultures. Virus isolation has
been reported to be easier from young puppies than
adult dogs. Uema et al. [30] reported that two to three
month-old infected dogs with or without vaccination
history can be used for CDV isolation. Co-cultivation
of mononuclear cells with B95a cells also yielded hig-
her CDV isolation rates [15,27]. False negative IFA
results were occasionally noted when compared with
antigen tests, especially when CPE was present in 100
% of the cells or when scraped infected B95a cells
were used. The chamber slide method [13] for IFA
showed better detection of CDV positive results than
the scraped IFA slide method in this study. The titers
of the two stock strains were 3.1×103
and 2.14×104
TCID50/mL, respectively. The titers were a little lower
than those of CDV isolated from lymph nodes, large
intestines, tonsil or spleen [19].
Recent extensive acquisition of molecular data
on the H gene of CDVs has allowed the identification
of six distinct phylogenetic clades with regard to their
geographic origin (Asia-1 [10,13,26], Asia-2 [10,26],
Europe [4,5,6,9,23,30], USA [4,20,30], Arctic [6,23],
and Old CDVs [9,17,28,31]. In a given geographical
region, two to three genotypes of CDVs can be present
[4,5,6,8,10,20,23,26,30]. Based on the phylogenetic
analysis of the H gene, six Taiwan strains were repor-
ted in this study, including NTU 1-2004, NTU 4-2003,
NTU 3-2004, NTU 2004, NTU 2005-1, and NTU
2005-2. These strains belong to the Asia-1 group
NucleotideandaminoacidsequenceidentitiesbetweentheCDVHgenes(%).Nucleotideidentitiesarepresentintheupperhalfofthematrixandaminoacid
identitiesinthelowerhalf
Nucleotide(%)
VirusNTU2005-1NTU2005-2NTU2004
Dog/Taichung
2003
TN-China
2003
Dog
Hamamatsu
Dog
KDK-1
Dog
98-002
Onderstepoort
Amino
Acid(%)
NTU2005-1*98.699.299.298.799.29892.790.8
NTU2005-298.8*98.498.498.398.797.792.190.3
NTU200499.298.2*99.598.69997.892.390.8
Dog/Taichung200399.398.499.3*98.69997.892.391.1
TN-China20039998.398.798.8*98.897.792.291.2
DogHamamatsu99.298.598.598.798.7*98.492.791.1
DogKDK-198.898.298.298.498.398.5*93.191.2
Dog98-00293.493.193.193.393.293.193.4*91
Onderstepoort90.590.590.59190.690.190.690.1*
*=100%

[10,13,26], which is distinct from the old CDVs and
Asia-2 groups [10,26].
The six CDV strains identified in this study were
tightly related to each other (< 2% amino acid varia
tion). This might be due to all the samples coming from
northern Taiwan during a limited period (2003-2005).
However, strains in the same clade of the phylogenetic
tree showing more than 95% amino acid similarity
(< 5 % amino acid variation) in the H gene may be
considered to belong to the same genotype [26]. The
8137th nucleotide on the viral genome (the 1059th one
on the H gene), a thymine (T) in the vaccine strains, is
substituted to cytosine (C) in the wild-type strains [6].
This point mutation was also found in all six Taiwa-
nese strains in this study. The H gene RFLP analysis
using EcoRV and SspI [30] may be useful for the de-
tection of Taiwanese field strains. The field CDV stra-
ins in this study possessed nine asparagines that were
Syncytium type of CPE in infected B95a cells (A) B95a cells infected with NTU 2005-2 CDV strain. Multinucleated
syncytial giant cells with more than 50 nuclei were observed. (B) Mock control of the B95a cells showed normal appear-
ance. Bar, 50 m.
1A 1B
2A 2B
Detection of virus antigen in B95a cells infected by a NTU 2005-1 strain (A) The positive green-color FITC labelling
was distinct in the cytoplasm of syncytial cells with red-color background. (B) Same field as in 2A, counterstaining of in-
fected B95a cells showed multinucleated syncytial CPE appearance. Bar, 40 m.

potential sites for N-linked glycosylation in their de-
duced H protein. The same nine potential sites were
also reported in Japanese strains [10,11,13,18,19].
Among them, the seventh (584-586) site is especially
characteristic of the Taiwanese strains in this report
and for recently reported Japanese strains.
Multiple alignment of deduced amino acid sequences of the H gene of the Taiwan CDV strains. CDV strains are
referenced in Table 1. Identical residues are shown by dots. The potential N-linked glycosylation sites are boxed. An aster-
isk indicates the seventh potential N-linked glycosylation site.

Neighbor-Joining tree of CDV strains analysed in this study based on the nucleotide alignment of the H protein.
CDV strains are referenced in Table1.The optimal tree with the sum of branch length = 0.39956060 is shown. The bootstrap
values indicate the number of times that each branching was found in 1000 bootstrap analyses. The evolutionary distances
were computed using the Kimura 2-parameter method and are in the units of the number of base substitutions per site.
Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated
from the dataset. There were a total of 1814 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4
[29].

In conclusion, the genetic variability among the-
se sites of the CDV isolates is an important consider-
ation for elucidating the immune response [9,13,19].
Furthermore, phylogenetic analysis of the H gene sho-
wed that the six isolates in this study were closely re-
lated to Chinese and Japanese isolates. These similari-
ties between Taiwanese, Chinese and Japanese isola-
tes might reflect the common prevalence of CD in Ta-
iwan, China and Japan.
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臺灣地區犬瘟熱病分離及血球凝集素基因分析
1,2
梁鍾鼎 2
闕玲玲 1
李泔泓 2
黃宣憲 3
Masashi UEMA 3
Akira WATANABE
3
Ryuichi MIURA 3
Chieko KAI 1
梁善居 1
余俊強 *2
劉振軒
1
財團法人國家實驗研究院國家實驗動物中心
2
國立臺灣大學獸醫專業學院獸醫學研究所
3
日本東京大學醫學研究所實驗動物中心
（收件日期：97 年 8 月 25 日。接受日期：97 年 11 月 5 日）
摘要於 2003-2005 年間自 17 隻未經疫苗注射之發病幼犬，應用患犬血液單核球與 B 95a 細胞株共同培養之技
術，分離出兩株具誘發融合細胞病變之病，經免疫螢光染色及抗原測試確認為犬瘟熱病。將此兩株病之血球凝
集素基因(H)，與同期間自台灣大學動物醫院臨床送檢病例之另外 4 株犬瘟熱病之 H 基因進行核酸定序。經比對序
列與樹狀圖分析發現，本土病株皆有 9 個 N 連結配醣位，其中第 7 個配醣位為日本或中國大陸流行之亞洲 1 型犬
瘟熱病所特有。本研究顯示臺灣地區所流行之犬瘟熱病，經其 H 基因分析屬亞洲 1 型。[梁鍾鼎、闕玲玲、李泔
泓、黃宣憲、Masashi UEMA、Akira WATANABE、Ryuichi MIURA、Chieko KAI、梁善居、余俊強、劉振軒。臺灣地
區犬瘟熱病分離及血球凝集素基因分析。台灣獸醫誌 34 (4): 198-210，2008。*聯絡人TEL/FAX: 886-02-2363 3289，
E-mail: chhsuliu@ntu.edu.tw]

2008Phylogenetic Analysis and Isolation of CDV

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