Rubella epidemic in Vietnam: Characteristic of rubella virus genes from pregnant women and their fetuses/newborns with congenital rubella syndrome

Abstract

Background:
Rubella remains poorly controlled in Southeast Asia, including Vietnam.

Objectives:
The aim of this study was to characterize rubella virus spread in Vietnam during 2011-2012.

Study design:
Amniotic fluid, throat swab and placenta samples were collected from 130 patients (110 cases from pregnant women with suspected rubella and 20 cases from fetuses/newborns). Viral RNA was obtained directly from clinical specimens, amplified by PCR, and then the E1 gene containing 739 nucleotides recommended by the WHO to identify the viral genotypes was sequenced.

Results:
By screening with real-time PCR, viral RNA was detectable in amniotic fluids from 103 out of 110 (93.6%) pregnant women with suspected rubella and in the throat swabs from all of 20 (100%) fetuses/newborns. In addition, viral RNA was also detected in the placenta from all cases of fetuses/newborns. All of 20 fetuses/newborns presented with congenital cataract. Twenty-four strains with the E1 gene were obtained by PCR. Using phylogenetic analysis with rubella reference sequences, all of the strains were found to be genotype 2B. Interestingly, 94% (30/32) of Vietnamese strains, including 9 strains from the database, formed an independent cluster within the genotype 2B suggesting that indigenous viruses are prevalent in this region.

Conclusions:
Rubella virus identified in Vietnam belonged to the genotype 2B. Importantly, the infection rate of rubella virus in fetuses/newborns was 100% and all of them had congenital cataract. Our results indicate an establishment of rubella prevention in this area is an urgent task in order to improve maternal and child health.

Full text

Journal
of
Clinical
Virology
57 (2013) 152–
156
Contents
lists
available
at
SciVerse
ScienceDirect
Journal
of
Clinical
Virology
jo
u
r
n
al
hom
epage:
www.elsevier.com/locate/jcv
Rubella
epidemic
in
Vietnam:
Characteristic
of
rubella
virus
genes
from
pregnant
women
and
their
fetuses/newborns
with
congenital
rubella
syndrome
Van
Hung
Phama,b,
Thong
Van
Nguyenc,
Truc
Thanh
Thi
Nguyenc,
Linh
Duy
Dangb,
Ngoc
Hieu
Hoangb,
Truong
Van
Nguyenc,
Kenji
Abea,d,∗
aBiomedical
Laboratory,
School
of
Medicine,
University
of
Medicine
and
Pharmacy
in
Ho
Chi
Minh
City,
Ho
Chi
Minh
City,
Viet
Nam
bLaboratory
for
Molecular
Diagnostics,
Nam
Khoa
Biotek
Co.,
Ho
Chi
Minh
City,
Viet
Nam
cDepartment
of
Pathology
and
Cytology,
Hung
Vuong
Hospital,
Ho
Chi
Minh
City,
Viet
Nam
dDepartment
of
Pathology,
National
Institute
of
Infectious
Diseases,
Tokyo,
Japan
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
8
November
2012
Received
in
revised
form
31
January
2013
Accepted
10
February
2013
Keywords:
Rubella
Rubella
virus
Virus
genotype
Congenital
rubella
syndrome
Vietnam
Southeast
Asia
a
b
s
t
r
a
c
t
Background:
Rubella
remains
poorly
controlled
in
Southeast
Asia,
including
Vietnam.
Objectives:
The
aim
of
this
study
was
to
characterize
rubella
virus
spread
in
Vietnam
during
2011–2012.
Study
design:
Amniotic
fluid,
throat
swab
and
placenta
samples
were
collected
from
130
patients
(110
cases
from
pregnant
women
with
suspected
rubella
and
20
cases
from
fetuses/newborns).
Viral
RNA
was
obtained
directly
from
clinical
specimens,
amplified
by
PCR,
and
then
the
E1
gene
containing
739
nucleotides
recommended
by
the
WHO
to
identify
the
viral
genotypes
was
sequenced.
Results:
By
screening
with
real-time
PCR,
viral
RNA
was
detectable
in
amniotic
fluids
from
103
out
of
110
(93.6%)
pregnant
women
with
suspected
rubella
and
in
the
throat
swabs
from
all
of
20
(100%)
fetuses/newborns.
In
addition,
viral
RNA
was
also
detected
in
the
placenta
from
all
cases
of
fetuses/newborns.
All
of
20
fetuses/newborns
presented
with
congenital
cataract.
Twenty-four
strains
with
the
E1
gene
were
obtained
by
PCR.
Using
phylogenetic
analysis
with
rubella
reference
sequences,
all
of
the
strains
were
found
to
be
genotype
2B.
Interestingly,
94%
(30/32)
of
Vietnamese
strains,
includ-
ing
9
strains
from
the
database,
formed
an
independent
cluster
within
the
genotype
2B
suggesting
that
indigenous
viruses
are
prevalent
in
this
region.
Conclusions:
Rubella
virus
identified
in
Vietnam
belonged
to
the
genotype
2B.
Importantly,
the
infection
rate
of
rubella
virus
in
fetuses/newborns
was
100%
and
all
of
them
had
congenital
cataract.
Our
results
indicate
an
establishment
of
rubella
prevention
in
this
area
is
an
urgent
task
in
order
to
improve
maternal
and
child
health.
© 2013 Elsevier B.V. All rights reserved.
1.
Background
Rubella
is
an
acute
infectious
disease
that
normally
has
a
mild
clinical
course.
However,
infections
during
pregnancy,
especially
before
week
12
of
gestation,
can
cause
severe
birth
defects
known
as
congenital
rubella
syndrome
(CRS).1Clinical
signs
of
CRS
include
cataract,
glaucoma,
heart
disease,
loss
of
hearing,
and
pigmentary
retinopathy.
Therefore,
it
is
very
important
to
control
the
rubella
in
order
to
improve
maternal
and
child
health
worldwide.
Rubella
virus
(RV)
has
no
host
other
than
humans
and
is
thought
to
consist
of
a
single
serotype.
However,
information
on
∗Corresponding
author
at:
Department
of
Pathology,
National
Institute
of
Infec-
tious
Diseases,
Toyama
1-23-1,
Shinjuku-ku,
Tokyo
162-8640,
Japan.
Tel.:
+81
3
4582
2702;
fax:
+81
3
5285
1189.
E-mail
address:
kenji@kih.biglobe.ne.jp
(K.
Abe).
the
epidemiologic
characteristics
of
the
virus,
such
as
antigenic
variation,
virulence,
and
phylogenetic
relationship
between
circu-
lating
strains,
is
in
short
supply.
In
fact,
there
have
been
only
a
very
few
epidemiologic
studies
conducted
in
Southeast
Asia.2For
the
prevention
of
CRS,
rubella-containing
vaccine
has
been
used
in
vaccination
programs
worldwide,
but
not
in
many
countries
in
the
Asian
continents.
The
RV
contains
three
structural
polypeptides:
envelope
polypeptides
E1
and
E2
and
capsid
polypeptide
(C).
It
has
a
single-stranded,
positive-sense
RNA
of
9762
nucleotides
as
its
genome.
The
5-terminal
two-thirds
of
the
genome
encode
the
nonstructural
polypeptides
in
a
single
open-reading
frame,
and
the
3-terminal
one-third
encodes
the
structural
polypeptides
in
a
single
open-reading
frame
in
the
sequence
of
5-C-E2-E1-3.
The
E1
glycoprotein
is
considered
immunodominant
in
the
humoral
response
induced
against
the
structural
proteins
and
contains
neu-
tralizing
and
hemagglutinating
determinants.3–7 A
739-nucletoide
1386-6532/$
–
see
front
matter ©
2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jcv.2013.02.008

V.H.
Pham
et
al.
/
Journal
of
Clinical
Virology
57 (2013) 152–
156 153
(nt)
region
within
the
E1
gene
(nt
8731–9469)
is
accepted
as
the
minimum
amount
of
sequence
information
required
for
molecular
epidemiological
purposes
and
genotypes.8,9 Therefore,
we
focused
on
the
E1
gene
for
the
molecular
based
epidemiologic
study
in
RV
sequences
from
Vietnamese
patients.
Although
information
about
genotypic
distribution
is
available
for
some
countries,
especially
those
pursuing
elimination,
data
on
the
genomic
characterization
of
RV
are
lacking
in
many
countries,
particularly
in
developing
countries
including
Vietnam.
2.
Objectives
The
aim
of
this
study
was
to
carry
out
genomic
characterization
of
RV
detected
in
pregnant
women
and
fetuses/newborns
with
CRS
in
Vietnam
during
the
period
from
2011
to
2012.
3.
Study
design
Clinical
samples.
Samples
obtained
from
110
pregnant
women
who
were
clinically
suspected
rubella
(18–40
years
old),
10
abor-
tion
fetus
(19–25
weeks
of
age)
and
10
newborns
from
suspected
disease
pregnant
women.
All
pregnant
women
patients
lived
in
Ho
Chi
Minh
City
and
its
environs
in
Vietnam.
Clinical
specimens
were
collected
during
the
period
from
2011
to
2012
and
kept
frozen
at
−80 ◦C
until
analyzed.
Clinical
specimens
consisted
of
an
amni-
otic
fluid,
throat
swab
(all
from
fetuses/newborns)
and
placenta
obtained
from
patients
at
the
Hung
Vuong
Hospital,
Ho
Chi
Minh
City,
Vietnam.
Samples
obtained
from
5
cases
of
fetuses/newborns
overlap
with
their
mothers
tested
in
this
study.
Determination
of
antibody
to
RV.
RV-specific
IgM
antibody
was
determined
in
cord
blood
using
the
Elecsys
Rubella
IgM
kit
(Roche,
Indianapolis,
IN,
USA).
Isolation
of
virus
by
tissue
culture.
To
isolate
viruses
by
tissue
culture,
amniotic
fluids
from
4
cases
were
inoculated
into
vero
cells.
The
vero
cells
inoculated
with
clinical
specimens
were
cultured
at
37 ◦C
for
7
days.
The
supernatant
and
cultured
cells
were
used
to
obtain
RV
genome
by
PCR
with
the
following
methods.
Determination
of
RV
gene
by
real-time
PCR.
For
screening
of
RV
RNA
determination,
the
real-time
PCR
was
used.
All
primers
were
designed
from
E1
gene
of
rubella
virus.
The
sequences
for
primers
and
probe
used
for
the
real-time
PCR
were
5-CAT
CTG
GAA
TGG
CAC
ACA
GC-3(rubella
tqF,
sense,
nt
8476–8495)
and
5-CTA
CAA
GCA
GTA
CCA
CCC
CAC-3(rubella
tqR,
antisense,
nt
8601–8581),
and
FAM/BHQ1-fluorescence
labeled
probe
(5-FAM-TGC
ACC
TTC
TGG
GCT
GTC
AAC
GC-BHQ1-3(rubella
tqPr,
sense,
nt
8501–8523).
Nucleotide
position
is
based
on
rubella
virus
vaccine
strain
wistar
RA
27/3
(accession
#
FJ211587).
Briefly,
total
RNA
was
extracted
from
clinical
specimens
and
cultured
cells
using
the
RNA
extrac-
tion
kit
(NKRNAPREP
kit,
Nam
Khoa
Biotek
Co.,
Ho
Chi
Minh
City,
Vietnam).
Viral
cDNA
was
synthesized
with
mixture
of
random
primer
and
oligo(dT)
primer
using
iScript
reverse
transcriptase
(Bio-Rad
Laboratories,
CA,
USA)
with
the
following
condition:
25 ◦C,
5
min,
42 ◦C,
30
min
and
85 ◦C,
5
min.
Five
microliters
of
cDNA
product
was
placed
in
the
real-time
PCR
buffer
containing
Platinum
Taq
(Invitrogen,
Carlsbad,
CA,
USA)
and
amplified
with
the
following
condition:
95 ◦C,
3
min
30
s
then
50
cycles
consisting
of
94 ◦C,
30
s
and
60 ◦C,
1
min.
The
sensitivity
of
this
real-time
PCR
method
was
270
copies
of
RV/ml.
Detection
of
RV
gene
by
nested
RT-PCR
and
sequence.
For
sequenc-
ing
and
genotyping
of
the
RV
gene,
using
the
amniotic
fluid
and
the
throat
swab,
nested
RT-PCR
was
carried
out
with
primers
designed
from
the
E1
gene
of
RV.
A
908-bp
fragment
was
amplified
by
the
primer
combination
of
RV8633F/RV9540R
in
the
E1
gene
containing
the
WHO-recommended
sequence
window
(739
bp;
nt
8731–9469).
The
sequences
of
all
primers
used
in
this
study
are
listed
in
Table
1.
As
shown
in
Table
1,
we
used
method
1
which
can
yield
a
908-bp
single
fragment
by
PCR.
Alternatively,
method
2
consisting
of
two
fragments
(485
bp
and
596
bp,
respectively)
was
also
used
when
method
1
was
not
successful.
Viral
RNA
was
heated
to
95 ◦C
for
1
min
then
cooled
on
ice
immediately
before
adding
pre-mixture
for
cDNA
synthesis.
The
cDNA
was
synthesized
by
the
same
method
as
mentioned
above.
Five
microliters
of
cDNA
was
placed
in
PCR
buffer
containing
Platinum
Taq
and
360
GC
Enhancer
(20%
v/v;
Applied
Biosystems,
Foster
city,
CA,
USA)
due
to
the
RV
genome
having
an
extremely
high
GC-rich
sequence.
Amplification
conditions
included
pre-incubation
at
95 ◦C,
5
min,
followed
by
40
cycles
consisting
of
94 ◦C,
30
s,
60 ◦C,
30
s
and
72 ◦C,
1
min
for
the
1st
round
PCR
and
94 ◦C,
30
s,
65 ◦C,
30
s
and
72 ◦C,
1
min
for
the
2nd
round
PCR.
Amplicons
were
analyzed
by
electrophoresis
on
1%
agarose
gels
staining
with
ethidium
bromide
and
recovered
using
the
promega
Wizard®SV
Gel
and
PCR
Clean-Up
System
(Promega,
Madison,
WI,
USA).
The
amplicons
were
subjected
to
direct
sequencing
using
the
ABI
PRISMTM Big
Dye
Terminator
Cycle
Sequencing
Ready
Reac-
tion
Kit
(Applied
Biosystems),
on
a
capillary
sequencer
model
3130
(Applied
Biosystems).
Characterization
of
RV
gene
by
phylogenetic
analysis.
For
phy-
logenetic
analysis,
obtained
nucleotide
sequences
were
multiple
aligned
with
CLUSTAL
W,
version
1.81.
The
distance
matrix
of
the
nucleotide
substitutions
among
each
sequence
was
estimated
by
the
eight-parameter
method10 and
phylogenetic
trees
were
con-
structed
by
the
neighbor-joining
method11 from
the
matrix.
These
procedures
were
computed
with
Phylo
win,
version
1.212 on
a
DEC
alpha
2000
server,
and
the
trees
were
drawn
with
TreeView,
version
1.5.2.13 To
confirm
the
reliability
of
the
pairwise
comparison
and
phylogenetic
tree
analysis,
bootstrap
resampling
and
reconstruc-
tion
were
carried
out
1000
times.
Bootstrap
values
greater
than
60%
were
considered
supportive
of
the
observed
groupings.
In
addition
to
our
sequences
recovered
in
this
study,
17
reference
strains
rec-
ommended
by
WHO
and
32
strains
obtained
from
database
were
used
as
reference
strains
of
known
genotypes.
Accession
numbers
submitted
to
database.
Nucleotide
sequence
data
of
RV
sequences
from
Vietnamese
patients
are
available
in
the
DDBJ/EMBL/GenBank
databases
under
the
accession
numbers
AB706298–AB706308
and
AB745027–AB745039
for
RV
E1
gene.
4.
Results
By
screening
with
real-time
PCR,
RV
RNA
was
detectable
in
amniotic
fluids
from
103
out
of
110
(93.6%)
pregnant
women
and
throat
swabs
in
all
of
20
(100%)
fetuses/newborns.
Virus-
specific
IgM
antibody
was
positive
in
cord
blood
in
19
of
20
(95%)
fetuses/newborns
tested.
In
addition,
RV
RNA
was
detected
in
the
placenta
tissues
in
all
cases
of
fetuses/newborns.
At
gross
examina-
tion,
all
of
20
fetuses/newborns
presented
with
congenital
cataract.
None
of
the
pregnant
women
in
this
study
had
an
obvious
history
of
vaccination
to
rubella.
Among
RV
RNA-positive
cases,
sufficient
amplicons
in
the
E1
gene
(908
bp)
covering
the
WHO
recommended
region
were
obtained
in
24
cases
(17
samples
of
amniotic
fluid
from
pregnant
women
and
7
samples
of
throat
swabs
from
fetuses/newborns)
by
nested
RT-PCR
and
then
sequenced.
Virus
strains
identified
were
named
according
to
the
WHO
systematic
nomenclature
for
RV.9
Vietnamese
RV
strains
recovered
in
this
study
were
analyzed,
in
comparison
with
the
WHO
reference
strains
and
strains
from
the
database
to
cover
all
genotypes.
The
nt
difference
between
the
Vietnamese
strains
ranged
from
0.2%
to
2.1%.
The
mean
divergence
within
all
Vietnamese
viral
sequences
was
2.2%
and
6.1%
relative
to
the
WHO
2B
reference
strains.
The
phylogenetic
tree
showed
all
Vietnamese
strains
sequenced
in
the
present
study
belonged
to
the
genotype
2B
group
with
the

154 V.H.
Pham
et
al.
/
Journal
of
Clinical
Virology
57 (2013) 152–
156
Table
1
Primers
used
for
detection
and
genotyping
of
rubella
virus
RNA
by
nested
RT-PCR.
AAA
5’
3’
p150p90CE2 E1
6512→7412→8258→9703
41→6391
9762 bases*
WHO recom
mend
ed sequ
ence
window
(nt 8731-9469; 739 bp)
Non-structura
l Str
uctura
l
Method 1
Method 2 A
B
908 bp
Method
1
RV8537F:
5-GGG
TAC
GCG
CAG
CTG
GCG
TC-3(sense,
nt
8537–556)
RV9572R:
5-AGG
TCT
GCC
GGG
TCT
CCG
AYA
C-3(antisense,
nt
9572–551)
RV8633F:
5-AGC
GAC
GCR
GCS
TGC
TGG
GG-3(sense,
nt
8633–652)
RV9540R:
5-TGT
GTG
CCR
TAC
ACC
ACG
CC-3(antisense,
nt
9540–521)
Primer
pairs:
RV8537F/RV9572R
(1036
bp)
for
the
1st
round
PCR
and
RV8633F/RV9540R
(908
bp)
for
the
2nd
round
PCR
Method
2
For
fragment
A
RV8537F:
5-GGG
TAC
GCG
CAG
CTG
GCG
TC-3(sense,
nt
8537–556)
RV9117R:
5-CAY
TTG
CGC
GCC
TGM
GAG
CC-3(antisense,
nt
9117–098)
RV8633F:
5-AGC
GAC
GCR
GCS
TGC
TGG
GG-3(sense,
nt
8633–652)
Primer
pairs:
RV8537F/RV9117R
(581
bp)
for
the
1st
round
PCR
and
RV8633F/RV9117R
(485
bp)
for
the
2nd
round
PCR
For
fragment
B
RV8945F:
5-TGG
GCC
TCY
CCG
GTT
TG-3(sense,
nt
8945–961)
RV9572R:
5-AGG
TCT
GCC
GGG
TCT
CCG
AYA
C-3(antisense,
nt
9572–551)
RV9540R:
5-TGT
GTG
CCR
TAC
ACC
ACG
CC-3(antisense,
nt
9540–521)
Primer
pairs:
RV8945F/RV9572R
(628
bp)
for
the
1st
round
PCR
and
RV8945F/RV9540R
(596
bp)
for
the
2nd
round
PCR
*Nucleotide
position
is
based
on
rubella
virus
vaccine
strain
wistar
RA
27/3
(accession
#
FJ211587).
0.01
• RV
s Miam
i FL
US
A 32
02 [1
j]
• RVi SLV 02 [1C]
• RV
i P
AN 99 [1C]
• RV
i To
yama JPN
67 [1a]
• RV
i Jerusalem
ISR
75 [1B]
• RV
i L
inq
ing
CHN 00 [1F]
• RV
i Tok
yo JPN
90 [1D]
• RV
i M
YS 01 [1E]
• RV
i M
ilan
ITA
46
92 [1
i]
• RVi Ontario CAN 27 05 [1G]
• RV
i Tomsk
RUS 05 [1h]
• RV
i Mo
scow RUS
67 [2C]
• RVi Moscow RUS 97 [2C] FJ875030_CHN
[2A
]
• RV
i Be
ijing
CHN 79 [2A]
DQ085338_ISR
[2B
]
• RV
i Tel
Aviv ISR
68 [2B]
AY16136
2_ITA
[2B
]
AF039134_IND
[2B
]
DQ085342_KOR [2B
]
• RV
i An
qing
CHN 00
2 [2B]
AY161370_ITA [2B]
* HQ893749_VNM [2B]
* HQ89
375
2_VN
M [2B]
JN635292_USA [2B]
AY96822
0_USA [2B
]
• RV
i Se
att
le US
A 16
00 [2B]
JN6352
96_USA
[2B
]
JN6352
95_USA
[2B
]
FN547017_FRA
[2B
]
GQ37457
2_CHN
[2B
]
EU240900_GBR
[2B
]
HM212630_BR
A [2B
]
JN5820
35_ARG [2B
]
FN547021_F
RA [2B
]
FR717207_BIH
[2B
]
FR717215_BIH
[2B
]
HM212632_BRA [2B]
GU254
251_BR
A [2B
]
GU254
253_BR
A [2B
]
* HQ89
375
1_VN
M [2B]
AB745034
_RVs/HoCh
iMinh.VNM
/43
.11 [2B]
CRS
AB745039
_RVi/Ho
ChiMinh
.VN
M/19.12 [2B]
* HQ89
375
0_VN
M [2B]
* HQ89
375
8_VN
M [2B]
* HQ893755_VNM [2B]
AB745029
_RVs/HoCh
iMinh.VNM
/23
.11 [2B]
AB745035
_RVs/HoCh
iMinh.VNM
/37
.11 [2B]
CRS
* HQ89
375
6_VN
M [2B]
* HQ89
375
7_VN
M [2B]
AB745028
_RVs/HoCh
iMinh.VNM
/23
.11 [2B]
AB706299
_RVs/HoCh
iMinh.VNM
/27
.11 [2B]
AB706301
_RVs/HoCh
iMinh.VNM
/26
.11 [2B
]
AB745038
_RVi/Ho
ChiMinh
.VNM
/5.12 [2B]
CRS
AB745027
_RVs/HoCh
iMinh.VNM
/19
.11 [2B]
AB745033
_RVs/HoCh
iMinh.VNM
/43
.11 [2B]
CRS
AB745030
_RVs/HoCh
iMinh.VNM
/23
.11 [2B]
AB745032
_RVs/HoCh
iMinh.VNM
/41
.11 [2B]
CRS
AB745037
_RVi/Ho
ChiMinh
.VNM
/41.11 [2B]
CRS
AB745031
_RVs/HoCh
iMinh.VNM
/40
.11 [2B]
AB745036
_RVi/Ho
ChiMinh
.VNM
/31.11 [2B]
AB706308
_RVs/HoCh
iMinh.VNM
/33
.11 [2B]
AB706300
_RVs/HoCh
iMinh.VNM
/27
.11 [2B]
* HQ89
375
4_VN
M [2B]
* HQ89
375
3_VN
M [2B]
AB706298
_RVs/HoCh
iMinh.VNM
/27
.11 [2B
]
AB706305
_RVs/HoCh
iMinh.VNM
/26
.11 [2B]
AB706303
_RVs/HoCh
iMinh.VNM
/29
.11 [2B]
CRS
AB706304
_RVs/HoCh
iMinh.VNM
/28
.11 [2B]
AB706306
_RVs/HoCh
iMinh.VNM
/32
.11 [2B]
AB706307
_RVs/HoCh
iMinh.VNM
/32
.11 [2B]
Evolutionary distance
Vietn
am
2B
2A
2C
2
1
64
100
70
100
65
89
100
92
100
64
96
73
88
65
100
86
85
86
Fig.
1.
Phylogenetic
tree
generated
by
neighbor-joining
analysis
of
genetic
distances
in
the
E1
gene
of
RV
based
on
WHO-recommended
sequence
window
(739
bp;
nt
8731–9469).
Tree
was
constructed
with
17
reference
strains
recommended
by
WHO
and
32
strains
obtained
from
database.
Vietnamese
strains
identified
in
this
study
are
underlined.
Vietnamese
strains
from
database
are
indicated
by
asterisk.
AB745032
(virus
identified
directly
by
PCR)
and
AB745037
(virus
isolated
by
tissue
culture)
are
strains
from
the
same
fetus.
WHO
reference
strains
are
indicated
by
black
dots.
Bootstrap
values
of
>60%
are
shown
at
the
branch
nodes.

V.H.
Pham
et
al.
/
Journal
of
Clinical
Virology
57 (2013) 152–
156 155
2B
reference
strains
(Fig.
1).
Interestingly,
except
for
2
Vietnamese
strains
from
the
database,
most
(31/33:
94%)
of
the
Vietnamese
2B
strains
formed
an
independent
cluster
within
the
same
genotype
2B
group,
suggesting
their
own
lineage.
This
finding
was
supported
by
the
high
bootstrap
value
of
73%.
5.
Discussion
Rubella
is
now
a
vaccine-preventable
disease,
and
live
atten-
uated
vaccines
have
been
available
since
the
late
1960s.14 The
vaccination
programs
have
dramatically
reduced
the
incidence
of
rubella
in
developed
countries.
In
2009,
they
were
in
use
in
more
than
67%
of
countries
worldwide,
but
vaccination
coverage
differs
widely.15 Only
a
few
Asian
countries
have
introduced
a
rubella-
containing
vaccine
into
their
national
immunization
programs.
So
far,
the
control
of
rubella
with
vaccination
has
been
achieved
only
in
Japan,
Taiwan
and
Singapore.
As
a
result,
rubella
still
remains
poorly
controlled
in
many
countries
in
Asia.
In
particular,
in
the
Southeast
Asian
continents,
the
vaccination
coverage
rate
was
only
4%
as
of
2009.15
Use
of
the
molecular
epidemiological
approach
has
contributed
to
the
understanding
of
the
worldwide
genetic
diversity
and
trans-
mission
routes
of
pathogens
and
is
considered
important
for
supporting
activities
aimed
at
control
and
elimination.
Based
on
partial
E1
gene
sequences,
so
far
nine
RV
genotypes
(1B,
1C,
1D,
1E,
1F,
1G,
2A,
2B
and
2C)
and
4
provisional
genotypes
(1a,
1h,
1i,
and
1j)
based
on
sequence
variation
in
the
739-nt
E1
region
have
been
established
by
WHO.8,9 Some
of
the
genotypes
are
geographically
restricted,
such
as
1C
which
is
endemic
only
in
Central
and
South
America,
and
others
are
more
broadly
distributed
such
as
1E
which
has
been
found
in
the
Americas,
Africa,
Europe
and
Asia.8,16,17 Geno-
typic
distribution
of
RV
has
been
reported
from
many
different
regions,
but
it
is
very
rare
from
Southeast
Asia.
Very
recently,
Tran
et
al.
first
reported
genomic
characterization
of
11
RV
strains
from
Vietnamese
infants
and
concluded
that
genotype
2B
virus
is
preva-
lent
in
Vietnam.2In
order
to
clarify
the
characteristics
of
rubella
in
Vietnam,
we
further
conducted
a
survey
in
detail
by
adding
a
num-
ber
of
pregnant
women
patients
included
their
fetuses/newborns.
From
these
two
studies
based
on
molecular
epidemiology,
we
con-
firmed
that
the
genotype
2B
virus
is
predominant
and
prevalent
in
Vietnam.
The
genotype
2B
virus
was
previously
known
to
be
preva-
lent
in
India,
China,
South
Korea,
South
Africa,
and
now
very
widely
distributed
worldwide.8,9,17–19
In
Vietnam,
vaccination
against
rubella
has
not
been
introduced
into
the
national
immunization
program.
As
a
result,
rubella
has
become
an
important
disease
in
maternal
and
child
health
in
this
country.
Prenatal
diagnosis
is
mainly
based
on
the
detection
of
viral
RNA
in
amniotic
fluid
by
RT-PCR20–23 or
less
frequently,
on
the
detection
of
virus-specific
IgM
antibody
in
fetal
blood.24,25 In
this
study,
we
mainly
used
amniotic
fluids
to
determine
the
viral
RNA
and
the
result
was
a
very
high
rate
of
RV
RNA
detection.
Further-
more,
RV-specific
IgM
antibody
was
positive
in
cord
blood
in
95%
of
fetuses/newborns
tested
in
this
study.
Our
data
reported
here
could
be
useful
in
efforts
to
control
rubella
and
its
related
CRS
in
Vietnam
and
adjacent
neighboring
countries.
Interestingly,
phylogenetic
analysis
revealed
that
genotype
2B
sequences
of
RV
recovered
in
Vietnam
formed
an
independent
cluster
within
the
same
genotype
2B.
They
have
not
been
found
elsewhere,
although
sequences
of
many
other
2B
strains
from
other
countries
are
available
for
comparison.
This
finding
suggests
indigenous
RVs
might
be
prevalent
in
this
region.
That
is,
the
Viet-
namese
strains
of
RV
may
have
arisen
from
mutations
and
random
genetic
drifts
that
conferred
a
selective
advantage
on
this
lineage
in
this
limited
geographic
region.
To
explore
the
characterization
of
the
RV
genome
circulating
in
Southeast
Asia,
we
are
now
planning
to
conduct
a
molecular-based
epidemiologic
survey
by
means
of
an
international
collaboration
with
Cambodia,
Laos,
Thailand
and
Myanmar.
Since
it
is
predicted
that
RV
could
have
originated
in
Asia26,
this
survey
is
very
important
for
elucidating
the
origin
and
nature
of
the
virus
and
how
it
spread
into
the
neighboring
countries
in
Asia.
In
fact,
although
rubella
has
been
controlled
in
Japan,
small
outbreaks
of
rubella
have
still
been
occurring
recently.
Interest-
ingly,
two
sequences
of
RV
recovered
from
Japanese
patients
in
2011
were
associated
with
the
same
cluster
in
the
Vietnamese
genotype
2B
strain
(data
not
shown).
This
suggests
some
virus
strains
spreading
in
Japan
have
been
imported
from
Vietnam.
No
data
about
CRS
in
Vietnam
are
available
so
far.27 Through
this
epidemiologic
study,
we
noted
a
very
high
frequency
of
CRS
in
fetuses/newborns
from
RV-infected
mothers
in
Vietnam.
Impor-
tantly,
viral
RNA
was
detected
in
throat
swabs
and
placenta
from
all
cases
of
fetuses/newborns
examined.
As
mentioned,
rubella
is
one
of
the
emerging
infectious
diseases
that
must
be
prevented
in
this
area
and
a
national
immunization
program
should
be
set
up
immediately
to
eliminate
it.
Studies
on
clinical
and
pathologi-
cal
characterization
of
RV
infection
in
pregnant
women
and
their
fetuses/newborns
are
now
in
progress
and
will
be
reported
sepa-
rately.
In
conclusion,
the
present
study
reported
here
showed
that
RVs
belonging
to
the
genotype
2B
group
were
prevalent
in
the
south-
ern
part
of
Vietnam.
Information
about
the
genetic
characterization
of
the
RV
in
Vietnam
has
improved,
which
should
aid
in
the
con-
trol
of
rubella
and
CRS
in
this
region.
Since
the
actual
conditions
of
rubella
and
its
related
CRS
occurrence
in
Southeast
Asia
are
poorly
understood,
a
detailed
survey
of
this
important
infectious
disease
is
needed
immediately
to
work
out
a
strategy
about
how
to
improve
maternal
and
child
health
in
this
region.
Funding
None.
Competing
interests
None.
Ethical
approval
Informed
consent
for
participation
in
this
study
was
obtained
from
all
patients
or
their
families.
This
study
conforms
to
the
ethical
guidelines
and
was
approved
by
the
ethics
committees
of
the
Hung
Vuong
Hospital,
Ho
Chi
Minh
City,
Vietnam.
Acknowledgements
We
thank
to
Dr.
Yoshio
Mori,
National
Institute
of
Infectious
Dis-
eases,
for
his
variable
advice
of
rubella
study,
Dr.
Hideki
Hasegawa,
National
Institute
of
Infectious
Diseases,
for
his
continuous
encour-
agement
during
this
study
and
Ms.
Thanh
Ngoc
Thi
Nguyen
and
Ms.
Trang
Thi
Thuy
Phan,
Nam
Khoa
Biotek
Co.,
for
their
technical
assistance.
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