Survey of important primate viralantibodies in Macaca fascicularis and M. nemestrina in Indonesia
T ~ ~ Indonesia,
J I
August 19" - 221'2006
~ o c e e d i n puf .47WbI(' ?OOh
S U R ~ E YOF IMPORTANT PRIMATE VIRAL ANTIBODIES
IN~ a c a c fascicularjs
a
AND M. nemestrina IN INDONESIA
kjus
Dish Iskand:iati, Raohmitasari Noviana. lsti Kartrka Sari.
Saepuloh, Maryati Suva, Silml Marlya and Joke Parmungkas
Prinlate Research Center at Bogor Agricultural Umversity
Jalan Lodaya 1 ~ 5Bogor
,
2 61 51 Indonesia; Email: p s S p - l ~ b @ ~ n dnet.id
o
!
Materials and Methods
Introduction
The benefits cf non-human primates
particularly macaques as ar.~maImodel in the
AlnS-related and 0 t h ~ ; infectious diseases
rasearch have been recognized for a long
time. A number af retroviruses that infect
macaques has s~mrlar characteristics as
human rmmclnodeficiency VlrUS (HIV). In an
experimental setting, certain retroviruses
infection in rhesus monkeys are wtdely spread
and produce many s~milarities with HIV
tnfection in human beings.
Among the Retraviridae family members,
simian immunodeficiency virus ( S W ) and type
D simian retrovirus (SRV) have been ~dentified
as the causal agents of immune system
disorders in macaque species which caused
clrnical symptoms similar to human AIDS, and
referred to as sim~anAIDS (SAIDS). Clinical
symptons induced by SRV include fever,
chronic. diarrhea, wasting syndrome, and
arlemia.
Sometimes
retroperitoneal
fibromatosis (RF) was found rn animals
infected by SRV. In additlon to the above
clinical symptoms, opportunistic infec!ions
were often found in SIV-~nfected anlma)s,
however, no RF was noted. Many rrlacaques
cotonres were naturally infected with simian
retroviruses that would confound experimental
data relevant to AIDS-related research and
other infectlous disease.
Simian T-lymphotroph~c virus type-?
( s ~ ~ v .is1 )a member of Retroviridae family,
which
was
reported
to
cause
immunosuppress~onin macaques.
Herpesvirus sirniae or Cercopifhecine
herpesvirus 7 (CHV-I), also known as herpes
B virus. is widely spread in macaques
populations and continues to represent a
significant health threat to personnel who
works closely with monkeys or handle their
tissue specimens. The B virus is the most
challenging ~nfectionto elminate from an SPF
population.
Here we report the results of retrospective
serological survey from 2004 to 2007 for the
presence of antibodies against SRV, SIV,
HTLV-1 and HSV-1 in macaques population :n
Indonesia.
Viral antigens
HTLV-1, S IV, ': ISV-l were purchased
from
Advanced
B~otechnologies fnc
(Columb~a, MD).
Purified
preparations
contained a tota; protein concentration of 1
rnglml,
SRV-2 was purified using ultracentrifugat~onand sepharose method. HTLV-1
and HSV-1 are serologically cross-reactive ic
STLV-1 and herpes 0 virus (B virus),
respectively.
Antigen preparation for EClSA and Western
blot
Viral antigens for coating in EtlSA were
prepared by 2:1 m~xingof the purified virus
preparat~on with 0.3% Sodium Dodecyl
Sulphate (SDS). While virus preparation for
Western blot were diluted in sample buffer
con\arnlng SDS, P-niercaptoethanol, glycerol
and bromphenol blue.
Monkey serum1 plasma samples
Thousands of serum or plasma samples
from Macaca fascrcularis and M. nemestrine
were tested by the Microbiology and
Immunology Laboratory a: the Primate
Research
Cer,ter,
Bogor
Agricultural
University, as part of the laboratory's
dragnostic services from 2004 through 2007.
The majority af samples source were M.
fascicularrs.
P - ~ c c d ~ c grTs .\Zh MU 21)08
R
:espectrvely.
2 1M
Di:uted antigens
wells
klocked with
milk in P8S
djluted in
buffer ( p ~
ELISA plate
at 4 " and
~
skim
For
samples were
the plates,
detected
by
rseradish peroxidase-labeled goat antieY IgG antibodes
with
(tetra
benzidine) chromogen
antibody
dliuted at 1.33 in
Bound antibod~es
In
case
an
differen
those
ELlSA
(
F
5
in
false
(1988) for SRV
ed
SIV anfibodies. The
in a sodium-
sa
res~
ass
SIV
In low
P O ~ Y - a c ~ l a m i d e gel
horesis (SDS-PAGE)
using
gel
the
from 7.50/~
SRV i
3C
samv\es
reac
for
2007
samples
sphatase-labeled
I ~ G
bdies (Sigma. USA)
BCIP-NBT (5m0-4-chloro-3-indolylpho~phat~
idinrum-nitroblue tetra-zolrum) chromogen
strate (Sigma.
Commercial HTLVWestern blol
Diagnostic,
Pore) was
test of
- This kits Were
to the
dfacturer's
The
CHV-1
30%-
il
in
70%, re:
optimization
as
of ~ t s
detectran
ST[
h
specificjb.
ELlSA
Several different ELlSA systems were
used, dspend~ngon the need and availability.
One system consisted of commercial HTLV112 EClSA kit (GenelabsB Diagnostics,
Singapore) was used for detection of STLV in
2004 and 2C05.The kits were used according
to the rnanufactilrer's instruction. For HTLV-1
(after 2005) and HSV-1 antibodies detection in
routine serology diagnostics performed at w
center, purltied whole vrral antigens obtainad
from ABI (Colurnb~a,MD) were used. Briefly,
EilSA plates were coated wilh viral lysates ob
HTLV
and
HSV
at
optimal protein
concentrations of 400 nglml and 220 n g h l ,
238
pla
fascrcularis and II
to 2007 we
accordir
239
Hugur. Indonevs, . l u g s t 1 9 ' ~ IFd:f~,,b
IY
PRlMATE VIRAL ANTIBODIES
Ica fascicrrlark
M.
Iskanuriati,
Noviana, lsti Kartika Sar~,
Marvatr Surya, Silmi Mac~ya
Parnungkas
Bogor
iU5, Bogor
iirnaf
lfectious
griized
iruses
:haracter~stics
tirus
4ain
iarilias
Un~versity
Ernail: pssp-~ph@indo.net.id
HTLV-1,
Advanced
MD).
mglml.
SRV-2
HIV
IISV-1
purchased
Biotechnologies
Inc
preparatiorls
of 1
pur~fied
ultraHTLV.1
cross-reactive to
B
respectively.
2
€LISA
(SIV)
~ v ebeer1 ident~fred
Western
Viral antigens
ELlSA
2:l mlxing
virus
0.39'0
Dodecyj
S u l ~ h a t e(SDS). While
for
dlluted
containing SDS, P-mercaptoethanol,
bromphencl
prepared
(SAIDS).
1V include
retroperitoneal
wnd
serum1 plasma
sampfes
from Macaca fascicularis
M. nemestrha
were
and
lnImunology Laboratov
the Primate
Research
Bogor
Agricultural
University,
d~agnostic
2007.
The majority of samples source were M
fascicularis.
unist~c
?tied
~und
ic
9troviridae
qua.
ELISA
Several
used,
One
112 ELISA
Slnga~ore)was
2004
to
(after 2005)
routine serology
Cercoprthecine
d
is
and
unst SRV, SIV,
~ e populat~on
s
~n
E L S A plates
HTLV and
HSV
concentrations
ranufacturer's instructions.
Results
Serum or plasma samples tested
n M.
fascicularis and M. nemestrina obtaqnerl from
2004 to 2007 were summarized in Table 1.
presented according to the viruses of interest.
Discussion
In the case of STLV, the significant
increase of its antibody prevalence in 2006
compared with the data from 2005 might be
caused by different assays that were used in
those years. The commercial HTLV antibody
ELlSA kit that was previously used In 2005 and
before was not as sensitive as the assay used
in 2006. Some samples from 2005 showed
false negative results when confirmed with
Western blot assay. There was notable
relatively constant posrtive detection of
antibodies against SRV, SIV and HSV.
Antibodies to SRV was detected In the range
of around 20 to 30% of all tested samples.
while SIV antibodies were detected constantly
in low percentage below I%,
except for the
samples from 2004, and HSV antibodies were
detected in quite high percentage of the
samples tested, reaching almost 70%, except
for the samples from 2007. The low
percentage of HSV antibodies detected in
2007 was probably due to the majority of
samples obtained from young animals under 2
years of age.
Conclusion
The prevalence of SRV, SIV and HSV-11
CHV-1 antibodies in M , fascicularis from
several locations in Indonesia was around 2030%, 1% and 70%. respectively;
Further optimizatron of the STLV-11 HTLV-1
antibody detection assay was done to better
reveal the data of its antibody detection. The
in-house kit for STLV-11 HTLV-1 antibody
detection proved to have a higher sensitivity
with better specifiaty.
Tabe I.Summary of antibodies testing In 2004-2007 in both M. fascicularis and M.nemesfrina
~n
according
For HTLV-1
detection in
at our
obtained
nglrnl
Western blot
~munoblo!t~ngtechnique was carried out by
Jsrng a modified techniques of Harlow and
,ane (1988) for SRV and SIV antibodies. The
;urified viral protein was run in a sodiumjodecyl-sulfate
poly -aerylamide
gel
electrophoresis (SDS-PAGE) system using
gradlent gel w ~ t hthe concentration from 7.5%
:J 17.5% at op!imal protein concen?rations at
530 nglstrip (SRV) and 150 n g l s t r i ~(SIV).
Sequentially, the proteins in the gel was
:?ansferred to nitrocellulose membranes and
socked with 5% blotto. For antibody detection,
serum samples were diluted at 1.50 in 5%
:lotto, then added to each test strip. Bound
antibodies were detected by
alkaline
;hosphatase-labeled goat anti-human lgG
antibodies (Sigma, USA) with BCIP-NET (5bromo-4-chloro-3-indolylphosphate
toluidinium-nitroblue tetra-zol~um) chromogen
substrate (Sigma, USA). Commercial HTLV::2 Western blot kits (MP Diagnostic,
Sogapore)
STLV
This was
kits used
Were for confirmatory
according lest
to the
of
HTLv.
IGenelabsB
Center.
from A61
~spectively. Viral antigens were diluted in
:1M carbonate- bicarbonate buffer (pH 9.6).
'Jliuted antigens were added to ELISA plate
*ells and incubated overn~ght at 4°C and
2ocked with 5% blotto (containing Soh skim
~ ; i k in p8S with 0 1% Tween-20). Far
aitlhdy detection, serum samples were
3, uted at 1.33 in 5% blotto added to the plates.
Zaund antibodies
were
detected
by
?oneradish peroxidase-labeled goat antironkey IgG ant~bodies (Sigma, USA) with
'MB (tetra methyl benzidine) chrornogen
wbstrate (Sigma, USA). The reaction Nas
s!opped by adding 50 ul 2 N sulfate acid
tizs04).
Color development was read at 450
~m filter measured as optical density (OD)
BloRad microplate reader model 3550).
were
ELISA
5
Ropor, III~PIIPSIA.
. \ u ~ u slgL"
~ - 2ZW 2UI)U
3et.dinps of ;ZZ'A?d41C' ZI'09
2004
Ag8n1s
% Posl!ive
:?v
2'74
23 09%
2270
3; rlerest
Iysates of
protein
ng/ml,
238
rl
2005
No of samples
2007
2066
No of samples
% Positive
No of samples
% Positive
24 58%
2907
20 P8To
2858
30.79%
tdo of s a , ~ p l e s 96 Positlve
3v
343
1.17%
1,043
0 96%
1422
o 98%
2.573
04 7 ;
STLV
503
9 74%
673
7 28%
1500
41 33%
1064
1 8.5Z01n
7 S'j
1771
67 64":0
375
70 74%
4911
69 59%
655
21 98%
B o p r . Indr~nesia.Auyla 19'
Prcxrcdlng of .UN,MC2008
- 22%!!dli
Spring Harbor Laboratory. Cold Spyin:
Harbor, New York. pp: 4 f 1-510
Lerche NW. Yee JL and Jennings MB. 1994
Establishing
specific
retrivirus-free
breeding colonies of
macaque5:an
approach to primary screening a ~ d
surveillance. Lab Anim Sci. 44(3): 2?7221
Rosenblum LL, Weiss UA and McCl~reMa.
2000.Virus Load and Sequence Variation
in Simian Retrovirus Type 2 Infection. J
References
B u ~ h lSJ, Keel~ngME and Vos WR. 19547.
EsBblishhg
specitc
pathagen-free
nonhuman primate colony llAR Journal
38(; ) h~p://dels.nas.edulilar.
niilariourllal
138-1 138-1 Establ~shing.shtml
Daniel MD, King NW, Let'din NL. Hunt RD.
Seghal P K and Deamsiers RC. 1984. A
new type D retrovirus isolated from
macaques with an immunodeficiency
syndrome. Science 2231602-605
H ~ ~ I QE
W and Lane D. 1908. Imm~noMoting.
In: Anfibodies: a Laboratory Manual. Culd
Vim, 74(8):3449-3454
RUMAH SAKlf HEWAN J A K A ~
(JAKARTA SMALL ANIMAL )IOSPI~
"Melayani Sepenuh ~ j w a
LOHASI
~ U M ~ H
Hew.*
I
240
Jam**r.
J I
August 19" - 221'2006
~ o c e e d i n puf .47WbI(' ?OOh
S U R ~ E YOF IMPORTANT PRIMATE VIRAL ANTIBODIES
IN~ a c a c fascicularjs
a
AND M. nemestrina IN INDONESIA
kjus
Dish Iskand:iati, Raohmitasari Noviana. lsti Kartrka Sari.
Saepuloh, Maryati Suva, Silml Marlya and Joke Parmungkas
Prinlate Research Center at Bogor Agricultural Umversity
Jalan Lodaya 1 ~ 5Bogor
,
2 61 51 Indonesia; Email: p s S p - l ~ b @ ~ n dnet.id
o
!
Materials and Methods
Introduction
The benefits cf non-human primates
particularly macaques as ar.~maImodel in the
AlnS-related and 0 t h ~ ; infectious diseases
rasearch have been recognized for a long
time. A number af retroviruses that infect
macaques has s~mrlar characteristics as
human rmmclnodeficiency VlrUS (HIV). In an
experimental setting, certain retroviruses
infection in rhesus monkeys are wtdely spread
and produce many s~milarities with HIV
tnfection in human beings.
Among the Retraviridae family members,
simian immunodeficiency virus ( S W ) and type
D simian retrovirus (SRV) have been ~dentified
as the causal agents of immune system
disorders in macaque species which caused
clrnical symptoms similar to human AIDS, and
referred to as sim~anAIDS (SAIDS). Clinical
symptons induced by SRV include fever,
chronic. diarrhea, wasting syndrome, and
arlemia.
Sometimes
retroperitoneal
fibromatosis (RF) was found rn animals
infected by SRV. In additlon to the above
clinical symptoms, opportunistic infec!ions
were often found in SIV-~nfected anlma)s,
however, no RF was noted. Many rrlacaques
cotonres were naturally infected with simian
retroviruses that would confound experimental
data relevant to AIDS-related research and
other infectlous disease.
Simian T-lymphotroph~c virus type-?
( s ~ ~ v .is1 )a member of Retroviridae family,
which
was
reported
to
cause
immunosuppress~onin macaques.
Herpesvirus sirniae or Cercopifhecine
herpesvirus 7 (CHV-I), also known as herpes
B virus. is widely spread in macaques
populations and continues to represent a
significant health threat to personnel who
works closely with monkeys or handle their
tissue specimens. The B virus is the most
challenging ~nfectionto elminate from an SPF
population.
Here we report the results of retrospective
serological survey from 2004 to 2007 for the
presence of antibodies against SRV, SIV,
HTLV-1 and HSV-1 in macaques population :n
Indonesia.
Viral antigens
HTLV-1, S IV, ': ISV-l were purchased
from
Advanced
B~otechnologies fnc
(Columb~a, MD).
Purified
preparations
contained a tota; protein concentration of 1
rnglml,
SRV-2 was purified using ultracentrifugat~onand sepharose method. HTLV-1
and HSV-1 are serologically cross-reactive ic
STLV-1 and herpes 0 virus (B virus),
respectively.
Antigen preparation for EClSA and Western
blot
Viral antigens for coating in EtlSA were
prepared by 2:1 m~xingof the purified virus
preparat~on with 0.3% Sodium Dodecyl
Sulphate (SDS). While virus preparation for
Western blot were diluted in sample buffer
con\arnlng SDS, P-niercaptoethanol, glycerol
and bromphenol blue.
Monkey serum1 plasma samples
Thousands of serum or plasma samples
from Macaca fascrcularis and M. nemestrine
were tested by the Microbiology and
Immunology Laboratory a: the Primate
Research
Cer,ter,
Bogor
Agricultural
University, as part of the laboratory's
dragnostic services from 2004 through 2007.
The majority af samples source were M.
fascicularrs.
P - ~ c c d ~ c grTs .\Zh MU 21)08
R
:espectrvely.
2 1M
Di:uted antigens
wells
klocked with
milk in P8S
djluted in
buffer ( p ~
ELISA plate
at 4 " and
~
skim
For
samples were
the plates,
detected
by
rseradish peroxidase-labeled goat antieY IgG antibodes
with
(tetra
benzidine) chromogen
antibody
dliuted at 1.33 in
Bound antibod~es
In
case
an
differen
those
ELlSA
(
F
5
in
false
(1988) for SRV
ed
SIV anfibodies. The
in a sodium-
sa
res~
ass
SIV
In low
P O ~ Y - a c ~ l a m i d e gel
horesis (SDS-PAGE)
using
gel
the
from 7.50/~
SRV i
3C
samv\es
reac
for
2007
samples
sphatase-labeled
I ~ G
bdies (Sigma. USA)
BCIP-NBT (5m0-4-chloro-3-indolylpho~phat~
idinrum-nitroblue tetra-zolrum) chromogen
strate (Sigma.
Commercial HTLVWestern blol
Diagnostic,
Pore) was
test of
- This kits Were
to the
dfacturer's
The
CHV-1
30%-
il
in
70%, re:
optimization
as
of ~ t s
detectran
ST[
h
specificjb.
ELlSA
Several different ELlSA systems were
used, dspend~ngon the need and availability.
One system consisted of commercial HTLV112 EClSA kit (GenelabsB Diagnostics,
Singapore) was used for detection of STLV in
2004 and 2C05.The kits were used according
to the rnanufactilrer's instruction. For HTLV-1
(after 2005) and HSV-1 antibodies detection in
routine serology diagnostics performed at w
center, purltied whole vrral antigens obtainad
from ABI (Colurnb~a,MD) were used. Briefly,
EilSA plates were coated wilh viral lysates ob
HTLV
and
HSV
at
optimal protein
concentrations of 400 nglml and 220 n g h l ,
238
pla
fascrcularis and II
to 2007 we
accordir
239
Hugur. Indonevs, . l u g s t 1 9 ' ~ IFd:f~,,b
IY
PRlMATE VIRAL ANTIBODIES
Ica fascicrrlark
M.
Iskanuriati,
Noviana, lsti Kartika Sar~,
Marvatr Surya, Silmi Mac~ya
Parnungkas
Bogor
iU5, Bogor
iirnaf
lfectious
griized
iruses
:haracter~stics
tirus
4ain
iarilias
Un~versity
Ernail: pssp-~ph@indo.net.id
HTLV-1,
Advanced
MD).
mglml.
SRV-2
HIV
IISV-1
purchased
Biotechnologies
Inc
preparatiorls
of 1
pur~fied
ultraHTLV.1
cross-reactive to
B
respectively.
2
€LISA
(SIV)
~ v ebeer1 ident~fred
Western
Viral antigens
ELlSA
2:l mlxing
virus
0.39'0
Dodecyj
S u l ~ h a t e(SDS). While
for
dlluted
containing SDS, P-mercaptoethanol,
bromphencl
prepared
(SAIDS).
1V include
retroperitoneal
wnd
serum1 plasma
sampfes
from Macaca fascicularis
M. nemestrha
were
and
lnImunology Laboratov
the Primate
Research
Bogor
Agricultural
University,
d~agnostic
2007.
The majority of samples source were M
fascicularis.
unist~c
?tied
~und
ic
9troviridae
qua.
ELISA
Several
used,
One
112 ELISA
Slnga~ore)was
2004
to
(after 2005)
routine serology
Cercoprthecine
d
is
and
unst SRV, SIV,
~ e populat~on
s
~n
E L S A plates
HTLV and
HSV
concentrations
ranufacturer's instructions.
Results
Serum or plasma samples tested
n M.
fascicularis and M. nemestrina obtaqnerl from
2004 to 2007 were summarized in Table 1.
presented according to the viruses of interest.
Discussion
In the case of STLV, the significant
increase of its antibody prevalence in 2006
compared with the data from 2005 might be
caused by different assays that were used in
those years. The commercial HTLV antibody
ELlSA kit that was previously used In 2005 and
before was not as sensitive as the assay used
in 2006. Some samples from 2005 showed
false negative results when confirmed with
Western blot assay. There was notable
relatively constant posrtive detection of
antibodies against SRV, SIV and HSV.
Antibodies to SRV was detected In the range
of around 20 to 30% of all tested samples.
while SIV antibodies were detected constantly
in low percentage below I%,
except for the
samples from 2004, and HSV antibodies were
detected in quite high percentage of the
samples tested, reaching almost 70%, except
for the samples from 2007. The low
percentage of HSV antibodies detected in
2007 was probably due to the majority of
samples obtained from young animals under 2
years of age.
Conclusion
The prevalence of SRV, SIV and HSV-11
CHV-1 antibodies in M , fascicularis from
several locations in Indonesia was around 2030%, 1% and 70%. respectively;
Further optimizatron of the STLV-11 HTLV-1
antibody detection assay was done to better
reveal the data of its antibody detection. The
in-house kit for STLV-11 HTLV-1 antibody
detection proved to have a higher sensitivity
with better specifiaty.
Tabe I.Summary of antibodies testing In 2004-2007 in both M. fascicularis and M.nemesfrina
~n
according
For HTLV-1
detection in
at our
obtained
nglrnl
Western blot
~munoblo!t~ngtechnique was carried out by
Jsrng a modified techniques of Harlow and
,ane (1988) for SRV and SIV antibodies. The
;urified viral protein was run in a sodiumjodecyl-sulfate
poly -aerylamide
gel
electrophoresis (SDS-PAGE) system using
gradlent gel w ~ t hthe concentration from 7.5%
:J 17.5% at op!imal protein concen?rations at
530 nglstrip (SRV) and 150 n g l s t r i ~(SIV).
Sequentially, the proteins in the gel was
:?ansferred to nitrocellulose membranes and
socked with 5% blotto. For antibody detection,
serum samples were diluted at 1.50 in 5%
:lotto, then added to each test strip. Bound
antibodies were detected by
alkaline
;hosphatase-labeled goat anti-human lgG
antibodies (Sigma, USA) with BCIP-NET (5bromo-4-chloro-3-indolylphosphate
toluidinium-nitroblue tetra-zol~um) chromogen
substrate (Sigma, USA). Commercial HTLV::2 Western blot kits (MP Diagnostic,
Sogapore)
STLV
This was
kits used
Were for confirmatory
according lest
to the
of
HTLv.
IGenelabsB
Center.
from A61
~spectively. Viral antigens were diluted in
:1M carbonate- bicarbonate buffer (pH 9.6).
'Jliuted antigens were added to ELISA plate
*ells and incubated overn~ght at 4°C and
2ocked with 5% blotto (containing Soh skim
~ ; i k in p8S with 0 1% Tween-20). Far
aitlhdy detection, serum samples were
3, uted at 1.33 in 5% blotto added to the plates.
Zaund antibodies
were
detected
by
?oneradish peroxidase-labeled goat antironkey IgG ant~bodies (Sigma, USA) with
'MB (tetra methyl benzidine) chrornogen
wbstrate (Sigma, USA). The reaction Nas
s!opped by adding 50 ul 2 N sulfate acid
tizs04).
Color development was read at 450
~m filter measured as optical density (OD)
BloRad microplate reader model 3550).
were
ELISA
5
Ropor, III~PIIPSIA.
. \ u ~ u slgL"
~ - 2ZW 2UI)U
3et.dinps of ;ZZ'A?d41C' ZI'09
2004
Ag8n1s
% Posl!ive
:?v
2'74
23 09%
2270
3; rlerest
Iysates of
protein
ng/ml,
238
rl
2005
No of samples
2007
2066
No of samples
% Positive
No of samples
% Positive
24 58%
2907
20 P8To
2858
30.79%
tdo of s a , ~ p l e s 96 Positlve
3v
343
1.17%
1,043
0 96%
1422
o 98%
2.573
04 7 ;
STLV
503
9 74%
673
7 28%
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Spring Harbor Laboratory. Cold Spyin:
Harbor, New York. pp: 4 f 1-510
Lerche NW. Yee JL and Jennings MB. 1994
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