Survey of important primate viralantibodies in Macaca fascicularis and M. nemestrina in Indonesia

T ~ ~ J I Indonesia, August 19" - 221'2006 P - ~ c c d ~ c g s rT .\Zh MU 21)08

~ o c e e d i n p uf .47WbI(' ?OOh R

S U R ~ E Y

OF

IMPORTANT PRIMATE VIRAL ANTIBODIES :espectrvely. djluted in

I N

~ a c a c a fascicularjs AND M. nemestrina IN INDONESIA

2

1

M

buffer ( p ~ pla

Di:uted antigens ELISA plate _{fascrcularis and}

II

Dish Iskand:iati, Raohmitasari Noviana. lsti Kartrka Sari. wells at 4 " ~ and to 2007 we

kjus Saepuloh, Maryati

Suva,

Silml Marlya and Joke Parmungkas klocked with skim accordir

milk in P8S For

Prinlate Research Center at Bogor Agricultural Umversity antibody samples were

Jalan Lodaya 1 ~ 5 , Bogor 2 61 51 ! Indonesia; Email: p s S p - l ~ b @ ~ n d o net.id dliuted at 1.33 in

the

plates, In case

Bound

antibod~es detected by _an

Materials

and

Methods rseradish peroxidase-labeled goat anti-

Introduction

The

benefits cf non-human

primates

eY IgG antibodes with differen

particularly

macaques

as ar.~maI model

in

the Viral antigens (tetra benzidine) chromogen those (

AlnS-related and 0 t h ~ ; infectious diseases HTLV-1, S IV, ': ISV- l were purchased ELlSA

F

rasearch have been recognized for a long from Advanced B~otechnologies fnc 5

time. A number af retroviruses that infect (Columb~a, MD). Purified preparations in sa

macaques has s~mrlar characteristics as contained a tota; protein concentration of 1 false r e s ~

human rmmclnodeficiency VlrUS (HIV). In an rnglml, SRV-2 was purified using ultra- ass

experimental setting, certain retroviruses centrifugat~on and sepharose method. HTLV-1 infection in rhesus monkeys are wtdely spread and HSV-1 are serologically cross-reactive ic

and produce many s~milarities with HIV STLV-1 and herpes 0 virus (B virus), SRV i

tnfection

in

human beings. respectively. 3C

Among the Retraviridae family members, (1988) for SRV SIV anfibodies. The SIV

simian immunodeficiency virus ( S W ) and type Antigen preparation for EClSA and Western

ed

in

a

sodium- In low

D simian retrovirus (SRV) have been ~dentified blot P O ~ Y - a c ~ l a m i d e gel

samv\es

as the causal agents of immune system Viral antigens for coating in EtlSA were horesis (SDS-PAGE) using

disorders in macaque species which caused prepared by 2:1 m~xing of the purified virus gel the from

7.50/~

reac

clrnical symptoms similar to human AIDS, and preparat~on with 0.3% Sodium Dodecyl

for

referred to as sim~an AIDS (SAIDS). Clinical Sulphate (SDS). While virus preparation for

symptons induced by SRV include fever, Western blot were diluted in sample buffer

2007

chronic. diarrhea, wasting syndrome, and con\arnlng SDS, P-niercaptoethanol, glycerol samples

arlemia. Sometimes retroperitoneal and bromphenol blue.

fibromatosis

(RF) was found rn

animals

infected by SRV. In additlon to the above Monkey

serum1

plasma samples

clinical symptoms, opportunistic infec!ions Thousands of serum or plasma samples The

were often found in SIV-~nfected anlma)s, from

Macaca

fascrcularis

and

M.

_nemestrine sphatase-labeled I ~ G _CHV-1 il

however, no RF was noted. Many

rrlacaques

were tested by the Microbiology and bdies (Sigma. USA) BCIP-NBT (5- in

cotonres

were naturally infected with simian Immunology Laboratory a: the Primate m0-4-chloro-3-indolylpho~phat~

30%-

70%, re:

retroviruses that would confound experimental Research Cer,ter, Bogor Agricultural idinrum-nitroblue tetra-zolrum) chromogen optimization data relevant to AIDS-related research and University, as part of the laboratory's strate (Sigma.

Commercial

HTLV- as

other infectlous disease. dragnostic services from 2004 through 2007. Western blol Diagnostic,

of

~ t s

Simian T-lymphotroph~c virus type-? The majority af samples source were

M.

Pore) was test of ST[

( s ~ ~ v . 1 )

is a member of Retroviridae family,

fascicularrs.

- This kits Were to the detectran h

which was reported to cause

dfacturer's

specificjb.

immunosuppress~on in macaques. ELlSA

Herpesvirus sirniae or Cercopifhecine Several different ELlSA systems were herpesvirus 7 (CHV-I), also known as herpes used, dspend~ng on the need and availability. B virus. is widely spread in macaques One system consisted of commercial HTLV- populations and continues to represent a 112 EClSA kit (GenelabsB Diagnostics, significant health threat to personnel who Singapore) was used for detection of STLV in works closely with monkeys or handle their 2004 and

2C05.

The kits

were

used according tissue specimens. The B virus is the most to the rnanufactilrer's instruction. For HTLV-1 challenging ~nfection to elminate from an SPF (after 2005) and HSV-1 antibodies detection

in

routine serology diagnostics performed at

w

population.

Here we report the results of retrospective center, purltied whole vrral antigens obtainad serological survey from 2004 to 2007 for the from ABI (Colurnb~a, MD) were used. Briefly, presence of antibodies against SRV, SIV, EilSA plates were coated wilh viral lysates

ob

HTLV-1 and HSV-1 in macaques population :n HTLV and HSV at optimal protein

Indonesia. concentrations of 400 nglml and 220 n g h l ,

Hugur. Indonevs, . l u g s t 1 9 ' ~ IFd :f~,,b

I Y PRlMATE VIRAL ANTIBODIES

Ica

fascicrrlark

M.

Iskanuriati, Noviana, lsti

Kartika

Sar~, Marvatr Surya, Silmi Mac~ya Parnungkas

Bogor Un~versity

iU5, Bogor Ernail: pssp-~ph@indo.net.id

iirnaf

lfectious HTLV-1, IISV-1 purchased

griized Advanced Biotechnologies Inc

iruses MD). preparatiorls

:haracter~stics

of

1

tirus mglml. SRV-2 pur~fied ultra-

4ain HTLV.1

cross-reactive to

iarilias HIV B

respectively. 2

(SIV) €LISA Western

~ v e beer1 ident~fred

Viral antigens ELlSA

prepared

2:l mlxing virus

0.39'0 Dodecyj

(SAIDS). S u l ~ h a t e (SDS). While for

1V include dlluted

containing SDS, P-mercaptoethanol,

retroperitoneal bromphencl

wnd

serum1 plasma

unist~c sampfes

from Macaca

fascicularis

M. nemestrha

were and

?tied lnImunology Laboratov the Primate

~ u n d Research Bogor Agricultural

University,

d~agnostic 2007.

ic The majority of samples source were

M

9troviridae fascicularis.

qua. ELISA

Cercoprthecine

Several ELISA were

used, and

d One HTLv.

112 ELISA

IGenelabsB

Slnga~ore) was ~n

2004 according

is

to For HTLV-1

(after 2005) detection in

routine serology at our

5 Center. obtained

from A61

unst SRV, SIV, E L S A plates Iysates

of

~ e s populat~on ~n HTLV and HSV protein

concentrations nglrnl ng/ml,

2 3 8

rl

3et.dinps of ;ZZ'A?d41C' ZI'09 Ropor, III~PIIPSIA. . \ u ~ u s ~ lgL" - 2ZW 2UI)U ~spectively. Viral antigens were diluted in Results

:

1M carbonate- bicarbonate buffer (pH 9.6). Serum or plasma samples tested n M.

fascicularis and M.

nemestrina

obtaqnerl from 'Jliuted antigens were added to ELISA

plate

*ells and incubated overn~ght at 4°C and 2004 to 2007 were summarized in Table 1. 2ocked with 5% blotto (containing Soh skim presented according to the viruses of interest. ~ ; i k in p8S with 0 1% Tween-20). Far

aitlhdy detection, serum samples were Discussion

3, uted at 1.33 in 5% blotto added to the plates. In the case of STLV, the significant Zaund antibodies were detected by increase of its antibody prevalence in 2006 ?oneradish peroxidase-labeled goat anti- compared with the data from 2005 might be ronkey IgG ant~bodies (Sigma, USA) with caused by different assays that were used in 'MB (tetra methyl benzidine) chrornogen those years. The commercial HTLV antibody wbstrate (Sigma, USA). The reaction

Nas

ELlSA kit that was previously used In 2005 and s!opped by adding 50 ul 2 N sulfate acid before was not as sensitive as the assay used tizs04). Color development was read at

450

in 2006. Some samples from 2005 showed

~m filter measured as optical density (OD) false negative results when confirmed with BloRad microplate reader

model

3550). Western blot assay. There was notable relatively constant posrtive detection of

Western blot antibodies against

SRV,

SIV and HSV.

~munoblo!t~ng technique was carried out by Antibodies to SRV was detected In the range Jsrng a modified techniques of Harlow and of around 20 to 30%

of

all tested samples. ,ane (1988) for SRV and SIV antibodies. The while SIV antibodies were detected constantly ;urified viral protein was run in a sodium- in

low

percentage below I%, except for the jodecyl-sulfate poly -aery lamide gel samples from 2004, and HSV antibodies were electrophoresis (SDS-PAGE) system using detected in quite high percentage

of

the gradlent gel w ~ t h the concentration from 7.5% samples tested, reaching almost 70%, except :J 17.5% at op!imal protein concen?rations at for the samples from 2007. The low 530 nglstrip (SRV) and

150

n g l s t r i ~ (SIV). percentage of HSV antibodies detected in Sequentially, the proteins in the gel was 2007 was probably due to the majority of :?ansferred to nitrocellulose membranes and samples obtained from young animals under 2 socked with 5% blotto. For antibody detection, years of age.

serum samples were diluted at 1.50 in 5%

:lotto, then added to each test strip. Bound Conclusion

antibodies were detected by alkaline The prevalence of SRV, SIV and HSV-11 ;hosphatase-labeled goat anti-human

lgG

CHV-1 antibodies in M , fascicularis from antibodies (Sigma, USA) with BCIP-NET (5- several locations in Indonesia was around 20- bromo-4-chloro-3-indolylphosphate 30%, 1 % and 70%. respectively;

toluidinium-nitroblue tetra-zol~um) chromogen Further optimizatron of the STLV-11 HTLV-1 substrate (Sigma, USA). Commercial HTLV- antibody detection assay was done to better ::2 Western blot kits

(MP

Diagnostic, reveal the data of its antibody detection. The Sogapore)

STLV This was kits used Were for confirmatory according lest to the of in-house kit for STLV-11 HTLV-1 antibody detection proved to have

a

higher sensitivity

ranufacturer's instructions. with better specifiaty.

Tabe I. Summary of antibodies testing In 2004-2007 in both

M.

fascicularis

and

M.

_nemesfrina

Ag8n1s 2004 2005 2066 2007

3; rlerest No of samples % Posl!ive tdo of s a , ~ p l e s 96 Positlve No of samples % Positive No of samples % Positive

:?v 2'74 23 09% 2270 24 58% 2907 20 P8To 2858 30.79%

3 v 343 1.17% 1,043 0 _{96% 1422}

o

_{98% 2.573} 0 _{4 7 ;}

STLV 503 9 74% 673 7 28% 1500 41 33% 1064 1 8.5Z01n

Prcxrcdlng of .UN,MC 2008 B o p r . Indr~nesia. Auyla 19'

-

22% !!dli

References Spring

Harbor Laboratory.

Cold

Spyin:

B u ~ h l SJ, Keel~ng

M E

and Vos WR. 19547. Harbor, New York. pp: 4 f 1-510

EsBblishhg

specitc pathagen-free Lerche NW. Yee

JL

and

Jennings MB. 1994 nonhuman primate colony llAR

Journal

Establishing

specific

retrivirus-free 38(; ) h~p://dels.nas.edulilar. niilariourllal breeding colonies of macaque5:an 138-1 138-1 Establ~shing.shtml

approach

to

primary

screening

a ~ d

Daniel

MD,

King

NW, Let'din NL.

Hunt

RD. surveillance. Lab Anim Sci. 44(3): 2?7-

Seghal

P K

and

Deamsiers RC.

1984. A 221

new type D retrovirus isolated from Rosenblum LL, Weiss UA and McCl~re Ma.

macaques

with an

immunodeficiency

2000.

Virus

Load

and Sequence

Variation

in Simian Retrovirus Type 2 Infection. J syndrome. Science

2231602-605

H ~ ~ I Q W

E

and

Lane

D.

1908.

Imm~noMoting. Vim, 74(8): 3449-3454

RUMAH

SAKlf

HEWAN

J A K A ~

In:

Anfibodies:

a

Laboratory

Manual.

Culd

(JAKARTA

SMALL ANIMAL

)IOSPI~

"

Melayani

Sepenuh

~ j w a

LOHASI

~ U M ~ H

Hew.* Jam**r.

I