Detection of chikungunya virus antigen in Aedes albopictus mosquitoes by enzyme-linked immunosorbent assay

Journal of Virological Methods, 12 (1985) 279-285

279

Elsevier JVM 01)459

DETECTION OF C H I K U N G U N Y A VIRUS ANTIGEN IN AEDES ALBOPICTUS M O S Q U I T O E S BY ENZYME-LINKED I M M U N O S O R B E N T ASSAY

EIJI KONISHI and J U N K O "I'AKAHASttI

Department of Medical Zoolog), Kobe Universit), School of Medicine, Kobe 650, Japan (Accepted 23 September 1985)

Double-antibody sandwich and modified sandwich systems of enzyme-linked immunosorbent assay for detecting chikungunya virus antigen present in female rnosquitocs, Aedes alhopic:us (Oahu strain), were evaluated as simple and rapid methods of selection of a highly susceptible mosquito line. Both assays were capable of detecting 3.9 × 10 ~ng (4.0 X 106 PFU) or more of the purified antigen. An inhibition system was less sensitive, and a direct system with adsorption of test specimens on the solid phase was not useful. Positive reactions were observed in 16 (48.5f~) of 33 infected mosquitoes with 10* to 10~ PFU, which correspond to the highly susceptible group of this strain. Mosquitoes with Iess than 106 PFU were all negative, indicating the usefulness of the sandwich techr~iques for identifying high-titered mosquitoes. chikungunya

xirus

enzyme-linked

immunosorbent assax

antigen detection

Aedes albopictus

vector mosquito

INTRODI'CTION

Although the virus-vector relationship is undoubtedly important for ecology and epidemiology of arthropod-borne viruses, reports on virus multiplication and transmission in vector mosquitoes have been limited as compared with cumulative studies dealing with mosquito cell culture systems (Rosen, 1980; Stollar, 1980; Hardy et al., 1983). Our previous screening method for identifying a mosquito line highly susceptible to oral infection with chikungunya (CHIK) virus (an alphavirus) for use in vector research, indicated that the Oahu strain ofAedes albopictus had the highest mean virus titer a m o n g several mosquito species and several geographic strains of A. albopictus (Yamanishi et al., 1983). However, it was also demonstrated that the strain was composed of 3 susceptible groups, which differed in their virus quantities 14 days after feeding on an infective blood meal of 1.3 X 105 PFU per individual: one group showed high virus titers of ]06 to 107 PFU and the others showed low (10 s to 104 PFU) and undetectable (<10 t PFU) titcrs (Yamanishi et al., 1984; Konishi and Yamanishi, 1986. For genetic selection of" the highly susceptible group, a simple a n d rapid virus detection m e t h o d would be indispensable as a substitute for infcctivity assays such as

plaque counting on tissue cultures. These conventional methods are laborious as well 0166-[}934/~5/$03 3(} ' 1985 [{lsL'~Icr %clencc Publishers B \ ' {Biomedical [ ) i ~ N o n l

280

as t i m e - c o n s u m i n g in a stud), which requires the testing of a large n u m b e r of m o s q u i t o specimens within a limited a m o u n t of time. E n z y m e - l i n k e d i m m u n o s o r b e n t assay ( E L I S A ) is recognized to be a suitable m e t h o d for antigen detection (Voller et al., 1976, 1979). The present p a p e r describes the e v a l u a t i o n o f the E L I S A technique for the detection o f C H I K virus antigen present in infected O a h u m o s q u i t o e s . MATERIAI.S ANt) M["I'HOD%

Virus An African strain o f C H I K virus p r o p a g a t e d on BHK-21 cells (Konishi a n d H o t t a , 1979) was used t h r o u g h o u t the stud\'. The culture fluid o b t a i n e d from infected m o n o l a y e r cultures was the seed virus for m o s q u i t o infection. The virus purified as described previously (Konishi and H o t t a , 1980)and dialyzed against p h o s p h a t e - b u f f e r ed saline (PBS), was used for i m m u n i / a t i o n of e x p e r i m e n t a l animals. The purified virus was also the s t a n d a r d antigen for E L I S A , after s o n i c a t i o n at 28 k11z for 1 min twice in an ice bath.

ELISA ./or antibody quantification A n t i b o d i e s to C I I l K virus in rabbit a n d mouse sera were m e a s u r e d by an E L I S A technique with a magnetic processing system (Konishi and T a k a h a s h i , 1983). The test p r o t o c o l was essentially the same as E I . I S A for .lapancse encephalitis virus (Konishi a n d Y a m a o k a , 1982). In brief, p o l y c a r b o n a t e - c o a t c d iron beads were sensitized at 4°C overnight with purified C H I K antigen in 0.1 M s o d i u m c a r b o n a t e buffer ( p l l 9.6) which had been a d j u s t e d to 13-19 }.tg/ml as d e t e r m i n e d by the m e t h o d of I.owry et al. (1951) with 0.1¢~ s o d i u m d o d e c y l sulfate in the reaction mixture, using bovine scrt, m a l b u m i n (BSA) as a s t a n d a r d . The first reaction with 100-fold diluted sera and the second reaction with 100-fold diluted goat a n t i - r a b b i t or anti-mou,~e i m m u n o g l o b u l i n (Ig) G or M (7- or bt-chain specific, r e s p e c t i v e l y ) c o n j u g a t e d with alkaline p h o s p h a t a s e (Engvall and P e r h n a n n , 1972), were p e , t o r m c d at 37:~C for 1 h. followed by the third reaction with 0 . 1 ~ p - n i t r o p h e n y l p h o s p h a t e at 37°C for 20 min. After s t o p p i n g the reaction with 1 N N a O l t , the a b s o r b a n c e at 410 nm was recorded.

Hyperimrnune sera A J a p a n white rabbit was p r i m e d by an i n t r a v e n o u s injection of 4 × 1()~ plaque f o r m i n g units ( P F U ) of the purified C H I K virus, and then b o o s t e d with 4 X 10" P F U on d a y 7, 4 )< 109 P F U on day 14, and 4 X 10 m P[-U on d a y 21. A n t i b o d y levels were m o n i t o r e d every week by E L I S A . and the rabbit was bled on day 28 when the m a x i m u m a n t i b o d v titer was observed. Mice were i n o c u l a t e d i n t r a p e r i t o n e a l l y with the virus at 4 X I(P P F U and bled 14 days after. W h o l e sera and their l g G fractions o b t a i n e d bv a m m o n i u m sulfate p r e c i p i t a t i o n were e m p l o y e d as c a p t u r e and d e t e c t o r a n t i b o d i e s for antigen by E L I S A . In some experiments, the a n t i b o d y a b s o r b e d with B H K cells was used. which was not different from u n a b s o r b e d a n t i b o d y in sensitivity o f EI,ISA.

281

Infected mosquitoes Adult female Aedes albopictus (Oahu strain) were orally infected with a blood-virus mixture at an infective titer of 6.7 × 107 P F U / m l as described previously (Yamanishi et al., 1984). Fourteen days after feeding, mosquitoes were individually homogenized in 125 lal of ELISA diluent (1% BSA, 0.05% Tween 20 and 0.02% NaN 3 in PBS), which had each been distributed in U-type microplate wclls. A 25-lal aliquot was diluted in 2.475 ml of culture medium (Eagle's minimum essential medium supplemented with 5% heat-inactivated calf serum, 0.03% glutamine, 0.03% sodium bicarbonate and 60 lag of kanamycin per ml), and was titrated for viral infectivity by the plaque assay method (Konishi and Hotta, 1979). The remaining 100 lal of the specimen were processed by the ELISA.

ELISA for antigen detection The principle of ELISA for antigen detection followed Voller et al. (1976, 1979). A double-antibody sandwich ELISA method was performed essentially as described previously (Konishi and Yamanishi, 1984). Beads were coated passively at 4°C overnight with a capture antibody, rabbit anti-CHIK virus at a dilution of 1 : 1,000 in sodium carbonate buffer (pH 9.6). They were reacted with test specimens made in ELISA diluent at 37°C for 1 h and then with a detector antibody, rabbit anti-CHIK virus conjugated with alkaline phosphatase at a dilution of 1 : 100 at 37°C for 1 h. The cnzyme activity on the beads was measured with p-nitrophenyl phosphate. In a modified sandwich system, mouse anti-CHIK virus was used as a capture antibody and rabbit anti-CHIK virus as a detector antibody, followed by incubation with enzyme-labelled goat anti-rabbit IgG. An inhibition assay was performed by incubating test fluids at 4°C ovcrnight with the standard anti-CHIK virus antiserum at a dilution of 1 : 100 or 1 : 1,000 and by measuring the level of the remaining antibody in ELISA with antigen-coated beads. A dircct ELISA system in which beads were directly sensitized with test specimens at 4°C overnight and antigens on the beads were dctectcd by an incubation with anti-CHIK virus conjugate at 37°C for 1 h, was also employed for comparison of sensitivity with the above antigen-detecting systems. RESI;I.TS

Comparison of four ELISAs for antigen detection Four ELISAs for detecting purified CHIK antigen were compared in their sensitivity, and the data are shown in Fig. 1. Two sandwich systems were able to detect a minimum of 3.9 X 10 ~ ng of thc antigen, corresponding to an infective titer of 4.0 X 10 6 PFU. Lower nonspecific rcactions were observed in ELISA with rabbit anti-CHIK virus as a capture antibody. Detection limit of the inhibition system was 2.6 lag or more, indicating less sensitivity than sandwich systems with a difference of ca. 100fold. The most sensitive method was the direct system where 2.6 ng of the antigen was detected. This system, however, failed to work when mosquito homogenate was added

282 10

II

LIJ Z r'n ,',.r" 0 133

Q

0.5 it--

o.o ~ 0

- -

t

10 -1

I

I

I

I

e

10 0

101

10 2

10 3

10 &

ANTIGEN

(rig)

Fig. 1. Dose-dependent absorbance curve for purified CHIK antigen as determined by EI,ISA with the sandwich system (o), the modified sandwich system (e), the inhibition system using antisera at 1 : 100 (n)and 1 : 1,000 dilutions (IlL and the direct system in which the solid phase was first coated with antigen (,:3 and the antigen mixed with an equal volume of mosquito homogenate prepared from 60 females in 3 ml of 0.1 M sodium carbonate buffer (A'L

to antigen specimens. The prolongation of incubation periods for the first and second reactions to 24 h or the use of 1-10% Tween 80 instead o1'0.05% Tween 20 in ELISA diluent, did not increase the sensitivity of the sandwich ELISAs.

Reaction to homogenate of infected mosquitoes Absorbance values obtained with homogenate specimens ot" 48 infected and 25 uninfected mosquitoes by the modified sandwich ELISA system using mouse antiC H I K virus as a capture antibody, were compared with their infective titers (Fig. 2). Mosquito specimens with 106 PFU or more ranged from 0.08 to 0.43 in absorbance, and the other specimens, including those uninfected, varied from 0.66 to 0.26. When a borderline was tentatively determined as 0.26, positive reactions were observed in 16 (48.5%) of 33 mosquitoes with 10 6 tO l 0 T PFU corresponding to the highly susceptible group of this strain. Mosquitoes with less than 106 PFU were all negative for ELISA reaction (0.26 or less). Accordingly, mosquitoes showing absorbances of more than 0.26 were at levels of 106 and 10: PFU. I)ISCUSSION

In genetic selection of mosquitoes for susceptibility to infection with C H I K virus, reduced egg production and high pupal mortality among the closely inbred lines have been reported (Tesh et al., 1976). Our previous attempt to select the highly susceptible group of the Oahu strain also resulted in decreased vigor, because the selection was initiated with a relatively small number of inbred pairs (Yamanishi and Konishi,

283

0.5

0.4 IdJ £.3

z

N

03

on n,-

on

0.2

.i %o

--

.'1'

.t

"... -.:. 't

0.1

t...._

O0

C

i

i

i

i

z,

5

6

7

LOG

PFU

Fig. 2. Relationship between ELISA reactions and infective titers in 48 infected mosquitoes and uninfected controls (C in abscissa).

25

unpubl, data). For the next step, which required a large number of outbred pairs, the ELISA seems to be favorable as a method to detect susceptibility, which dispenses with cumbersome procedures in infectivity assays, such as filtration of samples to remove contaminating bacteria, inoculation in indicator cells and cultivation for an appropriate number of days. The EEISA method for antigen detection includes four systems used here (Fig. 1). Comparison of these systems provided the highest sensitivity in the direct method. Shalev et al. (1980) proposed the theoretical detection of attograms of antigen using this principle in combination with a fluorogenic substrate. Crook and Payne (1980) described detection limits for dissolved capsules of baculoviruses in the direct system (25 ng/ml) and the indirect system (1 ng/ml). Our results are consistent with those of Greenstone (1983) who reported 1.57 ng of estimated sensitivity for Amblyospora antigen. However, this system was useful only for purified antigen, not for antigen contained in mosquito homogenate under the present condition. The lower limit of sensitivity of both the sandwich systems was 3.9 X 101 ng corresponding to 4.0 × l06 PFU. Previous reports indicated the detection of 40 PFU of herpes simplex virus (Pronovost et al., 1981) and 2 to 3 log~0 50% tissue culture infective dose units per ml (Hildreth et al., 1982). Attempts to enhance sensitivity by prolonged incubation periods were unsuccessful. The use of Tween 80 at high concentrations also had no cffect, though dissociation of viral membrane and delipidation of the membrane protein were cxpectcd to increase antigen potency (Mussgay and Rott, 1964: Helenius and S6derlund, 1973). A fluorogenic substrate was not employed because nonspccific reactions werc not complctely excluded even when the homolo-

284 g o u s a n t i b o d y was used in this s y s t e m ; the b a c k g r o u n d m a y also p r e v e n t the c l e a r - c u t d i f f e r e n t i a t i o n o f p o s i t i v e r e a c t i o n s f r o m n e g a t i v e r e a c t i o n s with f l u o r o m e t r i c d e t e c tion. M o s q u i t o e s p o s i t i v e f o r C H I K a n t i g e n by E L I S A had i n f e c t i v i t y titers o f 106 to 107 P F U (Fig. 2). P o s i t i v e r e a c t i o n s were o b s e r v e d in s o m e m o s q u i t o e s s h o w i n g titers o f less t h a n 4 ×

106 P F U , a d e t e c t i o n limit, p r o b a b l y d u e to the fact that the E L I S A

d e t e c t e d n o t o n l y i n f e c t i v e v i r i o n s m e a s u r e d by the p l a q u e assay b u t also o t h e r viral a n t i g e n s c o n t a i n e d in the h o m o g e n a t e . S o m e m o s q u i t o e s with h i g h e r i n f e c t i v i t y titers t h a n the d e t e c t i o n limit but n e g a t i v e in E L I S A m a y be a t t r i b u t e d to the i n h i b i t o r y effect o f h o s t m a t e r i a l s o n E L I S A r c a c t i o n ( K o n i s h i a n d Y a m a n i s h i , 1984). A l t h o u g h the p r o p o r t i o n

o f the h i g h l y s u s c e p t i b l e g r o u p w i t h 106 to l07 P F U in a selected

m o s q u i t o c o l o n y c a n n o t be e x a c t l y o b t a i n e d , these s a n d w i c h m e t h o d s p r o v e d to be useful for d e t e r m i n i n g the p a r e n t s w h o s e o f f s p r i n g w o u l d be used for the next g e n e r a tion. ACKNOWLEDGEMENTS W e wish to express o u r h e a r t y t h a n k s to Dr. H. Y a m a n i s h i for his k i n d n e s s in p r o v i d i n g the O a h u strain o f A. albopictus a n d t e c h n i c a l c o m m e n t s f o r m o s q u i t o i n f e c t i o n . T h i s i n v e s t i g a t i o n was s u p p o r t e d in p a r t by a G r a n t - i n - A i d for Scientific R e s e a r c h l ¥ o m the M i n i s t r y o f E d u c a t i o n , S c i e n c e a n d C u l t u r e o f J a p a n . REFERENCES Crook. N.E. and C.C. Payne, 1980, ,I. Gcn. Virol. 46.29. Fngvall, I-. and P. Perlmann, 1972, .1. Immunol. 109, 129. Greenstone, M.H., 1983, J. Invcrtebr. Pathol. 41. 250. Hardy, J.l.,., F.J. Houk, L.D. Kramer and W.C. Reeves. 1983, Ann. Rev. Fntomol. 28, 229. Helenius, A. and H. Sfderlund, 1973. Bioch~m. Biophys. Acta 307, 287. ltildreth. S.W., B.J. Beat','. ,I.M. Meegan. C.I,. Frazier and R.[-. Shope, 1982. ,I. Clin. Microbiol. 15,879. Konishi. E. and S. Hona, 1979, Microbiol. lmmunol. 23, 659. Konishi, E. and S. Hotta, 1980. Microbiol. Immunol. 24, 419. Konishi, E. and J. Takahashi, 1983, J. Clin. Microbiol. 17, 225. Konishi, E. and tl. Yamanishi. 1984, .1. Med. Fntomol. 21, 506. Konishi, E. and H. Yamanishi, 1986, .I. Mcd. It-ntomol. 23. 92. Konishi. E. and M. Yamaoka, 1982, J. Virol. Methods 5, 247. Lowry. O.1-t., N.J. Rosebrough, A.L. l:arr and R.J. Randall, 1951. ,I. Biol. Chem. 193, 265. Mussgay, M. and R. Rott, 1964. Virology 23, 573. Pronovost, A.D.. A. Baumgarten and G.D. llsiung, 1981, .I. Clin. Microbiol. 13. 97. Rosen, 1..., 1980. in: Arthropods as Hosts and Vectors of Alphaviruses and Flaviviruscs -Experimental Infections. The Togaviruscs. ed. R.W. Schlesingcr (Academic Press, Ne,.v York) p. 229. Shalcv, A., A.t|. Greenberg and P.J. McAIpinc, 198(I, J. Immunol. Methods 38. 125. Stollar. V., 1980. in: loga,.iruses in Cultured Arthropod Cells. The Toga',iruses, cd. R.W. Schlcsinger (Academic Press. Nev, York) p. 584. Tesh. R.B.. D.,I. Gubler and L. Rosen, 1976. Am. ,I. Trop. Med. llyg. 25. 326.

285 Yamanishi, H., E. Konishi, T. Sawayama and T. Matsumura, 1983, Jpn. J. Sanit. Zool. 34, 229 (in Japanese). Yamanishi, H., E. Konishi and T. Matsumura, 1984, Jpn. J. Sanit. Zool. 35, 361 (in Japanese). Voller, A., D.E. Bidwell and A. Bartlett, 1976, in: Microplate Enzyme Immunoassays for the lmmunodiagnosis of Virus Infections. Manual of Clinical Immunology, eds. N.R. Rose and H. Friedman (American Society for Microbiology, Washington, D.C.) p. 506. Voller, A., D.E. Bidwell and A. Bartlett, 1979, Enzyme Linked Immunosorbent Assay (Dynatech Laboratories, Inc., Alexandria).