Assay of arbovirus neutralizing antibody by micro methods

446 TRANSACTIONSOF THE ROYAL SOCIETYOF 'TROPICAL MEDICINE AND HYGIENE. Vol. 63. No. 4. 1969.

ASSAY OF ARBOVIRUS NEUTRALIZING ANTIBODY BY MICRO METHODS PAIRATANA SUKHAVACHANA, THOMAS M. Y U I L L AND P H I L I P K. RUSSELL Department of Virology, Medical Research Laboratory,~ SEA TO Medical Project, Rajavithi Road, Bangkok, Thailand and Faculty of Public Health, Rajavithi Road, Bangkok, Thailand Introduction Extensive serological surveys require simple, reproducible and specific antibody assay systems. The haemagglutination-inhibition (HI) test (CLARKE and CASALS, 1958) has often been used for arbovirus serological surveys, but it may be complicated by nonspecific reactions despite kaolin or acetone extraction of thesera (HOLDEN, MUTH and SHRINER, 1966). Kaolin extraction may remove 40-60% of the specific immunoglobulins from the sera (MANN et al., 1967). H I antibody is cross reactive within arbovirus groups and interpretation of results in terms of specific viruses is frequently difficult. To confirm the presence of antibody, sera positive by H I test must, therefore, be retested by virus neutralization. Neutralization tests are therefore needed which are as efficient and economic of materials as the H I test, but avoid the disadvantages of the H I test. We describe two neutralization tests developed for antibody assays with arboviruses in Thailand, including Japanese encephalitis (JEV), dengue (DV) types 1-4, chikungunya (CV), Sindbis (SV), Wesselsbron (WV) and Batai (BV) viruses. M a t e r i a l s and m e t h o d s Cell cultures and m e d i u m BHK-21 cells * were grown in medium (M) 199 with 5 o~o foetal bovine serum (FBS) inactivated at 56°C. for 1 hour, and 0"07~o NaHCO3, 200 units penicillin and'200 ~g. streptomycin. The propagation of LLC-MK2 cells has been described previously (RusSELL et al., 1967). Viruses The viruses used are listed in Table I. 1Alternate address : SEATO Medical Project, U.S. Component, APO San Francisco 96346. We acknowledge the skilled technical assistance of Miss Suwanna Vithanomsat, Mrs. Jiraporn Supavadee and Miss Laddawal Sukecharoen, Drs. Phinit Simasathien and Ananda Nisalak of this laboratory kindly furnished some of the reference antisera. * Obtained from Microbiological Associates, Bethesda; Maryland.

PAIRATANA SUKHAVACHANA~ THOMAS M. YUILL AND P H I L I P K. RUSSELL

TABLE I.

447

Viruses used in metabolic inhibition and microculture plaque-reduction neutralization tests. Virus

Strain

Passage history

Dengue 1

Hawaii

Dengue 2

New Guinea C

26

Mouse

Dengue 3

H-87

21

Mouse

Dengue 4

H-241

25

Mouse

Chikungunya

Ross

Chikungunya

Thai 25263

Japanese encephalitis '

Nakayama

14 Mouse

Japanese encephalitis

Thai CgLt 541

10 Mouse

Sindbis

Thai CgLt 599

10 Mouse

Wesselsbron

Thai BKM-367/66

5 Mouse

Batai

Thai BKM-457/66

5 Mouse

125 Mouse

182 Mouse 6 BHK-21 cell

I m m u n e sera. Antisera used and the method of preparation are presented in Table II. Sindbis (strain Ar 399) hyperimmune mouse ascitic fluid*, prepared by the method of BRANDT et al. (1967), was also used. M i c r o m e t a b o l i c inhibition (MMI) test

M 199, with 20% heated FBS and the usual concentrations of antibiotics and NaHCO3, was used as diluent for virus and sera. Tests were performed in disposable, "U"-bottomed flexible microtitre plates**. The plates were prepared by soaking in detergent followed by 3 rinses each in tap water and then distilled, demineralized water. After drying, they were wrapped with clear plastic wrapping and sterilized by ultraviolet irradiation for 4-8 hours. NonCtoxic droppers were made from 1 ml. plastic disposable tuberculin syringes fitted with rubber medicine dropper bulbs and 18-gauge needles from which the points and bevels had been ground. This dropper arrangement delivered very uniform volumes of approximately 0.025 ml. per drop. Test antisera were diluted to 1 : 4 with microtitre loops or in tubes and 0 . 0 2 5 ml. was transferred to each of 5 or more wells of the plate with a 0.1 ml. pipette. 2 of these wells per serum did not receive virus and served as controls for serum toxicity to the cells. To the other wells, 0.025 ml. of the virus test dose (30-100 TCLDS0) was added by dropper. 10-fold dilutions of the virus test dose were made in tubes and 0.025 ml. of each dilution was transferred to wells containing 20% heated FBS. Cell controls free of test virus or serum were included in each test. Plates were covered * Kindly furnished by Dr. W. E. Brandt, Walter Reed Army Institute of Research, Washington, D.C. **Obtained from Cooke Engineering Co., Falls Church, Va.

448

ASSAY OF ARBORIVUS NEUTRALIZING ANTIBODY BY MICRO METHODS

with plastic wrap after application of heavy, white mineral oil to the edges, and incubated at 37°C. for 2 hours. After this incubation period, 0.1 ml. of growth medium containing 4000 live BHK-21 cells was added to each well. Sufficient (about 0-12 ml.) mineral oil was added to each well to leave just enough room for the later addition of 0.05 ml. of growth medium. The plates were rewrapped with plastic wrap and incubated at 37°C. 4 days later they were unwrapped. Growth medium (0.05 ml.) was added to each well by dropper. The plates were reincubated for 2 more days and the results recorded. As in other M I tests, red colour indicated virus-induced cell death (and no neutralization) and yellow colour indicated cell life (virus absent or neutralized). Where any doubt of cell condition existed, the cells were observed by means of an inverted microscope. TABLE II. Immunizing virus

Production of reference immune sera.-

Vaccine source

Animal and route

Number of vaccinations and interval

Dengue 1

Mouse brain

Monkey, SC*

1

Dengue 2, 3 and 4

LLC-MK2 cells

Monkey, SC

1

Chikungunya (Ross)

Mouse brain

Rabbit, SC & IP**

3, 1-week

Japanese enceph. (Nakayama)

Mouse brain'

Rabbit, SC & IP

3, 1-week

Sindbis (Ar 339)

Mouse brain

Mouse, IP

2, 3-week

Sindbis (Thai CgLt 599)

Mouse brain

Mouse, IP

2, 3-week

Batai (Thai BKM457/66)

Mouse brain

Mouse, IP

2, 3-week

Wesselsbron (Thai BKM-367)

Mouse brain

Mouse, IP

2, 3-week

* SC = subcutaneous ** IP = intraperitoneal

Microculture plaque-reduction neutralization (MPRN) test Disposable plastic plates with flat-bottomed wells measuring 15 mm. in diameter were used. These plates were cleaned, wrapped in.plastic wrap and sterilized in the same fashion as the smaller M M I test plates. M 199 with 5% heated calf serum (CS) with the usual antibiotics and NaHCO3 to p H 8.3 was used as serum and virus diluent. LLC-MK~ cells were grown in sterile plastic plates. 70,000 live cells in 0.7 ml. of growth medium were added to each well. Glycerin was applied around the edges of the plates before they were wrapped with plastic wrap. The wrapped plates were incubated at 37°C. until monolayers formed (usually 4 days). On the day of the test, serum dilutions were made in tubes and 0.1 ml. of each dilution was transferred to a well of a disposable microfitre plate. An equal volume of virus diluted to contain 20-30 plaque-forming units (PFU) per 0.0125 ml. was added to each serum dilution. A titration of this virus dose was made and added to equal

PAIRATANA SUKHAVACHANA~ THOMAS M. YUILL AND PHILIP K. RUSSELL

469

volumes of diluent. Glycerin was added to the edges of the plate to insure a tight seal of the plastic wrap, and the wrapped plate was incubated at 37°C. for I hour. During the incubation of serum-virus mixtures, growth medium was removed from the cells by suction. The cells were washed with 0.5 ml. of Hanks balanced salt solution, pH 8.3. To each well 0.025 ml. each of diluent and of serum-virus mixture was added. This volume was adequate to prevent drying of the cells during adsorption for 1½ hours at 37°C. Frequent agitation of the plates uniformly dispersed the virus over the cell sheet. At the end of the adsorption period, 0.4 ml. of first agar overlay medium was added. The plates were tightly rewrapped and incubated at 37°C. for 3 (with JEV and WN) or 6 (with DV) days, when the same volume of second agar overlay containing neutral red was added. Plaques were counted after overnight incubation. Results Micro metabolic inhibitiorl tests

Since the M M I test is basically a cell culture tube neutralization test, parallel M M I and standard tube neutralization tests (SCHMID% 1964) were carried out for comparison. The incubation time and temperatures for serum-virus mixture neutralization were used for M M I and tube tests. Reference antisera or acute-convalescent-phase serum pairs from virologically or serologicaUy confirmed cases of arboviral disease were tested against the homologous JEV, CV, SV, WV and BV. Except for CV, titres of sera measured by the two methods were essentially similar (Tables III and IV). With CV TABLE III. Titres of paired sera from encephalitis patients tested comparatively against Japanese encephalitis virus by tube and by micrometabolic inhibition (MMI) neutralization tests. Patient

Serum phase

Tube test Titre*

acute

MMI test

Virus dose-~

Titre

Virus dose

< 5

300

30

70

convalescent

20

300

80

70

acute

15

300

convalescent

20

300

acute convalescent

7.5 20

300 300

7"5 80 <5 40

70 70 70 70

* Reciprocal of serum dilution giving 50% Cell protection. t Virus dose as cell culture LD50.

virus, however, M M I serum titres were considerably higher than tube test titres. In the tubes it appeared that any virus "break-through" in the established monolayers resulted in cell to cell spread as evidenced by widening cell destruction from initial small foci. This apparently did not occur in the M M I test, where cells did not form a monolayer until the fourth day after inoculation, reducing the opportunity for cell to cell spread. This phenomenon has also been observed by others with other viruses in the metabolic inhibition test (ScHMmT and LE~NETTE, 1961).

450

ASSAY OF ARBOVIRUS NEUTRALIZING ANTIBODY BY MICRO METHODS

TABLE IV.

Comparative homologous neutralization titres of sera by tube neutralization and micrometabolic inhibition (MMI) tests. Tube test

M M I test

Serum (host) Titre*

'Virus doset

Titre

Virus dose

Chikungunya (Rabbit 1)

10

200

/> 500

70

(Rabbit 2)

10

200

>/500

70

(Mouse)

30

200

~>500

70

200

120

70

(Human)

<5

(Mouse)

120

100

110

70

(Mouse)

60

100

20

70

(MouseIAF)$

30

100

110

70

Batai

(Mouse)

320

50

960

100

Wesselsbron

(Mouse)

240

2000

450

30

Sindbis

* Reciprocal of serum dilution giving 50% cell protection. t Virus doses as cell culture LDS0. :~ Immune mouse ascitic fluid. T o determine the relative specificity of the M I test, homologous and heterologous neutralization trials were conducted with 2 Group A viruses and antisera, CV and SV, and with 2 Group B viruses and antisera, JEV and WV. Antisera to DV i-4 and Tembusu were also tested against the Group B viruses. There was no cross neutralization between CV and SV viruses and antisera. Except for D V 1 and 4 antisera, there was no detectable Group B cross neutralization (Table V). There was low level cross neutralization of both JEV and WV by the DV 1 and 4 antisera. To test the effect of variation in virus doses on serum dilution titres, 2-fold dilutions of JEV antisera were tested against 5 concentrations of the homologous virus. There was an approximate 2-fold decrease in titre with each 3-fold increase in virus concentration (Table VI).

Microculture. plaque-reduction neutralization test A comparison was made of the specificity of neutralization of DV 1-4 and JEV and their anfisera in the microculture and standard 1 oz. prescription bottle culture (RussELL et al., 1967) systems. Plaques produced in microculture were smaller than, but comparable in clarity with, those in bottle cultures (Fig. 1). The results achieved in these two systems are essentially the same (Table VII). Both tests were highly specific and there was no difficulty in distinguishing t h e 4 dengue virus serotypes or JEV. Though some low-level cross neutralization was observed, in every case the homologous titres were more than 10-fold higher.

451

PAIRATANA SUKHAVACHANA, THOMAS M. YUILL AND PHILIP K. RUSSELL

TABLE g . Relative specificity of neutralization of certain Group B antisera tested against Japanese encephalitis (JE) virus and Wesselsbron (WV) virus by micrometabolic inhibition ( M M I ) test compared with the homologous plaque reduction neutralization test (PRNT) titre. P R N T titre* Homol. virus

M M I titre Antisera to WV virus

JE virus

<5

80

Japanese enceph. Wesselsbron

<10

480

Tembusu

<10

<5

Dengue 1

15

Dengue 2

<10

<5

400

Dengue 3

<10

<5

320

Dengue 4

15

1000 320

15

90

10

Reciprocal of the serum dilution neutralizing 50% of the virus dose in the plaque reduction neutralization test, TABLE VI.

Effect of virus dose variation on Japanese encephalitis virus-antiserum neutralization titres measured by micrometabolic inhibition test. Virus dose (TCLD50)

Serum titre (reciprocal)

TABLE VII.

2000

700

300

100

15

30

60

120

10 > 320

Homologous and heterologous plaque reduction neutralization titres measured in microculture and 1 oz. prescription bottle (macro) cultures. Antisera Dengue 1

Dengue 2

Dengue 3

Dengue 4

Jap. e------nceph*

_ Micro Macro Micro V1acr, lacro Micro Macro Micrc _Macr(

Viruses

Micro

Dengue 1

400

Dengue 2

<10

<10

Dengue 3

<10

<10

20

Dengue 4

<10

<10

Jap. enceph*

<10

<10

<10

lO

* Nakayama Strain.

<10

<10

I0

200

270

20

< 10

<10

<10

<10

<10

<10

<10

400 > 640

<10

<10

1280

2560

452

ASSAY OF ARBOVIRUS NEUTRALIZING ANTIBODY BY MICRO METHODS

FIG. 1. Plaques of dengue virus (DV) 1 through 4, Japanese encephalitis virus (JEV)in microculture plates (15 ram. wells) Of L L C - M K 2 cells (upper) and neutralization of DV-2 by dilution of reference antiserum (lower).

640

Serum Dilutions (Reciprocal) 160 40

10

The specificity of the microculture P R N T was rigorously tested by using 2 pairs of sera from dengue haemorrhagic fever patients, both of which had had prior Group B (probably dengue) infections. The convalescent-phase sera were broadly cross reactive by the H I test, but far less so by the microculture P R N T (Table VIII). It was therefore possible to rule out as aetiologicai agents DV 1 and 4 and JEV in one case, and DV 4 and JEV in the other. To determine the variability that might be expected from test to test, two workers carried out homologous titrations of a dengue type 2 antiserum on two occasions, employing a range of virus test doses calculated to contain 2-82 plaque-forming units (PFU). When a mean of less than 5 PFU per well was observed, inherent variability in plaque numbers obscured accurate calculation of plaque reduction. When large virus doses were used, plaque overlap resulted in an underestimate of the virus dose and plaque

PAIRATANA SUKHAVACHANA, THOMAS M. YUILL AND P H I L I P K. RUSSELL

453

reduction could not be accurately estimated. When observed plaque counts were compared with the numbers expected from the dilutions employed, it was apparent that only up to 15-20 P F U per well could be counted without significant overlap (Fig. 2), TABLE VIII.

Haemagglutination-inhibition (HI) and microculture plaque reduction neutralization test titres of acute and convalescent-phase sera from dengue haemorrhagic fever patients Neutralization titre

H I titre Patient

No,

1

2

Serum phase

2 Dengue 1 I,..._Dengue - - - ~ 80*

Acute

- Dengue 3

2560

<20

<20

<20

1280 <20

320

320

320

640

640

Acute

i

'--JE '

Dengue 1

160

160~

160

320

I

640

Conval.

Conval.

Dengue4

iii

Dengue 2 '

l

~

Dengue 4

160

40

20


> 2560

160

40

<10

160 10

< 10

< 10

10

<10

160

1000

100

10

<10

*Reciprocal of s e r u m dilution inhibiting 8 units of haemagglutinin. TReclprocal of serum dilution producing 50 % plaque reduction.

I008060 50 40 30'

/ /

20.

/

111 > 15'

/

./

.J

./

co

0

I0 9

8 o-

K

7

6

s •

•

O

O Test 2 ( N = 2 )

,------= • ......

[ •

,

FIGURE 2.

i

z

I

i

s 6

i i i

89,o

l

,5 2'0

Test I (N = 3 ) Test 3 [ N = 2 )

•

Test 4 ( N = 3 )

io odo do,;o

Plaques Expected Discrepancies of observed from expected plaque numbers with increasing virus concentration, due to plaque overlap inaccuracies.

although as a practical matter, somewhat more P F U per well could be used if (1) accurate dilution and assay of the test dose was made, and (2) the 50% (reduced) virus tkre was well within the countable P F U range. Within an observed range of 5-23 PFU, titres measured by the two workers were similar (Table IX). At 3 of the 7 doses employed, however, some variability resulted in mean plaque counts which did not fall on a straight

454

ASSAY OF ARBOVIRUS NEUTRALIZING ANTIBODY BY MICRO METHODS

line when plotted on log probit paper. Even in those cases, however, titres were only 2-5- to 5-fold higher than the means of titrations where linearity was good. TABLE IX. Reproducibility of microculture plaque reduction neutralization tests on two separate occasions, conducted by two workers, employing a variety of dengue type-2 virus doses and a single reference antiserum. Virus test dose PFU* observed) 15

14

P

S

P

P

S

P

P

Date

Nov 67

Feb 68

Feb 68

Nov 68

Feb 68

Nov 68

Feb 68

Titre

640-1280t

310

250

225

340t

160-640t

270

Worker

~

~

6

--y-

23

* Plaque-forming units per well. t Plaque numbers variable, plaque reduction not exactly linear. Discussion

The micrometabolic inhibition test and microculture plaque reduction neutraliz-i ation test were specific, reproducible, and sparing of reagents and, most importantly, they were found to be satisfactory for serological surveys for most arboviruses present in Thailand. The metabolic inhibition test has been used successfully by others as an efficient, specific neutralization test with a variety of virus and cell systems (ScaMmT, 1964). The test was successfully reduced to a microtitre system with polioviruses and monkey kidney cells by SEVER(1962). We have modified this system for the BHK-21 cell line, which is particularly suitable for arboviruses (KARABATSOS and BUCKLEY,1966) and modified the pH regulation by wrapping the plates in plastic wrap, which has the additional benefit of practically eliminating troublesome mould contamination (especially important in the tropics). The BHK-21 M M I test has, in our experience, been as efficient to conduct and nearly as sparing of sera and reagents as the H I test. The M M I test combines this efficiency and economy with specificity far greater than the H I test, however, particularly where the serological picture is confused by the presence of several endemic viruses in the same antigenic group. In Thailand where the presence of 7 or more Group B agents made serological surveys by HI tests impossible to interpret, the M M I test provided the required specificity. Although we used only 5 arboviruses in our investigations, the broad spectrum of susceptibility of BHK-21 cells may allow the test to be used with many other viruses. The BHK-21 M M I test is not without its limitations, however. Its sensitivity is not as great as HI or PRN tests. Though hyperimmune reference antisera may neutralize the test virus to high titre, sera from naturally infected animals obtained months or years after infection may have very low titres, or may not react at all. This is especially true when the cell cultures used are highly susceptible to the test virus. Thus, one cannot safely extrapolate from test results obtained with hyperimmune antisera to field-collected sera without risk of false negative results. A degree of sensitivity is therefore sacrificed to obtain results which are highly specific and reliable.

PAIRATANA SUKHAVAGHANA, THOMAS M. YUILL AND PHILIP K. RUSSELL

455

The MMI test is, of course, satisfactory only for those viruses producing rapid, complete cell destruction. For some viruses producing little or no cell death, or in instances when greater precision of antibody measurement as well as test efficiency is desired, the microculture P R N T is a satisfactory alternative and has been previously used by others with success (SALIM, 1966; MIUV,A and SCI-mgER, 1962; EARLY et al., 1967). The LLC-MK~ cell-dengue virus plaque system (SuKHAVACI-IANAet al., 1966) was adapted to disposable plastic plates to make the P R N T more efficient and economical. In adapting the P R N T to microculture, a certain degree of precision is sacrificed owing to the use of smaller plaque numbers, and inaccuracies due to plaque overlap. None the less, the test is reliable for detection of antibody in prevalence surveys and is sufficiently accurate to detect 5-fold differences in titre; thus it is suitable for measuring antibody in routine diagnostic serology. In our experience, use of the microcukure method enabled technicians to test 3 times the number of sera that could be tested by using the bottle culture system in the same time. The micro system has the additional advantage of requiring one quarter of the serum and one eighth of the reagents needed for the bottle P R N T .

Summary Efficient, reproducible and specific neutralizing antibody assays are needed, particularly for use in arboviral studies in areas where antigenic cross reactions make serological interpretation difficult. Accordingly, 2 methods were developed for this purpose. A micrometabolic inhibition (MMI) test with BHK-21 cells in disposable "U"-bottomed microtitre plates was used with Japanese encephalitis, Wesselsbron, Sindbis, chikungunya and Batai viruses. The M M I test was highly specific, of efficiency comparable with the haemagglutination-inhibition (HI) test, and was very sparing of reagents and sera. Although equal to or better in sensitivity than conventional cell culture neutralization tests, it was not as sensitive as H I or plaque-reduction neutralization (PRN) tests. The L L C - M K 2 cell microculture PRN test was devised for use with the dengue viruses or for use when a combination of precision of antibody measurement and efficiency of testing was required. The microculture and 1 oz. bottle culture PRNtests were of equal specificity. Although not as precise as the bottle PRN test, the microculture PRN test is suitable for routine serology and has the added advantages that it is more efficient and requires less serum and reagents. REFERENCES BRANDT, W. E., BUESCHER,E. L. & HETRICK,F. M. (1967). Am. ft. trop. ivied. Hyg., 16, 339. CLARKE, D. H. & CASALS,J. (1958). 1bid., 7, 561. EARLY, E., PERALTA,P. H. & JOHNSON, K. M. (1967). Proc. Soc. exp. Biol. Med., 125~ 741. HOLDEN, P., MUTH, D. & SHRINER, R. B. (1966). Am. ff Epidemiol., 84, 67. KARABATSOS,N. & BUCKLEY,S. M. (1967). Am. ft. trop. Med. Hyg., 16, 99. MANN, J. J., ROSSEN,R. D., LEHRICH,J. R. & KASEL,J. A. (1967). ft. lmmunol., 98, 1136, MIURA, T. & SCI-IERER,W. F. (1962). Am. ft. trop. Med. Hyg., 76, 197. RUSSELL, P. K., NISALAK, A., SUXHAVACHANA,P., & VIVOI,~A, S. (1967). ft. lmmunol., 99, 285. SALIM, A. R. (1967). Trans. R. Soc. trop. Med. Hyg., 61~ 259. SCHMIDT, N. J. (1964). In Lennette and Schmidt, Diagnostic Procedures for Viral and Rickettsial Diseases. 3rd Ed. New York: American Public Health Association. - • L E N N E T T E , E. H. (1961). Prog. Med. Virol., 3, 1. SEVER, J. L. (1962). ft. Immunol., 88, 320. SUKHAVACHANA, P., NISALAK, A., & HALSTEAD, S. g . (1966). Bull. Wld. Hlth. Org., 35, 65~