Heretofore, the most reliable way of measuring yellow fever virus antibody was to use the mouse ..... 1965. Pro- tection against Junin virus by immunization.
Vol. 16, No. 11 Printed in U.S.A.
APPLIED MICROBIOLOGY, Nov. 1968, p. 1770-1775 Copyright © 1968 American Society for Microbiology
Yellow Fever Virus I. Development and Evaluation of a Plaque Neutralization Test SHELDON SPECTOR AND NICOLA M. TAURASO Laboratory of Virology and Rickettsiology, Division of Biologics Standards, National Institutes of Health, Bethesda, Maryland 20014 Received for publication 18 June 1968
Heretofore, the most reliable way of measuring yellow fever virus antibody was to use the mouse neutralization (MN) test employing either suckling or weanling mice. Certain disadvantages (e.g., expense both of animals and of maintaining a mouse colony, allergic reactions of many laboratory workers, and the relatively long time, 21 days, before end points are reached) are inherent in any program with mice or other laboratory animal species and have discouraged the use of the MN test by many laboratories. A previously reported plaque neutralization (PN) test with primary chick embryo cell cultures could not be consistently reproduced by later investigators. We have developed a convenient and reproducible PN test employing the MA-104 embryonic rhesus monkey kidney cell culture and a single agar-overlay procedure. When compared with MN tests with newborn (1 to 3 days old) and weanling (16 to 20 g, 24 to 28 days old) mice inoculated by the intracranial route, the PN test was the most sensitive for measuring neutralizing antibody; it was also less variable, less costly, and it achieved results in the shortest period of time. End points could be determined in 5 to 6 days for the PN test as compared to 21 days for the MN test. There are several ways to measure neutralizing activity to yellow fever (YF) virus. Neutralization and protection tests in monkeys have been used successfully in the past but are impractical because the use of large numbers of monkeys is expensive and inconvenient (1). The introduction of the susceptible white mouse provided a convenient and less expensive alternative and a means of testing large numbers of sera (15, 16). The basic technique, employing a constant serumvarying virus dilution, has been studied with different routes of inoculation, such as intracranial (ic), intraperitoneal (ip), and intraperitoneal with concomitant cerebral trauma (10, 17). A plaque neutralization (PN) test could provide a handy in vitro method of assaying YF virus neutralizing activity, but its success has varied in different laboratories. The use of chick embryo monolayers by Porterfield (7, 8) could not be reproduced by Henderson and Taylor (4, 5) or by Schulze and Schlesinger (12). We have developed a PN test with the MA104 embryonic rhesus monkey kidney cell line and a simple single agar-overlay procedure. In this report, we describe this PN test and compare it with the more conventional mouse neutralization (MN) test performed by inoculating mice by the ic or ip route.
MATERIALS AND METHODS
Virus. Infectious stock YF virus (17D strain) used in the PN and MN tests was made from suckling mouse brains harvested when the animals became sick and was prepared as a 20% suspension in phosphate-buffered saline (PBS; 2) with 0.5% bovine plasma albumin (BPA). The 17D vaccine strain was passaged three times in 10- to 12-g mice and four times in suckling mice in our laboratory. All pools were stored in sealed glass ampoules at -70 C until used. Animals. Newborn mice (1 to 3 days old), suckling mice prior to weaning (10 to 18 days old), and weanling mice (16 to 20 g, 24 to 28 days old; Swiss albino, NIH General Purpose strain) used for the MN tests were obtained from the Rodent and Rabbit Production Section, Laboratory Aids Branch, National Institutes of Health (NIH). In our studies, we used complete litters (containing from 6 to 16 newborn or suckling mice) and 10 weanling mice at each dilution point. Rhesus monkeys (Macaca mulatta) obtained from India were immunized with YF vaccine either on the day of their arrival at NIH or after 6 weeks of quarantine. Some monkeys were inoculated with commercial RIF (9) virus-contaminated YF vaccine and others were inoculated with an RIF virus-free vaccine (14) being developed in our laboratory. Antisera. Sera used in our neutralization tests were obtained from monkeys before and 28 days after 1770
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inoculation with YF vaccine. The sera were stored at -20 C until used. Cell culture. The MA-104 embryonic rhesus monkey kidney cell cultures (originally obtained from Microbiological Associates, Inc., Bethesda, Md.) were prepared in the routine manner established by the Tissue Culture Section of our laboratory. Monolayer cell cultures in 32-oz (0.946 liter) bottles were dispersed with a solution containing 0.25% trypsin and 0.2% ethylenediaminetetraacetic acid in Dulbecco's saline (2) without calcium and magnesium. Prescription bottles [2 oz (0.059 liter)] were seeded with 7 ml of cell suspension (80,000 cells/ml) in a growth medium consisting of minimum essential medium containing Hanks' solution base (11), 10% fetal bovine serum, and neomycin (50 ,g/ml). Bottle cultures [2 oz (0.059 liter)] were received in our laboratory 3 to 5 days after initial seeding of cells; growth medium was replaced with maintenance medium consisting of Earle's balanced salt solution containing 0.5% lactalbumin hydrolysate, 2% fetal bovine serum, penicillin G (100 units/ml), streptomycin (100 ,ug/ml), and nysstatin (50 units/ml). PN test. The PN test was performed in the following manner. All reagents were kept in an ice-water bath before and during mixing; unheated serum diluted 1:2 in PBS with 0.5% BPA was added to equal amounts of serial 10-fold dilutions of virus in the same diluent. After incubation at room temperature (23 to 25 C) for 1 hr, the mixtures were retumed to an ice-water bath, and 0.2 ml of each mixture was inoculated into each of two to four 2-oz (0.059 liter) bottle cultures of MA-104 cells. After adsorption for 1 hr at 36 C, the cultures were overlaid once with 5 ml of an agar medium (13). Plaques were read after incubation at 36 C for 5 to 6 days. End points were usually determined from dilutions containing 10 to 50 plaque-forming units (PFU). The difference in titer between serum samples taken before and 28 days after immunization represents the neutralizing capacity of the serum and is expressed as neutralization indices (NI) in this paper. MN tests. A sample of the same virus-serum mixtures used in the PN test was inoculated into mice. The standard inoculum was 0.03 ml inoculated ic and 0.2 ml inoculated ip. Deaths occurring over a period of 21 days were recorded; LD5o end points were determined by the Karber method (6) and were expressed as decimal exponent (Dex) values (3).
TABLE 1. Plaque neutralization (PN) and intracranial weanling mouse neutralization (WMN) tests with sera from monkeys immuntized with yellow fever vaccinte Neutralization index Monkey
no.
a
PN test
WMN testb
1
53 .5c
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
53.3 53.1 53.3 52.2 52.5 52.4 5:2.9 3.9 3.8 4.0 4.2 4.2 3.4 3.5 3.8 5:5.4 4.5 5:5.3 3.6 4.1 5:5.7
53.1 53.2
5:5.5 4.2
52.5 52.8 51.9 51.7 52.1 52.0 2.3 2.2 3.7 2.9 1.2 2.3 2.6 1.6 2.6 1.8 2.8 1.8 2.2
5:3.2 53.5 5:3.4
a Monkey no. 1 to 12 were immunized with RIF (9) virus-contaminated YF primary seed (lot no. AB 237); no. 13 to 24 were immunized with the RIF virus-free candidate primary seed (14; National Drug lot no. 17D-51). b The mice weighed 16 to 20 g and were 24 to 28 days old. c Neutralization index, expressed as Dex values (3), represents the difference in titer between serum samples taken before and 28 days after immunization.
larger than yellow fever virus plaques by several millimeters. Comparison of PN and intracranial weanling neutralization (WMN) tests. Table 1 shows mouse RESULTS the results of PN and intracranial WMN tests Characteristics of YF virus plaques. Plaques performed with sera from 24 different monkeys (no. 1 to 24). Neutralization end points could are best observed with indirect fluorescent lighting from an oblique angle. A plaque develops as not be determined from tests performed with a tiny (0.5 mm) white speck on day 5, progressing postimmunization sera from monkey no. 1 to 8 to 3 to 4 mm and developing a "foamy" appear- because the virus preparation had a low titer ance and an irregular border on day 7 to 8. As (4.1 to 4.5 Dex PFU and 3.2 to 4.3 Dex mouse the plaque becomes larger, it can be seen as a LD50/0.1 ml). (Titers have been corrected to 0.1 clear area by direct lighting. In our experience, ml for comparison.) These tests could not be only two other viruses (West Nile and Murray repeated because of a lack of serum. A higher Valley encephalitis) produce a similar "foamy" infectious titer (6.4 to 6.7 Dex PFU and 5.7 to plaque in the MA-104 cell culture but they are 6.8 Dex mouse LDto/0.1 ml) of a subsequently
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TABLE 2. Plaque neutralization (PN) and intracranial newborn mouse neutralization (NBMN) tests with sera from monkeys immunized with yellow fever vaccine
APPL. MICROBIOL.
TABLE 3. Effect of age and route of inoculation upon the mouse neutralization test and comparison with the plaque neutralization (PN) test Neutralization index
Neutralization index
Monkey
Monkey no." PN test
NBMN testb
54.1c
3.3 3.0 4.5 3.4 4.0 5.1 3.2 4.2 2.5 2.3 1.9 2.3 2.1 3.1 1.9 3.1 1.6 2.2 1.8 2.2 0.9 1.6 2.3
NBMN test"
no.
icb
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
54.1 54.0 54.0 54.0 54.0 53.7
54.1 55.3 4.3 2.8 2.9 3.6 3.3 3.8 3.5 2.8 3.4 3.2 3.0 3.8 3.2 2.6
a Monkey no. 25 to 36 were immunized with RIF (9) virus-contaminated YF secondary seed (National Drug lot no. 5731); no. 37 to 47 were immunized with an RIF virus-free candidate secondary seed (14; National Drug lot no. 6676). b The mice were I to 3 days old. , Neutralization index, expressed as Dex values (3), represents the difference in titer between serum samples taken before and 28 days after immunization.
prepared virus pool enabled us to determine neutralization end points in tests performed with postimmunization sera from monkey no. 9 to 24. Because end points were obtained, these results are more meaningful for purposes of comparing the relative sensitivity of both neutralization tests. The PN test was consistently more sensitive than the intracranial WMN test for measuring neutralizing activity in the postimmunization sera.
Comparison of PN and intracranial newborn (NBMN) tests. Table 2 shows the results of the PN and intracranial NBMN tests performed with sera from 23 different monkeys (no. 25 to 47). The PN test was more sensitive than the intracranial NBMN tests for measuring neutralizing activity. Effect of age and route of inoculation upon MN mouse neutralization
SMN
test5
WMN
test5
ic
ip
ic
ip
PN test
48 49 50
2.4c >5.0 2.7
1.3 2.1 1.2
ip
>2.9 1.1 2.4 2.3 >2.9 1.5
1.9 3.4 2.0
a NBMN, newborn (1 to 3 days old) mouse neutralization test; SMN, suckling (15 to 18 days old) mouse neutralization test; WMN, weanling (16 to 20 g, 24 to 28 days old) mouse neutralization test. b Intracranial, ic; intraperitoneal, ip. c Neutralization index, expressed as Dex values (3), represents the difference in titer between negative serum control and postimmunization serum samples.
test and comparison with PN test. Results of PN, NBMN, suckling mouse neutralization (SMN), and WMN tests performed simultaneously with postimmunization sera from three different monkeys are shown in Table 3. Owing to a shortage of preinoculation sera, the negative serum control was performed with a pool of sera from the same three experimental monkeys. The NBMN test performed with mice inoculated by the ip route and the PN test were the most sensitive; from tests with sera from monkey no. 48 and 50 (Table 3), it appeared that the intraperitoneal NBMN test may have been more sensitive than the PN test if end points could have been obtained in the former. Suckling and weanling MN tests were unsuccessful with mice inoculated ip because the virus strain used was not highly lethal to mice inoculated by this route. Table 4 compares the virus infectivity end points obtained in the above-described experiment and demonstrates the effect of age and route of inoculation upon virus titer in the MN tests; a comparison is made with the MA-104 cell culture plaquing system. With nonimmune control serum, virus titers in ic inoculated newborn, suckling, and weanling mice were comparable as was the titer in the plaquing method, considering the fact that the inoculum was almost 10-fold greater in this latter system. However, differences in virus titers occurred with the immune sera; this indicated that the systems varied in their ability to measure neutralizing capacity of the sera. To resolve further the sensitivity of the various MN tests and the PN test, additional experiments
were performed with a virus preparation having a higher infectious titer. Table 5 shows the results of this experiment with paired pre- and postimmunization serum samples from four different monkeys. The intraperitoneal NBMN test was consistently the most sensitive test for TABLE 4. Effect of age and route of inoculation upon virus titer in the mouse neutralization test and comparison with the MA-104 cell culture plaquing system Virus titer (LDio) Monkey no. PFJ4U no. PF Monkey
Controld 48 49 50
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VoL. 16, 1968
Newborn
Suckling miceb
Weanling mice6
icc
ic
ic
miceb
ip
ip
ip
5.8e 4.8 3.4 4.6
