Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/23/14 For personal use only.
A rapid fluorescent focus-inhibitiontest for determining the neutralizing-antibody response to lymphocytic choriomeningitis virus' W. LANIERTHACKER A N D VESTERJ. LEWIS VircrlZootroses Brunch, Virology Diifision, Centerfor Disecise Control, Depcrrtment of Healill, Edlrccrtion, crrrd W e f i r e , Atlcintci, G A , U . S . A .30333 GARYJ . HALLER Georgici Strrte University, Atlcrntci, G A , U . S . A .30303 AND
VircrlZoonoses Brciticll, Virology Dh~ision,Centerfor Diseerse Control, Depcirtment ofHeciltll, Ed~rccrtion,and Welfare, Atlantci, G A , U . S . A .30333 Accepted January 18, 1977
T H A C K E RW. , L., V. J. LEWIS,G. J. H A L L E Rand , G. M. BAER. 1977. A rapid fluorescent focus-inhibition test for determining the neutralizing-antibody response to lymphocytic choriomeningitis virus. Can. J. Microbiol. 23: 522-526. Levels of neutralizing antibody to lymphocytic choriomeningitis (LCM) virus in the sera of 66 infected persons were assayed by a rapid fluorescent focus-inhibition test (RFFIT). T h e test was more sensitive than the mouse-neutralization (MN) test and could be completed in less than 24 h. The R F F I T titers were compared with titers obtained by the indirect fluorescent-antibody (IFA) and complement-fixation (CF) tests. Neutralizing antibody detected by the R F F I T remained positive after IFA, C F , and MN antibodies had disappeared. T h e R F F I T for detection of LCM antibody is specific and reproducible and seems especially useful for determining the incidence and epidemiology of LCM virus infections. T H A C K E RW. . L.. V . J. LEWIS,G. J . H A L L E Re t G. M. BAER. 1977. A rapid fluorescent focus-inhibition test for determining the neutralizing-antibody response to lymphocytic choriomeningitis virus. Can. J . Microbiol. 23: 522-526. A partirdu serum de 66personnes infectees on a mesure le taux d'anticorps neutralisantcontre le virus de la choriomeningite lymphocytaire (LCM) par une technique rapide d'inhibition des foyers de fluorescence (RFFIT). Cette methode est plus sensible que I'epreuve d e neutralisation chez la souris (MN) et peut etre complktee en moins de 24 h. Les titres RFFIT sont compares aux titres obtenus par immunofluorescence indirecte (IFA) et fixation du complement (CF). La methode R F F I T permet de detecter des anticorps neutralisants meme aprts la disparition des anticorps IFA, C F et MN. Cette mtthode R F F I T d e detection d e s anticorps LCM est specifique e t reproductible et semble particulierement utile dans la determination de I'incidence et de I'epidemiologie des infections a virus LCM. [Traduit par le journal]
Introduction In the past, investigators have relied mainly on the complement-fixation (CF) and mouse-neutralization (MN) tests for the serological diagnosis of lymphocytic choriomeningitis (LCM) virus infection. However, the C F test frequently fails to detect antibody after the infection, and neutralizing antibody detectable by the M N test may require several weeks to develop (8). The indirect fluorescent-antibody (IFA) test 'Use of trade names is for identification only and does not constitute endorsement by the Public Health Service o r by the U.S. Department of Health, Education, and Welfare.
(4) and, more recently, a microplaque-reduction (MPR) test (6) have also been used for determining antibody levels to L C M virus infection. Although the IFA test detects antibody very early after LCM virus infection, its usefulness is limited in epidemiological surveys, because I F A antibody disappears before neutralization antibody (6). With the MPR test, the diagnostic laboratory cannot report results promptly because an incubation period of 4 d is required before plaques can be read. Recently a rapid fluorescent focus-inhibition test (RFFIT) was developed for the detection of neutralizing antibodies to rabies virus in human and animal sera (12). Winn et al. (13) have
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/23/14 For personal use only.
THACKER E T AL.
reported the use of a similar test for detecting neutralizing antibodies to Tamiami virus in mouse sera. The purpose of the present study was to determine whether the RFFIT could be used for measuring neutralizing antibody against LCM virus in human serum, and to compare the RFFIT with other tests for the diagnosis of LCM virus infections. Materials and Methods Sera Human sera tested were submitted to the Viral Zoonoses Branch, Center for Disease Control (CDC), for diagnosis of present or past infection with LCM virus. The 64 serologically positive patients studied were infected by pet or laboratory hamsters from hamsteries which contained LCM virus-infected animals. Also included were multiple sera from two individuals with laboratory-acquired infections. Sera from 50 individuals with no known exposure to LCM virus were used as negative controls. All sera were stored at - 20°C and were not heat-inactivated before testing. Indirect Fluorescent-antibody Test The preparation of antigen slides for use in the IFA test (4, 9) and the procedure used to stain the cells (3) have been previously described. Known positive and negative sera were included with each test. Each lot of fluorescein-conjugated anti-human globulin was titrated against positive and negative control sera to determine its optimal dilution before it was used in the IFA test. Cotnplernent-fixationTest The CDC Laboratory Branch complement-fixation (LBCF) microtiter method (2) was used for the C F test, with reagents from the Biological Products Division, CDC, Atlanta, Georgia. Mouse-neutralization Test A 10% suspension of mouse brain infected with a recent isolate of LCM virus was diluted to obtain 100 intracerebral median lethal doses (ICLD50)/0.03 ml for 3week-old mice (ICR strain). This dilution was mixed with equal volumes of serial twofold dilutions of test serum, the mixtures were incubated at 37°C for 1 h, and 0.03 ml was then inoculated intracerebrally into each of five mice. The number of mice that died between 3 and 21 days after inoculation was used to calculate the 50% neutralization end-point dilution of the serum by the Karber method (7). Rapid Fluorescerzt Focus-inhibition Teclmique The procedure developed was based on the test used to determine rabies-neutralizing antibody (12). Twofold serum dilutions (1 :5 thru 1 :640) were prepared in eightchamber tissue culture slides (Lab-Tek Products, Westmont, IL). MacPherson and Stoker medium (10) containing 10% fetal bovine serum was modified by adding Fe (NO,), (0.1 mg/ml) for use as diluent. For challenge virus, the WE strain (11) of LCM virus was used after six suspension culture passages (9) in baby hamster kidney (BHK)-21 cells. The virus titer in the medium from the last passage was 106.5 ICLDSO/mlin 3-week-old mice. A 1 :20 dilution of the virus-infected
523
medium produced adequate infection of the cells after 20 h and was chosen as the challenge dose. A 0.1-ml sample of the diluted virus was added to 0.1 ml of each serum dilution in a slide chamber, and the serum-virus mixtures were than incubated for 90 min at 35°C in a humidified atmosphere of 4% C 0 2 . Next, 1.5 x 10' BHK-21 cells suspended in 0.2 ml of diluent were added to each chamber. After an incubation period of 20 h, the medium was removed and the plastic chamber walls were detached from the slides. The slides were rinsed once in 0.01 M phosphate-buffered saline (PBS), pH 7.4, once in acetone at room temperature, and then fixed for 5 min in acetone at -20°C. The slides were dried for 15 min at -20°C and placed at room temperature for 15 min before fluorescein-conjugated antiserum to LCM virus was added. The conjugate was prepared by routine methods used at C D C (5) with guinea pig antiserum obtained from the Biological Products Division, CDC. After 30 min of incubation at 37°C in a humidified chamber, the slides were washed for 10 min in PBS, dipped briefly in distilled water, and allowed to dry at room temperature. The slides were examined with a Zeiss RA microscope equipped with a BG-12 exciter filter and Nos. 65 and 41 barrier filters and illuminated with an HBO-200 mercury vapor lamp. Twenty low-power (160 x ) fields were examined in each chamber that had received serum-virus mixtures. The challenge virus control slides (virus diluted 1 :20 plus normal serum diluted 1 :5) showed at least 19 of 20 fields with two to three fluorescent cells per field. A serum was considered to contain neutralizing antibody if a 50% or greater reduction was noted in the number of fields containing fluorescing foci.
Results The challenge LCM virus inoculum in the RFFIT resulted in two o r three fluorescing cells in each microscopic field of the stained monolayers; fluorescence was predominantly granular, and was restricted to the cytoplasm. The infected cells were readily distinguishable, and in most cases less than a minute was needed for examining 20 fields in each slide chamber. Reproducibility of the RFFIT was verified by testing dilutions of the same positive human serum six times with a constant virus dose. The reproducibility was within acceptable limits; the greatest variation observed was at the 1 :80 serum dilution, for example, with which the minimum number of 20 microscopic fields containing fluorescent foci was 5 and the maximum was 11. Table 1 presents L C M virus-antibody titers obtained with .the RFFIT, IFA, CF, and M N tests for sera from two persons having laboratory-acquired cases with known dates of onset. In both cases IFA antibody was detected first; maximal titers were obtained about 1 month after clinical onset of infection. The C F test
524
CAN. J. MICROBIOL. VOL. 23. 1977
TABLE1. LCM virus-antibody titers of sera from two laboratory-acquired cases as determined by four serological tests LCM virus-antibody titer* Case No. 2
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/23/14 For personal use only.
Case No. 1 Serum collectedt
RFFIT
IFA
CF
MN
RFFIT
IFA
CF
MN
*Dilution factor of serum end-point dilution. tDay after onset when serum collected. $Not tested.
became positive about a week after the IFA test. Throughout both cases the C F antibody remained at a low level and disappeared in a few months. Neutralizing antibody was detected almost a month earlier by the RFFIT than by the M N test; in one case it was detected simultaneously with the CF antibody. Neutralizing antibody was detectable in the last serum sample drawn from each patient by the RFFIT but was detectable in only one of these sera by the M N and IFA tests. All 50 control sera were negative by the IFA, CF, and M N tests, although one was positive by the RFFIT at a 1 :10 dilution. We further compared the sensitivities of these four tests by examining sera from 28 recent LCM cases with known dates of onset (Table 2). With the IFA test antibody was detected in all 28 cases, whereas CF, MN, and RFFIT antibodies were detected in 24, 8, and 18 cases, respectively. The RFFIT was positive in 10 cases in which the M N test was negative, but in no case was the M N test positive and the RFFIT negative. In addition, 36 human sera from individuals employed in hamsteries having LCM virusinfected animals were tested by each of the four methods (Table 3). The times between infection and collection of the sera were not precisely known, but for most of the sera they were longer than the times recorded in Table 2. All 36 sera were positive by both the RFFIT and the IFA-
test, whereas only 33 were positive by the M N and 17 by the C F test. The geometric means of antibody titers obtained by the RFFIT, IFA, MN, and C F tests were 49, 28, 13, and
A rapid fluorescent focus-inhibitiontest for determining the neutralizing-antibody response to lymphocytic choriomeningitis virus' W. LANIERTHACKER A N D VESTERJ. LEWIS VircrlZootroses Brunch, Virology Diifision, Centerfor Disecise Control, Depcrrtment of Healill, Edlrccrtion, crrrd W e f i r e , Atlcintci, G A , U . S . A .30333 GARYJ . HALLER Georgici Strrte University, Atlcrntci, G A , U . S . A .30303 AND
VircrlZoonoses Brciticll, Virology Dh~ision,Centerfor Diseerse Control, Depcirtment ofHeciltll, Ed~rccrtion,and Welfare, Atlantci, G A , U . S . A .30333 Accepted January 18, 1977
T H A C K E RW. , L., V. J. LEWIS,G. J. H A L L E Rand , G. M. BAER. 1977. A rapid fluorescent focus-inhibition test for determining the neutralizing-antibody response to lymphocytic choriomeningitis virus. Can. J. Microbiol. 23: 522-526. Levels of neutralizing antibody to lymphocytic choriomeningitis (LCM) virus in the sera of 66 infected persons were assayed by a rapid fluorescent focus-inhibition test (RFFIT). T h e test was more sensitive than the mouse-neutralization (MN) test and could be completed in less than 24 h. The R F F I T titers were compared with titers obtained by the indirect fluorescent-antibody (IFA) and complement-fixation (CF) tests. Neutralizing antibody detected by the R F F I T remained positive after IFA, C F , and MN antibodies had disappeared. T h e R F F I T for detection of LCM antibody is specific and reproducible and seems especially useful for determining the incidence and epidemiology of LCM virus infections. T H A C K E RW. . L.. V . J. LEWIS,G. J . H A L L E Re t G. M. BAER. 1977. A rapid fluorescent focus-inhibition test for determining the neutralizing-antibody response to lymphocytic choriomeningitis virus. Can. J . Microbiol. 23: 522-526. A partirdu serum de 66personnes infectees on a mesure le taux d'anticorps neutralisantcontre le virus de la choriomeningite lymphocytaire (LCM) par une technique rapide d'inhibition des foyers de fluorescence (RFFIT). Cette methode est plus sensible que I'epreuve d e neutralisation chez la souris (MN) et peut etre complktee en moins de 24 h. Les titres RFFIT sont compares aux titres obtenus par immunofluorescence indirecte (IFA) et fixation du complement (CF). La methode R F F I T permet de detecter des anticorps neutralisants meme aprts la disparition des anticorps IFA, C F et MN. Cette mtthode R F F I T d e detection d e s anticorps LCM est specifique e t reproductible et semble particulierement utile dans la determination de I'incidence et de I'epidemiologie des infections a virus LCM. [Traduit par le journal]
Introduction In the past, investigators have relied mainly on the complement-fixation (CF) and mouse-neutralization (MN) tests for the serological diagnosis of lymphocytic choriomeningitis (LCM) virus infection. However, the C F test frequently fails to detect antibody after the infection, and neutralizing antibody detectable by the M N test may require several weeks to develop (8). The indirect fluorescent-antibody (IFA) test 'Use of trade names is for identification only and does not constitute endorsement by the Public Health Service o r by the U.S. Department of Health, Education, and Welfare.
(4) and, more recently, a microplaque-reduction (MPR) test (6) have also been used for determining antibody levels to L C M virus infection. Although the IFA test detects antibody very early after LCM virus infection, its usefulness is limited in epidemiological surveys, because I F A antibody disappears before neutralization antibody (6). With the MPR test, the diagnostic laboratory cannot report results promptly because an incubation period of 4 d is required before plaques can be read. Recently a rapid fluorescent focus-inhibition test (RFFIT) was developed for the detection of neutralizing antibodies to rabies virus in human and animal sera (12). Winn et al. (13) have
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/23/14 For personal use only.
THACKER E T AL.
reported the use of a similar test for detecting neutralizing antibodies to Tamiami virus in mouse sera. The purpose of the present study was to determine whether the RFFIT could be used for measuring neutralizing antibody against LCM virus in human serum, and to compare the RFFIT with other tests for the diagnosis of LCM virus infections. Materials and Methods Sera Human sera tested were submitted to the Viral Zoonoses Branch, Center for Disease Control (CDC), for diagnosis of present or past infection with LCM virus. The 64 serologically positive patients studied were infected by pet or laboratory hamsters from hamsteries which contained LCM virus-infected animals. Also included were multiple sera from two individuals with laboratory-acquired infections. Sera from 50 individuals with no known exposure to LCM virus were used as negative controls. All sera were stored at - 20°C and were not heat-inactivated before testing. Indirect Fluorescent-antibody Test The preparation of antigen slides for use in the IFA test (4, 9) and the procedure used to stain the cells (3) have been previously described. Known positive and negative sera were included with each test. Each lot of fluorescein-conjugated anti-human globulin was titrated against positive and negative control sera to determine its optimal dilution before it was used in the IFA test. Cotnplernent-fixationTest The CDC Laboratory Branch complement-fixation (LBCF) microtiter method (2) was used for the C F test, with reagents from the Biological Products Division, CDC, Atlanta, Georgia. Mouse-neutralization Test A 10% suspension of mouse brain infected with a recent isolate of LCM virus was diluted to obtain 100 intracerebral median lethal doses (ICLD50)/0.03 ml for 3week-old mice (ICR strain). This dilution was mixed with equal volumes of serial twofold dilutions of test serum, the mixtures were incubated at 37°C for 1 h, and 0.03 ml was then inoculated intracerebrally into each of five mice. The number of mice that died between 3 and 21 days after inoculation was used to calculate the 50% neutralization end-point dilution of the serum by the Karber method (7). Rapid Fluorescerzt Focus-inhibition Teclmique The procedure developed was based on the test used to determine rabies-neutralizing antibody (12). Twofold serum dilutions (1 :5 thru 1 :640) were prepared in eightchamber tissue culture slides (Lab-Tek Products, Westmont, IL). MacPherson and Stoker medium (10) containing 10% fetal bovine serum was modified by adding Fe (NO,), (0.1 mg/ml) for use as diluent. For challenge virus, the WE strain (11) of LCM virus was used after six suspension culture passages (9) in baby hamster kidney (BHK)-21 cells. The virus titer in the medium from the last passage was 106.5 ICLDSO/mlin 3-week-old mice. A 1 :20 dilution of the virus-infected
523
medium produced adequate infection of the cells after 20 h and was chosen as the challenge dose. A 0.1-ml sample of the diluted virus was added to 0.1 ml of each serum dilution in a slide chamber, and the serum-virus mixtures were than incubated for 90 min at 35°C in a humidified atmosphere of 4% C 0 2 . Next, 1.5 x 10' BHK-21 cells suspended in 0.2 ml of diluent were added to each chamber. After an incubation period of 20 h, the medium was removed and the plastic chamber walls were detached from the slides. The slides were rinsed once in 0.01 M phosphate-buffered saline (PBS), pH 7.4, once in acetone at room temperature, and then fixed for 5 min in acetone at -20°C. The slides were dried for 15 min at -20°C and placed at room temperature for 15 min before fluorescein-conjugated antiserum to LCM virus was added. The conjugate was prepared by routine methods used at C D C (5) with guinea pig antiserum obtained from the Biological Products Division, CDC. After 30 min of incubation at 37°C in a humidified chamber, the slides were washed for 10 min in PBS, dipped briefly in distilled water, and allowed to dry at room temperature. The slides were examined with a Zeiss RA microscope equipped with a BG-12 exciter filter and Nos. 65 and 41 barrier filters and illuminated with an HBO-200 mercury vapor lamp. Twenty low-power (160 x ) fields were examined in each chamber that had received serum-virus mixtures. The challenge virus control slides (virus diluted 1 :20 plus normal serum diluted 1 :5) showed at least 19 of 20 fields with two to three fluorescent cells per field. A serum was considered to contain neutralizing antibody if a 50% or greater reduction was noted in the number of fields containing fluorescing foci.
Results The challenge LCM virus inoculum in the RFFIT resulted in two o r three fluorescing cells in each microscopic field of the stained monolayers; fluorescence was predominantly granular, and was restricted to the cytoplasm. The infected cells were readily distinguishable, and in most cases less than a minute was needed for examining 20 fields in each slide chamber. Reproducibility of the RFFIT was verified by testing dilutions of the same positive human serum six times with a constant virus dose. The reproducibility was within acceptable limits; the greatest variation observed was at the 1 :80 serum dilution, for example, with which the minimum number of 20 microscopic fields containing fluorescent foci was 5 and the maximum was 11. Table 1 presents L C M virus-antibody titers obtained with .the RFFIT, IFA, CF, and M N tests for sera from two persons having laboratory-acquired cases with known dates of onset. In both cases IFA antibody was detected first; maximal titers were obtained about 1 month after clinical onset of infection. The C F test
524
CAN. J. MICROBIOL. VOL. 23. 1977
TABLE1. LCM virus-antibody titers of sera from two laboratory-acquired cases as determined by four serological tests LCM virus-antibody titer* Case No. 2
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/23/14 For personal use only.
Case No. 1 Serum collectedt
RFFIT
IFA
CF
MN
RFFIT
IFA
CF
MN
*Dilution factor of serum end-point dilution. tDay after onset when serum collected. $Not tested.
became positive about a week after the IFA test. Throughout both cases the C F antibody remained at a low level and disappeared in a few months. Neutralizing antibody was detected almost a month earlier by the RFFIT than by the M N test; in one case it was detected simultaneously with the CF antibody. Neutralizing antibody was detectable in the last serum sample drawn from each patient by the RFFIT but was detectable in only one of these sera by the M N and IFA tests. All 50 control sera were negative by the IFA, CF, and M N tests, although one was positive by the RFFIT at a 1 :10 dilution. We further compared the sensitivities of these four tests by examining sera from 28 recent LCM cases with known dates of onset (Table 2). With the IFA test antibody was detected in all 28 cases, whereas CF, MN, and RFFIT antibodies were detected in 24, 8, and 18 cases, respectively. The RFFIT was positive in 10 cases in which the M N test was negative, but in no case was the M N test positive and the RFFIT negative. In addition, 36 human sera from individuals employed in hamsteries having LCM virusinfected animals were tested by each of the four methods (Table 3). The times between infection and collection of the sera were not precisely known, but for most of the sera they were longer than the times recorded in Table 2. All 36 sera were positive by both the RFFIT and the IFA-
test, whereas only 33 were positive by the M N and 17 by the C F test. The geometric means of antibody titers obtained by the RFFIT, IFA, MN, and C F tests were 49, 28, 13, and
