Indirect Fluorescent-Antibody Technique for Serological Diagnosis of La Crosse (California) Virus Infections

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 192, p Vol. 15, No /2/ $02.00/0 Indirect Fluorescent-Antibody Technique for Serological Diagnosis of La Crosse (California) Virus Infections B. J. BEATY,'* J. CASALS,1 K. L. BROWN,2 C. B. GUERSEN,2 D. NELSON,3 J. T. McPHERSON,3 A W. H. THOMPSON4 Yale Arbovirus Research Unit, Department of Epidemiology and Public Health, New Haven, Connecticut ; Gundersen Clinic-La Crosse Lutheran Hospital, La Crosse, Wisconsin ; and Virology Section3 and Zoonosis Research Unit,4 State Laboratory of Hygiene, Madison, Wisconsin Received 7 August 191/Accepted 21 September 191 A clinically relevant indirect fluorescent-antibody technique (IFA) was developed for the serological diagnosis of La Crosse virus infections. The IFA (67%) was as sensitive as the hemagglutination inhibition (5%) and neutralization (5%) tests in the detection of antibodies in acute-phase specimens. Immunoglobulin M antibodies were detected by the IFA test in 4% (11 of 23) of these specimens. Diagnostically significant increases in IFA titer were detected in 6% (19 of 22) of the paired samples. Antibodies were detectable in some patients 7 years after infection; however, the IFA test was not as sensitive as the other two tests in the detection of previous infections. La Crosse virus (LAC), an arbovirus in the California group of the family Bunyaviridae, is a major cause of encephalitis in the United States (11). Currently, serological diagnosis of LAC infections is accomplished by demonstration of >4-fold changes in hemagglutination inhibition (HI) or virus neutralization (Nt) titers between acute- and convalescent-phase specimens (14). The complement fixation (CF) test is also used in the diagnosis of LAC infections. However, CF antibodies appear later in the illness, and diagnostic changes in titer are typically obtained only in widely spaced serum samples (10). The HI and Nt tests also present difficulties in the diagnosis of LAC infections. Production of hemagglutination antigens for California group viruses is a difficult and laborious procedure (4, 13). Although Nt is the preferred test for diagnosis, few laboratories can afford the expense of maintaining tissue culture production and virus containment facilities. In practice, specimens from suspected arbovirus cases are forwarded to centralized facilities, such as state laboratories or the Centers for Disease Control. Thus, none of these three classic diagnostic tests (CF, HI, and Nt) can be readily applied in the clinical setting to provide information to assist the physician in prescribing patient care. Indirect fluorescent-antibody (IFA) tests have been reported for the serological diagnosis of several arbovirus diseases (1, 3, 7). In the initial report on IFA testing for the diagnosis of LAC infections, antibodies to LAC were detected in 2% of acute-phase patient specimens. Specific immunoglobulin M (IgM) antibodies were detected in 20% (5 of 20) of acute-phase specimens in that study, but the authors concluded that counterimmunoelectrophoresis was the preferred method for diagnosis (9). We report here the development of a more sensitive IFA technique for the diagnosis of LAC infections in humans. MATERIALS A METHODS Serum. Serum specimens were obtained from patients admitted to the Gundersen Clinic-La Crosse Lutheran Hospital, La Crosse, Wisconsin, with symptoms compatible with encephalitis. Specimens were obtained from patients that became ill during the arboviruses season (June through October, 1979). Some specimens were also obtained from patients that presented with central nervous system manifestations in the months before the arbovirus season. IFA test. The procedures used are essentially the same as those reported previously for arenaviruses (12, 15), with modifications for California group viruses. Production of infected cells. BHK-21 cells were seeded in Corning flasks (150 cm2; approximately 107 cells per flask). After 2 to 3 days, cells were inoculated with the respective viruses at a multiplicity of infection of approximately 100. When cytopathic effect was approximately 2+, usually 2 days postinfection, the maintenance medium was removed, and the monolayer was washed with Hanks balanced salt solution. A total of 10 ml of 0.25% trypsin was added, and the cells were incubated at 37 C for 3 min. The trypsin suspension was removed, and the monolayer was incubated at 37 C for an additional 20 to 30 min. Detached cells were collected in 2 to 3 ml of phosphate-buffered saline and gently pipetted to ensure dispersion. Cells from 6 to 10 flasks were pooled and centrifuged at 1,500 rpm for 5 min, washed three times in phosphate-buffered saline, and suspended in an amount of phosphatebuffered saline equal to 2 ml per flask used. This 429

2 430 BEATY ET AL. suspension was then adjusted to 3 x 106 cells per ml; a 150-cm2 flask typically supplied 3 to 10 ml of adjusted cell suspension. Preparation of spot slides. Microscope slides were obtained from Cel-Line Associates, Minotola, N.J. These slides (75 by 25 mm) were coated with Teflon, except for 12 5-mm-diameter spots in two rows of six spots (5 mm diameter) each. Infected and uninfected cells were mixed in a 3:1 ratio. One drop of mixed cells was placed on the first five spots of each row, using a 27-gauge needle mounted on a 0.25-ml tuberculin syringe. Each drop contained approximately 0.01 ml of cell suspension, or 3 x 104 cells. This procedure ensured that at least onefourth of the cells were not infected, providing an internal control in each spot. To the final spot in each row, only uninfected cells were added, serving as a negative control. After dispensing the cells, the slides were air dried, fixed in acetone, and stored at -70 C. Slides were brought to room temperature in sealed plastic bags to prevent condensation and subsequent loss of cells from the spots. Staining. The IFA technique used was similar to that of Gardner and McQuillin (). Serum samples were diluted, and a drop of the dilution was placed on the appropriate spot. The slides were incubated for 30 min at 37 C and then washed three times for 10 min each in 0.02 M phosphate-buffered saline. After air drying, the slides were stained with the appropriate dilution of a commercially prepared goat anti-human immunoglobulin conjugated with fluorescein isothiocyanate. To detect IgM, the spots were stained with goat antihuman IgM. The conjugates contained Evans blue at a dilution of 1:10,000 as a counterstain. Fluorescence. Either a Zeiss diagnostic immunofluorescence microscope or an Olympus Vanox incident light microscope with an HBO OSRAM 200-W mercury vapor light source and a BG 12 excitation filter was used to examine the slides. Slides were examined at magnification x200 and 400 (oil) for the presence of fluorescence. The amount of fluorescence was measured on a 1-to-4+ scale for both the number of cells infected and the intensity of fluorescence in the cells. HI and CF tests. The procedure of Clarke and Casals (5) was used to perform the HI assays. Sera were acetone extracted and adsorbed with goose erythrocytes before testing. LAC hemagglutinins were prepared in BHK-21 spinner cultures (4, 13). The Centers for Disease Control Laboratory Branch Complement Fixation technique was used to determine CF titer. Nt test. A microneutralization test *as used to determine Nt titers (10). Serial dilutions of serum specimens (0.025 ml) were incubated for 1 h at 37 C with between 30 and % tissue culture infective doses per ml of the virus. The suspension was then combined with 0.05 ml of BHK-21 cells (approximately 104 cells), and the mixture was added to the respective well of the microtiter plate. The plates were sealed, incubated at 37 C, and observed for cytopathic effect after 4 to 5 days. The same procedure was used to determine Nt titers for the other California group viruses known to be present in Wisconsin: snowshoe hare, Jamestown Canyon, and trivittatus. J. CLIN. MICROBIOL. RESULTS Between the months of June and October, 1979, 76 patients presented at the Gundersen Clinic-La Crosse Lutheran Hospital with symptoms compatible with clinical encephalitis. Of these, 67 were diagnosed as having encephalitis based on signs, symptoms, and the presence of leukocytes in the cerebrospinal fluid. Twentyfive patients were serologically diagnosed as having LAC infections. Eight more patients had LAC antibodies, but serological conversions were not demonstrable by the HI, Nt, or IFA test; one of these was a suspected case, but a convalescent-phase specimen was not obtained. Thirteen patients were diagnosed as having had encephalitis of enterovirus etiology, and an additional eight encephalitis cases were suspected to be enterovirus infections but were unconfirmed. Eighteen cases were not diagnosed. The serological results obtained for those 26 individuals diagnosed as, or suspected of, having LAC encephalitis in 1979 are shown in Table 1. IFA antibody became detectable soon after onset. Case 5 had detectable antibody on the day of onset and admission, and nine other individuals had detectable antibodies by 3 days after onset. Specific IgM antibodies were detected in 4% (11 of 23) of the acute-phase serum specimens. Diagnostic (-4-fold) titer changes occurred in at least one test for all individuals with paired sera. Of those sera examined by the IFA test, 19 of 22 (6%) had.4-fold titer changes. For the Nt test, 1 of 22 (2%) seroconverted, and 20 of 24 (3%) of those sera examined by HI had.4-fold titer changes. Of the 24 sera tested by CF, (33%) seroconverted, but in many instances, acute- and convalescent-phase specimens may have been too closely spaced to detect rises in CF titer. The IFA test was as sensitive as the HI and Nt tests in the detection of antibody in acute-phase specimens (Table 2) of patients subsequently diagnosed by the Nt and HI test as having LAC encephalitis. There was complete concordance between the detection of antibody by the IFA technique and the subsequent detection of antibodies to LAC by the HI or Nt test. The specificity of the IFA and Nt tests in the differentiation of antibodies to California group viruses in convalescent-phase sera is shown in Table 3. In all cases, there were -4-fold IFA titer differences between LAC and trivittatus virus. Diagnostic differences in titer were obtained between LAC and Jamestown Canyon virus in all but one instance. In case 1, the IFA titer was 1:12 for LAC, snowshoe hare, and Jamestown Canyon viruses. The IFA test was able to differentiate between LAC and snowshoe hare virus in 5% (7 of 12) of the specimens. To determine the longevity of IFA antibodies, selected serum samples from cases from previ-

3 VOL. 15, 192 IFA TEST FOR LAC ENCEPHALITIS 431 TABLE 1. Results of serological testing of human LAC infections Serological titers obtained with following testa: Age (yr) Date No. of days Date IFA Case no. and sex (mo/day/yr) 0.0er ofday/yrn ofpatentofonertof samnide TtlNt HI CF immunoglobulin IgM 1 7, M 9/2/ , M /7/ , M 7/6/ , M 9/16/ , F /4/ , M 9/17/ , M /6/ , M 10/11/ , M 10/4/ , M 7/1/ , F 9/7/ , M /6/ , F 9/23/ , F /14/ , M 9/31/ , F /17/ , M /5/ , M 10/9/ , F /11/ /29/79 10/1/79 11/5/79 /11/79 1/1/0 7//79 /3/79 9/1/79 10/11/79 /4/79 /24/9 9/21/79 10//79 /1OM9 /17/79 10/13/79 11/1/79 10/7/79 2/13/0 7/6/9 /2/79 9/10/79 9/19/9 /10/79 1/16/0 9/23/79 9/27/79 10/24/79 /16/79 11/15/79 10/12/79 10/30/79 1/2/0 /20/79 /29/79 3/5/0 /14/79 10/23/79 1/16/0 /12/79 124/ ,04 2,04 2,04 2, <10 <10 < b < <10 <10 < < <10 <10 < <10 <20 < 4 1, < < < < < < <10 <10 < , < 0 < 0 0 < <10 <10 < < < <10 < <10 <10 < < < < < < < 40 < <10 <20 < <

4 432 BEATY ET AL. J. CLIN. MICROBIOL. TABLE 1-Continued Age (yr) Date NoD of days Date Serological titers obtained with following testa: IFA of patient of onset of sample Total immunoglobulin IgM Nt HI CF 20 4, M 9/21/ /24/79 10/12/ < < Case no. and sex (mo/day/yr) No.e ofnday (mo/day/yr) Nt HI CF_ 21 5, M 7/2/79 4 /1/79 <10 <20 < 13 /10/ < 22 7, F 9/1/10 Unknown 7/30/79 <10 < /6/ , F 9/5/79 5 9/10/79 <10 <20 < 15 9/20/ < 44 10/19/ , M 9/5/79 5 9/10/ < 16 9/21/ < 25 5, M 10/6/ /11/ < 16 10/22/ < 26 12, M /3/79 3 /6/ < 24 /27/ < a Reciprocal titer. b, Not determined. ous years were obtained from the serum bank of the Zoonosis Research Unit. Specific LAC antibodies were detected in all patients 1 year after infection (Table 4). All individuals sampled at 3, 6, and 7 years postinfection had detectable antibody. At years postinfection, neither of the individuals sampled had detectable IFA antibody. Total antibody titers did decline with time, but IgM antibody to LAC declined precipitously. Most individuals had low titers after 1 year (Table 4), and only one patient (no. 6) had detectable IgM after 3 years. DISCUSSION The IFA test seems to be a suitable technique for the serological diagnosis of LAC infections. It was as sensitive as the HI and Nt tests in the detection of antibody to LAC in acute-phase sera (Table 1). Antibody was detectable soon after onset of disease, and seroconversions were detectable by the IFA test in 6% of the cases (Table 1). Furthermore, the IFA test seemed as capable as the Nt test in distinguishing between antibodies to the various California group viruses (Table 3). The apparent equivalent sensitivity of the IFA and Nt tests in the detection of antibody in acute-phase specimens was somewhat surprising. The Nt test is considered to be the most sensitive technique for the detection of LAC antibodies (10). Perhaps the sensitivity of the IFA test is in part due to the nature of the antigens available in the test. The HI and Nt tests detect antibodies to the glycoproteins of LAC (2). CF is thought to detect antibodies to the nucleocapsid protein. All viral antigens as well as intact virions are present in various stages of maturation in BHK-21 cells. Thus, the IFA test can detect all groups of antibodies instead of only those participating in one reaction. Alternatively, by the time of admission, patients already have or rapidly develop substantial IFA, Nt, and HI antibody titers. Therefore, the tests would seem to be of equivalent sensitivity, based on results obtained with the acute-phase specimens alone. However, the IFA test detected antibody in only one of nine specimens considered to be from previous infections. All nine specimens contained Nt antibody, and most had 1:10 or 1:20 HI titers. Thus, the HI and Nt test do seem to be more sensitive TABLE 2. Detection of antibodies to LAC in acutephase serum specimens Serological test No. of specimens No. (%) examined positive IFA (67) Nt (5) HI (5) CF 24 0 (0)

5 VOL. 15, 192 IFA TEST FOR LAC ENCEPHALITIS 433 TABLE 3. Case no. Cross-reaction of convalescent-phase serum specimens with other California group viruses as determined by the IFA and Nt techniques Titer of following virus type obtained with indicated testa: California LAC Snowshoe hare Jamestown Canyon Trivittatus IFA Nt IFA Nt IFA Nt IFA Nt , < < <10 4 2, < ob 10 0 < < <10 2, < ,04 22,04 a Reciprocal titer. b 0, Less than 4. C, Not determined. and would be preferred tests in retrospective studies. This is compatible with the beforementioned hypothesis; CF antibody, which is principally directed to the nucleocapsid protein, is known to be short-lived. CF antibodies would no longer be present, which could account in part for the decreased sensitivity of the IFA test in the detection of previous infections. The detection of specific IgM by the IFA test further enhances the rapid diagnostic capability of the test. However, the detection of IgM, albeit at low levels, in specimens from patients at 1 year after infection may present difficulties in the establishment of unequivocal diagnoses. Detection of IgM in one patient 3 years postinfection is of special concern. However, none of these specimens were assayed for the presence of rheumatoid factor (6). The detection of IgM after 3 years may be owing to the presence of IgM-IgG aggregates which could complex with specific IgG, resulting in false IgM positives. In future studies, sera to be tested for IgM will be routinely treated to remove rheumatoid factor. The unexpectedly high titers obtained in the studies to determine the persistence of IFA antibodies may be due to long-term frozen storage of the serum samples. In summary, the IFA test proved to be a rapid, reliable, and sensitive technique for the serological diagnosis of LAC encephalitis. The test was remarkably sensitive in the detection of acute-phase antibodies and was also rapid. If the spot slides are prepared in advance, results can be obtained in approximately 3 h. Furthermore, the test is inexpensive and readily adaptable to most clinical settings currently possessing immunofluorescence capability. TABLE 4. Persistence of antibodies to LAC in patients at 1 to years after infection IFA titer' Case no. Date (mo/day/yr) Total immunoglobulin 1 7/20/ 5/26/65, /10/ /27/65 4,096 b 2/17/66 2,04 3 7/27/65 2, /31/66 2,04 4 7/6/ /29/65 4,096 5/14/66 2,04 5 9/6/71 2, /21/71,192 10/24/72,192 10/11/77 2, /22/ /24/ /11/77 7 9/27/ /26/65 2,04 10/2/66 10/24/ /13/ /11/ a Reciprocal titer. b, Not determined.

6 434 BEATY ET AL. ACKNOWLEDGMENTS We acknowledge the technical expertise and assistance of Joan Schallern, Stanley Horner, Les Lorenz, and the personnel in the Immunology Department of La Crosse Lutheran Hospital. We are also indebted to the physicians, house staff, and personnel of the Gundersen Clinic-La Crosse Lutheran Hospital and the Gundersen Medical Foundation for their advice, assistance, and cooperation in these studies. This research was supported by Public Health Service grants R01-AI-151 and R01-AI from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Aaskov, J. G., and C. Davies An immunofluorescence assay for human antibodies to Ross River virus. J. Immunol. Methods 25: Bishop, D. H. L., and R. E. Shope Bunyaviridae, p In H. Fraenkel-Conrat and R. Wagner (ed.), Comprehensive virology, vol 14. Plenum Publishing Corp., New York. 3. Boonpucknavig, S., 0. Vuttivirojana, J. Siripont, P. Futrakul, and S. Nimmannitay Indirect fluorescent antibody technique for demonstration of serum antibody in dengue hemorrhagic fever cases. Am. J. Clin. Pathol. : Chappel, W. A., P. E. Halonen, R. F. Tolle, C. H. Calisher, and L. Chester Preparation of La Crosse virus hemagglutinating antigen in BHK-21 suspension cell cultures. Appl. Microbiol. 1: Clarke, D. H., and J. Casals Techniques for hemagglutination and hemagglutination inhibition with arthropod-borne viruses. Am. J. Trop. Med. Hyg. 7: Cremer, N. E., and J. L. Riggs Immunoglobulin classes and viral diagnosis, p In E. H. Lennette J. CLIN. MICROBIOL. and N. J. Schmidt (ed.), Diagnostic procedures for viral, rickettsial, and chlamydial infections. American Public Health Association, Washington, D.C. 7. El Mekki, A. A., G. Van Der Groen, and S. R. Pattyn Evaluation of immunofluorescence and immunoperoxidase methods for antibody determination against Chikungunya, West Nile, and Yellow Fever viruses. Ann. Soc. Belg. Med. Trop. 59: Gardner, P. S., and J. McQuillin Rapid virus diagnosis. Application of immunofluorescense. Butterworth and Co., London. 9. Kalis, J. M., A. C. Burgess, and H. H. Balfour Serological diagnosis of California (La Crosse) encephalitis by immunofluorescence. J. Clin. Microbiol. 1: Lindsay, H. S., C. H. Calisher, and V. J. H. Mathews Serum dilution neutralization test for California group virus identification and serology. J. Clin. Microbiol. 4: Monath, T. P Arthropod-borne encephalitides in the Americas. Bull. WHO 57: Peters, C. J., P. A. Webb, and K. M. Johnson Measurement of antibodies to Machupo virus by indirect fluorescent technique. Proc. Soc. Exp. Biol. Med. 142: Rabson, M., B. J. Beaty, S. W. Hildreth, and R. E. Shope Production of hemagglutinating antigens of La Crosse virus using polyethylene glycol precipitation. J. Clin. Microbiol. 13: Shope, R. E., and G. E. Sather Arboviruses, p In E. H. Lennette and N. J. Schmidt (ed.), Diagnostic procedures for viral, rickettsial, and chlamydial infections. American Public Health Association, Washington, D.C. 15. Wulff, H., and J. V. Lange Indirect immunofluorescence for the diagnosis of Lassa fever infections. Bull. WHO 52: Downloaded from on September 19, 201 by guest