type H3N8 isolate A/equine 2/Miami/ ... Equine Respiratory Disease Research Group, Western College of Veterinary Medicine, University of Saskatchewan, ... A2/Miami influenza virus in studies ..... Foundation and the technical assistance.
Effect of Influenza A/equine/H3N8 Virus Isolate Variation on the Measurement of Equine Antibody Responses Jaret R. Bogdan, Paul S. Morley, Hugh G.G. Townsend and Deborah M. Haines
ABSTRACT
RESUME
This study has tested the effect of using homologous or heterologous equine influenza A virus isolates to evaluate serum antibody levels to influenza A virus in vaccinated and naturally-infected horses. In addition, the potential effect of antigenic selection of virus variants in egg versus tissue culture propagation systems was studied. Serum antibody levels in samples from horses recently infected with a local influenza A virus isolate (A/equine 2/Saskatoon/1/90) or recently vaccinated with a prototype isolate (A/equine 2/Miami/1/63) were assessed by hemagglutination inhibition and by single radial hemolysis using cell or egg-propagated A/equine 2/Saskatoon/1/90, A/equine 2/Miami/1/63 or A/equine 2/Fontainebleau/1/79. There were no significant differences in hemagglutination inhibition or single radial hemolysis antibody levels obtained with homologous or heterologous isolates or between viruses propagated in either eggs or cell culture. However there was a trend to higher titers in the hemagglutination inhibition assay when cell-propagated virus was used. These results suggest that antigenic variation in equine influenza A virus isolates and host-cell selection of antigenic variants during virus propagation may not be of sufficient magnitude to influence serological evaluation of antibody responses by hemagglutination inhibition or single radial hemolysis.
Dans cette etude, 1'effet de l'utilisation d'isolats homologues et heterologues du virus influenza A equin, pour evaluer le taux d'anticorps serique contre le virus influenza A, a ete etudie chez des chevaux vaccines et des chevaux infectes naturellement. De plus, 1'effet potentiel d'une selection antigenique de variants, suite a la propagation sur oeufs ou en culture cellulaire, a ete etudie. Le taux d'anticorps serique dans les echantillons provenant des chevaux recemment infectes avec un isolat local du virus influenza A (A/equin 2/ Saskatoon/1/90) ou recemment vaccines avec un isolat de reference (A/equin 2/ Miami/1/63) a ete evalue par inhibition d'hemagglutination et par hemolyse radiale simple en utilisant les isolats A/equin 2/Saskatoon/1/90, A/equin 2/ Miami/1/63, ou A/equin 2/ Fontainebleau/1/79 propages sur oeufs ou cellules. II n'y avait pas de difference significative dans les taux d'anticorps mesures par inhibition d'hemagglutination ou par hemolyse radiale simple avec les isolats homologues ou heterologues ou entre les isolats viraux propages sur oeufs ou en culture cellulaire. Toutefois, les titres en inhibition d'hemagglutination avaient tendance a etre plus eleves lorsque du virus propage en culture cellulaire 'etait utilise. Ces re'sultats suggerent que la variation antigenique d'isolats du virus influenza A equin, ainsi que la selection,
par la cellule, lors de la propagation virale, de variants antigeniques ne seraient pas sufflsantes pour influencer l'evaluation serologique d'anticorps par inhibition d'hemagglutination ou hemolyse radiale simple. (Traduit par Drs R. Magar et J.P. Lavoie)
INTRODUCTION Influenza A viruses have been shown to undergo changes in antigenicity. Influenza viruses sporadi-
cally undergo major antigenic changes or shifts in surface antigens and the more common minor changes in antigenicity referred to as antigenic drift (1). A major antigenic shift was recorded in equine influenza A virus isolates in 1963 when the hemagglutinin (H) and neuraminadase (N) antigens underwent significant antigenic change. Until that time all equid
influenza A virus isolates had similar HN antigens designated H7N7. The prototype isolate of the H7N7 equine influenza A isolates is A/equine I/Prague/1/56 (Al/Prague), while the major shift in equid influenza antigens is represented by the 1963 prototype H3N8 isolate A/equine 2/Miami/ 1/63 (A2/Miami) (2). In addition, as the result of antigenic drift, more recent equine isolates of influenza A virus have shown antigenic differences to the A2/Miami prototype virus (3). The 1979 Fontainebleau isolate (A/equine 2/Fontainebleau/1/79)
Equine Respiratory Disease Research Group, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N OWO.
Reprint requests to J.R. Bogdan, Department of Veterinary Microbiology, WCVM. Supported by a research grant from the Max Bell Foundation. Submitted August 28, 1992.
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Can J Vet Res 1992; 57:126-130
(A2/Fontainbleau) (4) was recommended by the World Health Organization as an additional prototype strain based on its significant antigenic drift from the original A2/Miami prototype. There is also evidence that the host cell-culture system used to propagate influenza isolates selects for certain antigenic variants, resulting in restricted antigenic populations for in vitro study, as compared to the virus spectrum which may be provoking the immune response in vivo (5). A study comparing egg-grown equine influenza A virus isolates with MadinDarby canine kidney (MDCK) cellgrown isolates reported that MDCKcell grown isolates were more reactive in hemagglutination inhibition (HI) reactions with postinfection and postvaccination equine sera (5). This suggests that egg-grown equine-2 influenza A virus differs antigenically from virus isolated in MDCK cells from the same clinical sample. Similarly, studies comparing HI tests of clinical isolates of human influenza A (H3N2) virus cloned in egg-culture with isolates grown in cynomolgus monkey kidney (MK) cells using postinfection ferret antisera and panels of monoclonal antibodies suggested that only the MK culture propagated the dominant antigenic variants found in the patient (6). Currently vaccines most commonly used in the prevention of equine influenza A virus-induced respiratory disease and test methods used to evaluate immune responses contain egggrown type 2 influenza A virus isolates similar to the A2/Miami prototype. The significant antigenic drift in recent isolates and suggestion of selection of antigenic variants by the cell-culture system raises questions as to the suitability of egg-propagated A2/Miami influenza virus in studies evaluating humoral immunity to more recent isolates of influenza virus in horses. This study compares egg-cultured and monkey kidney cell-cultured A2/Miami and A2/Fontainebleau influenza A virus isolates with the influenza virus A/equine 2/Saskatoon/l/90 (A2/Saskatoon) isolated from diseased horses in HI 7) and single radial hemolysis assays (8,9) to determine antibody levels in vaccinated and clinically diseased horses.
MATERIALS AND METHODS INFLUENZA VIRUS ISOLATES
The A/equine 2/Miami/l/63 (A2/Miami) isolate first described in 1963 was obtained from American Type Culture Collection (ATCC, Rockville, Maryland) at the sixth egg passage (E6) The A/equine 2/Fontainebleau/1/79 (A2/Fontainebleau) 1979 isolate was obtained from Dept. of Health and Human Services (Centers for Disease Control, Atlanta, Georgia) at an egg passage greater than three (E,3). The 1990 equine influenza A virus isolate A2/Saskatoon was obtained through nasal swab isolation from clinically diseased animals in primary rhesus monkey kidney (PRMK) cell cultures. This isolate as well as an egg-grown isolate of the same virus were typed using HI and neuraminidase inhibition tests (NI) by the Medical Research Council (National Institute for Medical Research, Mill Hill, London, England). These tests employed hyperimmune rabbit sera to Al/Prague, A2/Miami, A/equine 2/Sachyama/l/71 (A2/Sachyama), A2/Fontainebleau and two monoclonal antibodies prepared against A2/Fontainebleau. The A2/Saskatoon isolate was found to closely resemble A2/Fontainebleau. These two isolates gave identical titers with hyperimmune rabbit sera specific to A2/Fontainebleau and one monoclonal antibody raised to A2/Fontainebleau, while a second monoclonal antibody demonstrated a twofold titer decrease in recognition of the Saskatoon isolate. Egg (PRMK3, El) and MDCK-grown (PRMK3, MDCKI) A2-Saskatoon virus gave identical results with the rabbit sera and monoclonal antibodies, however MDCK-grown virus had a twofold higher reaction in the HI tests. VIRUS CULTURES
Egg-passaged virus was obtained by inoculating nine-day-old chicken embryos with A2/Miami (E6) virus, A2/Fontainebleau (Ex+3) virus, and A2/Saskatoon virus (PRMK3, E3). The allantoic fluid was harvested by standard procedures (7), clarified by centrifugation (800 g, 10 min), and tested for hemagglutinin activity with a 1% suspension of chicken erythrocytes (7).
Mammalian-cell passaged virus was obtained by inoculation of PRMK cell cultures (Viromed Laboratories, Inc. Minneapolis, Minnesota) with 200 4L of infected fluid (nasal swab fluid or allantoic fluid). Cells were maintained in RPMI 1640 media with L-glutamine (Gibco, Grand Island, New York) containing 2% fetal bovine serum, 100 units/mL penicillin G, 100 ,ug/mL streptomycin sulfate, 2.5 ,ug/mL amphotericin B, 2.05 pLg/mL sodium desoxycholate, 50 mg/mL gentamicin sulfate. Since no A2/Miami or A2/Fontainebleau mammalian cell grown isolates were available (originally isolated in egg embryos), infected allantoic fluid of egg-passaged isolates was used to infect PRMK cultures. The A2/Saskatoon virus (PRMK3) was the only one of the three PRMK cell grown isolates grown exclusively in a mammalian cell line. HEMAGGLUTINATION AND HEMAGGLUTINATION INHIBITION TESTS
Virus concentration was determined by hemagglutination (HA) titration utilizing 0.05 mL diluted virus and 0.05 mL of 1% chicken erythrocytes (in phosphate buffered saline (PBS) 0.1 M, pH 7.4), incubated at 4°C. Virus concentration was expressed as the reciprocal of the highest dilution of virus showing agglutination and represents one HA unit/0.05 mL. The virus was diluted in PBS to 8 HAU/0.05 mL for use in the HI test. Hemagglutination inhibition tests were performed in 96-well round bottom polyvinyl chloride microtitration plates (Dynatech Laboratories, Virginia). Sera were incubated at 56°C for 30 min before testing to inactivate complement. Serially diluted test serum (0.025 mL) was added to wells containing 0.025 mL of diluted virus (4 HAU/0.025 mL) and incubated for 30 min at room temperature. Subsequently 0.05 mL of 1% erythrocytes in PBS (7) was added and the plate incubated at 4°C until the erythrocytes had settled (1-2 h). Hemagglutination inhibition titers were recorded as the reciprocal of the maximum dilution giving total inhibition of hemagglutination. Results of the HI test are considered accurate to within one doubling dilution (7).
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TABLE I. Influenza A antibody measurements by hemagglutination-inhibition" with egggrown and cell-grown influenza A virus isolates
Egg-passaged virus Cell-passaged virus A2/Font A2/Sask A2/Fontc A2/Saskb Sera Vaccinated horses 40 80 Y 80 160 40 80 40 Z 160 Race horses 0 0 0 0 IA 160 160 640 320 IC 0 0 0 0 2A 160 160 640 320 2C 10 0 20 20 3A 160 320 160 640 3C 20 20 40 80 4A 160 160 320 320 4C 40 80 40 5A 160 320 160 320 640 5C 10 20 40 6A 40 80 160 80 6C 320 20 10 40 7A 40 160 320 320 640 7C a Reciprocal of the highest dilution of the serum showing hemagglutination-inhibition bA2/Sask = A/equine 2/Saskatoon/1/90 cA2/Font = A/equine 2/Fontainebleau/1/79 dA2/Miami = A/equine 2/Miami/1/63 A = acute, C = convalescent
A2/Miamid 40 40 0 160 0 160 0 160 20 160 40 160 10 80 20 160
The effect of high albumin concen- mixture only, gently mixed, and the trations found in infected allantoic suspension allowed to sit at room fluid on the HI test was investigated. temperature for 5 min (CrCl 3 step is Equal virus concentrations of both omitted if chicken erythrocytes are cell-grown (PRMK cell lysate clari- used). The virus-sensitized cell susfied by centrifugation at 1500 g for pension was sedimented by centrifu5 min) and egg-grown virus (clarified gation (750 g for 5 min), resuspended allantoic fluid) were diluted with in HEPES buffered saline, washed varying levels of normal allantoic once in PBS, and resuspended with fluid (undiluted and doubling dilu- PBS to a final concentration of 15%. tions to 1 in 8 in PBS), or with bovine The SRH plates were prepared by serum albumin (BSA, Fraction V, combining 3 mL of virus-sensitized Sigma Chemicals, St.Louis, Missouri) erythrocytes, 1 mL of fresh undiluted (4.0% and doubling dilutions to 0.5% guinea pig serum, and 26 mL of in PBS). Virus was diluted in PBS as agarose gel (1.5% Seakem ME agarose, a control. These dilutions of cell and in 0.15 M NaCl + 0.01 M phosphate egg-grown virus were then tested for buffer + 0.1% sodium azide, final pH hemagglutination. adjusted to 7.4). The agarose was first melted by microwaving then placed in SINGLE RADIAL HEMOLYSIS (SRH) TEST 43°C water bath to cool. VirusThe SRH test was modified from sensitized erythrocytes and guinea pig that previously described (8,9). A serum were combined, heated at 430C 10% suspension of sheep or chicken for approximately 30 seconds, then erythrocytes was made in HEPES buf- mixed quickly into the agarose. The fered saline (0.05 M, pH 6.5) and final suspension was poured into 150 mixed with clarified allantoic or tis- x 15 mm plastic petri plates (Falcon sue culture fluid containing A2/Miami 1058). Wells of 2 mm diameter were virus (500 hemagglutinating units cut in the gel and 3 ,uL of heat-inacti(HAU) per mL of 10% erythrocyte vated (560C, 30 min) serum added. suspension). Chromium chloride Samples were placed randomly on the (CrCI3) was freshly diluted 1/400 in plate and were tested in duplicate. unbuffered physiological saline from Zones of hemolysis were measured a 2.25 M solution (10). After gentle using a calibrating viewer (Transidyne periodic mixing at 4°C for 10 min a General Corp., Ann Arbor, Michigan) half volume of diluted CrCl3 was after incubating 21 h in a 37°C moist added to the sheep erythrocyte virus chamber. Antibody levels are
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expressed as hemolysis zone diameters and as hemolysis zone area. Results obtained with the SRH test used in this report have been shown to have a standard deviation of 0.5 mm for hemolysis zone diameters and 15% for calculated hemolysis zone areas (data not shown). SERUM SAMPLES
Sera tested by HI and SRH included two mares vaccinated three times at two week intervals with vaccine containing egg-cultured Al/Prague and A2/equine/Lexington/ 1/63 (considered identical to the A2/Miami virus strain) killed viruses (Dr. David Hustead, Fort Dodge Labs., Fort Dodge, Iowa, personal communication). Also tested were acute and convalescent samples from seven thoroughbred race horses which showed signs consistent with acute, infectious, upper respiratory tract disease. Acute samples were collected at onset of clinical symptoms and convalescent samples three weeks later. RESULTS The results of HI assays comparing influenza antibody measurement utilizing A2/Miami, A2/Fontainebleau, and A2/Saskatoon virus grown in egg culture and A2/Fontainebleau and A2/Saskatoon virus grown in PRMK cell culture are shown in Table I. Accepting that the HI test is read accurately to within one doubling dilution the titers of vaccinated and naturally-infected horses are similar among virus isolates within each culture system. However, between the two culture systems, the HI titers were consistently higher in both vaccinated and naturally-infected horses when tested with virus cultured in PRMK cell cultures as compared to virus grown in embryonated eggs. Using SRH tests influenza A virus antibody levels in sera from vaccinated and diseased horses were determined. Sera were tested with plates sensitized with the three egg-cultured influenza A virus isolates (Table II). The PRMK cell-cultured A2/Saskatoon and egg-cultured A2/Saskatoon were used to compare serum antibody levels as shown in Table III. The data shows SRH results are similar (within the
TABLE II. Influenza A antibody measurementsa by single radial hemolysis with egg cultured influenza virus isolates Sera Vaccinated Horses Y Z Race Horses 1 Acute Convalescent 2 Acute
A2/Saskd
Diameter (mm)b A2/Fonte A2/Miamil
A2/Sask
Area (mm2)c A2/Miami A2/Font
ND 8.0
ND 7.5
ND 7.4
ND 50
ND 44
ND 43
0.0 13.9 0.0 13.7 4.4 13.6 5.0 11.7 8.2 13.2 4.4
0.0 13.8 0.0 14.1 4.1 13.0 5.9 11.2 8.6 12.9 5.4
0.0 13.8 0.0 13.3 4.1 12.5 6.0 11.0 8.3 12.6 5.0
0 152 0 147 15 145 20
0 150 0 156 13 132 27 99 58 131 23
0
Convalescent 3 Acute Convalescent 4 Acute Convalescent 107 53 5 Acute 137 Convalescent 15 6 Acute 87 80 Convalescent 10.5 10.1 10.5 23 7 Acute 5.4 5.4 5.2 23 135 Convalescent 13.6 13.1 12.7 145 aResults expressed as hemolysis zone diameter and calculated hemolysis zone area bS.D. = 0.5 mm S.D. 15% of calculated area dInfluenza A/Equine 2/Saskatoon/90 Influenza A/Equine 2/Fontainebleau/79 I Influenza A/Equine 2/Miami/63
1 r%A
0 139 13 122
28 95 125 20 87
27
c
TABLE III. Influenza A antibody measurementsa by single radial hemolysis with eg g and cell cultured A/equine 2/Saskatoon/l/90 influenza virus Area (mm2)c Diameter (mm)b Cell-cultured Sera Cell-culturedd Egg>-cultured Egg-cultured Vaccinated Horses 113 12.0 113 y 12.0 z 98 11.2 11.2 98 Race Horses 0 0 1 Acute 0.0 0.0 177 174 Convalescent 14.9 15.0 0 0 2 Acute 0.0 0.0 196 15.3 15.8 184 Convalescent 3 Acute 8.5 8.7 57 58 14.2 Convalescent 15.3 184 59 4 Acute 9.4 8.7 69 154 14.2 Convalescent 14.0 158 90 10.7 83 5 Acute 10.3 14.7 14.9 170 Convalescent 31 5.8 6.3 26 6 Acute 143 135 Convalescent 13.1 13.5 71 9.5 71 7 Acute 9.5 177 174 14.9 15.0 Convalescent aResults expressed as hemolysis zone diameter and calculated hemolysis zone area bS.D. = 0.5 mm S.D. 15% of calculated area dCultured in primary rhesus monkey kidney cells
c
accuracy of the test method) among isolates of influenza A virus cultured in eggs as well as between PRMK-cultured and egg-cultured A2/Saskatoon virus in both vaccinated and naturally infected horses. Differences seen between data in Tables II and III using egg-cultured A2/Saskatoon virus reflect day to day test variability. In HA tests, dilution of egg-grown virus with normal allantoic fluid
caused fourfold decrease in I IA titers compared to the PBS contrnol in all four allantoic fluid concen trations tested. Similarly, egg-growin virus diluted in four concentrations ,of BSA showed twofold decreases in ti ter compared to the PBS control. The HA titer of cell-grown virus also de(creased. The titer diminished more th[an fourfold in undiluted normal a llantoic fluid, fourfold in allantoiic fluid
diluted 1/2, and twofold in higher dilutions of allantoic fluid. The HA titer of cell-grown influenza virus decreased twofold when diluted in either 2 or 4% BSA.
DISCUSSION This study has examined the effect of using different influenza A virus isolates in HI and SRH tests for determining equine serum antibody levels to type 2 equine influenza A viruses. While only a small number of horses were tested the results demonstrate that horses mount humoral immune responses which cross-react to recognize similar immunodominant epitopes on three H3N8 equine influenza A virus isolates separated widely geographically and temporally: A/equine 2/Miami/i /63, A/equine 2/Fontainebleau/1/79 and A/equine 2/Saskatoon/ horses suggest that while antigenic variation among these influenza A virus isolates may be detected by monoclonal antibodies, the polyclonal serological responses of horses, if examined by HI and SRH tests, can be reliably detected and quantified using prototype virus isolates of the appropriate HN subtype. However, this conclusion may apply only to previously exposed and/or vaccinated horses such as those tested in the present study, since naive ponies have been shown to produce isolate-specific antibodies which are poorly reactive to other influenza A viruses of the same subtype (11). In addition, other more sensitive test methods, may demonstrate that a proportion of the humoral immune response in horses may be isolate specific (12). However, while the role of cross-reactive antibody has been clearly demonstrated, the clinical significance, if any, of isolate-specific antibody is uncertain (1 1). While it does not appear to significantly alter interpretation of test results if virus isolates used for antibody determination are grown in egg or mammalian cell culture, differences were noted in results obtained between the two culture systems. These findings are similar to some studies with human influenza A virus in which isolates were considered to be a mixture of virus variants, the rel-
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ative proportions of which can change depending upon the isolation method (6). This variability has been reported to significantly alter the results of HI testing of postvaccinal immunity in ferrets (3), however other studies have shown that the antigenic variability that occurs within and between human HINI influenza isolates is detectable with monoclonal antibodies but not with polyclonal antisera (A.R. Douglas, National Institute for Medical Research, London, personal communication). The findings of this study suggest that any effect of culture system selection may not be of practical concern for serological testing in horses. In a previous report the tendency for equine sera to give higher HI titers when tested with cell-cultured rather than egg-cultured virus (5) was inferred to mean that growth of influenza in eggs selected for a subpopulation of less reactive influenza variants (13). In the present study this variation in HI titers was shown to be associated with factors present in allantoic fluid and BSA rather than evidence for selection of antigenic variants since the addition of uninfected allantoic fluid or BSA similarly decreased HA reactivity of cell-cultured virus. Since factors in allantoic fluid interfere with virus-induced hemagglutination, to achieve equivalent HAU concentrations between cell and egg-cultured virus for use in HI assays higher concentrations of egg-cultured virus must be added. This higher level of egg cultured virus would require higher antibody levels to be neutralized in the HI assay, resulting in apparently lower HI titers. Therefore the diminished HI titers measured using eggcultured virus may not be due to antigenic differences in the virus, but instead to decreased hemagglutinating activity of egg-cultured virus. This concept is supported by other studies in which homologous egg and tissue culture isolates of H3N2 viruses were examined with a panel of monoclonal antibodies and while the tissue-culture isolates consistently gave much higher values in HI tests than their egg-grown counterparts, no antigenic differences were discernible (A.R. Douglas, National Institute for Medical Research, London, personal communication). The finding in the
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present study of the inhibitory effect of allantoic fluid protein on HI results may also explain the findings of Cook et al (5) in which HI titers were consistently lower when egg-cultured viruses were compared to homologous viruses cultured in cells, however solid phase binding assays demonstrated that polyclonal sera and monoclonal antibodies to the hemagglutinin molecule bound equally well to egg and cell-cultured homologous isolates. The finding that the addition of trypsin to egg cultured virus increases HI reactivity by cleavage of the hemagglutinin (14), may be partially due to enzymatic degradation of hostderived inhibitory factors which would otherwise interfere with virus hemagglutination. Serological tests such as HI and SRH which employ whole virus to detect antibody levels in polyclonal sera of the naturally-infected horses and the two vaccinated horses give largely the same results regardless of whether homologous or prototype viruses of the appropriate HN specificity are used. In addition, either cell or egg propagated virus may be used to assess antibody titers however HI titers will be lower in all sera if eggcultured virus is employed suggesting that the use of egg-cultured virus in the SRH test may be more appropriate than in the HI test. These findings may be helpful in standardizing serological assays for influenza A virus antibody and in comparing studies in which different strains of equine influenza virus were used in serological studies of vaccinated or naturally infected horses.
ACKNOWLEDGMENTS The authors are grateful for the financial support of the Max Bell Foundation and the technical assistance of Wendy Glossop, WCVM Equine Respiratory Disease Group, Western College of Veterinary Medicine, Saskatoon, Canada.
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