Myxoviruses: Virus Hemadsorption Assay

Transcription

1 APPLIED MIcRooOGY, Apr. 1973, p Copyright 1973 American Society for Microbiology Vol. 25, No. 4 Printed in U.SA. Quantitative Assessment of Hemadsorption by Myxoviruses: Virus Hemadsorption Assay NICHOLAS HAHON, JAMES A. BOOTH, AND HERBERT L. ECKERT Appalachian Laboratory for Occupational Respiratory Diseases, Morganton, West Virginia 2655 Received for publication 17 January 1973 The standardiation and quantitative evaluation of an assay for myxoviruses, based on the enumeration of individual infected clone 1-5C-4 cells manifesting hemadsorption ithin 24 h of infection, are described. Hemadsorption as detectable earlier than immunofluorescence in infected cells or hemagglutinins in culture medium. The relationship beteen virus concentration and cells exhibiting hemadsorption as linear. The assay as highly precise, sensitive, and reproducible. The phenomenon of hemadsorption, first described by Vogel and Shelokov (15), ith influena viruses gron in cell cultures subsequently proved cardinal to the discovery and serological characteriation of the parainfluena viruses (1). The applicability of the phenomenon for assaying myxoviruses, hoever, has been based on techniques that either lacked simplicity of performance, adequate quantitative evaluation, or, relying on multiple cycles of virus propagation in host cells, required prolonged incubation periods. Some hemadsorption assay procedures that have been employed ith myxoviruses include counting clumps of agglutinated erythrocytes in 48-h infected-cell cultures (13), the virus dilution end-point method of hemadsorption hich requires incubation of infected cell cultures for 5 days (9), and a 48-h hemadsorption plaque assay (3). An attempt as made to develop a virus hemadsorption assay, similar in principle to immunofluorescent cell-counting virus assays (5, 6), that is rapid, quantitative, and dependent on a single cycle of cell infection. This report describes the application and standardiation of a quantitative assay for myxoviruses based on the enumeration of individual infected cells manifesting hemadsorption ithin 24 h of infection. MATERIALS AND METHODS Cell culture. The principal cell line used as clone 1-5C-4 derived from a variant line of Chang's conjunctival cell (16). Cells ere propagated ith Eagle minimum essential medium containing 1% fetal calf serum and maintained ith minimal essential medium plus 5% fetal calf serum. For the virus hemadsorption assay, cells ere cultivated on circular cover slips (15-mm diameter) inserted in flat-bottomed 595 glass vials (19 by 65 mm). One milliliter of cell suspension, containing approximately 1' cells, as introduced onto each cover slip hich as then incubated at 35 C for 24 h or until a complete cell monolayer as formed. Virus. Influena A/PR8 and parainfluena 3 C-243 virus strains employed in this study ere obtained from the American Type Culture Collection, Rockville, Md. Influena virus as gron in the allantois of 11-day-old chicken embryonated eggs. After incubation at 35 C for 48 h, eggs ere chilled (6 C) for 5 or more h. Allantoic fluids ere then harvested asceptically and individually assayed for hemagglutinin (HA), and those ith the highest HA titer ere pooled. The stock virus preparation as stored in 1-ml amounts in glass vials at -7 C. To propagate parainfluena 3 virus, HeLa cell monolayers in 25-ml, plastic, tissue culture flasks (Falcon Plastics, Div. of B-D Laboratories, Inc., Los Angeles, Calif.) ere each inoculated ith 3 ml of an appropriate virus dilution. After incubation at 35 C for 2 h, residual inoculum as removed, and 15 ml of maintenance medium as added per flask. Cell cultures ere then incubated at 35 C until a cytopathic effect as observed. The contents of each flask ere then froen (-7 C) and thaed (35 C) three times, pooled, and centrifuged at 17 x g for 5 min. The supernatant fluid as dispensed and stored as described for the influena virus pool. Erythrocyte suspension. Guinea pig blood obtained by cardiac puncture as mixed ith an equal volume of Alsever solution and stored at 4 C. Erythrocytes ere usable for up to 1 eek. For each day's test, cells ere ashed three times in phosphate-buffered saline (PBS), ph 7.2. The final ashing as carried out in a graduated centrifuge tube at the recommended conditions of sedimentation (9). Packed cells ere resuspended in PBS to make a.4% erythrocyte suspension that as calculated by volume. Virus hemadsorption assay. Virus dilutions prepared in saline (.2 M NaCI) buffered by.1 M Donloaded from on September 26, 218 by guest

2 596 HAHON, BOOTH, AND ECKERT APPL. MICROBIOL. phosphate (PBS), ph 7.2, ere introduced in.2-mi manner described earlier for virus assay. The amount volumes directly onto cover slip cell monolayers held of virus that as attached to cells at a given time as in rotor chamber inserts (6). These ere employed expressed as a percentage of the virus input. The because they ithstand the centrifugal force required latter as the sum of the amounts of attached and to sediment virus. Rotor chamber inserts placed in a unattached virus. singing-bucket SB-11 rotor ere centrifuged in a Determination of virus penetration. Virus penetration into cells as measured by the insensitivity of model B-5 ultracentrifuge (International Equipment Co., Needham Heights, Mass.) at 1, rpm (9,838 to attached virus to antiserum. After inoculum as 16,155 x g, depending on the distance of the chamber attached to cells by centrifugation (6 C), cell cultures insert in the arm of the rotor from the axis of rotation) ere ashed ith PBS, overlaid at designated intervals of incubation ith.5 ml of a prearmed 1:15 for 12 min at 6 C. Residual inoculum as removed after centrifugation, the cover slip cell monolayers dilution of virus antiserum, and then incubated at 35 ere placed into glass vials, and 1 ml of maintenance C for 2 h. The antiserum as removed and replaced medium as then added to each vial. After incubation ith maintenance medium, and incubation of cell at 35 C for 2 to 24 h, cover slip cell monolayers ere cultures as continued for an additional 2 h. The rinsed ith PBS. A.4% suspension of guinea pig quantity of virus that penetrated into cells at a given erythrocytes in PBS as added in a.5-ml volume time as expressed as a percentage of the input virus. onto each cell monolayer hich as then held at 6 C for 2 min. RESULTS For enumerating cells exhibiting hemadsorption, cover slip cell monolayers ere examined ith a Standardiation of the assay. The efficiency Nikon inverted microscope. With this optical system of procedures employed to initiate virus infection of cells by using hemadsorption as the at a magnification of x 2, the number of microscopy fields contained in the area of a 15-mm diameter cover monitor of cell infection as investigated. The slip as 365. For each cover slip cell monolayer, 3 rate of influena and parainfluena 3 virus microscopy fields ere examined. To calculate the attachment as determined during stationary number of hemadsorption cell units of virus per incubation (35 C) and during centrifugation (6 milliliter, the average number of cells shoing hemadsorption per field as multipled by the number of C). Aided by centrifugal force, approximately field per cover slip, the reciprocal of the dilution of 98% of the influena virus inoculum as attached ithin 1 min; after stationary incuba- virus inoculum, and the volume factor (for conversion to milliliters). tion for 2.5 h, only 37% of the inoculum as Virus immunofluorescence assay. This procedure attached (Table 1). is based on counting fluorescent cells in infected cell The rate of penetration of influena virus into monolayers that have been stained ith viral antiserum conjugated ith fluorescein isothiocyanate. insensitivity of attached virus to antiviral cells at 35 C as folloed by determining the In all aspects, the infection and incubation conditions serum at designated times. Results sho that for cell monolayers ere identical to that described for penetration proceeded at a linear rate and as the virus hemadsorption assay. The direct fluorescent-antibody method as used to demonstrate immunofluoresence of viral antigens in infected cells. influena virus attachment and penetration complete ithin 15 min (Fig. 1). The findings on Details of the staining procedure, materials, microscope equipment, and enumeration of infected cells fluena 3 virus. ere comparable to those noted ith parain- have been described elsehere (7). The incubation period is defined as the time HA titration. Test samples ere assayed for HA by using the standard Microtiter system ith plastic "V"-bottomed plates. In.5-ml amounts, test for attachment of influena (PR8) virus onto clone TABLE 1. Centrifugation versus stationary incubation preparations ere serially diluted in PBS in tofold steps. Each ell then received.5 ml of a 1-5C-4 cell monolayers.5% chicken erythrocyte suspension, and after incubation at room temperature from 1 to 2 h, test Inoculum attached (%) patterns ere read. The HA titer as the reciprocal of Min Stationary Centrifugationa (35 C) the highest dilution of test sample that shoed incubation complete agglutination of cells. Determination of virus attachment. Attachment NDb as measured by folloing the disappearance of virus ND from inoculum after its addition to cell monolayers ND Virus inoculum at multiplicity of infection of.1 in a 3 ND ml volume as introduced onto cells. After designated intervals of incubation or centrifugation, resid- 9 ND ND 19.6 ual inoculum as removed, cell cultures ere immediately ashed ith PBS, and residual inoculum as 15 ND ND 3. introduced onto fresh cell monolayers to measure unattached virus. Cover slip cell monolayers exposed aconditions: 16,155 x g; 6 C. to initial or residual inocula ere treated in the b Not determined. 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3 VOL. 25, 1973 I-!a. C,) I I~~~~~~ MINUTES FIG. 1. Rate ofpenetration of influence (PR8) virus into clone 1-5C-4 cells at 35 C. interval beteen virus inoculation of cell monolayers and the development of hemadsorption capability by maximal numbers of infected cells. This period as established from sequential sampling of cell monolayers inoculated ith either influena or parainfluena 3 viruses at a multiplicity of infection of.1 and incubated at 35 C. Cell monolayers ere then periodically tested for hemadsorption. At the same time TABLE 2. Time after HEMADSORPTION BY MYXOVIRUSES. I 597 interval, additional infected-cell monolayers ere froen (-7 C) and thaed (35 C) tice and tested for hemagglutinin and the production of infectious virus. Hemadsorption as detected ithin 7 h after virus inoculation of cell monolayers (Table 2). The general pattern of erythrocyte adherence to infected cells as in the form of rosettes or clusters (14). The maximal number of cells exhibiting hemadsorption occurred beteen 2 and 24 h of the primary cycle of infection. The production of infectious virus and hemagglutinin as not detected earlier than 48 h. The susceptibility of five established cell lines to infection by influena and parainfluena 3 viruses as determined by the virus hemadsorption assay. Virus infectivity for each cell line as carried out in triplicate. Results indicate that clone 1-5C-4, LLC-MK,, and BHK-21/C13 cell lines ere equally susceptible to infection by influena virus; L-929 and HeLa cell lines ere less susceptible. The folloing cell lines ere susceptible to parainfluena 3 virus infection (in order of decreasing susceptibility): BHK-21/C13, LLC-MK2, 1-5C-4, HeLa, and L-929. The BHK-21/C13 cell line as not suitable for the hemadsorption assay of virus. With this cell line, cells infected by either influena or parainfluena 3 viruses exhibited a "streamer effect" (14) in the presence of guinea pig erythrocytes, making it difficult to count accurately individual cells exhibiting hemadsorption. To determine the optimal incubation conditions for adsorption of erythrocytes to infected cells, the reaction as observed at varied temperatures (6 and 22 C) and time intervals (5 to 3 min) in cell monolayers infected ith either influena or parainfluena 3 viruses. Results (Table 3) sho that maximal hemadsorption occurred in influena or parainfluena 3 virus- Sequence of viral antigen formation in clone 1-5C-4 cells infected ith myxoviruses at a multiplicity of infection of.1 infection (h) Hemadsorp-., Influena (PR8) Parainfluena 3 Infectious Infectious tion Hemaggiutvi' Hemadsorption Hemagglutsinu x x 1' x 1' 5. x 1' x 1' 4.3 x 1' 2 3. x 1' 7.5 x 1' x 1' 1.6 x 1' x 1' NDd 1.3 x 1' 7. x 1' x 14 a Number of cells exhibiting hemadsorption on a 15-mm cover slip cell culture per.2 ml of inoculum. b Reciprocal of highest dilution of.2-ml test sample shoing complete hemagglutination. c Number of cells ith fluorescent antigen on a 15-mm cover slip cell culture per.2 ml of inoculum. d Not determined. Donloaded from on September 26, 218 by guest

4 598 HAHON, BOOTH, AND ECKERT APPL. MICROBIOL. TABLE 3. Hemadsorption of guinea pig erythrocytes to virus-infected clone 1-5C-4 cell monolayers at varied conditions Incubation Influena (PR8) HCUa Parainfluena 3 HCU ith per ml per ml erythrocytes (min) 6 C 22 C 6 C 22 C x 1' 7.3 x 1' 4.2 x 1' 3.6 x 1' x 1' 1.2 x x 1' 8.9 x 1' x x x 1' 1.4 x 1' x x x x 1' a Hemadsorption cell units. infected cell monolayers ithin 2 min at either 6 or 22 C. Specific attachment of erythrocytes to virus-infected cells has been reported to be independent of temperature ithin the range of to 37 C (12). Quantitative evaluation of the assay. A linear relationship as obtained beteen the number of cells manifesting hemadsorption and the relative concentration of influena virus throughout the inoculum range of 1.5 log1 units (Fig. 2). These data suggest that each hemadsorption cell unit as the consequence of infection by a single virus particle or aggregate not divisible by dilution. To estimate the precision of the hemadsorption assay for influena and parainfluena 3 viruses, 13 determinations ere performed ith each virus by the prescribed manner. The number of HCU of influena virus per ml of inocuhum ranged from 1.2 x 17 to 1.7 x 17, ith a mean of 1.4 x 17 and standard deviation (SD) of Expressed as a percentage, the SD as 1.7% of the mean. The number of hemadsorption cell units of parainfluena 3 virus per milliliter of inoculum ranged from 5. x 1' to 5.9 x 1', ith a mean of 5.3 x 1' and SD of The SD as 5.4% of the mean. The reproducibility of the assay ith the use of tofold dilutions of virus inoculum in each of the six determinations is shon in Table 4. The results attest to the reproducibility of the assay at the virus concentration levels employed. The mode of distribution of cells exhibiting hemadsorption on a cover slip monolayer as determined by examining 145 random microscopy fields. The frequencies of fields containing cells shoing hemadsorption correspond closely to the expected frequencies (Fig. 3). The chisquare test of goodness-of-fit of the observed data to the theoretical Poisson distribution shoed no significant deviation (P-.95 ith 6 df). Cells manifesting hemadsorption ere randomly distributed in cell monolayers. The sensitivity of the hemadsorption and immunofluorescence assays for influena and parainfluena 3 viruses as compared. Although these procedures involve different indicator systems of cell infection, they are both based on infective cell counting. For both assays, inoculation and incubation of infected clone 1-5C-4 cell monolayers ere performed in a similar manner. Results in Table 5 sho that the immunofluorescence assay for both viruses as approximately tice as sensitive as the -J 4 Co/ C-) Co 4 C-) - 3- (n -J -j M L. a- 2- C') CI RELATIVE VIRUS CONCENTRATION FIG. 2. Linear function beteen the number of cells exhibiting hemadsorption and concentration of influena (PR8) virus in inoculum. TABLE 4. Reproducibility of hemadsorption assay for influena (PR8) virus Assay Dilution of virus inoculum HCUa/ml no. 1:4 1:8 1:1,6 1:3,2 (x 17) b Mean ahemadsorption cell units. b Average number of cells exhibiting hemadsorption per 3 microscopy fields ith.2 ml of inoculum. Donloaded from on September 26, 218 by guest

5 VOL. 25, Expected SJ 4-4 Observed 2 NUMBER OF HEMADSORPTI ON CELLS PER MlICROSCOPIC FIELD FIG. 3. Frequency of distribution of cells exhi biting hemadsorption on 1-,C-4 cell monolayer inoculated ith influena (PR8) virus. TABLE 5. HEMADSORPTION BY MYXOVIRUSES. I Comparison of infective cell counting assays for influena (PR8) and parainfluena 3 viruses Influena virus Parainfluena virus Determi- Immuno- Immunonationain fluores- flors sopto Hemad- fluoressrto f Hemad- cence (X 1on cence ( 1 ClUa/Ml }HCU5/ml) CIU/ml) HCU/ml) Assay no Mean SDC ±.54 ±.18 ± SEd ±.27 ± a Cell-infecting units. Cell monolayers ere infected ith influena or parainfluena viruses at a multiplicity of.1 in.2 ml of inoculum. After incubation at 35 C for 22 h, cell monolayers ere stained ith fluorescein-conjugated viral antiserum. bhemadsorption cell units. Cell monolayers ere infected ith influena or parainfluena viruses at a multiplicity of.1 to.2 ml of inoculum. After incubation at 35 C for 22 h,.4% guines pig erythrocytes ere introduced onto cell monolayers. c Standard deviation. dstandard error of mean. hemadsorption assay. The latter, hoever, exhibited slightly less variability than the former. 599 DISCUSSION The feasibility of employing an assay for myxoviruses, i.e., influena and parainfluena 3, based on the enumeration of individual infected cells manifesting hemadsorption as established by the studies described here. Hemadsorption as detected approximately 7 h after virus inoculation of cell monolayers and as comparable in time to that observed previously (8). The maximal number of cells exhibiting hemadsorption occurred beteen 2 and 24 h of the primary cycle of infection. By using hemadsorption as the indicator of cell infection, resultant rates of attachment and penetration of influena virus ere similar to that attained by other means (7). The hemadsorption phenomenon appeared earlier than the production of hemagglutinins and the production of infectious virus demonstrable by immunofluorescence staining. Hemadsorption by virusinfected cells has been noted in the absence of detectable virus filaments and infectious virus particles (2, 8, 1). This suggests that the phenomenon may be attributable to an immunological structure alteration of the cell surface (1) or, more specifically, the presence of viral envelope proteins on the cell surface in the form of a "precursor" to the projections seen on virions (2). The use of primary monkey kidney cell cultures or monkey continuous cell lines for virus assays based on hemadsorption involves serious disadvantages. A specific reaction beteen erythrocytes and infected cells may occur, due to the presence of myxoviruses of simian origin that occur naturally in these cell cultures (11). In addition, nonspecific tissue hemadsorption (not only common to monkey kidney cells, but hich may also be manifested in kidney cell strains or lines derived from other animal species) may result (4). Although the established cell lines, clone 1-5C-4 and LLC-MK2 (rhesus monkey kidney), appeared to be equally susceptible to myxovirus infection, in vie of the above circumstances clone 1-5C-4 cells ere preferred as the cell line for the myxovirus hemadsorption assay. Quantitative evaluation of the assay shoed that it as highly precise, reproducible, and almost equivalent in sensitivity to virus assays based on immunofluorescent cell counting (5). Limited trials performed to compare the sensitivity of the hemadsorption assay and the hemagglutination test for assessment of myxoviruses confirmed previous findings of the superiority of the former (13). In assaying influena virus, e found that the hemadsorption assay as more than 3. log1 units higher in sensitivity than the hemagglutination test. LITERATURE CITED 1. Chanock, R. M., R. H. Parrott, K. Cook, B. E. Andres, J. A. Bell, T. Reichelderfer, A. Z. Kapikian, F. M. Mastrota, and R. J. Huebner Nely recognied myxoviruses from children ith respiratory disease. N. Engl. J. Med. 258: Compans, R. W., N. J. Dimmock, and H. Meier-Eert An electron microscopic study of the influena Donloaded from on September 26, 218 by guest

6 6 HAHON, BOOTH, AND ECKERT virus-infected cell, p In R. D. Barry and B. W. J. Mahy (ed.), The biology of large RNA viruses. Academic Press Inc., Ne York. 3. Deibel, R., and J. Hotchin Groth characteristics of hemadsorption virus type 1 (myxovirus parainfluena 3) in tissue culture. Virology 14: Dodle, W. R., and R. Q. Robinson Non-specific hemadsorption by rhesus monkey kidney cells. Proc. Soc. Exp. Biol. Med. 121: Fraser, K. B Immunological tracing: viruses and rickettsiae, p In R. C. Nairn (ed.), Fluorescent protein tracing. The Williams & Wilkins Co., Baltimore. 6. Hahon, N Fluorescent cell-counting assay of yello fever virus. J. Infect. Dis. 116: Hahon, N., J. A. Booth, and H. L. Eckert Cell attachment and penetration by influena virus. Infect. Immunity 7: Hahon, N. and H. L. Eckert Cell surface antigen induced by influena virus. Infect. Immunity 6: Hoskins, J. M. (ed.) Virological procedures. Butterorths and Co., London. 1. Hotchin, J. E., S. M. Cohen, H. Ruska, and C. Ruska Electron microscopical aspects of hemadsorption APPL. MICROBIOL. in tissue cultures infected ith influena virus. Virology 6: Hull, R. N., J. R. Minner, and J. W. Smith Ne agents recovered from tissue cultures of monkey kidney cells. Amer. J. Hyg. 63: Marcus, P. I Dynamics of surface modification in myxovirus-infected cells. Cold Spring Harbor Symp. Quant. Biol. 27: Roee, K. R., G. L. Williams, and C. E. van Rooyen Detection and titration of Asian influena A virus by HeLa cell and monkey kidney cell cultures. Science 128: Shelokov, A., J. E. Vogel, and L. Chi Hemadsorption (adsorption-hemagglutination) test for viral agents in tissue culture ith special reference to influena. Proc. Soc. Exp. Biol. Med. 97: Vogel, J., and A. Shelokov Adsorption-hemagglutination test for influena virus in monkey kidney tissue culture. Science 126: Wong, S. C., and E. D. Kilbourne Changing viral susceptibility of a human cell line in continuous cultivation. I. Production of infective virus in a variant of the Chang conjunctival cell folloing infection ith sine or N-WS influena viruses. J. Exp. Med. 113: Donloaded from on September 26, 218 by guest