Sensitive Plaque Neutralization Assay for Parainfluenza Virus

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1981, p /81/ $02.00/0 Vol. 13, No. 4 Sensitive Plaque Neutralization Assay for Parainfluenza Virus Types 1, 2, and 3 and Respiratory Syncytial Virus JOYCE D. HAWTHORNEt AND PAUL ALBRECHT* Divison of Virology, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland Received 5 September 1980/Accepted 31 December 1980 A sensitive plaque neutralization assay for parainfluenza virus types 1, 2, and 3 and respiratory syncytial virus was developed in Vero and MA 104 cell cultures. The tests were performed in semimicrotiter trays containing 24 wells, 16 mm in diameter. Parainfluenza virus type 1 formed plaques in Vero and MA 104 cells only when trypsin was added to the overlay medium. Plaquing of parainfluenza virus type 1 was more sensitive and technically reproducible in MA 104 cells than in Vero cells. Parainfluenza virus types 2 and 3 and respiratory syncytial virus readily formed plaques in Vero cells. Plaques with all viruses were necrotic in character, except for plaques produced by parainfluenza virus type 3, which appeared red due to an increased uptake of neutral red by infected cells. Different conditions for plaquing of the four viruses had to be used to obtain plaques of suitable size. Antibody titers of commercially prepared guinea pig typing sera were 5- to 50-fold higher by the plaque neutralization test than by complement fixation. The addition of guinea pig immunoglobulin G antiglobulin to the serumvirus mixtures enhanced the conventional neutralization test 5- to 10-fold. The sensitivity and specificity of the plaque neutralization test was also determined with sera of marmosets experimentally infected with parainfluenza virus types 1 and 3. The generally low postinfection titers could be enhanced, on the average, 40-fold by using human immunoglobulin G antiglobulin in the neutralization test. A low degree of cross-reactivity was shown between parainfluenza virus types 1 and 3 both in the conventional neutralization test and in the anti-immunoglobulin enhanced neutralization test. Since their isolation in the late 1950s, parainfluenza viruses (5, 12, 13, 19) and respiratory syncytial virus (RSV) (6, 15) have been recognized as the two leading causes of lower respiratory tract infection in infancy and early childhood. Studies concerning the epidemiology of these infections and the effect of preventive measures led to the development of tissue culture techniques for virus isolation and antibody measurement. For parainfluenza viruses, primary monkey kidney cells proved to be most suitable (4), whereas primary human embryonic kidney cultures were equally sensitive but less readily available (26). The restriction in the supply of primates for biological investigation led to the search for equally sensitive but more available cell types for viral propagation and testing. Although parainfluenza viruses have been reported to grow and form plaques in a number of cell strains (10, 21) and cell lines (8, 20, 26, 27; F. A. Miller and R. G. Brackett, Bacteriol. Proc., p. 155, 1963), primary monkey kidney cells are still t Present address: Microbiological Associates, Bethesda, MD routinely recommended for their isolation and for the serological identification of infection (4). RSV, on the other hand, is routinely isolated and identified in HEp-2 cell cultures (1, 4, 17). In this paper we describe the successful use of two continuous cell lines of monkey kidney cells for virus plaque titration and antibody measurement with RSV and parainfluenza viruses. The antibody test was made highly sensitive by potentiating the virus-antibody reaction with heterologous anti-immunoglobulin (AIG). MATERIALS AND METHODS Cell cultures. Vero cells at passage levels 140 through 200 and MA 104 cells at passage levels 215 through 250 were supplied by the Cell Culture Section of the Division of Virology, Bureau of Biologics, Food and Drug Administration. The cells were grown in Eagle minimum essential medium supplemented with 10% inactivated fetal or newborn bovine serum, 0.03% glutamine, 50 gg of gentamicin per ml, and 0.4,ug of amphotericin B per ml. Solid overlay medium for parainfluenza viruses types 2 and 3 and RSV consisted of minimum essential medium containing 5% fetal calf serum and 0.5% agarose (Seakem; Marine Colloids, 730

2 VOL. 13, 1981 Rockland, Maine). The overlay medium for parainfluenza virus type 1 consisted of minimum essential medium containing 0.5% agarose, 0.5% gelatin (Difco Laboratories, Detroit, Mich.), and a final concentration of 1.6 to 2.0 pg of trypsin (lx crystallized; Worthington Diagnostics, Freehold, N.J.) per ml. Viruses. The following viruses were used: strains Sendai and HA-2 for parainfluenza virus type 1, strains Greer and DA for parainfluenza virus type 2, strain C243 for parainfluenza virus type 3, and strain Long for RSV. Ail strains were obtained from the American Type Culture Collection, Rockville, Md. All parainfluenza virus strains were passed one additional time in primary African green monkey kidney cells before being used in the neutralisation (Nt) test. RSV was passed once in HEp-2 cells. The titers of the stock suspensions are given in Table 1. Sera. Monospecific guinea pig sera against parainfluenza virus types 1, 2, and 3 and RSV were purchased from two commercial sources; Flow Laboratories, Inc., Rockville, Md., and Microbiological Associates (MA) Bioproducts, Walkersville, Md. Additional sera were obtained from the Centers for Disease Control (CDC), Atlanta, Ga., and the Research Resources Branch, National Institute of Allergy and Infectious Diseases (NIAID), Bethesda, Md. Guinea pig sera obtained from commercial sources and NIAID were produced by two inoculations of the respective live viruses. The first was administered intranasally, and the second was administered intraperitoneally 2 to 3 weeks later. The animals were bled approximately 2 weeks after the booster inoculation. The immunizing viruses were grown in embryonated TABLE 1. Virus (stramn) ASSAY FOR PARAINFLUENZA VIRUSES AND RSV 731 eggs for parainfluenza virus type 1, in primary vervet monkey kidney cells for parainfluenza virus types 2 and 3, and in HEp-2 cells for RSV. The sera obtained from CDC were prepared in a similar manner. The immunizing viruses for intranasal inoculation were grown in embryonated eggs for parainfluenza virus types 1, 2, and 3 and in HEp-2 cells for RSV. The intraperitoneally administered viruses were grown in Vero cells for parainfluenza virus types 1, 2, and 3 and in HEp-2 cells for RSV. All sera were produced by using virus strains Sendai, Greer, C243, and Long. No adjuvants were administered. Pre- and post-immunization sera were obtained from moustached marmosets (Saguinus mystax) inoculated intranasally with parainfluenza viruses. One group of marmosets was inoculated with 107` plaqueforming units of strain Sendai per dose, and another group was inoculated with 1065 plaque-forming units of strain C243 per dose. These animals were bled at regular intervals after infection. AIG. Rabbit immunoglobulin G (IgG) against guinea pig IgG (heavy and light chains) and rabbit IgG against human IgG (heavy and light chains) were purchased from Cappel Laboratories, Cochranville, Pa. The concentrations of specific antibody for the two AIGs as given by the manufacturer were 2.5 to 2.8 and 4.0 mg/ml, respectively. Titers of AIG preparations were determined in our laboratory by the Ouchterlony double-diffusion test in plates with 0.35% agarose in distilled water. The precipitation titer of the anti-human IgG was 1:8 with homologous (human) serum and 1:4 with marmoset serum. It was negative with rabbit serum. The titer of the anti-guinea pig IgG was 1:8 with homologous serum. Virus plaque Nt test. Serial dilutions of serum were mixed with an equal volume (80 pl) of virus diluted so as to yield 20 to 30 plaque-forming units per well. After this mixture was incubated for 1 h at 37 C, 80 pl of tissue culture medium (conventional Nt test) or 80 1l of AIG (enhanced Nt test) was added, and the mixture was incubated for 20 min at room temperature. Samples of 0.1 ml were inoculated into each of two wells 16 mm in diameter on a 24-well disposable plastic tray (Linbro, Hamden, Conn., or Costar, Cambridge, Mass.) carrying either Vero or MA 104 cell monolayers. After adsorption for 60 min, the inoculum in each well was replaced with 1.0 ml of agarose overlay medium, and the trays were incubated in a humidified, 5% C02 atmosphere. At given time intervals after infection, each well received an additional 0.5 ml of agarose overlay supplemented with neutral red (final dilution, 1:30,000). The time for adding the neutral red overlay, the total incubation time, and the temperature of incubation are given in Table 1. Upon termination, the overlay was removed from the wells, and the trays were dried. The virus Nt titer was determined as the dilution of serum reducing the number of viral plaques per well by 50%. The dilution was calculated by the method of Karber (16). A virus, a virus-aig, an AIG, and a cell control were included in each test. RESULTS Virus plaque formation. The parainfluenza viruses and RSV plaqued readily in Vero or MA Optimal conditions for plaque formation under agarose byparainfluenza viruses and RSV Cell culture Incuba- Neutral Test ter- Titer of tion red. Character of Plaque size stock virus tep temp added mmated 'd5` plqe plaques (m) (mm) (gi (log,o ( C) (day) (day, PFUa/ml) Parainfluenza 1 (Sendai) MA Necrotic' Parainfluenza 1 (HA-2) MA Necrotic' Parainfluenza 2 (Greer) Vero Necrotic Parainfluenza 2 (DA) Vero Necrotic Parainfluenza 3 (C243) Vero Red RSV (LongJ Vero Necrotic a PFU, Plaque-forming units. b Develop only in the presence of 2.0 ug of trypsin per ml in the overlay.

3 732 HAWTHORNE AND ALBRECHT 104 cells under the conditions listed in Table 1. Under these conditions, the plaques had a distinct and reproducible morphology, and their size did not exceed 2 mm in diameter. A total of 20 to 40 plaques of this size could be readily counted in 16-mm wells as required in antibody titration by plaque Nt in 24-well trays. The plaque morphology achieved with individual viruses is shown in Fig. 1. Parainfluenza virus type 1, both strain Sendai and strain HA-2, required the addition of trypsin in the overlay, evidently for cleavage of the viral hemagglutinin (24). The optimal concentration of trypsin for MA 104 cell monolayers was from 1 to 2 fig/ml. The cells did not seem to be damaged even at higher trypsin concentrations. The concentration range of trypsin for Vero cell cultures was much narrower, between 1.2 and 1.6 gg/ml. Higher concentrations of trypsin had a tendency to destroy the Vero cell monolayers, and lower concentrations of trypsin were not sufficient to cause plaquing. Fetal bovine serum had to be replaced with 0.5% gelatin in both cell systems so as not to interfere with the activity of the trypsin. Since MA 104 cells yielded better t- J. CLIN. MICROBIOL. and more reproducible plaque morphology than did Vero cells, they were considered the substrate of choice for parainfluenza virus type 1 plaquing. Parainfluenza virus type 1 gave approximately 50% higher titers when plaqued at 34 C compared with 37 C. Parainfluenza virus type 2, on the other hand, yielded higher titers at 37 C. Dense Vero cell sheets at infection gave higher plaque counts than did lightly sheeted monolayers for parainfluenza virus type 2. Parainfluenza virus types 1 and 2 and RSV produced necrotic plaques. Parainfluenza virus type 3 produced "red plaques," consisting of infected cells which absorbed an increased amount of neutral red and therefore appeared darker than the surrounding uninfected cell sheet. The red plaques were much more distinct when incubated at 34 C than when incubated at 37 C. RSV was plaqued in both Vero and HEp-2 cell monolayers. Even though plaques developed slower in Vero cells, the final titer of the virus preparation was higher in Vero than in HEp-2 cells. As fetal bovine serum became scarce and expensive, an attempt was made to replace it with U '.. r--- > -«:- FiG. 1. Plaques produced by parainfluenza viruses in monolayer cultures in 16-mm wells on plastic trays. (A) Parainfluenza virus type 1, strain Sendai; (B) parainfluenza virus type 2, strain Greer; (C) parainfluenza virus type 2, strain DA; (D) parainfluenza virus type 3. All wells stained supravitally with neutral red.

4 VOL. 13, 1981 newborn calf serum in the diluent as well as in the agarose overlay. Ten percent newborn calf serum reduced the number of parainfluenza virus type 3 plaques by 95%. Plaques of parainfluenza virus types 1 and 2 were reduced by less than 10%, and plaques of RSV were reduced in size but not in number. This was probably due to the presence in the newborn calf serum of cross-reacting antibody to bovine parainfluenza viruses. Two lots of newborn calf serum have been tested so far. Gamma globulin-free newborn calf serum has not been tested yet. The results of the following experiments were obtained using fetal bovine serum exclusively. Virus Nt with immune guinea pig sera. Monospecific guinea pig sera against parainfluenza virus types 1, 2, and 3 and RSV were titrated with homologous and heterologous viruses in the conventional and enhanced Nt tests. The results of tests with parainfluenza virus type 1 antiserum are given in Table 2. Sera from all three sources had a high homologous antibody titer in the range 1:1,000 to 1:2,000. Potentiation of Nt with AIG resulted in a 5- to 10-fold increase in the Nt titers. There was a moderate but reproducible cross-reactivity between type 1 antiserum and parainfluenza virus type 3. Table 3 shows the antibody titers obtained with parainfluenza virus type 2 antiserum. The homologous titers were again mostly in the range 1:1,000 to 1:2,000 and were potentiated by about 10-fold with AIG. Cross-reactivity with the other parainfluenza viruses was low and not reproducible. Parainfluenza virus type 3 guinea pig serum gave the highest homologous Nt titers (Table 4). Three of the four sera showed some cross-reactivity with parainfluenza virus type 2, and one of three showed some cross-reactivity with parainfluenza virus type 1. Potentiation by AIG was TABLE 2. ASSAY FOR PARAINFLUENZA VIRUSES AND RSV 733 about 2.5- to 4-fold. Antibody titers with RSV antiserum are given in Table 5. The sera were highly specific, showing only an occasional low-level cross-reaction with parainfluenza viruses and only in the enhanced Nt test. As evaluated by the t test on paired samples, the enhanced Nt titers were significantly higher than the conventional Nt titers at P < 0.001, P < 0.01, P < 0.001, and P < 0.05 for parainfluenza virus types 1, 2, and 3 and RSV, respectively. Complement fixation (CF) antibody titers determined by the serum manufacturers are listed in each table. In ail instances, they were considerably lower than the titers measured in the plaque Nt test. Antibody titers in experimentally infected marmosets. Before being infected, marmosets were screened for antibody against parainfluenza virus types 1 and 3 and found to be negative. After intranasal inoculation, the animals developed antibody to the respective virus 10 to 14 days after infection (Tables 6 and 7). Conventional Nt titers in marmosets were considerably lower than the titers of the hyperimmune guinea pig sera. On the other hand, they were enhanced by rabbit anti-human AIG, on the average, 40-fold (range, <6- to 90-fold). Antibody titers in marmosets infected with parainfluenza virus type 3 were appreciably higher than antibody titers in animals infected with parainfluenza virus type 1. This is similar to the homologous titers of the guinea pig sera, suggesting that parainfluenza virus type 3 is a better antigen for both animal species. Parainfluenza virus type 1 antibody titration in Vero and MA 104 cell cultures. A comparative titration of selected marmoset and guinea pig sera was performed in Vero and MA 104 cell cultures. MA 104 cells provided a more Homologous and cross-reactive antibody titers ofparainfluenza virus type I guinea pig serum Cross-reactive titer with following virus (strain): Homologous Nt test Antiseruma Parainfluenza 1 Parainfluenza 2 Parainfluenza RSV (Long) CF titerb (Sendai) (Greer) 3 (C243) Conventional Flow 1,050 (3)C <8 (2) 32 (2) <8 (2) 80 NIAID 2,270 (3) <8 (2) 27 (2) <8 (2) 320 CDC 1,860 (1) <8 (1) 88 (1) <8 (1) >64 Enhanced Flow 12,360 (3) 240 (2) 500 (2) <128 (2) NIAID 24,350 (3) <128 (2) 940 (2) <160 (2) CDC 8,900 (1) <128 (1) 700 (1) <128 (1) a See the text for an explanation of serum origin. b Titer given by serum manufacturer. c Reciprocal of mean 50% plaque Nt titer (number of tests).

5 734 HAWTHORNE AND ALBRECHT J. CLIN. MICROBIOL. TABLE 3. Homologous and cross-reactive antibody titers ofparainfluenza virus type 2 guinea pig antiserum Cross-reactive titer with following virus (strain): Nt test Antiserum' Parainfluenza 1 Parainfluenza 2 Parainfluenza 3 R (Long) Homologous CF titerb (Sendai) (Greer) (C243) Conventional Flow <8 (3) 660 (2) <8 (2) <8 (2) 80 NIAID 15 (3) 2,480 (2) c16 (2)d <8 (2) 160 CDC <8 (1) 2,860 (1) <8 (1) <8 (2) >64 MA <8 (1) 1,860 (1) ND' ND NA' Enhanced Flow 220 (3) 6,330 (2) <200 (2) <128 (2) NIAID 660 (2) 15,290 (2) -160 (2) <128 (2) CDC <128 (1) 12,680 (1) <128 (1) <128 (1) MA <128 (1) 7,740 (1) <128 (1) <128 (1) a See the text for an explanation of serum origin. b Titer given by serum manufacturer. ' Reciprocal of mean 50% plaque Nt titer (number of tests). d c indicates that the mean was calculated from values which included one titer which is less than 8 in the conventional NT test or less than 128 in the enhanced Nt test. e ND, Not determined. f NA, Not available. TABLE 4. Homologous and cross-reactive antibody titers ofparainfluenza virus type 3 guinea pig antiserum Cross-reactive titer with following virus (strain): Homologous Nt test Antiserum" Parainfluenza 1 Parainfluenza 2 Parainfluenza 3 RSV (Long) CF titer" (Sendai) (Greer) (C243) Conventional Flow <8 (3)c <8 (2) 3,960 (2) <4 (1) 80 NIAID 27 (2) -16 (2)d 5,750 (2) <4 (1) 80 CDC <8 (1) <8 (1) 8,063 (2) <4 (1) >64 MA NDe il (1) 15,390 (2) <8 (1) NAf Enhanced Flow -250 (3) 470 (2) 10,470 (2) <128 (1) NIAID 570 (2) 2,110 (2) 19,090 (2) <128 (1) CDC <128 (1) <128 (1) 63,648 (2) <128 (1) MA ND 130 (1) 96,132 (2) <128 (1) See the text for an explanation of serum origin. b Titer given by serum manufacturer. Reciprocal of mean 50% plaque Nt titer (number of tests). d c indicates that the mean was calculated from values which included one titer which is less than 8 in the conventional Nt test or less than 128 in the enhanced Nt test. e ND, Not determined. f NA, Not available. sensitive test system than did Vero cells (Table 8). DISCUSSION Antibodies to parainfluenza viruses are routinely measured by hemagglutination inhibition or the Nt test (4). Antibodies to RSV are measured by CF or the Nt test (14, 18). The Nt test is generally preferred for both viruses due to its greater sensitivity and reproducibility. In most instances, primary monkey kidney cell cultures are used as the cell substrate for the parainfluenza viruses, and HEp-2 cell cultures are used for RSV. Because of the scarcity of primary cell cultures of primate origin, parainfluenza viruses and RSV have been propagated in human diploid cell strains, but higher titers were obtained with primary kidney cells of primate origin. Parainfluenza virus type 1 could also be grown in avian cells (7) and primary calf kidney cells (21), and parainfluenza virus type 3 was grown in primary bovine kidney cultures (9). Sugita et al. (27) reported the use of an established monkey kidney cell line, LLC-Mk2, for plaquing parainfluenza virus type 1. Deibel (8) used the human amnion cell line FL for parainfluenza virus type 2. Miller and Brackett (Bacteriol. Proc., p. 155, 1963) reported the appearance of plaques in KB

6 VOL. 13, 1981 ASSAY FOR PARAINFLUENZA VIRUSES AND RSV 735 TABLE 5. Homologous and cross-reactive antibody titers ofrsvguinea pig antiserum Cross-reactive titer with following virus (strain): Nt test Antiserum' Parainfluenza 1 Parainfluenza 2 Parainfluenza 3 Homologous CF titer' (Sendai) (Greer) (C243) RSV (Long) Conventional Flow <8 (1)C <8 (1) <8 (1) >1,825 (1) 64 NIAID <8 (1) <8 (3) <8 (2) 356 (2) 80 MA <8 (1) <8 (1) <8 (1) >1,850 (1) NAd Enhanced Flow <128 (1) 128 (1) 220 (2) 4,700 (1) NIAID s230 (2)e 150 (3) s190 (2) 4,185 (1) MA <128 (1) <128 (1) <128 (1) 9,640 (1) a See the text for an explanation of serum origin. b Titer given by serum manufacturer. c Reciprocal of mean 50% plaque Nt titer (number of tests). d NA, Not available. e < indicates that the mean was calculated from values which included one titer which is less than 8 in the conventional Nt or less than 128 in the enhanced Nt test. TABLE 6. Antibody titers in marmosets infected with parainfluenza virus type I Titer on following day post in- Nt test Marmoset fection: Conventional M62 <8a <8 < M85 <8 <8 < M101 <8 < Enhanced M62 <64 < ,274 M85 <64 < ,589 2,455 M101 <64 <64 <64 1,231 3,171 areciprocal of 50% plaque Nt titer. TABLE 7. Antibody titers in marmosets infected with parainfluenza virus type 3 Titer on following day post infec- Nt test Marmoset tion: Conventional M52 <8a <8 < M68 <8 < M103 <8 < , Enhanced M52 <128 <128 <128 <128 6,690 M68 <128 <128 < ,456 M103 <128 <128 6,958 21,015 17,681 areciprocal of 50% plaque Nt titer. cells infected with parainfluenza virus type 3. The human cell lines HeLa and HEp-2 were also tested by Smorodintsev (26) for parainfluenza virus types 1, 2, and 3, but were found to be less sensitive than primary human embryonic kidney cells. It was our goal to adapt the Nt test with ail parainfluenza virus strains and RSV to a continuous cel line. One obvious candidate for this purpose was Vero cells, which are already used extensively in many laboratories for work with viruses. Our second aim was to make such TABLE 8. Comparison of serum antibody titers to parainfluenza virus type 1 in Vero and MA 104 cell cultures Titer Serum MA 104 Vero Conven- En- Con- Entional hanced ven- hanced Nt test Nt test Nttealt Nt test Guinea Pig Flow 1,050-12, ,046 NIAID 2,270 24,350 1,784 21,847 CDC 1,860 8,900 3,512 6,137 Marmoset M62 (day 10) <8 409 <8 <64 M85 (day 10) <8 649 <8 <64 M101 (day 10) 12 <64 8 <64 M62 (day 14) <64 M85 (day 14) 26 1, M101 (day 14) 43 1, <64 M62 (day 21) 25 2, <64 M85 (day 21) 42 2, ,033 M101 (day 21) 161 3, ,850 a Reciprocal of 50% plaque Nt test. tests simple and economical by using compact 24-well trays. These trays were not suitable for use with primary cell cultures, which in our hands yielded plaques too large to be assayed in the 16-mm weils, a fading in agreement with the work of Shibuta et al. (25). Antibody titers of guinea pig antisera, utilizing the assays described here, were 5- to 50-fold higher than CF titers reported by the serum manufacturers. In one instance the serum titer was determined by the manufacturer by cytopathic effect Nt; it was fourfold lower than that determined in our conventional plaque Nt test. The increase in virus Nt caused by the addi-

7 736 HAWTHORNE AND ALBRECHT tion of AIG varies widely. For instance, antibodies against measles virus, poliovirus, or yellow fever virus are potentiated little or not at all (P. Albrecht, unpublished data). On the other hand, antibody titers against mumps and rubella viruses are enhanced 50- to 200-fold with AIG (22, 23). It seems that parainfluenza viruses and RSV take an intermediate position in terms of potentiation by AIG. The initial serum dilution used in the enhanced Nt tests was 1:64. Lower serum dilutions caused a prozone phenomenon, as described originally for the enhanced mumps Nt test (22). It seems that at low serum dilutions nonspecific human IgG binds too much of the AIG to leave effective concentrations for potentiating the virus-specific antibody complex. However, the prozone phenomenon was not seen with fluids of low protein content, e.g., cerebrospinal fluid (23). A practical application of the enhanced Nt test would therefore be in measuring low levels of antibody present in the cerebrospinal fluid, nasal, or other secretions. It has been suggested that AIG enhances virus Nt by firming up the specific virus-antibody complex or by adding an additional layer of protein around the sensitized virus or by both (2). It was established that antibodies with low antigen-binding affinity are potentiated by AIG more readily than are antibodies with high affinity (22, 28). The higher degree of potentiation of marmoset sera as opposed to guinea pig sera seen in this study could in part be due to an impaired immune response in marmosets which results in low-affinity antibody production (1). On the other hand, delayed IgM-to-IgG conversion in the course of the immune response in marmosets (11) may explain the failure to detect in some instances early (days 10 and 14) antibody in the enhanced Nt test, using AIG against IgG (Table 7). Cross-reactivity among members of the parainfluenza group was low and probably detected because of the high sensitivity of the Nt tests. The cross-reactions could also be due, in part, to hyperimmunization of the guinea pigs (3). Work on cross-reactivity of sera from reconvalescent marmosets is in progress. ACKNOWLEDGMENT The skillful technical assistance of Elizabeth Boone in developing the RSV test is gratefully acknowledged. LITERATURE CITED 1. Albrecht, P., D. Lorenz, M. J. Klutch, J. H. Vickers, and F. A. Ennis Fatal measles infection in marmosets: pathogenesis and prophylaxis. Infect. Immun. 27: Almeida, J. D., B. Cinander, and H. Howatson The structure of antigen-antibody complexes. A study by electron microscopy. J. Exp. Med. 118: J. CLIN. MICROBIOL. 3. Bellanti, J. A Immunology 11, p The W. B. Saunders Co., Philadelphia, Pa. 4. Chanock, R. M Parainfluenza viruses, p In E. H. Lennette and N. J. Schnmidt (ed.), Diagnostic procedures for viral and rickettsial infection. 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8 VOL. 13, 1981 ASSAY FOR PARAINFLUENZA VIRUSES AND RSV 737 antibody. I. Mumps antibody. Arch. Virol. 58: Sato, H., P. Albrecht, S. Krugman, and F. A. Ennis Sensitive neutralization test for rubella antibody. J. Clin. Microbiol. 9: Scheid, A., and P. W. Chopin Identification of the biological activities ofparamyxovirus glycoproteins. Activation of cell fusion, hemolysis, and infectivity by proteolytic cleavage of an inactive precurosr protein of Sendai virus. Virology 57: Shibuta, H., M. Akami, and M. Mataumoto Plaque formation by Sendai virus of parainfluenza virus group, type 1 on monkey, calf kidney, and chick embryo cell monolayers. Jpn. J. Microbiol. 15: Smorodintsev, A. A., Jr Experiences with the isolation and propagation of parainfluenza viruses. Acta Virol. 6: Sugita, K., M. Maru, and K. Sato A sensitive plaque assay for Sendai virus in an established cell line of monkey kidney cells. Jpn. J. Microbiol. 18: Svehag, S. E Formation and dissociation of virusantibody complexes with special reference to the neutralization process. Prog. Med. Virol. 10:1-62.