Fowlpox Virus-Infected Cells - NCBI

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Apr 13, 1983 - conducted for 8 h with a constant current of 25 mA and then for another ..... Frank, M. M. 1979. ... Tripathy, D. N., L. E. Hanson, and W. L. Mayers.
INFECTION AND IMMUNITY, Nov. 1983, P. 721-727

Vol. 42, No. 2

0019-9567/83/110721-07$02.00/0 Copyright © 1983, American Society for Microbiology

Activation of Chicken Alternative Complement Pathway by Fowlpox Virus-Infected Cells HIROAKI OHTA,1 CHIEKO KAI,2 YASUHIRO YOSHIKAWA,2 AND KAZUYA YAMANOUCHI2* Bio-Pharmaceuticals Inc., Tochigi Laboratory, Imaichi City, Tochigi 321-11,1 and Laboratory Animal Research Center, Institute of Medical Science, University of Tokyo, Minato, Tokyo 108, Japan2

Received 13 April 1983/Accepted 8 August 1983

Fresh normal chicken serum (NCS) which lacked virus-neutralizing antibody to fowlpox virus (FPV) was found to inhibit the appearance of the cytopathic effect of the virus, virus growth, and plaque formation in chicken embryo cells. Immunofluorescent examination revealed the deposition of the third component of complement (C3) on FPV-infected chicken embryo cells incubated with fresh NCS. The inhibitory activity of fresh NCS on viral cytopathic effect was independent of the Ca2' ion and was abrogated by treatment of fresh NCS with inulin or zymosan. Similarly, deposition of C3 on FPV-infected cells occurred independently of the Ca2+ ion and was inhibited by treatment of fresh NCS with inulin or zymosan but was not inhibited by absorption with FPV-infected cells. These results suggest that antibody-independent activation of complement by FPV-infected cells via the alternative pathway caused the inhibition of the virus growth as well as the C3 deposition. Involvement of complement activation as nonspecific host response to virus infection was also suggested by the demonstration of the C3 deposition in the skin lesions of FPV-infected chickens.

Fowlpox, distinguished by characteristic pock lesions on the skin, is an important infectious disease of chickens. In the severe form of the disease, the mucous membranes of the digestive and respiratory tracts are also involved. The skin lesions first appear as nodules 2 to 8 days after virus infection. This is followed by the formation of vesicles. The lesions heal by scab formation. Histopathologically, hyperplasia of the epithelial cells, which are frequently associated with eosinophilic inclusions and infiltration of inflammatory cells, is observed (1). Both humoral and cellular immunity are considered to play important roles in recovery from this virus infection (7, 10). However, the significance of complement activation in this virus infection has not been examined because complement components in chickens have not yet been identified. Recently, we prepared monospecific antiserum to the third component of complement (C3) of Japanese quail and found that this antiserum cross-reacted with chicken C3 as well (5). In the present study, we used this antiserum to demonstrate the antibody-independent activation of chicken complement via an altemative complement pathway (ACP) by fowlpox virus (FPV)infected cells. A possible role of complement as a nonspecific host response at an early stage of FPV infection is also suggested. 721

MATERIALS AND METHODS Virus. A vaccine strain of FPV (Chick-N-Pox strain 946) which was propagated in specific-pathogen-free chicken embryo (CE) cells was obtained from Salsbury Laboratories (Charles City, Iowa). The virus was cloned three times by the plaque method in CE cells. To prepare stock virus, CE cells were inoculated with the virus, and the supernatant fluid of the infected cells was obtained with an infectivity titer of 1060 50%o tissue culture infective doses per ml. Cells. Primary cultures were prepared in plastic dishes or plates (Falcon 3002 or 3008, Becton-Dickinson Labware, Oxnard, Calif.) from 10- to 11-day-old specific-pathogen-free CEs. Eagle minimal essential medium supplemented with 5% fetal bovine serum (inactivated by heating at 56°C for 30 min), 5% tryptose phosphate broth (Difco Laboratories, Detroit, Mich.), and antibiotics was used as a culture medium. Normal chicken serum. Specific-pathogen-free chicken eggs were purchased from SPAFAS, Norwich, Conn. Hatched chicks were raised in an isolator system without any vaccination. At 4 to 10 weeks of age, three or four chickens were bled by heart puncture. The serum was separated at 4°C, pooled, and stored at -80°C. The serum, designated normal chicken serum (NCS), was confirmed to be free of neutralizing antibody to FPV. Antiserum. Anti-quail C3 monospecific serum was prepared by immunizing rabbits with purified Japanese quail C3 as described previously (5). Anti-FPV serum was prepared in chickens. Briefly, chickens at 3 weeks of age were intradermally inoculated with 1045 50%7o tissue culture infective doses of FPV and were further

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OHTA ET AL.

challenged twice with 108.5 50% tissue culture infective doses of the virus as a booster immunization. One week after the final injection, serum was collected and stored at -80°C until use. The antibody titer of this serum to FPV was 1:160 when measured by indirect immunofluorescence technique described by Trypathy et al. (14). Titration of anti-C3 serum. Chicken hemolysin to sheep erythrocytes was prepared as described by Fukuda (3). Sheep erythrocytes were washed three times in phosphate-buffered saline and were suspended in isotonic gelatin Veronal-buffered saline (pH 7.5) containing 0.15 mM CaCl2, 0.5 mM MgC92, and 0.1% gelatin, or were suspended in phosphate-buffered saline. Sensitized erythrocytes were prepared by mixing the washed erythrocytes with the chicken hemolysin. The resulting sensitized erythrocytes were further reacted with fresh NCS at a final concentration of 5% at room temperature for 5 min, washed 3 times in PBS, and used as erythrocytes with C3 receptor (EAC). The anti-quail C3 serum was inactivated by heating at 56°C for 30 min, and two fold dilutions of it in phosphatebuffered saline were mixed with an equal volume of sensitized erythrocyte or EAC suspension in phosphate-buffered saline (6 x 107 cells per ml) on microtest plates (Nunc, Copenhagen, Denmark). After incubation at 37°C for 1 h, reciprocals of the highest dilutions of serum showing distinct pattern of hemagglutination were taken as hemagglutinating antibody titers. Crossed immunoelectrophoresis. Crossed immunoelectrophoresis was performed as previously described (5). Briefly, 20 ,ul of fresh NCS was applied to small holes on the agarose gel. Electrophoresis was conducted for 8 h with a constant current of 25 mA and then for another 1.5 h at a right angle to the first run. Half of the agarose plate was removed and replaced with agarose suspension containing 10% anti-C3 serum. The electrophoresis was continued overnight. Virus neutralization test. The plaque neutralization test described by Morita (8) was used with slight modifications. Briefly, 0.5 ml of virus was mixed with 0.5 ml of serum and incubated at 37°C for 1 h. For enhanced neutralization by anti-C3 serum (11), the virus-serum mixture incubated as above was further mixed with 0.5 ml of anti-C3 serum at dilutions of 1:10, 1:30, and 1:100 and was incubated for another 1 h. CE cells in plastic dishes were inoculated with 0.2 ml of the mixture of virus, serum, and anti-C3 serum, left at 37°C for 1 h for virus adsorption, and overlaid with agar medium consisting of Eagle minimal essential medium, 2% fetal bovine serum, and 0.8% agar (Difco). Four days later, a second agar medium consisting of Eagle minimal essential medium, 0.8% agar (Difco), and 0.006% neutral red was overlaid. Plaques were counted on the next day. Decomplementation from fresh NCS. Procedures for the inactivation of fresh NCS were previously described in detail (16). In brief, 10 mM EDTA (Tokyo Kasei Co., Ltd., Tokyo, Japan) was used to chelate both Ca2' and Mg2" ions from serum, and 10 mM EGTA [ethylene glycol-bis(P-aminoethyl ether)N,N,N',N'-tetraacetate; Sigma Chemical Co., St. Louis, Mo.] plus 2 mM MgCl2 (Wako Co., Ltd., Tokyo, Japan) was used to chelate the Ca2+ ion. Zymosan A (Sigma) or inulin (Merck Sharp & Dohme, West Point, Pa.) was used as a complement inactiva-

INFECT. IMMUN.

tor. Zymosan (2 mg) or inulin (20 mg) was added to 1 ml of fresh NCS, incubated at 37°C for 1 h, and removed by centrifugation at 4°C for 1 h. Fresh NCS was also heated at 56°C for 30 min to inactivate

complement. Immunofluorescent technique. CE cells were inoculated with FPV. At intervals, the cells were collected and fixed in acetone at -20°C for 5 min. The fixed cells were incubated with fresh or heated NCS diluted to 1:4 in Veronal-buffered saline at 37°C for 90 min and then were reacted with anti-FPV serum or anti-C3 serum for 30 min to detect virus antigen or C3, respectively. Subsequently, the cells were stained with rabbit antichicken immunoglobulin G or goat anti-rabbit immunoglobulin serum conjugated with fluorescein isothiocyanate. To detect membrane antigen, unfixed cells were incubated with fresh or heated NCS diluted to 1:2 at 37°C for 15 min. The cells were collected by washing with 0.1 mM EDTA and 0.01% trypsin solutions followed by low-speed centrifugation. They were then stained for 15 min by the same method as that used for fixed cells. To examine skin lesions, 1-day-old chickens were inoculated with FPV intradermally on the back or at the wing web. The skin lesions with pocks were collected 6 and 14 days post-inoculation (p.i.), which corresponded to the progressive and regressive stages of the lesions, respectively. The tissues were examined by the same method as that used for CE cells (see above). Cytotoxicity test. CE cells grown in microtest plates (Costar no. 3506, Cambridge, Mass.) at a concentration of 4 x 104 cells per 0.1 ml per well were labeled with [51Cr]sodium chromate (New England Nuclear Corp., Boston, Mass.; specific activity, 208.56 Ci/g; 2.5 ,uCi/0.1 ml per well) and were inoculated with FPV at a multiplicity of infection of 1.0. After incubation at 37°C for 1 h, the cells were washed three times with medium and were further incubated in Eagle minimal essential medium containing 5% tryptose phosphate broth (Difco) and 25% fresh or heated NCS. An 0.1-ml sample of the culture fluid was collected 5 h p.i. from five cultures per sample, and the radioactivity in the supernatant was measured with a gamma counter. The maximum release was determined by adding 0.1 ml of distilled water to the cells after washing. The cytotoxicity was expressed as the percentage of specific 51Cr release by subtracting the background release in the presence of medium instead of NCS.

RESULTS Cross-reaction of anti-C3 serum to chicken C3. The hemagglutinating antibody titers of the antiC3 serum were