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Indian Journal of Experimental Biology Vol. 39, December 2001, pp. 1314-1317 Virus enhancement following infection with antibody-coated infectious bursal disease virus (IBDV) in chickens Rajesh Kumar & Shiv Charan* Deptt. of Veterinary Microbiology, College of Veterinary Sciences, Chaudhary Charan Singh Haryana Agricultural University, Hisar 125 004, India Received /6 March 2001; revised 13 August 2001 A novel concept of vaccination, employing virus-antibody complex has been reported for the control of infectious bursal disease in chickens. A comparison of virus replicati on, serum neutralizing antibody response and pathogenicity in chickens inoc ulated with the antibody coated virus, prepared by mixing virus and antibody in different ratios (1:1, 1:0.1, 1: 0.01) and virus alone without antibody, has been made. Antibody coated virus (when mixed in certain crucial ratios) replicated to a hi gher magnitude in the target organ, caused more severe pathogenesis but induced a primary serum neutrali zing antibody response almost comparable. The results may have important implications in understanding of pathogenesis and development of co ntrol strategies against infectious bursal disease vi rus, specially employ ing immune complex vaccine. Infectious bursal disease virus (IBDV) mainly infects the B-cells of bursa of Fabricius. However, other lymphoid organs e.g. caecal tonsils, thymus, spleen and Harderian glands are also affected 1• The virus is known to produce severe combined B- and T -cell immuno-suppression 2 .3 . To control IBD, both killed and live vaccines have been used 45 . Recently, usc of virus-antibody complex has been suggested as prophylactic measure6-8. Although , the exact mechanism of induction of protection by the antibody complex vaccine has not been fully understood, it has been suggested that better immune response with antibody complex vaccine over the conventional vaccines is associated with delayed release of virus, lesser degeneration of lymphoid organs and higher localization of IBDV antigen on bursal and splenic follicular dendritic cells 9 . In these studies we have investigated the replication kinetics of antibody coated virus vs virus alone and we report that the antibody-coated IBDV may lead to enhancement of the virus replication in vivo under certain conditions. This is probably the first report in IBDV on antibody mediated enhancement of virus replication in vivo. Therefore, these studies may have important implications in the field applications of immune-complex vaccines. Chicks-Broiler chicks (18 days old; 80) from a farm with the hi story of no outbreak were procured. *Correspondent author: Phone: +91 1662 44460; Fax: +91 1662 34952; E. mail: [email protected] The chicks were tested for anti-IBDV antibody status. For raisi ng polyclonal anti-IBDV antibodies 7-9 weeks old chickens were used. Pre-immune status of chicks for IBD antibodyAlthough the chicks were obtained from a farm with the history of no outbreak of IBD, the chicks were tested for anti-IBDV antibody possibly for any maternal antibody. The sera were collected randomly from 5-6 chicks and pooled for the assessment of antibody employing SN test and the fter was found to be I: 160. Virus-Georgia strain (intermediate) of IBDV was procured from M/s Indovax Ltd, Hisar (Haryana) and was grown and titrated in chickens embryo fibroblast (CEF). Titration of virus-Micro-titration method was used for titration of the virus. Various dilutions of the virus ranging from 10"1 to 10"8 were made in the growth medium-199. 100 Jll of each dilution of the virus was added to the well starting from highest dilution. This was followed by 100 Jll of cell suspension (l x 106 cells/ml) to all the wells. Proper cell controls were also set up simultaneously. The plates were observed daily for CPE. After 4-5 days supernatant was discarded and the cells were stained with crystal violet (1 %). The virus titer was calculated by the method of Reed and Munch (10). The titer of stock virus was found to be 5x I 0 6 TCID 5ofml. Production and titration of hyper-immune serumAntiserum against the Georgia strain of IBDV, propa- NOTES gated in CEF, was raised in 7-9 weeks old 6 chickens by inoculating three doses of lx106 TCID 50 (approx.) virus at 15 day interval by ocular-oral route. The chickens were bled 10 days after the last injection. Hyper-immune serum raised in White Leghorn chickens against Georgia strain grown in CEF was titrated by serum neutralizing test and titer was found to be 1 : 5000 (approx.), whereas the serum from control chickens had a titer of less than I :5. The antibody titer of the hyper-immune serum against IBDV was further confirmed in a commercial ELISA and was found to be 5300. Preparation of Virus antibody complex-For preparing virus-antibody complex for immunization, appropriate dilutions of anti-serum were mixed in equal volume of virus 1x 105 TCID 50 . This virus antibody was kept at room temperature (23°-25°C) for I hr with frequent gentle mixing. Various preparations were prepared so as to give l x l05 TCID 50 of IBDV and unit activity of IBDV antibody so that the final ratio of I : I; 1:0.1 ; 1:0.01 was achieved between IBDV and unit activity of IBDV antibody (unit activity of antiserum has been defined as reciprocal of serum neutralizing antibody titer per milliliter of antiserum, ref 8). Experimental design-Chicks (80) were divided into 5 groups of 16 birds each. These groups were inoculated with different ratio of virus and antibody i.e. Group I with I: I , Group II with I : 0.1, Group III with I: 0.0 I, Group IV with virus with normal chickens serum, while Group V with phosphate buffered saline (PBS) alone. After 6 weeks these groups were re-inoculated with the virus via ocular-oral route. Various parameters i.e. clinical signs, mortality, gross lesions, bursa/ body weight ratio (b:b ratio) including microscopic examination of organs (bursa, thymus, spleen) were recorded. In addition, virus isolation from bursa specimen was al so done. Blood samples were collected at 0, 3, 6, I 0, 15, 21 and 28 days after primary infection as well as 5 and 10 days after second infection for serum neutralization test. Bursal preparation for virus titration-Bursa\ samples from 2-3 birds at various time intervals from Group I to V were pooled, grounded in Dounce's homogenizer in Hank's balanced salt solution (HBSS) to make 10% (w/v ) homogeneous suspension and subjected to antibiotic treatment for I hr at 37°C. The suspension was then subjected to three cycles of freezing and th awing. Finally, it was centrifuged at 2000g for 15 min . The supernatant was passed 1315 through 0.22 Jl Millipore membrane. The titer was determined as described earlier and expressed as per g of bursal tissue. Neutralization test (SN)-Constant virus varying serum method was followed for performing SN test in 96-well micro tissue culture plates (Nunc, Denmark). Sera were inactivated at 56°C for 30 min. in water bath. Test was run in triplicate. Two-fold dilutions of each serum was made starting from 1:10 to 1:5120 in 50 )li volume using medium M-199 . IBDV was diluted to give titer of 100 TCID 5o/50f.ll and added to each well in 50 111 amount, except in wells marked for cell control. In the wells marked for cell control , 100 111 of growth medium (M-199 supplemented with 10% bovine serum, !-glutamine, sodium bicarbonate and antibiotics) was added. The plates were incubated at 37°C for 1 hr to allow serum to neutralize the virus. CEF cells were diluted in growth medium (M-199) to give a concentration of 1x 106 cells/ml and added in I 00 f.ll amounts in each well. The plates were incubated at 37"C in 5% C0 2 in air. After 5 days, the supernatant was discarded and cells were stained with crystal violet and examined . Microscopic examination of bursa- Samples of bursal tissues were collected in 10% formal saline from each group at various time post inoculation and the tissue sections were examined after Haematoxi lin and Eosin staining for comparison of di sease severity. Virus titration in the bursa- The results of virus titration in bursa (pooled from 2-3 birds) of chickens inoculated with IBDV antibody complex or virus alone at various time intervals have been shown in Fig 1. The maximum virus replication was found in Gp II inoculated with virus antibody ratio (1 :0.1). The degree of virus replication was minimum in Gp I which had maximum amount of antibody. The virus replication in chickens inoculated with virus along with normal chickens serum (Gp IV) or with virus antibody ratio of 1:0.01 (Gp III) were almost similar. A direct correlation between concentration of antibodies and inhibition of virus replication in bursa was not seen in these studies, virus titers in bursae of Gp II chickens reached highest peak with virus titre 7 log 1o/g (approx.) of tissue and was detectable till day 28. Thus, in Gp II ( 1:0.1) maximum titre of virus in bursa suggests that probably thi s ratio of virus and antibody was very crucial and may support enhanced viral replication. Serum neutralization (SN) antibody responseAfter primary immunization, SN antibodies appeared INDIAN J EXP BIOL, DECEMBER 2001 1316 -o--Group I :;"'"' -ft-- Group Ill [v+Ab)1:0.01)) .0 ~ Group 0 E - g> IV [v+N) Normal serum) - * -Group V [PB Scontrol] C)~ Q; (v+Ab) ~ : 1)J -<>-Group II [v+Ab)1:0.1)] 4 Q._J ~- .,. 2 > Days post inoculation Fig. 1- Effect of coating infec ti ons bursal di sease virus with antibodies on in vivo replication of virus [Groups of chickens were inoc ulated with 1x I 0 5 TCID 50 of virus coated with the antibodies in different ratios (Group f, If, Ill) of virus alone in (Group IV) or no virus (Group V) and in vivo replication of the virus was assessed at various time points between day 3 and day 28] between day 10 and 15 post-inoculation and declined to basal level between day 28 and 35 in all the virus inoculated groups (I-IV). After secondary immunization, there was rapid increase in the level of SN antibody titers (2560) in I, III and IV groups, whereas in Gp II where virus antibody ratio was (1 :0.1), SN antibody titers remained low (20) at day 5 and 10 days post secondary immunization. This was the same group where there was maximal replication of virus. Gross and microscopic lesions-The group inoculated with IBDV alone showed mild lesions characterized by petecheal haemorrhages in thigh muscles. However, the lesions were more severe in the groups inoculated with virus antibody complex. The highest degree of the haemorrhages in thigh and pectoral mu scle and splenomegaly was found in Gp II at 15 day post infection . This was the same group in which virus replication was maximal and SN antibody titers were lowest after secondary immunization. Similarly, histopathological lesions in the group of chickens inoculated with virus alone, showed milder reaction of heterophil infiltration and depletion of lymphocytes in follicles of bursa of Fabricius between day 3 and 6, as compared to the groups inoculated with viru s antibody complex which showed moderate to severe lesions characterized by atrophy of follicles and necrosis of lymphocytes, thickening of interfollicular space due to connective ti ssue proliferation and infiltration of reticular cells and leucocytes . The max imum lesions were also found on day 15 as was the virus titers in bursa. With the virulent strain of IBDV, generally the maximum lesions and virus replication in the bursae have been reported around day 4. This delayed reaction in the present studies may be because of the use of vaccine virus and due to the effect of coating with antibodies in these experiments . In the present study, the virus replication, SN antibody responses and pathogenesis of the li ve IBDV alone (as in case of a conventional live vaccine) and after mixing with the antibodies (as in case of an immune-complex vaccine) were compared. It was observed that in chickens (Gp II) inoculated with the virus coated with the antibody, there was about 100fold more virus replication and the severity of the lesions was markedly higher than in chickens inoculated with the virus alone. Primary SN antibody responses, however, were almost comparable in the groups of chickens whether the virus was gi ven alone or together with the antibodies. Interestingly, in the chickens showing higher virus replication there was a remarkably suppressed secondary SN antibody response. Such a remarkably lower secondary antibody response may be attributable to degeneration of bursal cells after primary inoculation since the target cells for the replication of IBDV are bursal B-cell leading to poor priming of B and/or T cells during immunization. The immunosuppressive feature of IBDV has been well documented with respect to B-cells resulting into lower antibody responses against a number of infectious agents and vaccines in chickens which have been previously exposed to IBDV 2' 11.1 2 . Suppression ofT cells responses as a result of IBDV infection has been reported more recently and is of tran sient nature3. Possibility of such phenomenon was recognized si nce the findings that the sera from human immune deficiency virus (HJV) infected individual can enhance HIV infection in vitro as well as in vivo where antibody dependent enhancement could be demonstrated at high dilutions (up to I :65000) while SN antibody activity can rarely be demonstrated at dilution s higher than 1: 1000 13' 14 • Similarly, in studies with feline immunodeficiency virus (FIV) enhancement of FIV infection has been reported. The cats vaccinated with FIV-env showed accelerated acute infection , early appearance of viraemia and greater vi ral load 15 16 after challenge than the control cats ' • In our studies, an enhanced virus replication in the group of chickens inoculated with virus antibody ratio of I :0. 1 may reflect a similar phenomenon. To the best of our knowledge this appears to be the first report with respect to IBDV . NOTES Thus, the present studies highlight the importance of routine monitoring the level of IBDV antibodies in commerci al flocks since lower levels of SN antibodies may rather support replication of IBDV . Therefore, acceleration or enhancement of IBDV infection by antibodies during field vaccination employing live vaccines and more particularly when immunecomplex vaccine are used could be very crucial. The results of these studies will therefore provide a novel bas is for understanding pathogenesis and formulating effi cient vaccination program against IBDV critically. The authors wish to thankfully acknowledge the valuable help of Dr R P Gupta, Deptt. of Veterinary Pathology for the interpretation of histopathological slides. RK was a recipient of a scholarship from Chaudhary Charan Singh Haryana Agricultural Uni versity, Hisar. References 1 All an W H, Faragher J T & C ull en G A, Immunosuppression by the infecti ous bursal agent in chickens immuni zed against Newcastl e di sease, Vet Rec, 90 ( 1972) 511. 2 Saif Y M, Immunosuppression induced by infectious bursal disease virus, Vet lmmunollmmunopathol, 30 ( 1991 ) 45 . 3 Kim I J & Sharma J M , Infec tio us bursal disease virus induced bursal T lymphocytes inhibit mitogenic response of normal splenocy tes, Vet lmmunol Immunopathol, 74 (2000) 47 . 4 Naq i S A, Millar D L & Grumbles L C, An evaluation of three co mmercially avai lable infecti ous bursal disease vaccines, Avian Dis, 24 (1 980) 233 . 5 Tsukamoto K, Tanimura N, Kakita S, Ota K, Mase M, lmai K & Hihara H, Effi cacy of three li ve vaccines against hi ghly vi rulent infectio us bursal disease vi ru s in chickens with or without maternal antibodi es. Avian Dis, 39 (1995 ) 2 18. 6 Whitfill C E, Haddad E E, Ricks C A, Skeeles J K, Newberry L A, Beasley J N, Andrews P D, Thoma J A & Wakenell P S, Determination of optimal formul ation of a novel infectious 1317 7 8 9 10 II 12 13 14 15 16 bursal di sease virus (IBDV) vacc ine constructed by mi xing bursal di sease antisera with IBDV, Avian Dis, 39 ( 1995) 687. Whitfill C E, Avaki an A P, Haddad E E, Vanden Wijngaard J C, Bai nes D, Chettle N & Lehrbach P R, Control of infec tious bursal disease in bro iler by in ovo vaccination as demonstrated by laboratory and controlled fi eld trials using a viru s antibody co mplex vacc ine. In : Proceeding XI Intern ational Congress of the World Veterinary Poultry Association, 1997, Budapest, Hungary. Haddad E E, Whitfill C E, Avaki an A P, Ricks C A, Andrews P D, Thoma J A & Wakenell P S, Effi cacy of a novel infecti ous bursal disease virus immune complex vaccine in broiler chickens, Avian Dis, 41 ( 1997) 882. Jeurissen S H M, Janse E M, Lehrbach P R, Haddad E E, Avaki an A & Whitfill C E, The working mechanism of an immune complex vacci ne that protects chi ckens agai nst infec tious bursal disease, Immunology, 95 ( 1998) 494. Reed L J & Muench H, A simple method of estimat ing fifty per cent end points, Am J Hyg, 27 ( 1938) 493. Lucio B & Hitchner S B, Immunosuppression and acti ve response induced by infect ious bursal di sease virus in chickens with passive antibodies, Avian Dis, 24 (1980) 189 . Nakamura T, Okaki N & Nunoya T, Immunosuppressive effec t of a virulent infec tious bursal disease virus isolated in Japan, Avian Dis, 36 (1 992) 891. Robinson WE, Montefiori D C , Mitchell W M, Prince AM , Alter H J, Dressman G R & Eichberg J W , Antibody dependent enhancement of human immunodeficiency virus type-! (HIV -I ) infection in vitro by serum fro m HI V- I infected and passively immunized chimpanzees, Proc Natl Acad Sci USA , 86 (1989) 4710. Schutten M, Andeweg A C, Bosch M L & Osterhaus A D M E, Enhancement of infectivity of a non sy ncytium inducing HIV - I by SCD4 and by human antibodies that neutralize syncytium inducing HIV- 1, Scand J lmmunol, 41 (1 994) 18. Hosie M J, Osborne R, Reid G, Neil J C & Jarret 0 , Enhancement after feline immunodefi ciency virus vaccination, Vet Immunollmmunopathol, 35 ( 1992) 19 1. Richardson J, Moraillon A, BaudS, Cuisinier AM, Soni go P & Pancino G, Enhancement of Feline Immunode fi ciency Virus (FIV) infection after DNA vaccination with the FIV envelope, J Viral, 71 ( 1997) 9640.