Inactivation of infectious bursal disease virus by binary ethylenimine and formalin

Inactivation of infectious bursal disease virus by binary ethylenimine and formalin 1 2 first-author †E-mail:mudasserhabib@yahoo.com †E-mail:mudasserhabib@yahoo.com mudasserhabib@yahoo.com 1 2 3 2 1 Department of Veterinary Microbiology, University of Agriculture, Faisalabad 38040, Pakistan Department of Veterinary Microbiology, University of Agriculture, Faisalabad 38040, Pakistan Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310029, China Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310029, China Department of Livestock and Fisheries, Animal Husbandry Sindh, Hyderabad 71000, Pakistan Department of Livestock and Fisheries, Animal Husbandry Sindh, Hyderabad 71000, Pakistan In this experiment conducted to study the inactivation dynamics of infectious bursal disease virus (IBDV) by binary ethylenimine (BEI) in comparison with formalin, IBDV was isolated from the bursa of infected chickens and its confirmation was done by agar gel precipitation test. Viral suspensions were subjected to inactivation with BEI and formalin for pre-set time intervals. BEI was employed at concentrations of 0.001 and 0.002 mol/L while formalin was used at 0.1% and 0.2%. Sampling was done at 6, 12, 24, 36 and 48 h of incubation and samples were tested for their inactivation status in 9-day-old embryonated eggs and 3-week-old broiler chickens. IBDV was completely inactivated by 0.001 and 0.002 mol/L BEI after 36 h of incubation at 37 °C, whereas formalin at 0.1% and 0.2% concentrations inactivated IBDV in 24 h. Infectious bursal disease virus (IBDV) Binary ethylenimine (BEI) Inactivation INTRODUCTION 1 Infectious bursal disease virus (IBDV) belongs to the genus Avibirnavirus , family Birnaviridae (ICTV, 2004 2004 ), with its genome composed of two segments of double stranded RNA (Lukert and Saif, 1991 1991 ). It is a non-enveloped 60 nm diameter icosahedral particle (Özel and Gelderblom, 1985 1985 ; Kibenge et al., 1988 1988 ; ICTV, 2004 2004 ). IBDV is the causative agent of acute or immunosuppressive disease in chickens. Serotype 1 strains are pathogenic, with the target organ being bursa of Fabricius (BF). While studies demonstrated that serotype 2 strains do not cause disease or protect against infection (Jackwood et al., 1982 1982 ; Ismail et al., 1988 1988 ; Zierenberg et al., 2004 2004 ). IBDV is responsible for a highly immunosuppressive disease in young chickens and causes significant economic losses to the poultry industry worldwide (Kibenge et al., 1988 1988 ). Due to the high resistance of IBDV to environmental exposure, hygienic measures alone are ineffective and vaccination is thus essential. The economical impact of both clinical and sub-clinical diseases warrants search for and the use of efficient vaccines. Satisfactory protection can be achieved by immunization with live or inactivated vaccines. Classical live vaccines achieve lifelong and broad protection but possess residual pathogenicity and a proportional risk of reversion to virulence. Inactivated vaccines, although costly, are used successfully (Box, 1989 1989 ). In order to obtain an inactivated immunologic or vaccine composition, the pathogen is harvested and subjected to clarification by chemical treatment and inactivation using different inactivants, for example formalin or formaldehyde, β -propiolactone, ethylenimine, binary ethylenimine or thimerosal. Most inactivated viral vaccines are prepared by the reaction of viruses with formaldehyde (Brown, 1995 1995 ). Formalin reacts with many chemical groupings of proteins that lead to the phenomenon of the “membrane effect” in which the reaction “closes” the outer protein shell of the virus before the nucleic acid of the infectious genome is destroyed. Even after prolonged incubation of the inactivated antigen infectious nucleic acid can emerge and lead to a replication of the virulent virus. This can cause a sub-clinical infection or even lead to disease. The membrane effect alters the surface proteins of the virus and modifies and reduces the antigenicity of the antigen (Bahnemann, 1990 1990 ). Binary ethylenimine (BEI), member of a group of alkylating substances “aziridines” reacts very little with proteins and therefore does not alter the antigenic components of the virus. BEI has an inactivation reaction that is more specific for the nucleic acid and it produces antigenically superior vaccine (Bahnemann, 1990 1990 ). By the time BEI has been used to inactivate different viruses i.e. rabies (Larghi and Nebel, 1980 1980 ) foot-and-mouth disease virus (Dilovski and Tekerlekov, 1983 1983 ), bluetongue virus (Stott et al., 1979 1979 ), porcine parvovirus (Buonavoglia et al., 1988a 1988a ), African horse sickness virus (Soliman et al., 1996 1996 ) and Newcastle disease virus (King, 1991 1991 ) etc. In the present study IBDV was subjected to inactivation with BEI for comparison with formalin inactivation. MATERIALS AND METHODS 1 Collection of samples 2 Infected bursae were collected from an outbreak of infectious bursal disease at a local poultry farm. Complete history of outbreak was taken. These samples were stored at −20 °C till used. Field virus isolation and purification 2 A 10% (w/v) suspension of infected bursae was made by chopping and grinding them in sterilized pestle and mortar with sterilized sand after the method of Reddy et al.( 1977 1977 ). The suspension was made in phosphate buffered saline (PBS) containing antibiotics (100 IU penicillin-G/ml and 50 μg gentamicin sulfate/ml). This suspension was later centrifuged at 5000 r/min for 20 min and the supernatant was collected. The supernatant fluid was mixed with chloroform (1:1, v/v) in centrifuge tubes and centrifuged at 5000 r/min for 20 min. Three distinct layers were obtained: top layer containing virus, middle one containing bursal tissue debris and bottom layer was containing chloroform. The clear supernatant was collected in sterilized screw capped test tubes. Confirmation of virus 2 The presence of IBDV in the clear fluid was checked by agar gel precipitation test as described by Sulochana and Lalithakunjamma ( 1991 1991 ). The hyperimmune serum used in the test was raised against live IBDV vaccine D-78 in rabbits according to the method of Barnes et al.( 1982 1982 ). Titration of virus 2 Egg infective dose 50 ( EID 50 ) of the virus was calculated by the method of Reed and Muench ( 1938 1938 ). Fertile eggs with 9 day-of-age embryos were used and injected with virus through chorioallantoic membrane (CAM) route. Ten fold dilutions of the virus suspension were made from 10 −1 to 10 −10 in sterile normal saline and each dilution was injected in a batch of five eggs. The eggs were inoculated with each dilution at rate of 0.1 ml per egg. Ten eggs were inoculated with sterile saline and were kept as control. After inoculation all the eggs were sealed with melted wax and were re-incubated at 37.5 °C. Inoculated eggs were candled daily. Mortality during first 24 h was discounted as non-specific. After 96 h the eggs were opened and embryos were checked for lesions. Signs of infection were characteristic IBDV lesions. Typical lesions in embryos were petechiae and congestion of skin and ecchymotic haemorrhages in the toe joints. The EID 50 calculated was 10 3.48 . Inactivation of virus 2 After confirmation and titration of virus, the supernatant fluid was divided into fractions and subjected to treatment with formalin and binary ethylenimine for inactivation. 1. Formalin treatment Thirty-seven percent formaldehyde solution (BDH Chemicals Ltd. Pool, England) was added to viral suspensions ( EID 50 =10 3.48 ) to make final formalin concentration equal to 0.1% and 0.2%. A control with no added formalin was included. Vials were capped, mixed and incubated at 37 °C. Sampling was done at 6, 12, 24, 36 and 48 h of treatment. 2. Binary ethylenimine (BEI) treatment BEI was prepared as a 0.1 mol/L solution by cyclization of 0.1 mol/L 2-bromoethylamine hydrobromide (Sigma) in 0.175 mol/L NaOH solution at 37 °C for one hour following the method of Bahnemann ( 1990 1990 ). BEI was used soon after its preparation. BEI of 0.1 mol/L was added to virus suspension ( EID 50 =10 3.48 ) at 1% and 2% for final BEI concentrations 0.001 and 0.002 mol/L respectively. A control without addition of BEI was included. Virus suspensions were incubated at 37 °C and sampling was done at 6, 12, 24, 36 and 48 h of treatment. The residual BEI was hydrolysed in samples by the addition of 1 mol/L sterile Na-thiosulfate (Merck) solution at 10% of the volume of the BEI used. Pathogenicity tests to check the inactivation of virus 2 Formalin and BEI treated IBDV was tested for its pathogenicity in: (1) un-vaccinated susceptible chicks of three weeks of age (Allan et al., 1972 1972 ); (2) ten-day-old embryonated eggs (Hitchner, 1970 1970 ). 1. Inoculation of 3-week-old susceptible broiler chicks Samples from both formalin and BEI treated virus collected at different time intervals were checked for their pathogenicity by their inoculation into 3-week-old susceptible broiler chicks. Each sample was inoculated in 3 birds at rate of 0.3 ml/bird subcutaneously. Birds were observed for 5 d post inoculation (Helmboldt and Garner, 1964 1964 ). 2. Inoculation of ten-day-old embryonated eggs Samples from both formalin and BEI treated viruses collected at different time intervals were also checked for their pathogenicity by their inoculation into ten-day-old embryonated eggs through chorioallantoic membrane (CAM) route. Each sample was inoculated into 3 eggs at rate of 0.1 ml/egg. After inoculation all eggs were sealed with melted wax and were re-incubated at 37.5 °C. Inoculated eggs were candled daily. Mortality during first 24 h was regarded as non-specific. After 96 h the eggs were opened and embryos were checked for lesions (Rosenberger et al., 1985 1985 ). RESULTS 1 Vaccine samples that were incompletely inactivated led to development of pathological lesions both in susceptible birds and embryonated eggs upon inoculation, whereas no lesions were observed in the case when vaccine samples were completely inactivated. Typical lesions in birds were observed in bursa, kidney, thymus and muscles while cutaneous congestion was a prominent sign in embryos. BEI of 0.001 and 0.002 mol/L completely inactivated the virus after 36 h, whereas 0.1% and 0.2% formalin inactivated the virus after 24 h of incubation at 37 °C. DISCUSSION 1 This study aimed at testing the inactivation ability of BEI for IBDV in comparison with conventional inactivant formalin. Most inactivated viral vaccines are prepared by reacting the viruses with formaldehyde (Brown, 1995 1995 ). Formalin reportedly reacts with many chemical groupings of proteins that lead to the phenomenon of the “membrane effect” in which the reaction “closes” the outer protein shell of the virus before the nucleic acid of the infectious genome is destroyed. Binary ethylenimine (BEI), a member of a group of alkylating substances “aziridines” reacts very little with proteins and therefore does not alter the antigenic components of the virus (Bahnemann, 1990 1990 ). In this study BEI was successfully used to inactivate IBDV. BEI produces antigenically superior vaccine because it does not alter the antigenic components of the virus as compared with other inactivants. Blackburn and Besselaar ( 1991 1991 ) used a panel of 23 monoclonal antibodies (mAbs) to study the effect of formalin, beta propiolactone (BPL) and binary ethylenimine (BEI) on the epitopes of the Rift Valley fever virus glycoproteins. After the inactivation period BEI had very little adverse affect on the epitopes whereas BPL significantly altered and formalin partially changed the conformation of most of the epitopes. Larghi and Nebel ( 1980 1980 ) studied the inactivation activity of binary ethylenimine on rabies virus and immunogenic properties and stability of the vaccines prepared using this agent. He concluded that rabies tissue culture vaccine inactivated with BEI was highly immunogenic and stable. Pyke et al.( 2004 2004 ) also used BEI for inactivation of different viruses. Buonavoglia et al.( 1988b 1988b ) inactivated Newcastle disease virus (NDV) with BEI. The BEI inactivated vaccine had almost twice the efficiency when compared to a vaccine prepared with formalin. Keeping in view the above findings promising results can be anticipated by using BEI-inactivated IBDV in chickens. In future studies BEI and formalin-inactivated vaccines will be inoculated in broiler birds to check their comparative efficacy. 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Generation of serotype 1/serotype 2 reassortant viruses of the infectious bursal disease virus and their investigation in vitro and in vivo 10.1631/jzus.2006.B0320 Project (No. PSF/Res/P-AU/Bio (246)) supported by Pakistan Science Foundation (PSF) 2005-10-29 2006-02-20 2006-03-13 2022-01-05 Copyright © 2006, Journal of Zhejiang University Science 2006 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 04 07