Department of Poultry Diseases, National Veterinary Research Institute, Pulawy, Poland

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1 Bull. Vet. Inst. Pulawy 47, , 2003 CHARACTERIZATION OF POLISH INFECTIOUS BURSAL DISEASE VIRUS STRAINS BY REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION COMBINED WITH RESTRICTION ENZYME ANALYSIS KATARZYNA '20$6.$.5=<6=72) 0,(7$1.$ AND ZENON MINTA Department of Poultry Diseases, National Veterinary Research Institute, Pulawy, Poland Received for publication August 28, Six Polish infectious bursal disease virus (IBDV) strains isolated from two epidemics on the turn of 1970/80 (early IBDV) and after 1990 (very virulent vvibdv) were characterized by a reverse transcription/polymerase chain reaction (RT/PCR) and restriction enzyme analysis (REA) method. They were compared to classical IBDV (cibdv): the F52/70 reference strain for European virulent IBDV, vaccine intermediate S706 strain, and intermediate plus 228E strain. The primers pair designed to amplify a 723 base pair (bp) fragment of the VP2 gene was used. The restriction enzymes SacI, BspMI, AccI and PdiI were used to differentiate the examined strains. SacI only cleaved cibdv RT/PCR products, BspMI cleaved vvibdv, AccI cleaved both vvibdv and cibdv, none of the RT/PCR products of early Polish strains was digested by these three enzymes. PdiI cuts all examined strains but only one cibdv strain (S706). The results demonstrate that RT/PCR can be used to rapidly detect IBDV infections in chickens, and combined with REA, to differentiate Polish IBDV strains. Key words: infectious bursal disease virus, reverse transcription, PCR, restriction enzyme analysis. Infectious bursal disease virus (IBDV) is a member of the genus Avibirnavirus within the family Birnaviridae (20). All IBDV strains are immunosuppressive but they can be classified according to differences in virulence and antigenicity (24). Since the first report in the USA (1), the disease became widespread all over the world and appeared in Europe for the first time in the middle of the 1960s. In Poland the first real epidemic of IBD occurred at the end of the seventies (14). Despite the wide spread of IBDV, the clinical form of the disease was rarely observed and the field strains isolated in 1978 from broiler chickens exhibited a low pathogenicity for SPF chickens (15, 16). However, these strains caused economic losses due to impaired growth of infected chickens and acquired immunodeficiency (4, 9, 16, 17, 19, 25). The first cases of the acute form of IBD in Poland were diagnosed at the end of During the next year the disease spread rapidly throughout the whole country and affected broiler and laying pullet flocks with mortality rates up to 50% and 70%, respectively (12). Pathogenic

2 350 characterization of the IBDVs isolated in revealed that their mortality rates in experimentally infected 4-week-old SPF chickens ranged from 40 to 100% (13). Polish IBDV strains were recently characterized by antigenic analysis and nucleotide sequencing of the variable region of VP2 gene (vvp2) (2, 3). These strains could be classified as either very virulent or early IBDVs representing a previously unrecognized group among serotype 1 IBDVs. Early IBDV strains sharing special antigenic and genetic features and causing mainly immunosuppression could be present in the mid seventies prior to the introduction of the F52/70-like viruses, which in turn have been replaced by vvibdvs in the late eighties. However, it cannot be ruled out that strains similar to the early isolates still exist in Europe and cause immunosuppression only, but such cases have hardly been investigated for some years as most of the efforts have been focused on the control of vvibdvs. The laboratory diagnosis of IBDV infections can be carried out by electron microscopy and serological assays such as agar gel precipitation (AGP), serum neutralisation (SN), immunofluorescence (IF) or immunoperoxidase (IP) tests and enzyme-linked immunosorbent assay (ELISA) (11, 21). These techniques are laborious, time-consuming and more or less sensitive. The differentiation of serotype 1 IBDV strains could be reached by molecular characterization of IBDV strains or using a panel of monoclonal antibodies in an antigen capture (AC)-ELISA (5). Recently, a method involving the use of reverse transcription/polymerase chain reaction-restriction enzymes analysis (RT/PCR-REA) has been developed. Zierenberg and co-workers used RT/PCR to amplify 723 base pair (bp) fragment of the VP2 gene and restriction enzymes SacI and BspMI to differentiate IBDV strains (26). Using this method, it could be possible to distinguish rapidly between classical (c) and very virulent (vv) strains of IBDV. Differentiation between vv and c and variant strains could be reached by using the enzyme SspI (8, 22). The absence of the specific NgoMIV cleavage site was consistent with some level of attenuation and its presence appears to be consistent with virulent IBDV strains (7). The aim of the present study was to develop a RT/PCR combined with REA for the detection of IBDV in infected chickens and rapid differentiation of Polish isolates obtained from field outbreaks in 1978 and after Material and Methods Virus strains and genome extraction. The Polish IBDV strains used in this study are listed in Table 1. The data on the year of the first isolation of these strains, virulence, source, and geographic origin are mentioned. Table 1 also includes the vaccine strains S706 (intermediate, cell-culture adapted), 228E (intermediate plus), and F52/70 (reference European strain) which can be grouped within the cibdvs. Viral RNAs were extracted from the bursa of Fabricius of infected birds (cibdv strain F52/70, the early Polish, and vvibdv strains) or from prepared, lyophilized vaccine (cibdvs S706, 228E) with the Rneasy Mini Kit (Qiagen, CA, USA) as recommended by the supplier. cdna synthesis and polymerase chain reaction. Approximately 2 JRI RNA was randomly primed to produce cdna. RT was performed for 50 min in 42 0 C in total volume 20 O. Reaction mixtures contained 4 O 5 x first strand buffer, 2 O of 0.1 M DTT, 1 O of 10mM dntps, 200 U SuperScript II H - reverse transcriptase (Invitrogen, USA), and 0.5 J of random hexamers (Promega, USA). To examine the

3 351 cleavage site of some restriction enzymes used in previous experiments (7, 8, 26) it was necessary to generate a product covering the vvp2 region. The sense primer VP2F (5 - CGC CAG GGT TTT CCC AGT CAC GAC AAC AGC CAA CAT CAA CG-3 ) and antisense primer VP2R (5 -TCA CAC AGG AAA CAG CTA TGA CGC TCG AAG TTR CTC ACC C-3 ) were used (26). The expected fragment size was 723 bp. PCR was set out in a total volume of 50 O containing 5 O cdna, 5 O PCR buffer 10x, 1 O dntps (25 mm each), 4 O MgCl 2 (25 mm), 1.5 U Taq DNA polymerase (MBI, Fermentas, Lithuania), and 2.5 O each of primers (10 mm). Reactions were started with initial denaturation step of 2 min at 94 0 C. The temperature profile of the first 10 cycles consisted of 30 s at 94 0 C for denaturation, 30 s at 58 0 C for annealing and 1 min at 70 0 C for elongation each. The elongation time of the following 30 cycles was increased to 2 s per cycle. The PCR was terminated by a final elongation step by 7 min at 70 0 C. The PCR products were separated on a 1.5% agarose gel stained with ethidium bromide and were visualized by ultraviolet (UV) transillumination. Restriction enzymes analysis. PCR products were digested with either AccI, SacI, BspMI or PdiI in a final volume of 20 O DFFRUGLQJ WR WKH PDQXIDFWXUHU V instructions (MBI, Fermentas, Lithuania or New England Biolabs, MA, USA), allowing 6 h for digestion to ensure that all reactions went to completion. Digested DNA was visualized on 2% ethidium bromide-stained agarose gels. The sizes of the REA bands were estimated by comparing them with a 100 bp DNA ladder inserted into each gel (MBI, Fermentas, Lithuania). Table 1 Virulence, date of isolation, source and origin of the studied IBDV isolates Lanes on agarose gels in Figures Strain designation Virulence (based on clinical signs) Date of isolation Source Geographic origin 1 S706 intermediate - - USA 2 228E intermediate plus France 3 F52/70 classical virulent Great Britain 4 78/GSz subclinical 1978 broilers Poland 5 78/GSi subclinical 1980 broilers Poland 6 93/35 very virulent 1993 broilers Poland 7 94/48 very virulent 1994 broilers Poland 8 00/40 very virulent 2000 broilers Poland 9 02/199 very virulent 2002 farm chickens Poland Results When the primers VP2F and VP2R in PCR were used all six Polish IBDVs as well as three reference strains (S706, 228E, and F52/70) produced a band of 723 bp. The RT/PCR products for all IBDV samples were digested with four restriction enzymes (SacI, BspMI, Acc or PdiI) and REA pattern compared. The cibdv, vvibdv and early Polish strains could be differentiated by the application of the restriction

352 a) M 1 2 3 4 5 6 7 8 9 723 bp 400 bp 320 bp b) M 1 2 3 4 5 6

4 352 a) M bp 400 bp 320 bp b) M bp 490 bp 240 bp c) M bp 530 bp 200 bp d) M bp 450 bp 270 bp Fig. 1. Electrophoresis of the RT/PCR products after digestion with the restriction enzymes: SacI (a), BspMI (b), AccI (c), PdiI (d). Lanes 1 to 9 IBDV strains as listed in Table 1. DNA size marker 100bp DNA ladder.

5 353 enzymes SacI, BspMI and AccI in REA. With the restriction enzyme SacI, two fragments of approximately 400 and 320 bp were observed in the case of the cibdv strains S706, 228E, and F52/70 (Fig. 1a, lanes 1 to 3), whereas the PCR product was not cleaved in the case of the early Polish strains and vvibdv strains (Fig. 1a, lanes 4 to 5 and 6 to 9, respectively). After digestion with BspMI, two fragments of approximately 490 and 240 were observed in the case of the vvibdv strains (Fig. 1b, lanes 6 to 9), no digestion of PCR products was detected in the case of early Polish IBDV strains (Fig. 1b, lanes 4 to 5) and cibdv strains (Fig. 1b, lanes 1 to 3). The AccI cleavage site is present in RT/PCR products of cibdv strains (Fig. 1c, lanes 1 to 3) and vvibdv strains (Fig. 1c, lanes 6 to 9). After digestion with AccI two fragments of approximately 530 and 200 bp were detected in the case of these two groups of IBDVs. No cleavage site of AccI was present in RT/PCR products of early Polish strains. This observation could be a valuable, confirmative tool in differentiation between vvibdvs, early Polish strains, and cibdv strains. The PdiI cleavage site was present in all the studied IBDV strains (Fig. 1d, lanes 2 to 9) but one cibdv strain (S706) (Fig. 1d, lane 1). Discussion Several researchers previously used REA as a method of differentiating IBDV strains (6-8, 10, 21-24). The majority of studies focused on the VP2 gene but different laboratories amplified different regions of the VP2 gene. The comparison of these studies results were further complicated by the use of different restriction enzymes. There is a lack of more universal system enabling direct comparison between various studies. Zierenberg and co-workers have shown that, using RT/PCR-REA to analyse 723 bp fragment of VP2 gene, it is possible to differentiate between cibdv and vvibdv strains (26). They validated their method on 11 strains isolated in Europe or in Asia and belonging to both serotypes. They reached IBDVs differentiation by using of two enzymes SacI and BspMI: SacI only cleaved cibdvs RT/PCR products, whereas products obtained with vvibdvs were only cleaved with BspMI. In the presented study vvibdv, cibdv, and early Polish strains were included. Similarly, cibdvs were cut by SacI and vvibdvs by BspMI, however, the early Polish strains were not digested by any of these enzymes. Thus, it should be outlined that RT/PCR- REA method could be a rapid and reliable tool for the differentiation between cibdv, vvibdv, and early Polish strains but only using both enzymes SacI and BspMI at the same time. The lack of the presence of the BspMI and SacI markers could indicate early Polish-like strains. Digestion with AccI produced two groups of REA pattern: the first group consisted of RT/PCR products of cibdvs and vvibdvs which were cut by this enzyme, RT/PCR products of early Polish strain included in the second group were not digested by AccI. The using of AccI could be the confirmative tool helpful in differentiation made with SacI and BspMI. A specific PdiI cutting site in S706 product of RT/PCR, contrary to the RT/PCR products of the other studied strains was observed. PdiI recognized the same nucleotide (nt) sequence as its NgoMIV isosyme but the products of PdiI digestion had

6 354 blunt ends while NgoMIV had sticky ends. Initially, the presence of NgoMIV restriction enzyme site was described as a marker for wild-type potentially pathogenic IBDV strains (6). However, the same authors demonstrated later that some of vaccine strains (hot intermediate) also contained the NgoMIV marker, thus it cannot be used to differentiate field strains and vaccine strains. However, the absence of the NgoMIV digestion site could be a marker for the identification of strains with some level of attenuation (7). This observation is consistent with our results because the only strain without PdiI (NgoMIV) cutting site was the S706 strain which possessed the ability to grow in cell culture. Field isolates acquired in 1978 during the first IBD epidemic and after 1990 from acute form of the disease in Poland were detected by the use of RT/PCR and differentiated by the REA method. The RT/PCR-REA method provides fast and reliable tool for the screening and differentiation of IBDV isolates. Six studied strains isolated in Poland could be classified by this method into two molecular groups: vvibdv strains and early Polish strains; additionally, the third group containing cibdv could be also differentiated. The obtained results of RT/PCR-REA confirmed previous results indicating that early Polish strains form a IBDVs group different from the classical and very virulent ones. Acknowledgments: This paper was granted by the Open Society Institute ZUG Foundation, Project Code: (Log-in ID: ). References 1. Cosgrove A.S.: An apparently new disease of chickens - avian nephrosis. Avian Dis., 1962, 6, 'RPDVND. Rivallan G., PLHWDQND. Toquin D., de Boisseson C., Minta Z., Eterradossi N.: Genetic characterisation of Polish infectious bursal disease virus strains. Bull Vet. Inst. Pulawy, 2003, 48, 'RPDVND. Rivallan G., PLHWDQND. Toquin D., Minta Z., Eterradossi N.: Antigenic characterisation of Polish infectious bursal disease virus strains. Bull. Vet. Inst. Pulawy, 2002, 46, Eterradossi N.: Progress in the diagnosis and prophylaxis of infectious bursal disease in poultry. Comprehensive reports on technical items presented to the International Committee or to regional Commissions. OIE, Paris, 1995, pp Eterradossi N., Arnaud C., Tekaia F., Toquin D., Le Coq H., Rivallan G., Guittet M., Domenech J., van den Berg T., Skinner M.: Antigenic and genetic relationship between European very virulent infectious bursal disease viruses and an early West-African isolate. Avian Pathol., 1999, 28, Jackwood D., Byerley E., Sommer S.: Use of a genetic marker for wild-type potentially pathogenic infectious bursal diosease viruses. Avian Dis., 2001, 45, Jackwood D., Sommer S.: Virulent vaccine strains of infectious bursal disease virus not distinguishable from wild-type viruses with the use of a molecular marker. Avian Dis., 2002, 46, Jackwood D., Sommer S., Knoblich H.: Amino acid comparison of infectious bursal disease viruses placed in the same or different molecular groups by RT/PCR-RFLP. Avian Dis., 2001, 45,

7 Lin Z., Kato A., Otaki Y., Nakamura T., Sasmaz E., Ueda S.: Sequence comparison of a highly virulent infectious bursal disease virus prevalent in Japan. Avian Dis., 1993, 37, Liu H., Giambrone J., Dormitorio T.: Detection of genetic variations in serotype 1 isolates of infectious bursal disease virus using polymerase chain reaction and restriction endonuclease analysis. J. Virol. Method., 1994, 48, Lukert P., Saif Y.: Infectious bursal disease. In: B.W. Calnek, H.J. Barnes, C.W. Beard, L.R. McDougald &Y.M. Saif (Eds.) Diseases of Poultry, Ames, I.A.: Iowa State University Press, 1997, pp Minta Z., Daniel A.: Infectious bursal disease in Poland: current situation and vaccinal control. In: Proceedings from international symposium of infectious bursal disease and chicken infectious anaemia, Rauischholzhausen 1994, pp Minta Z., Daniel A.,.R]DF]\VNL:&KDUDFWHUL]DWLRQRILQIHFWLRXV bursal disease virus strains isolated in Poland from acute outbreaks. In: Proceedings from World's Poultry Congress, New Delhi, 1996, pp Minta Z., Karczewski W., Roszkowski J.: Badania nad krajowym szczepem wirusa choroby Gumboro. Med. Wet., 1982, 38, Minta Z., Roszkowski J., Karczewski W.,.R]DF]\VNL : 3URSHUWLHV RI indigenous strains of infectious bursal disease virus. I. Identification. Bull. Vet. Inst. Pulawy, , 28-29, Minta Z., Roszkowski J., Karczewski W.,.R]DF]\VNL : 3URSHUWLHV RI indigenous strains of infectious bursal disease virus. II. Pathogenicity. Bull. Vet. Inst. Pulawy, , 28-29, Minta Z., Roszkowski J., Karczewski W.,.R]DF]\VNL : 3URSHUWLHV RI indigenous strains of infectious bursal disease virus. III. Immunosuppressive effect. Bull. Vet. Inst. Pulawy, , 28-29, Nunoya T., Otaki Y., Tajima M., Hiraga M., Saito T.: Occurrence of infectious bursal disease with high mortality in Japan and pathogenicity of field isolates in SPF chickens. Avian Dis., 1992, 36, Pitcovski J., Goldberg D., Levi B., Di-Castro D., Azriel A., Krispel S., Maray T., Shaaltiel Y.: Coding region of segment A sequence of a very virulent isolate of IBDV-comparison with isolates from different countries and virulence. Avian Dis., 1998, 42, Pringle CR.: The universal system of virus taxonomy of the International Committee on Virus Taxonomy (ICTV), including new proposals ratified since publication of the Sixth ICTV Report in Arch. Virol., 1998, 143, Rosenberger J.K.: Infectious bursal disease. In: H.G.Purchase, L.H. Arp, C.H.Dommermuth & J.E.Pearson (Eds.) A laboratory manual for the isolation and identification of avian pathogens, 1989, pp Sapats S., Ignjatovic J.: Restriction fragment length polymorphism analysis of the VP2 gene of Australian strains of infectious bursal disease virus. Avian Pathol., 2002, 31, Tham K., Young L Moon C.: Detection of infectious bursal disease virus by reverse transcription-polymerase chain reaction amplification of the virus segment A gene. J. Virol. Method., 1995, 53, van den Berg T.P.: Acute infectious bursal disease in poultry: a review. Avian Pathol., 2000, 29,

8 van den Berg T.P., Meulemans G.: Acute infectious bursal disease in poultry: isolation and characterisation of a highly virulent strain. Avian Pathol., 1991, 20, Zierenberg K., Raue R., Müller H.: Rapid identification of "very virulent" strains of infectious bursal disease virus by reverse transcription-polymerase chain reaction combined with restriction enzyme analysis. Avian Pathol., 2001, 30,

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