Molecular Typing of Field Isolates from two outbreaks of Infectious Bursal Disease Virus from Pakistan

A reverse transcriptase polymerase chain reaction restriction fragment length polymorphism (RT-PCR/RFLP) technique was used for the identification and characterization of Pakistani field isolates of infectious bursal disease virus (IBDV). A total of 8 bursa samples were collected from two outbreaks during September and October 2003 from Tehsil Sumandri, Dist. Faisalabad with 40-50% mortality in commercially reared broiler chicken flocks experiencing signs typical of infectious bursal disease (IBD). Four samples were found to contain IBDV genome by One Step RTPCR using VP2 gene specific primers. The assay amplified a 743 bp fragment from 701-1444 nucleotides. RT-PCR product was further subjected to restriction digestion using MboI and MvaI restriction enzymes. A third enzyme SspI was used to identify the very virulent phenotype. The RFLP profile was found similar for all four isolates with MvaI enzyme but different for one isolate when digested with MboI. All three MvaI-positive viruses were further found positive for SspI digestion and yielded RFLP profile similar to vvIBDV in Europe whereas one isolate was SspI negative and had a RFLP profile similar to classic IBDV strains. The clinical history of high mortality and SspI restriction enzyme positivity revealed that vvIBDV strains exist in Pakistan.


Introduction
outbreaks resulting in 30% mortality in broilers & 60-70% mortality in layers and then spread to Middle Infectious bursal disease is an acute, highly East, Asia, Africa and South America (1,8,19,21,22).contagious viral disease of young chickens that causes Infectious bursal disease virus (IBDV) is a member of significant losses to the poultry industry worldwide the genus Avibirnavirus in family Birnaviridae.(

12). After infection, the virus multiplies in the
The viral genome consists of two segments of developing B-lymphocytes in the bursa of Fabricius dsRNA, designated as A and B, which are enclosed (BF) leading to immuno-suppression and susceptibility within an icosahedral, non-enveloped capsid of 60 nm to the other infections (16).Two distinct serotypes of + in diameter (19).The smaller segment B ( 2800 bp) IBDV designated as serotype I and serotype II have encodes VP1, the viral RNA polymerase whereas been identified.The serotype I strains are pathogenic segment A contains two ORFs of 3039 bp and 438 bp, to chickens and vary in their virulence, whereas / which partially overlap at the 5 end of the genome.serotype II strains isolated from turkeys are apathogenic The larger ORF encodes the polyprotein (NH -VP2to both chickens and turkeys (12).Pathotypic 2 VP4-VP3-COOH), which is cleaved in to three serotype-I IBDV strains can be grouped into classical proteins designated VP2, VP3 and VP4 while small virulent (cv), antigenic variants and very virulent (vv) partially overlapping ORF encodes VP5, a small (18).Antigenic variant strains have been reported in USA, Central America (9) and in Australia (17).The 17KDa protein with regulatory function (15).VP2 is the major structural capsid protein that carries highly classical virulent IBDV strains cause bursal damage and lymphoid necrosis resulting into 20-30% conformational epitopes responsible for the induction mortality (12).In the mid-1980s very virulent (vv) of neutralizing antibodies (5).

IBDV strains emerged and caused devastating
The RT-PCR/ RFLP assay has been described to RNA, 25 l of 2 X Reaction mix and 1 l of RT/ Taq.

Materials and Methods
Nuclease free DEPC water was added up to 50 l.MgCl concentration was kept at 2.2 mM.The reaction (Stratagene, USA) after ethidium bromide staining @ Isolation of Viral RNA: The bursa samples and the 0.5g/ ml.The size of the bands was confirmed with the vaccine vials were processed in TNE buffer [Tris HCl help of a 100-bp DNA ladder as a molecular size (pH 8.3) 10mM, NaCl 100mM and EDTA 1mM] (9).marker (Inivtrogen, Life Technologies).To homogenate, chloroform was added and centrifuged Restriction fragment length polymorphism 0 @ 10000 g at 4 C for 10 minutes.The aqueous phase, 0.5 analysis: The Restriction enzymes i.e.MvaI, MboI ml of the TRIzol (LS-Reagent, Life Technologies Inc., and SspI were selected for their ability to differentiate Frederick, MD) and 0.1 ml of the chloroform were IBDV strains as described (1,9,18).A total of 20 l of mixed and centrifuged @ 10000 g for 10 minutes at reaction volume was prepared according to the 0 4 C (4).The aqueous phase was mixed with 0.5 ml of manufacturer's instructions (MBI Fermentas, was confirmed with the help of a 100-bp DNA ladder Molecular characterization of IBDV strains by as a molecular size marker (Inivtrogen Life Technologies).molecular techniques such as RT-PCR/ RFLP has been described in recent years as a good tool to identify and

Results
classify virus isolates (8, 9, 10, 11, 13, 14, 18).In the present study four isolates of IBDV were Bursa samples collected from two suspected IBD outbreaks were examined.Four out of eight tested characterized and classified by RT-PCR of a 743-bp samples were found to contain IBDV genome as fragment of the hypervariable region of the VP2 gene evidenced by the amplification of a 743-bp region of and RFLP of this region with restriction enzymes the VP2 gene using One-Step RT-PCR.The agarose MvaI, MboI and SspI.gel electrophoresis of the amplified fragments showed A One-Step RT-PCR technique was used for the similar band size.The procedure was repeated to molecular diagnosis of IBDV in bursa samples using ensure the reproducibility of the results.No difference the VP2 gene specific primers.Primers used in the was found in the length of fragments.The specificity present study amplified a 743 bp fragment from the of the primers was confirmed by finding the amplified nucleotide 701 to 1444 of VP2 gene that has been product in case of Bursine plus IBDV commercial considered to be the hypervariable region and vaccine whereas none in case of bronchitis vaccine as conserved among all field strains (3).These primers shown in Figure -1.
are licensed by IDEXX laboratories Inc. (Westbrook, The RFLP profile obtained by digestion of the ME USA) for commercial use and have been RT-PCR product with MboI, MvaI and SspI showed successfully used (9).that majority of the prevalent strains belonged to Two different restriction profiles were obtained vvIBDV phenotype.The restriction pattern of three when MboI enzyme was used.Three of the isolates isolates (AZ1, VMb1 and QMK2) when digested with (AZ1, VMb1 and QMK2) had identical RFLP profile SspI enzyme yielded two fragments of approximately and found similar to the RFLP profile reported in 470 bp and 273 bp in size similar to the vvIBDV in previous studies (1,9,18).The PK03 isolate found Europe, while one isolate (PK03) was found SspI SspI negative and had similar profile to the classic negative.The MvaI restriction enzyme generated three IBDV strains as described (9).No classic strains fragments with sizes of 424 bp, 172 bp and 119 bp having similar restriction profile as of Lukert/ Edgar whereas MboI yielded two different restriction IBDV strains have been detected outside the USA.The profiles i.e. three isolates (AZ1, VMb1 and QMK2) present study reported that classic strains are present in with similar fragment lengths of 362 bp and 229 bp Pakistan.It is possible that these viruses might have while one isolate (PK03) with 480 bp and 229 bp been introduced in to the flocks by vaccination.fragments as shown in Table 1.
When RT-PCR products of the PK03, VMb1,

Abbreviations used in this paper IBD=
Abbreviations used in this paperIBD= Infectious bursal disease; IBDV= Infectious bursal disease virus; PCR= Polymerase chain reaction; RFLP= Restriction fragment length polymorphism; RT= Reverse transcriptase; vv= Very virulent Viruses: A total of 8 bursa samples were collected from 2 0 two outbreaks during September and October 2003 from conditions consisted of incubation at 50 C for 30 0 Tehsil Sumandri, Dist.Faisalabad that caused high minutes for once then denaturattion at 94 C for 30 sec, 0 0 mortality (40-50 %) in commercially reared broiler annealing at 53 C for 30 sec and extension at 72 C for 1 0 birds.The Bursine Plus commercial IBDV vaccine min.A final extension was given at 72 C for 10 min.(ForteDogde Animal Health, Forte Dodge, IA 50501, The reaction steps were repeated for 35 cycles.A 10 l f USA) as a positive control and an unrelated vaccine of the PCR product was electrophoresed (80 V for 40 infectious bronchitis (Bronchitis Vaccine Massachusetts minutes) on 1.5% agarose gel.The amplified bands type-Forte Dogde Animal Health, Forte Dodge, IA were visualized under UV light at a wavelength of 254 50501, USA) as a negative control were included for nm with Eagle Eye Gel Documentation System specificity test.