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Molecular Characterization of Pakistani Field Isolates of Infectious Bursal DiseaseVirusAuthor(s): Nazir Ahmed Lone, Shafqat Fatima Rehmani, Shahana Urooj Kazmi, Rana Muzaffar, TaseerAhmed Khan, Adnan Khan, Shakeel Ahmed Khan, and Ayaz AhmedSource: Avian Diseases, 53(2):306-309. 2009.Published By: American Association of Avian PathologistsDOI: http://dx.doi.org/10.1637/8325-042108-Reg.1URL: http://www.bioone.org/doi/full/10.1637/8325-042108-Reg.1

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Research Note—

Molecular Characterization of Pakistani Field Isolates ofInfectious Bursal Disease Virus

Nazir Ahmed Lone,A Shafqat Fatima Rehmani,BE Shahana Urooj Kazmi,A Rana Muzaffar,C Taseer Ahmed Khan,D

Adnan Khan,A Shakeel Ahmed Khan,A and Ayaz AhmedB

ADepartment of Microbiology, University of Karachi, Karachi 75270, PakistanBSindh Poultry Vaccine Centre, Animal Science Complex–Korangi, Karachi 74900, Pakistan

CSindh Institute of Urology and Transplantation, Civil Hospital, Karachi 74200, PakistanDDepartment of Physiology, University of Karachi, Karachi 75270, Pakistan

Received 30 April 2008; Accepted and published ahead of print 11 February 2009

SUMMARY. The reverse transcriptase-polymerase chain reaction followed by restriction fragment length polymorphism (RT-PCR/RFLP) technique was used to identify and characterize Pakistani field isolates of infectious bursal disease virus (IBDV). Theseisolates have caused heavy losses to the poultry industry (mortality up to 60%) during the period between 1999 and 2005. Tensamples (five local isolates and five commercial vaccines) were examined for IBDV. Nine samples were positive for IBDV asevidenced by the amplification of the 743-bp region of the VP2 gene by RT-PCR. The RT-PCR products were subjected torestriction enzyme digestion with BstNI, MboI, and SspI. The RFLP profiles of all samples on digestion with the MboI enzymeyielded a fragment size of 229 and 362 bp except for vaccine strain Bursine Plus, which yielded a profile of 229 and 480 bp.However, digestion with BstNI yielded two distinct RFLP patterns. The first profile was detected in field isolates ML-1/SPVC/2001 and NP2/SPVC/2002 with four fragments of 119, 154, 172, and 209 bp, resembling RFLP profiles of molecular group 4isolates. NL-3/SPVC/2003, NK-4/SPVC/2004, and NPK-5/SPVC/2005 generated a different RFLP profile with fragments of119, 172, and 424 bp, resembling the profiles of molecular group 6 isolates. However, all the field and vaccine strains showed theabsence of SspI restriction sites in their genome. It can be concluded that the Pakistani isolates can be grouped in molecular groups4 and 6 of IBDV.

RESUMEN. Nota de Investigacion—Caracterizacion molecular de aislamientos de campo Pakistanıes del virus de la enfermedadinfecciosa de la bolsa.

La transcripcion reversa y la reaccion en cadena de la polimerasa seguida del analisis del polimorfismo de la longitud de losfragmentos de restriccion (de las siglas en ingles RT-PCR/RFLP) fueron utilizadas para identificar y caracterizar aislamientos decampo del virus de la enfermedad infecciosa de la bolsa Pakistanıes. Estos aislamientos han causado perdidas significativas a laindustria avıcola (mortalidad de hasta 60%) durante el periodo comprendido entre 1999 y 2005. Diez muestras (cinco aislamientoslocales y cinco vacunas comerciales) fueron examinadas para detectar al virus de Gumboro. Nueve muestras fueron positivas para elvirus de la enfermedad de la bolsa, tal como se evidencio por la amplificacion por RT-PCR de un producto de 743 pares de basesque incluye al gene VP2. Los productos de RT-PCR fueron sometidos a la digestion con las enzimas de restriccion BstNI, MboI ySspI. Los perfiles de RFLP de todas la muestras con la digestion con la enzima MboI mostraron fragmentos de 229 y 362 pares debases respectivamente, con excepcion de la cepa vacunal Bursine-Plus que indujo un perfil con fragmentos de 229 y 480 pares debases. Sin embargo, la digestion con BstNI indujo dos patrones de RFLP distintos. El primer perfil fue detectado en los aislamientosde campo ML-1/SPVC/2001 y NP2/SPVC/2002 con cuatro fragmentos de 119, 154, 172 y 209 pares de bases, que es similar alperfil de aislamientos pertenecientes al grupo molecular 4. Mientras que los aislamientos NL-3/SPVC/2003, NK- 4/SPVC/2004, yNPK-5/SPVC/2005 generaron diferentes perfiles de RFLP con fragmentos de 119, 172 y 424 pares de bases semejantes a losperfiles del grupo molecular 6. Sin embargo, todos los aislamientos de campo y las vacunas mostraron la ausencia del sitio derestriccion para la enzima SspI. Se puede concluir que los aislamientos Pakistanıes pueden ser agrupados en los grupos moleculares 4y 6 del virus de la enfermedad infecciosa de la bolsa.

Key words: IBDV, Pakistan, RT-PCR, RFLD

Abbreviations: IBD 5 infectious bursal disease; IBDV 5 infectious bursal disease virus; RFLP 5 restriction fragment lengthpolymorphism; RT-PCR 5 reverse transcriptase-polymerase chain reaction

Infectious bursal disease (IBD) is an acute, highly infectiousdisease of young chickens with significant economic importance.IBD virus (IBDV) is a nonenveloped; double-stranded RNA viruswith a bi-segmented (A, 3300 bp, and B, 2800 bp) genome (12).The segment A encodes a precursor poly-protein that is processed inthree mature proteins; VP2, VP3, and VP4. The VP2 is the majorstructural protein that contains antigenic regions, that is, those

responsible for the production of neutralizing antibodies in chickens,and a hyper-variable region in terms of amino acid sequencevariation between strains (5,14). The segment B of the IBDVgenome that is a double-stranded polymer encodes a protein, VP1.There are two serotypes of IBDV, but only serotype I causesinfection in poultry. Serotype I has been divided into several groupson the basis of antigenic variation and virulence: classical virulentstrains, antigenic variant strains, and very virulent strains. For thecharacterization of antigenic differences among IBDV strains, severalin vitro and in vivo tests have been used; these include: virusECorresponding author. E-mail: dr_shafqat@hotmail.com

AVIAN DISEASES 53:306–309, 2009

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neutralization assay, antigen capture enzyme-linked immunosorbentassay, in vivo cross-protection, and reverse transcriptase polymerasechain reaction (RT-PCR) followed by restriction fragment lengthpolymorphism (RFLP) (3,4,15). The antigenic variation amongIBDV strains is the main hurdle in designing a strategy forvaccination, because vaccine from one antigenic type does notprovide protection to chickens against other strains (11). The RT-PCR/RFLP is widely used to diagnose the disease and to identify themolecular differences in the VP2 gene of IBDV strains (9).Molecular characterization of IBDV is carried out by digestingRT-PCR fragments with restriction enzymes BstNI, MboI, and SspI.Restriction enzyme BstNI and MboI are used to generate the RFLPpattern to distinguish viruses into molecular groups; however, SspI isused to predict the vvIBDV phenotype (1). Jackwood and Sommer(8,9) have classified the field and vaccine strains of IBDV into sixmolecular groups. Variant strains of IBDV are placed into 2 groups:1 and 2; while the classic strains are in groups 3 and 4. The Lukertstrain is in group 5 and vvIBDV is in group 6. In molecular group 6a subset of viruses has also been identified that possess a SspIrestriction site present only in highly virulent strains. Since its firstisolation in 1957 the IBDV has been continuously mutating, and thevvIBDV strains were isolated from many countries of Europe, Asia,and Middle East (4,10,13). In Pakistan, IBD continues to be aserious problem as in other countries. Severe outbreaks of the diseaseoccurred in commercial broiler flocks, causing up to 60% mortalitydespite vaccination. The diagnosis of the disease until now hasdepended mainly on clinical signs, gross pathology, and serologicaltests, and there has been a lack of information about the prevalentIBDV strains in Pakistan. RT-PCR/RFLP assay is superior toconventional techniques in terms of sensitivity and specificity. Theassay is also used to identify the vaccine strains of IBDV in order toensure their authenticity and to evaluate vaccination programs andepidemiological studies. Therefore, the present study was initiated toidentify and characterize the local isolates at the molecular level.

MATERIALS AND METHODS

Some isolates of IBDV and available commercial vaccines wereselected for molecular characterization during 1999–2005, as listed inTable 1.

RNA isolation and RT-PCR. The IBDV isolates were inoculated in 4-wk-old susceptible chickens, and bursa tissue specimens were collectedaseptically after an incubation period of 4 days. The bursa were halved andstored in liquid nitrogen until needed. The SV Total RNA IsolationSystem (Promega, Madison, WI) was used according to the manufacturer’sinstructions for isolation of the viral RNA of IBDV. The Access Quick RT-PCR Kit (Promega) was used according to the manufacturer’s instructions.The primers designated 700-59 and 700-39 were used to amplify a 743-bpfragment of the VP2 gene of IBDV (6). A reaction mixture of 50 ml wasprepared consisting of 25 ml Access Quick Master Mix containing PCRbuffer, dNTPs, and MgCl2. An aliquot (3.0 ml) of primer JL1-JL2 (10pmoles; MWG, Ebersberg, Germany), 1 ml (5U) of enzyme AMV-RT, 8.0ml nuclease-free water, and 10 ml extracted RNA was used. An aliquotcontaining all the reagents as mentioned except 10 ml of nuclease-free water(instead of RNA) served as a negative control. A previously amplified PCRproduct served as a positive control. RT-PCR was performed in anautomated thermal cycler (Perkin Elmer 9600, Waltham, MA). The viralRNA was reverse transcribed by incubating at 48 C for 45 min and aninitial denaturation at 95 C for 2 min followed by 35 cycles of 95 C for 30sec, 53 C for 30 sec, and 72 C for 45 sec. The cycling program ended witha final extension at 72 C for 7 min. The amplified products wereelectrophoresed on 1.5% agarose gel (Roche, Mannheim, Germany)containing ethidium bromide (20 mg/ml) and examined on a Chemi Doc(Bio Rad, Hemel Hempstead, U.K.). Amplicons of appropriate size (743)were identified by comparison with a DNA marker (Roche, XIV) in thegel.

Purification of amplicon and RFLP analysis of gene 743 bpof IBDV. The amplified products were cleaned with rapid PCRpurification system (Marligen, Life Technologies, U.S.A.) as per themanufacturer’s instructions. The purified product was stored in arefrigerator until needed. RFLP was performed by digesting the purifiedamplicons with the restriction enzymes BstNI, MboI, and SspI (MBI,Fermentas, Vilnius, Lithuania) according to the manufacturer’sinstructions.

RESULTS AND DISCUSSION

IBD, which started in Pakistan in the early 1980s as an epidemic,has now become an endemic and is listed as a major disease ofpoultry. Live and killed vaccines produced around the world areimported into Pakistan because there are no regulations for theimportation of poultry vaccines; therefore, mild, intermediate, andhot strains are available in the local market. As there was no

Table 1. IBDV isolates and commercial vaccines tested in RT/PCR-RFLP assay.

IBDVisolate/strain

Serotype/subtypeA Source Host

Virus isolation(yr)

Mortality%

Age(wk) Breed

ML-1/SPVC/2001 Serotype I/ND Field virus (Karachi, Pakistan) Chick bursa 2001 15 3 and 5 BroilerNP-2/SPVC/2002 Serotype l/ND Field virus (Karachi, Pakistan) Chick bursa 2002 15 4 BroilerNL-3/SPVC/2003 Serotype l/ND Field virus (Karachi, Pakistan) Chick bursa 2003 30 4 Broiler and

indigenouschicken

NK-4/SPVC/2004 Serotype l/ND Field virus (Karachi, Pakistan) Chick bursa 2005 25 3.5 BroilerNPK-5/SPVC/2005 Serotype l/ND Field virus (Karachi, Pakistan) Chick bursa 2005 40 4 BroilerGumboroD78 Serotype l/classic Intervet International, the

NetherlandsVaccine — — — —

Tad Gumboro VacForte

Serotype l/classic Lohman Animal Health, Germany Vaccine — — — —

Gumboro 228-E Serotype/ND Intervet International, theNetherlands

Vaccine — — — —

Bursine Plus Serotype I/ND Fort Dodge Animal Health, IA Vaccine — — — —AK-3 Serotype/ND Sindh Poultry Vaccine Centre,

Karachi, PakistanVaccine 1997 — — —

AOther scientists previously determined the serotype and classic or variant subtype of some vaccine viruses. ND 5 Not determined.

Molecular characterization of Pakistani isolates of IBDV 307

information available on the IBDV strains of Pakistan, this study wascarried out to evaluate the molecular characteristics of the IBDVstrains circulating in Pakistan.

Fifty samples based on post mortem findings were collected frompoultry farms experiencing IBDV. Only five samples designated asMLI/SPVC/2001, NP2/SPVC/2002, NL3/SPVC/2003, NK4/SPVC/2004, and NPK5/SPVC/2005 were positive for RT-PCR reaction. In asimilar study carried out by Jackwood and Nielson (7), 48 samples outof 151 were positive by RT-PCR assay. Similar results have also beenreported by Gomma et al. (4). It was noted earlier that the performanceof individual RT-PCR test might vary from one sample to anotherbecause of the presence of inhibitors (2,16). The tissue type and thequality of extracted RNA have been noted to affect tests performance;therefore, commercially available vaccines (Gumboro D-78, TadGumbo Vac Forte, Gumboro 228-E, Bursine Plus, and AK-3) wereused as an internal control in this study.

The IBDV can be classified into six molecular groups on the basisof RT-PCR of the 743-bp region of VP2 gene followed by RFLPusing the three restriction enzymes, BstNI, MboI, and SspI. Thesamples were subjected to RT-PCR and RFLP analysis. The VP2region (701–1444 bp) of the IBDV gene was analyzed, and a 743-bpproduct was obtained using an already defined primer, JL1/JL2 (7).As opposed to the previous studies, a MgCl2 concentration of 6.0mM was used to yield better results.

Out of 10 samples analyzed, nine were positive for IBDV on amolecular basis. These samples were then subjected to restrictionanalysis. Digestion with the BstNI enzyme differentiated the isolatesinto two profiles. The first profile was detected in two isolates and threevaccine strains that had four fragments of 119, 154, 172, and 209 bp insize, which resemble the RFLP pattern of the vaccine (Gumboro-D-78,Tad Gumboro Vac Forte, and Gumboro 228-E) strain. The secondprofile was detected in three isolates and one vaccine strain that hadthree fragments of 119, 172, and 424 bp in size (Table 2), whichresembles the RFLP pattern of previously published (9) Puerto Rico,Mexico, El Salvador, South Africa, and Spain IBDV isolates. Digestionwith the MboI enzyme yielded two fragments with a size of 229 and362 bp for five isolates and three vaccine strains that resemble theRFLP profiles of New Zealand and Venezuela isolates (9), whereas thevaccine strain Bursine Plus yielded two fragments of 229 and 480 bp,which resembles the RFLP profile of molecular 5 isolates. The resultsrevealed that none of the samples were positive for the SspI restrictionenzyme (Table 2). Therefore, it can be concluded that the Pakistaniisolates can be grouped into molecular groups 4 and 6. Although theisolates (NL3, NK4, and NPK5) are classified in molecular group 6,they are negative for the SspI restriction enzyme; however, this groupcontains a subset of viruses that had a SspI marker for virulence (9).

Similar results were reported by Sapats and Ignjatovic (13), whoconcluded that Australian isolates were negative for the SspI marker,which is contrary to the findings of Jackwood and Sommer (9). Thissupports the arguments that the SspI marker is unreliable in predictingan isolate as vvIBDV. Since the RFLPs of most vaccine strains areknown (9), hence viruses detected in a flock with new RFLPs could bea wild-type strain (4).

The present study was first of its kind in Pakistan, wherevaccination programs are prescribed unduly without monitoring theimmune status of breeding flocks. This may lead to a flock’sbecoming fully susceptible to disease. This gives some explanation ofthe severe outbreaks of IBD in commercial poultry after vaccination.The study has provided information on the molecular status ofPakistani isolates, although the sample size was too small. Weencourage molecular studies to be carried out on a larger sample sizefor further confirmation.

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Table 2. RFLP band sizes for IBDV strains from Pakistan.

Sample

Restriction enzymeA

BstNI MboI

SspI424 350 209 172 154 119 480 403 362 280 269 229

ML1/SPVC/2001 3 3 3 3 3 3

NP2/SPVC/2002 3 3 3 3 3 3

NL3/SPVC/2003 3 3 3 3 3

NK4/SPVC/2004 3 3 3 3 3

NPK5/SPVC/2005 3 3 3 3 3

GumboroD78 3 3 3 3 3 3

Tad Gumboro Vac Forte 3 3 3 3 3 3

Gumboro 228-E 3 3 3 3 3 3

Bursine Plus 3 3 3 3

AEach value indicates the length in base pairs of the restriction fragment. Checked items designate which restriction fragments were obtainedfollowing digestion with the BstNI and MboI enzymes.

308 N. A. Lone et al.

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