Molecular and in vivo Characterization of an Iranian Infectious Bursal Disease Virus Containing a Mixed Virus Population

Document Type : Infectious agents- Diseases- Surgery


1 Department of Avian Diseases, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

2 Department of Avian Diseases Faculty of Veterinary Medicine University of Tehran Tehran, Iran


BACKGROUND: Infectious bursal disease (IBD) is a highly contagious immunosuppressive disease of young chickens caused by infectious bursal disease virus (IBDV). IBDV consists of a two-segmented double-stranded RNA genome which can easily undergo genomic recombination or reassortment during mixed infections.
The objectives of this study were aimed to characterize a previously identified Iranian IBDV strain (JRMP29IR) in specific-pathogen-free (SPF) chickens, evaluate the presence of a mixed and/or reassortant virus popula-tion in this strain, and examine the frequency of genomic recombination and reassortment in publicly available IBDV genomes through bioinformatics study.
The SPF chickens were challenged with the JRMP29IR strain via oral and intraocular routes. Bursal tissues were extracted for histopathological evaluation and reverse transcription-polymerase chain reaction (RT-PCR) followed by Sanger sequencing. Putative recombinations and reassortments were evaluated using the Recombination Detection Program 5.
Through genomic sequencing of the viruses from the bursas of infected chickens, the JRMP29IR strain was found which contained viruses from the classic, variant and very virulent IBDV genotypes. Through bioinformatics, nu-merous putative recombination and reassortment events were identified that naturally occur throughout the IBDV genome.
Parental JRMP29IR appears to be derived from a flock undergoing a mixed IBDV infection. High frequency of recombination and reassortment among IBDVs suggests that these events are evolutionarily beneficial for the virus

Article Title [فارسی]

تعیین هویت ملکولی و بالینی یک سویه ایرانی ویروس بیماری بورس عفونی حاوی جمعیت مخلوط ویروسی

Authors [فارسی]

  • امیر قربانی 1
  • سید مصطفی پیغمبری 2
  • جمشید رزمیار 1
1 گروه بیماری‌های طیور، دانشکد، دامپزشکی، دانشگاه تهران، تهران، ایران.
2 گروه بیماریهای طیور دانشکده دامپزشکی دانشگاه تهران تهران، ایران
Abstract [فارسی]

زمینه مطالعه: بیماری بورس عفونی پرندگان، یک بیماری بسیار واگیردار و سرکوب‌کنندۀ سیستم ایمنی در مرغ‌های جوان است که به‌وسیلۀ ویروس بیماری بورس عفونی ایجاد می‌شود. ویروس بیماری بورس عفونی حاوی ژنوم دوقطعه‌ای از نوع RNA دو رشته‌ای است. پدیدۀ نوترکیبی و یا بازآرایی ژنتیکی در خلال عفونت همزمان با دو یا چند ژنوتیپ از این ویروس محتمل می‌شود.  
هدف: اهداف این مطالعه، شامل تعیین هویت یک سویه از قبل شناسایی‌شده ویروس بیماری بورس عفونی (JRMP29IR) در مرغ‌های عاری از پاتوژن مشخص (SPF)، ارزیابی حضور جمعیت‌های مختلف ویروسی و یا واجد بازآرایی ژنتیکی در این سویه، و آزمایش فراوانی نوترکیبی یا بازآرایی های ژنومی در ویروس بیماری بورس عفونی به‌وسیلۀ بررسی توالی ژنومی موجود در بانک جهانی ژن از طریق روش‌های بیوانفورماتیک بوده است.
روش کار: مرغ‌های SPF از طریق خوراکی و داخل چشمی با سویۀ JRMP29IR چالش داده شدند. بافت بورس پرندگان بیمار برای ارزیابی‌های هیستوپاتولوژیکی و تعیین توالی ژنوم ویروسی پس از انجام واکنش زنجیره‌ای پلیمراز رونویسی معکوس استخراج گردید. نوترکیبی و بازآرایی‌های ژنتیکی محتمل توسط نرم‌افزار Recombination Detection Program 5 ارزیابی شدند.
نتایج: نتایج تعیین توالی ژنومی ویروس‌های جداشده از بورس‌های عفونی مرغ‌ها، حاکی از آن بود که سویۀ JRMP29IR حاوی ویروس‌هایی از ژنوتیپ‌های کلاسیک، واریانت و بسیار حاد بیماری بورس عفونی است. از طریق بیوانفورماتیک، تعداد قابل توجهی از وقایع محتمل نوترکیبی و بازآرایی، که به نظر می‌رسد به‌طور طبیعی در ژنوم ویروس بیماری بورس عفونی اتفاق افتاده‌اند، شناسایی گردید.
نتیجه‌گیری نهایی: به نظر می‌رسد که سویه JRMP29IR از گله‌ای در خلال عفونت همزمان با چندین ژنوتیپ ویروس بیماری بورس عفونی جدا شده است. با توجه به فراوانی بالای نوترکیبی و بازآرایی در بین ویروس‌های بیماری بورس عفونی به نظر می‌رسد که این وقایع نقش پر اهمیتی در تکامل این ویروس‌ها در گذر زمان ایفا می‌کنند

Keywords [فارسی]

  • بازآرایی
  • تعیین هویت ملکولی
  • نوترکیبی
  • ویروس بیماری بورس عفونی



Infectious bursal disease (IBD) is a highly contagious immunosuppressive disease of young chickens with active lymphoid tissues (Hemida et al., 2019; Eterradossi and Saif, 2020). IBD was first recognized as avian nephrosis in 1962. Almost after a decade, the causative agent of IBD was proposed as infectious bursal disease virus (IBDV). Owing to its lymphotropic nature, IBDV causes huge economic loss to the poultry industry both through high levels of mortality and severe immunosuppression which often leads to secondary infections and vaccination failure in the infected flocks (Hemida et al., 2019; Eterradossi and Saif, 2020).

IBDV consists of a two-segmented double-stranded RNA genome (segments A and B), which encodes the five viral proteins designated as VP1-VP5 (Hemida et al., 2019; Eterradossi and Saif, 2020). Although IBDVs are divided into two main serotypes (serotypes 1 and 2), so far, all pathogenic IBDVs have been characterized as serotype 1 (Eterradossi and Saif, 2020). Within the IBDV serotype 1, antigenic drifts have caused a significant change in virus antigenicity mainly through amino acid changes in the mid-third of viral protein 2 (VP2), also known as the VP2 hypervariable domain (Eterradossi and Saif, 2020). While the antigenic changes in VP2 hypervariable domain appear to play a major role in generation of the viral antigenic variants, larger antigenic shifts through genetic recombination and/or reassortment (mixing of the entire or parts of the genes between two different viruses) are likely to occur in the case of mixed infections (Hemida et al., 2019; Touzani et al., 2019; Eterradossi and Saif, 2020).

Genetic reassortments can play a vital role in the evolution of viruses with segmented genomes by creating viruses with novel genetic and antigenic makeups (McDonald et al., 2016; Jackwood et al., 2018). With further advancement of rapid molecular sequencing techniques, a putative reassortant IBDV strain from Europe was described for the first time in 1996 (Brown and Skinner, 1996). Since then, several studies have described additional reassortment events in IBDVs from across the world (Yamaguchi et al., 1997; Kong et al., 2004; Le Nouen et al., 2006; Wei et al., 2006; Chen et al., 2012; Kasanga et al., 2013; Pikuła et al., 2018). Majority of the genetic reassortments in the previous studies occurred betwee-n the very virulent and attenuated or classic IBDV strains. Therefore, vaccination with live-attenuated IBDV strains is likely to be involved in these reassortment events. However, the exact mechanisms governing the generation of reassortant IBDVs and their clinical attributes are yet to be elucidated.

In a series of previous studies, a number of IBDV viruses were identified from the infected poultry farms in Iran during 2005-2006 (Razmyar and Peighambari, 2008; Razmyar and Peighambari, 2009; Ghaniei et al., 2011). Our first study conducted by Razmyar and Peighambari (2008 and 2009) using combination of reverse transcriptase-polymerase chain reaction/restriction fragment length polymorphisms (RT-PCR/RFLP) and nucleotide sequence analyses of the VP2 hypervariable region, revealed that the majority of Iranian IBDVs (34 out of 37) belong to the very virulent IBDV genotypes (encoded by segment A) (Razmyar and Peighambari, 2008; Razmyar and Peighambari, 2009). Only 3 out of 37 st-udied viruses from Iran were identified as classic or standard IBDV genotypes based on their VP2 gene (Razmyar and Peighambari, 2008). Next, Ghaniei performed a second RT-PCR/RFLP analysis on the VP1 (encoded by segment B) of the previously identified viruses using a primer set specific to the very virulent IBDV strains (Ghaniei et al., 2011). Interestingly, one of the studied viruses, JRMP29IR, which was originally identified as a classic strain based on its VP2 gene could be identified as a very virulent strain based on its VP1 gene. Of note that, JRMP29IR was collected from an IBDV-vaccinated farm with high mortality (~15%) and clinical signs similar to infection with very virulent IBDV strains (e.g., extensive hemorrhage in cloacal bursa) (Razmyar and Peighambari, 2008). Collectively, these results suggested that JRMP29IR may contain a mixed or reassortant IBDV population.

Mixed infections may result in the emergence of recombinant or reassortant viruses with novel antigenic and/or pathogenic makeups (Patel et al., 2016; Jackwood et al., 2018; Kim et al., 2018). For instance, viruses with novel antigenic makeups can effectively break through the host immunity developed against the previous vaccinations (Jackwood, 2017; Jackwood et al., 2018). A better under-standing of possible recombination and reassortment events within the virus genome and their effects on the virus phenotype can help on designing novel vaccines and therapeutics. The aim of this study was to further characterize the JRPM29IR strain in SPF chickens and evaluate the presence of a mixed and/or reassortant IBDV population in this strain. Further, we evaluated the frequency of genomic recombination and reassortment in publicly available IBDV genomes through bioinformatics, pro-viding a strong evidence for the presence of numerous naturally occurring recombinant and reassortant IBDV strains.


Materials and Methods


The JRMP29IR strain was collected from a broiler flock with 15% mortality, previously vaccinated with a live-attenuated IBDV vaccine, located in Tabriz, Iran in 2006, as previously described (Razmyar and Peighambari, 2008; Razmyar and Peighambari, 2009). Aliquots of the JRMP29IR infected bursal tissues (100 mg) were obtained from the Department of Avian Diseases, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. Bursal aliquots were homogenized using the sterile scalpel blades, mixed with 5 mL sterile PBS, and filtered through sterile 0.2 µm filters to remove cell debris and bacterial contaminants. Chicken bursal passage (CBP) subclones of JRMP29IR were collected from the bursa of individual infected chickens in this experiment.

Chicken Experiment and Ethics Statement

Three-week-old white leghorn SPF chickens were obtained from the Razi Vaccine and Serum Research Institute (Karaj, Alborz, Iran). The birds were divided into two subgroups of 5 chickens and kept inside large cages (700 cm2) for the experiment duration. IBDV challenge was performed at 3 weeks of age in SPF chickens via oral and intraocular routes (1:1 ratio, v/v) with 600 µL of the JR-MP29IR bursal tissue homogenates per bird. The health status of chickens was monitored twice daily throughout the experiment. Chic-kenswere humanely euthanized when they displayed severe symptoms such as ruffled feathers,reluctance to move, and respiratory distress. Blood and tissue collection from the euthanized or dead birds were performed immediately after euthanasia or daily checks.


Histopathology and bursal lesion scoring were performed as previously described (Jackwood et al., 2011). Briefly, bursal tissues were fixed in 10% neutral buffered formalin, sectioned at 4-5 μm and stained using hematoxylin and eosin (H&E). Bursal tissues were scored under a light microscope based on the extent of lymphocyte necrosis, follicular depletion and atrophy. The lesion scores from 0 to 4 indicate relative degree of severity with the score of ‘0’ indicating no lesions, and scores of 1 to 4 indicating lesions in <25%, 25 to 50%, 50 to 75% and >75% of the bursal follicles, respectively (Jackwood et al., 2011).

RNA Extraction and RT-PCR

Bursal tissues were collected from the experimentally infected birds at 3-4 days post-challenge. The collected tissues were cut in half and pressed onto Whatman FTA cards (GE Healthcare Life Sciences, Pittsburgh, PA). Viral RNA was extracted from the FTA cards as previously described in detail (Michel and Jackwood, 2017).Briefly, FTA card punches were vortexed with 300 μL of TE buffer pH 8.0 with 1 mg/mL of proteinase K (Invitrogen, Carlsbad, CA) and 0.5% SDS (Sigma-Aldrich, St. Louis, MO) and incubated at 56°C for 60 min. The samples were lysed by adding 300 μL of RNA Lysis Buffer (Zymo Research, Irvine, CA) followed by incubation at room temperature for 9 min. Total RNA was extracted using the Quick-RNA™ MiniPrep Kit (Zymo Research) according to the manufacturer’s instructions, precipitated with 3 M sodium acetate buffer and ethanol, and resuspended in 25 μL of 90% dimethyl sulfoxide (Sigma-Aldrich) (Michel and Jack-wood, 2017).The RT-PCR was performed to amplify a 579-bp fragment of the VP2 hypervariable region using the Qiagen One-Step RT-PCR Reagents Kit (Qiagen, Valencia, CA) by 743-F (5´-GCC CAG AGT CTA CAC CAT -3´) and 1331-R (5´-ATG GCT CCT GGG TCA AAT CG-3´) primers, as previously described (Michel and Jackwood, 2017). The RT-PCR was performed at 48°C for 30 min and 95°C for 10 min, followed by 35 cycles of 95°C for 30 s, 57°C for 90 s and 72°C for 90 s, with a final extension at 72°C for 5 min (Michel and Jackwood, 2017).

Nucleotide and Amino Acid Sequence Analysis

The RT-PCR products were extracted from the agarose gel using Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI) and Sanger sequenced at the Molecular and Cellular Imaging Center, The Ohio State University (Wooster, OH). BioEdit (Version 7.2.5) was used to assemble and translate the nucleotide sequences into putative protein sequences. Nucleotide and amino acid ali-gnments were performed with the NCBI GenBank reference sequence UK661 (GB#: NC-004178) using the ClustalW alignment option in MEGA-X (Version 10.0.5) (Kumar et al., 2018).Phylogenetic analysis was carried out for 182 amino acids of the VP2 hypervariable region using the Maximum Likelihood method and JTT matrix-based model with 1000 bootstrap replicates.

Recombination Analysis

A total of 113 nucleotides of complete IBDV genomes were downloaded from the NCBI’s Virus Database as of July 2020. To analyze the putative intra-segment recomb-ination events, segments A (n=58) and B (n=49) of each complete genome were separately aligned using MUSCLE alignment option in MEGA-X (Version 10.0.5) (Kumar et al., 2018). To explore the potential presence of inter-segment recombination events (i.e., genomic reassortments), genomic sequences of segments A and B from each strain was pooled (n=51) and MUSCLE aligned before being used for the downstream analysis. Putative recombination events were identified using Recombination Detection Program 5 (RDP5; Version 5.5), which uses multiple methods to minimize the false recombination discovery (Martin et al., 2015). All genomes were analyzed using 3Seq, Chimaera, SiScan, MaxChi, Bootscan, Geneconv, and RDP detection methods (cut-off value P≤0.001) and only the recombination events predicted by at least six out of the seven methods were considered positive (Hoxie and Dennehy, 2020).

Statistical Analysis

Statistical analysis and data visualization were performed using GraphPad Prism ver-sion 8 (GraphPad Software, San Diego, CA). The statistical analyses were performed through one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. A P-value less than 0.05was considered significantly different.



Pathogenicity of JRMP29IR in SPF Chickens

We determined and confirmed the pathogenicity of the JRMP29IR strain in 3-week-old SPF chickens. At 2 days post-challenge, all infected chickens became depressed and showed the typical signs of IBD including ruffled feathers, pasty vents, diarrhea and a reluctance to move. Mortality started from the second day after challenge and reached to 60% at 4 days post-challenge (Figure 1). At necropsy, the bursas from all birds were found enlarged and edematous, and either contained yellowish exudates or varying degrees of hemorrhages, resembling an intermediate clinical profile for JRMP29IR between the variant, classic, or very virulent IBDV viruses. In addition, ecchymotic hemorrhages were observed on the mucosal surface at the proventriculus and gizzard junction, on the heart fats, and thigh muscles. Microscopic lesions including follicular lymphoid depletion and atrophy with severe lymphoid necrosis were observed in the bursal tissues of all infected chickens (Figure 2). The extent of microscopic lesions in bursas also suggested an intermediate pathogenicity index for JRMP29IR between the variant, classic and very virulent IBDV strains.



Figure 1. Mortality of chickens inoculated     with the JRMP29IR strain. Three-week-old SPF chickens were challenged with     the JRMP29IR-infected bursal homogenates via the oral and intraocular     routes, as described in the materials and methods. Percent survival of chickens     was calculated with 10 birds per group and the counting number of birds     that died each day for 5 days post challenge.