Continuous circulation of an antigenically modified very virulent infectious bursal disease virus for fifteen years in Egypt

Journal Pre-proof Continuous circulation of an antigenically modified very virulent infectious bursal disease virus for fifteen years in Egypt

Ahmed Samy, Céline Courtillon, François-Xavier Briand, Mohamed Khalifa, Abdullah Selim, Abd El Satar Arafa, Ahmed Hegazy, Nicolas Eterradossi, Sébastien M. Soubies PII:

S1567-1348(19)30325-9

DOI:

https://doi.org/10.1016/j.meegid.2019.104099

Reference:

MEEGID 104099

To appear in:

Infection, Genetics and Evolution

Received date:

31 July 2019

Revised date:

11 October 2019

Accepted date:

29 October 2019

Please cite this article as: A. Samy, C. Courtillon, F.-X. Briand, et al., Continuous circulation of an antigenically modified very virulent infectious bursal disease virus for fifteen years in Egypt, Infection, Genetics and Evolution(2018), https://doi.org/10.1016/ j.meegid.2019.104099

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© 2018 Published by Elsevier.

Journal Pre-proof

Continuous circulation of an antigenically modified very virulent infectious bursal disease virus for fifteen years in Egypt. Ahmed Samya1 , Céline Courtillon b , François-Xavier Briand b , Mohamed Khalifa a, Abdullah Selima, Abd El Satar Arafa a, Ahmed Hegazya, Nicolas Eterradossib and Sébastien M. Soubiesb* a

Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research

Institute, Dokki, Giza 12618, Egypt. b

Avian and Rabbit Virology Immunology and Parasitology Unit,French Agency for Food,

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Environmental and Occupational Health and Safety, OIE reference laboratory for Infectious

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Bursal Disease, Ploufragan, 22400, France.

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Correspondence: *

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Sébastien Mathieu Soubies:

Phone: 33 2 96 01 01 57 / Fax : 33 2 96 01 62 63 1

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Zoopole – rue des Fusillés BP 53 - 22440 Ploufragan – France

Present address: The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF.

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Abstract:

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UK.

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Infectious bursal disease virus (IBDV), the agent of an immunosuppressive and sometimes lethal disease in chickens, is causing recurrent outbreaks in broiler chickens in Egypt. In particular, an antigenically modified isolate of very virulent IBDV (vvIBDV) called 99323 was detected in Egypt nearly twenty years ago; this isolate was shown to be experimentally controlled by an antigenically classical live vaccine. However, acute IBD is still reported, even in vaccinated flocks, and little is known about the genetic and antigenic properties of viruses currently circulating in Egypt. In the present study, ten samples collected in Egyptian broiler farms in 2015 as well as five samples collected in 2001 were analyzed. Genetic analyses of partial VP2 sequences revealed that 8 isolates clustered with vvIBDV strains, and 5 with tissue culture adapted and vaccine strains. Similar results were observed for partial VP1 sequences with the exception of isolate 160019, for which VP2 clustered with the vaccine strain Bursine while VP1 clustered with vvIBDV, suggesting reassortment. For isolates genetically related to vvIBDV, antigenic profiling revealed two patterns: while some isolates exhibited typical European vvIBDV reactivity with lack of binding of mAbs 5, other

Journal Pre-proof revealed extensive antigenic modifications, with lack of binding of mAbs 3, 5, 6, 8 and 9, similar to isolate 99323. These different patterns were associated with a single amino acid mutation at position 321 of VP2 that is located within peak P HI. Full genome sequencing was performed for three isolates, among which two were representative of the two antigenic patterns observed for vvIBDV as well as the reassortant isolate 160019. This study highlights the co-circulation of both antigenically typical and modified vvIBDV during the last fifteen years in Egypt.

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bursal disease virus, chicken, Egypt, antigenic variation, evolution.

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Keywords : infectious

1. Introduction:

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Infectious bursal disease (IBD) is a viral disease of young chickens that represents a persistent

(Eterradossi et

al.,

1992;

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issue for poultry production worldwide. Some viral strains can induce high mortality Eterradossi &

Saif,

2013).

Infection also

induces an

immunosuppression that facilitates infection by other avian pathogens and results in

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vaccination failures (Rautenschlein and Alkie, 2016). The etiologic agent of this disease,

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called IBDV, is a bisegmented double-stranded RNA virus that belongs to the Birnaviridae family, genus Avibirnavirus (Delmas et al., 2019). IBDV segment A encodes four viral

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proteins (VP2, VP3,VP4 and VP5) while its segment B only encodes VP1, the RNAdependent RNA polymerase (Müller and Becht, 1982; von Einem et al., 2004). The mature VP2 is formed from 3 domains, the helical base domain (B-domain), the shell domain (S domain) and the projection domain (P domain) (Coulibaly et al., 2005). VP2 is the major target of protective humoral immunity and VP2 major antigenic sites are located within the P domain, which represents the most exposed part and contains the hypervariable region (HVR). In particular, the HVR exhibits four exposed loops at the extremity of the P domain, named loop PBC (positions 212 to 224), loop PDE (positions 249 to 256), loop PFG (positions 283 to 287) and loop PHI (positions 314 to 325), respectively (Coulibaly et al., 2005).Vaccination failures correlated with mutations found in VP2 HVR, which include amino acids critical for the binding of neutralizing monoclonal antibodies (mAb) (Coulibaly et al., 2005; Eterradossi et al., 1998; Snyder et al., 1988a).

Journal Pre-proof Based on the lack of in vitro cross-neutralization, IBDV strains are classified into two serotypes, named serotype 1 and 2, respectively; only some serotype 1 strains are pathogenic to chickens (Ismail et al., 1988; McFerran et al., 1980). Serotype 1 viruses are classified depending on their pathogenicity into pathotypes that are classic virulent, that induce mortality and

immunosuppression,

immunosuppressive antigenic variant (Snyder et al.,

1988a), that induce immunosuppression in the absence of mortality and very virulent IBDV (vvIBDV) which emerged in Europe in the late 1980s and induce at least twice the mortality rates observed with classical strains (Chettle et al., 1989; Eterradossi et al., 1992; Müller et al., 1992). In addition, natural reassortant viruses with different pathogenic properties have

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been isolated in different continents (Abed et al., 2018; Jackwood et al., 2011; Kasanga et al.,

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2013; Le Nouen et al., 2006; Li et al., 2015; Soubies et al., 2017). A genogroup classification

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was recently proposed based on the sequences of VP2 (Michel and Jackwood, 2017). In Egypt, very virulent IBDVs were first reported in 1989 (El-Batrawy, 1990; Zierenberg et

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al., 2000). In 1999, an antigenically atypical vvIBDV isolate was detected in this country. A

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live-attenuated, intermediate plus, classical strain-based vaccine, extensively used in Egypt was demonstrated to confer clinical protection against this Egyptian isolate (Eterradossi et al., 2004). During the last twenty years, Egypt has experienced repeated IBDV outbreaks with

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high mortality, despite the intensive use of live attenuated, inactivated, immune complex-

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based and vectored-based vaccines (Abdel-Alim et al., 2003; Hassan et al., 2002; Mawgod et al., 2014; Metwally et al., 2009; Mohamed et al., 2014; Shehata et al., 2017), but thorough

not available.

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genetic, antigenic and pathogenicity data of currently circulating IBDV strains in Egypt are

In order to better understand IBDV epidemiology in Egypt, samples collected in Egyptian farms in 2015 and in 2001 were analyzed. Samples that tested positive for IBDV were characterized genetically by partial sequencing of VP2 and VP1; additionally, full-genome sequencing was performed on a subset of the analyzed samples. Finally, antigenic profiling was performed by antigenic-capture ELISA (AC-ELISA) using a panel of VP2 neutralizing mAbs directed against VP2. 2. Material and methods:

2.1. Sample collection: Fifty bursae of Fabricius were collected from a total of ten vaccinated broiler farms located in three Egyptian governorates (5 bursae per farm, each farm being considered as one

Journal Pre-proof epidemiological unit) located in Northern Egypt that experienced outbreaks of IBD based on clinical diagnosis during summer 2015 (Table 1). Additionally, samples from five Egyptian farms (each 3 to 5 bursae of Fabricius) that experienced clinical IBD episodes in 2001 and which were delivered to the OIE reference laboratory for IBD for antigenic analysis in the context of a world-wide survey were included in the genetic analysis. Isolate 00154, which is genetically and antigenically identical to isolate 99323 (Eterradossi et al., 2004), was included as a second Egyptian reference strain in addition to 99323. 2.2.Sample preparation:

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Bursal samples were prepared as previously described (Eterradossi et al., 1992). Briefly,

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frozen pools of bursae of Fabricius, each pool corresponding to one epidemiological unit, were weighed, cut with a sterile scalpel and homogenized with an equal mass of phosphate

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buffer saline (PBS). An equal volume of 1,1,2-trichloro-1,1,2-trifluoroethane was added to

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delipidate the homogenate, which was then mixed again and centrifuged at 1260 g for 30 min before collecting the supernatant. Potential contaminating enveloped viruses were inactivated

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by overnight incubation of the supernatant with an equal volume of chloroform with endover-end rotation (20 rounds per minute) at 4 °C followed by centrifugation at 3000 g for 10

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min. Supernatant was collected and stored at -70 °C.

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2.3. RT-PCR and partial sequencing of VP1 and VP2: Viral RNA was extracted using QIAamp viral RNA mini kit (Qiagen) according to the

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manufacturer’s instructions. Complementary DNA (cDNA) was produced using Maxima minus reverse transcriptase kit (Thermo Scientific) and reverse primers specific for segment A (VP2) and segment B (VP1) as described in the OIE manual (Le Nouen et al., 2006; OIE, 2008). Two micro liters of cDNA were PCR-amplified using the Expand high fidelity kit (Roche) according to the manufacturer’s instructions and forward and reverse primers for segments A or B, respectively, as previously described (Le Nouen et al., 2006) . PCR products were subjected to electrophoresis in tris-acetate EDTA (TAE) gel containing 2 % agarose; the specific PCR products were excised and purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey Nagel) according to manufacturer’s instructions. The Big dye terminator V.3.1 sequencing kit was used to prepare the sequencing reactions with 4 primers for each gene (available upon request). The prepared sequencing reactions were analyzed using an Applied Biosystem 3130 genetic analyzer (Life technology,USA).

Journal Pre-proof Contigs were generated using vector NTI software (Thermofisher) and their identity was confirmed by a Nucleotide BLAST analysis.

2.4. Phylogenetic and evolutionary analysis: To perform phylogenetic analysis, partial VP1 and VP2 gene sequences obtained during the present study were aligned with VP1 and VP2 sequences from representative IBDV strains (Abed et al., 2018; Le Nouen et al., 2006) (Supplementary Table 1). Alignments were performed with the ClustalW algorithm in MEGA software version 7 (Kumar et al., 2016).

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Phylogenetic trees were constructed using the neighbor joining method with 1000 bootstrap replications and Kimura 2 parameter model; bootstrap values lower than 75 % were

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2.5. Antigenic characterization:

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considered as non-significant.

AC-ELISA was performed as previously described (Eterradossi et al., 1997b). The results were compared for each monoclonal antibody (mAb) with the results previously obtained

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with reference IBDV strains: Faragher 52/70 (UK, classical), 89163 (France, antigenically

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typical vvIBDV), 91168 (France, antigenically atypical vvIBDV) and 99323 (Egypt, antigenically atypical vvIBDV) that were isolated and previously characterized (Eterradossi et

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al., 1999; Eterradossi et al., 1998; Eterradossi et al., 1997a; Eterradossi et al., 1997b). 2.6. PCR amplification and full-length sequencing of segments A and B: Viral RNA was extracted using QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer’s instructions. First strand cDNA for segments A and B were synthesized using complementary primers for the entire coding region of segment A and segment B (available upon request) and Maxima H Minus Reverse Transcriptase (Thermofisher). Segments A and B were amplified in two overlapping segments using Phusion High-Fidelity PCR Kit (Thermofisher) and two forward and two reverse primers (available upon request). Specific amplified bands were excised precisely from 2% agarose gel, then were purified using the PCR Clean-up kit (Macherey Nagel) according to manufacturer’s instructions. The Big dye terminator V.3.1 sequencing kit (ThermoFisher Scientific) was used to prepare the sequence reaction with specifc primers for the entire coding sequences of segments A and B (Soubies et

Journal Pre-proof al., 2017). The prepared sequence reactions were analyzed by the Applied Biosystem 3130 genetic analyzer (Life technology, USA). Retrieved sense and antisense sequences were assembled using the Vector NTI software (Thermofisher) to produce consensus sequences. Nucleotide and amino acid sequences alignments were done using the Clustal W algorithm in MEGA software version 7 (Kumar et al., 2016). Representative strains for all IBDV segment A and B genotypes were retrieved from GenBank data base (Supplementary Table 1) and aligned with isolates of the present study. Phylogenetic trees were constructed by neighbor joining method based on the Kimura

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2-parameter model with 1000 bootstrap replicates implemented in MEGA software version 7 .

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2.7. Accession numbers:

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The partial sequences obtained in this study were submitted to Genbank under the following accession numbers: KY597845 and KY597833 (isolate 160017); KY597846 and KY597834

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(isolate 160018); KY597847 and KY597835 (isolate 160019); KY597848 and KY597836 (isolate 160021); KY597849 and KY597837 (isolate 160022); KY597850 and KY597838

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(isolate 160023); KY597851 and KY597839 (isolate 160024); KY597852 and KY597840 (isolate 160025); KY597853 and KY597841 (isolate 02049F2); KY597854 and KY597842

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(isolate 02049F12); KY597855 and KY597843 (isolate 02049H21); KY597856 and KY597844 (isolate 02049H22); KY597858 and KY597857 (isolate 02049F4) for VP1 and

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VP2 respectively (Table 1).Complete coding sequences were submitted to GenBank with the following accession numbers for segments A and B respectively: KY610528 and KY597859 (isolate 00154), KY610529 and KY597863 (isolate 160019), KY610530 and KY597860 (isolate 160021), KY610531 and KY597861 (isolate), KY610532 and KY597862 (strain Bursine).

3. Results:

3.1. Nucleotide sequence and phylogenetic analysis: Out of the ten samples that were collected upon IBD clinical episodes during Summer 2015, nine tested positive for IBDV using conventional PCR. The nine positive samples as well as the five positive samples from 2001 were subjected to partial VP1 and VP2 gene sequencing.

Journal Pre-proof Phylogenetic analysis based on partial VP2 sequences revealed that 8 isolates (160017, 160021, 160022, 160023, 160024, 160025, 02049F2 and 02049H22) were in the vvIBDVrelated clade and clustered with the antigenically modified vvIBDV Egyptian isolates 99323 and 00154 (Figure 1A, bootstrap value 97 %) as well as more recent Egyptian isolates (USC2004, USC2007, USC2010 and USC201 (Shehata et al., 2017), (Figure 1A, red box, genogroup 3 according to (Michel and Jackwood, 2017)). It is noteworthy that the recent vvIBDV samples (160017, 160021, 160022, 160023, 160024 and 160025) clustered with the old Egyptian isolates 99323 and 00154 but in a distinct subgroup while vvIBDV-related sequences from 2001 (samples 02049F2 and 02049H22) were closely grouped with isolates

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99323 and 00154 (Figure 1A, bootstrap value 97 %). In contrast, 4 sequences (160018,

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160019, 02049H21 and 02049F4) clustered with cell-culture adapted and vaccine IBDV strains; one sequence (02049F12) clustered with Australian strains such as 002/73 and V877

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(bootstrap value : 100 %) and vaccine strain Poulvac Bursa, which is based on strain V877

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(Figure 1A, blue box). Sample 160026 was not included in the phylogenetic tree since chromatogram analysis revealed the presence of double peaks (data not shown), indicating the

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presence of at least two viral populations in the bursal sample. The phylogenetic analysis based on VP1 sequences produced results that were consistent with

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VP2-based analysis for 12 out of 13 isolates: 8 isolates (160017, 160021, 160022, 160023,

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160024, 160025, 02049F2 and 02049H22) clustered with vvIBDV (Figure 1B, red box) while 3 sequences (160018, 02049H21 and 02049F4) clustered with cell-culture adapted

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IBDV strains (Figure 1B, blue box) and one sample (02049F12) clustered with Australian strains (Figure 1B, green box).

Interestingly, for sample 160019, the VP2 sequence clustered with cell-culture adapted IBDV strains while the VP1 sequence clustered with vvIBDV (Figure 1, red square and red arrow), indicating that this isolate could be a reassortant. The deduced amino acid sequences of VP2 for the studied samples were aligned with a classical strain (F52/70), a typical vvIBDV strain (89163), the Egyptian isolates 99323 and 00154 as well as attenuated and/or cell-culture adapted IBDV strains (strains Bursine, CEVAC-IBDL, V877, CT and Cu1M) (Figure 2). Two samples from 2001 (02049F2 and 02049H22) and two samples from 2015 (namely 160021 and 160024) were 100 % identical to 99323 and 00154, with three unusual mutations compared to typical vvIBDV such as strain 89163: Y220F, located within PBC (Figure 2, blue frame), G254S, within PDE (Figure 2,

Journal Pre-proof green frame) and A321T, within P HI (Figure 2, red frame), as previously described for isolate 99323 (Eterradossi et al., 2004). Interestingly, samples 160017, 160022, 160023 and 160025 VP2 sequences differed from those of the above-mentioned isolates by the single mutation A321T, making them more similar to typical vvIBDV on this precise position. Although samples 02049H21 and 166019 VP2 amino acid sequences were highly similar to that of strain Bursine 2, they exhibited some differences that were S222L, H253Q, G254D (02049 only), D258N and S317N. Sample 02049F4 differed from strain Ct by the single H253Q mutation. Samples 160018 and 02049F12 showed 100 % identity with strains CEVAC-IBDL and Poulvac Bursa, respectively.

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The deduced amino acid sequences of the sequence portion of VP1 were also aligned to cognate reference sequences (Supplementary Figure 1). For vvIBDV-related sequences,

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only 3 mutations were observed compared with strain 89163: mutation V141I was observed on all Egyptian isolates while mutations D238N and G241R were only seen for isolate 99323

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and 02049H22, respectively. The VP1 from samples 02049H2, 160018, 02049F2 and

IBDL, Cu1M and V877, respectively.

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02049F12 were 100 % identical to their cognate vaccine strains, that are Bursine, CEVAC-

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To confirm that the results obtained for sample 160019 were due to reassortment and not to co-infection, 3 rounds of cloning by limiting dilutions on primary chicken embryonic

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fibroblasts were performed. The obtained viral isolate, as well as a cloned isolate of strain Bursine, two recent (samples 160021 and 160023) and one older (isolate 00154) Egyptian

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vvIBDV-related isolates underwent full-length genome sequencing. Phylogenetic analysis based on the complete sequences confirmed the results obtained based on partial sequences (Supplementary Figures 2 and 3), concomitantly excluding the occurrence of co-infection in sample 160019 and recombination in all samples. In particular, this phylogenetic analysis confirmed that isolate 160019 is a reassortant whose segment A is related to Bursine and sample 02049H21 and whose segment B is related to Egyptian vvIBDV. Additionally, alignment of deduced amino acid sequences was performed for all viral proteins followed by a comparison of residues differing between isolates (Table 2). Strain Lukert, for which a complete sequence is available in Genbank, and which showed 99.25 % identity with isolate 160019, was presented as an additional reference. For vvIBDV-related sequences, which were compared with the typical vvIBDV strain 89163, only few mutations were observed on segment A. On VP5, isolates 160021 and 160023 showed the E14K change compared with isolates 00154 and 89163 and all Egyptian isolates showed a Y118H change compared with

Journal Pre-proof strain 89163. On VP2 amino acid sequence, no further change was observed compared with partial sequence analysis. Isolate 160021 presented a single change in VP4 (mutation L548I, numbering based on the polyprotein). For isolate 160019 segment A, apart from changes already documented on VP2, a single mutation in VP5 (H138Y) and one mutation in VP3 (L922Q, numbering based on the polyprotein) were observed compared with the cloned Bursine strain. Comparison of VP1 confirmed the V141I change already seen in partial sequence-based analysis and further revealed the V4I and Y243F changes in the three Egyptian sequences. In particular, VP1 sequence from isolate 160019 showed a D393E

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change.

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3.2. Antigenic characterization:

Eight Egyptian isolates, as well as four reference strains, were characterized antigenically.

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Isolates 02049F4 and 02049F12, which are genetically related to classical strain-based vaccines, reacted with all 8 mAbs, similarly to F52/70. Typical vvIBDV (89163) did not react

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with mAbs 3 and 4. Furthermore, Egyptian isolates 99323 and 00154 did not react with mAbs 3,4,5,6 and 9 and showed reduced reaction with mAb 8 as previously described (Eterradossi et

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al., 2004). Two samples from 2001 (F2 and H22) and sample 160021 displayed the same

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antigenic profile as isolates 99323 and 00154. In contrast, samples 160023 and 02049H21

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reacted with mAbs 6, 8 and 9 in addition to mAbs 1 and 7 (Table 3).

4. Discussion:

IBDV represents a persistent concern for poultry production worldwide as a direct and/or indirect cause of heavy losses. In Egypt, the first case of vvIBDVwas described in 1989 (ElBatrawy, 1990; Zierenberg et al., 2000). Ten year later (1999), during an international survey of acute IBD cases, isolate 99323 was collected and characterized genetically and antigenically. This isolate was genetically related to typical vvIBDV but showed extensive antigenic changes that correlated with amino acid changes in the region encoding VP2 projection domain. A classical live IBDV vaccine, commonly used in Egypt, provided clinical protection against this atypical Egyptian vvIBDV (Eterradossi et al., 2004). Since that time and despite the intensive vaccination strategies used, including classical live IBDV vaccines, Egypt has experienced several outbreaks (Abdel-Alim et al., 2003; Hassan et al., 2002;

Journal Pre-proof Mawgod et al., 2014; Mohamed et al., 2014; Shehata et al., 2017). The present study aimed at performing a genetic and antigenic characterization of samples collected in Summer 2015 in vaccinated farms from Northern Egypt (Nile delta area) and to compare these viruses with isolates collected in 2001 from clinically suspected cases, to monitor evolution and diversity of IBDV in Egypt. Three main genetic types were identified based on partial sequencing of VP1 and VP2 genes: viruses genetically related to vvIBDV (8 samples), vaccine-related viruses (4 samples) and one reassortant. Eight samples indeed clustered with vvIBDV based on partial phylogenetic analyses of segments A and B (Figure 1). In particular, for segment A partial analysis, 6

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samples from 2014-2015 and two samples from 2001 clustered with isolate 99323 within the vvIBDV sequences group (boostrap value: 97 %), consistently with their geographic origin.

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The two samples from 2001 were more closely related to isolate 99323 (bootstrap value: 96 %) than more recent samples, suggesting genetic drift along time. This pattern of clustering

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was not found in segment B-based sequence analysis (Figure 1B). Alignment of the deduced

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amino sequences for VP2 revealed that the eight sequences exhibit the unusual Y220F and G254S mutations that are found in isolate 99323. Interestingly, mutation A321T, which is seen in isolate 99323, is only found in 4 out of the 8 sequences. Antigenic profiling of

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selected samples revealed that the atypical antigenic profile already described for isolate

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99323, where only 2 (mAb 1 and mAb7) out of 8 neutralizing anti-VP2 mAbs retain reactivity towards the capsid protein, is only observed when the 3 mutations (Y220F, G254S and

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A321T) are simultaneously present (Table 3), as in the case of sample 160021 and of the earlier isolates 00154 and 99323. The mutation of residue 321 back to an alanine is associated with a typical vvIBDV-like reactivity, with a restoration of mAbs 6, 8 and 9 binding, like in the case of sample 160023. Mutations in the region of loop P HI,which comprises residue 321(Coulibaly et al., 2005), were previously associated to antigenic modifications in VP2: in the case of strain 99432, which exhibits a A321V change, a lack of binding of mAbs 5 and 6 (Escaffre et al., 2013) is seen; strain 91168 has a Q324L change which is associated to the loss of mAbs 7 and 8 binding (Eterradossi et al., 1997b). Complete VP2 amino acid sequence comparison between samples 160021 and 160023 confirmed that their VP2 sequences only differ by position 321. These data thus strongly suggest that mutating residue 321 is sufficient to induce a dramatic change in antigenicity provided that the Y220F and G254S changes preexist. Reverse genetics based experiments would be necessary to formally demonstrate this hypothesis. This AlaThr change is obtained through a single GA mutation, suggesting

Journal Pre-proof that Egyptian vvIBDV-related isolates can easily switch from typical to atypical antigenicity or vice versa. Interestingly, careful examination of the sequencing chromatograms did not reveal the presence of subpeaks in any of the studied sequence for the position coding for residue 321, suggesting that viruses circulate as either typical or atypical but are not mixes of these phenotypes; if variants are present in the isolates, their relative frequency is too low to allow their detection by Sanger sequencing. Our data show that antigenically typical and atypical vvIBDV-related viruses are co-circulating in Egypt. Interestingly, both typical (such as 160023 and 160025) and atypical (such as 160021 and 160024) isolates were found in the 2015 survey in the same governorates, such as 160021 (atypical) and 160023 (typical) in

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Damietta governorate, which covers approximately 1000 km², or 160024 (atypical) and

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160025 (typical) in Kafr El Sheikh, indicating that co-ciculation in geographically limited areas indeed takes place. Our results are in agreement with other recent studies that detected

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Egyptian IBDV isolates with the Y220F and G254S changes with or without the A321T

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change (Michel and Jackwood, 2017; Shehata et al., 2017).

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Another question raised by our study is the potential benefits of antigenic variation in vvIBDV. Mutations in VP2 HVR, responsible for changes in IBDV antigenicity in response to vaccine immune pressure have been documented in several instances (Coulibaly et al.,

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2005) such as the emergence of US antigenic variants in the mid-eighties (Snyder et al.,

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1988b). A recent study revealed the existence of the variation of VP2 HVR from vvIBDVrelated isolates in relation with the geographic origin of samples, with particular amino acid

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signatures found in viruses from Russia (Michel and Jackwood, 2017) or Indonesia (Rudd et al., 2002) in addition to Egypt. Few antigenically modified vvIBDV such as strains 91168 and 94432 (Eterradossi et al., 1997b), have been described so far but, unlike Egyptian antigenically atypical vvIBDV, they were not reisolated (with the possible exception of Polish strain 93/35 that seems similar to 91168, (Domanska et al., 2004; Escaffre et al., 2013). This suggests that any advantage provided by antigenic variation was not sufficient to confer a major evolutionary benefit and make the spread of these isolates possible. In the case of 94432, reverse-genetics based studies even showed that the A321V change, responsible for its atypical antigenic profile, was partly responsible for the decreased pathogenicity and replication of this isolate (Escaffre et al., 2013), which suggests that antigenic variation can have a detrimental effect on viral fitness. The persistence of antigenically atypical vvIBDV for 15 years in Egypt argues against an evolutionnary detrimental effect of the Egyptian specific mutations on viral fitness. Alternatively, mutations affecting antigenicity may only be

Journal Pre-proof beneficial in the context of the pressure of a vaccinal immune response, which may explain the concomitant presence of antigenically typical and atypical viruses in Egypt. Previous experiments showed that the antigenically atypical Egyptian vvIBDV isolate 99323 could be clinically controlled by vaccination using a classical virulent-based intermediate-plus live vaccine (Eterradossi et al., 2004), which indicates that antigenically atypical Egyptian vvIBDV do not escape from active, vaccine-induced immunity. It would be interesting to investigate whether the potential evolutionary advantage conferred by those mutations might only exist in the context of the passive immunity provided by maternally-derived antibodies in young chicks .

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Complete sequencing of isolates 00154 (which is genetically and antigenically similar to isolate 99323 and was collected in 2000) and samples 160021 and 160023 (collected in 2015)

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provide the first complete sequences of Egyptian vvIBDV. Amino acid sequences comparison highlighted the remarkable stability of these viral sequences in viruses collected nearly 15

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years apart. Very few mutations were observed in Egyptian sequences in comparison with

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European vvIBDV (Table 2). Among these, the V4I mutation in VP1 has been previously associated to the reduced pathogenicity of some Chinese IBDV isolates (Yu et al., 2013).

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Two vaccine-related samples from this study, samples 02049F4 and 02049H21, as well as the reassortant isolate 160019, presented the H253Q mutation in VP2. This change was observed

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when a vaccine strain was serially passaged on chickens (Noor et al., 2014), or in vaccinerelated isolates obtained from the bursa of vaccinated chickens in the field and exhibiting

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microscopic bursal lesions (Olesen et al., 2018); this change was also associated with an increase in the residual pathogenicity of vaccine strains (Jackwood and Sommer-Wagner, 2011). Residue Q253, along with A284, is found in field viruses while tissue culture adapted strains exhibit residues H253 and T284; moreover, introducing the Q253H and A284T changes using reverse genetics was sufficient to adapt the vvIBDV strain UK661 to tissue culture (van Loon et al., 2002), highlighting the importance of those positions on viral tropism. Finally, isolate 160019 was confirmed, by repeated limiting-dilution cloning and full genome sequencing, to be a reassortant virus whose segment A is related to vaccinal strain Bursine while its segment B is related to Egyptian vvIBDV. Phylogenetic analysis based on partial segment A sequence revealed that isolate 160019 was more closely associated to sample 02049H21 (bootstrap value: 89 %) than to Bursine. This might indicate that isolate 160019

Journal Pre-proof segment A originates from a virus closely related to 02049H21, that might itself originate from the vaccine strain Bursine. Apart from the H253Q change in VP2 compared to Bursine , 02049H21 and isolate 160019 share the S222L (located in loop P BC), and S317N (located loop PHI) mutations in VP2. Antigenic profiling using AC-ELISA of isolate 02049H21 revealed a lack of binding of mAbs 3, 4 and 5, which is consistent with the L222 and D254 residues; interestingly, binding of mAbs 6, 7 and 8 was conserved, which indicates that the S317N change does not alter the antigenicity of peak P HI. It would be of interest to compare this antigenic profile with that of strain Bursine, which is unfortunately not feasible using ACELISA due to the low amount of viral antigen in the viral stocks. Full-length sequencing

f

revealed few mutations between isolate 160019 segments A and B and their cognate related

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“parental” segments. Reassortment can result in drastic reduction of viral pathogenicity (Le Nouen et al., 2006; Soubies et al., 2017); on the contrary, it can enhance pathogenicity (Abed

pr

et al., 2018; Li et al., 2015; Pikula et al., 2018). Two reassortant IBDV isolates with a

e-

vaccine-derived segment A and a vvIBDV-derived segment B were previously described (Wei et al., 2006; Wei et al., 2008). In both cases, the pathogenicity of the reassortant was

Pr

increased compared with the vaccinal parental strain. Pathotyping of isolate 160019, which extends beyond the scope of this study, is currently underway in the laboratory.

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In conclusion, genetic and antigenic studies revealed the persistence for 15 years of

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antigenically modified vvIBDV-related viruses in Egypt, along with the co-circulation of viruses antigenically closely related to typical vvIBDV and the existence of a reassortant

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virus.

Competing interests

The authors have no conflict of interest.

Acknowledgments Ahmed Samy's postdoctoral studies in VIPAC supported by a fund from the Science & Technology Development Fund in Egypt (STDF) and Institut Français d'Egypte (IFE), project ID 18551.

Journal Pre-proof ANSES laboratory is grateful for the financial support provided by the “agglomeration de Saint-Brieuc”, the “Conseil départemental des Côtes d’Armor” and by “Région Bretagne”. The authors thank Dr. Sylvain Comte, Ceva Santé Animale and Dr. Mohamed Said Soliman for collecting and providing support to Anses for the study of the 2001 Egytian IBDV samples.

References:

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Abdel-Alim, G.A., Awaad, M.H.H., Saif, Y., 2003. Characterization of Egyptian field strains of infectious bursal disease virus. Avian diseases 47, 1452-1457. Abed, M., Soubies, S., Courtillon, C., Briand, F.X., Allee, C., Amelot, M., De Boisseson, C., Lucas, P., Blanchard, Y., Belahouel, A., Kara, R., Essalhi, A., Temim, S., Khelef, D., Eterradossi, N., 2018. Infectious bursal disease virus in Algeria: Detection of highly pathogenic reassortant viruses. Infection, genetics and evolution 60, 48-57. Chettle, N., Stuart, J., Wyeth, P., 1989. Outbreak of virulent infectious bursal disease in East Anglia. Veterinary Record 125, 271-272. Coulibaly, F., Chevalier, C., Gutsche, I., Pous, J., Navaza, J., Bressanelli, S., Delmas, B., Rey, F.A., 2005. The birnavirus crystal structure reveals structural relationships among icosahedral viruses. Cell 120, 761-772. Delmas, B., Attoui, H., Ghosh, S., Malik, Y.S., Mundt, E., Vakharia, V.N., Ictv Report, C., 2019. ICTV virus taxonomy profile: Birnaviridae. The Journal of general virology 100, 5-6. Domanska, K., Mato, T., Rivallan, G., Smietanka, K., Minta, Z., De Boisseson, C., Toquin, D., Lomniczi, B., Palya, V., Eterradossi, N., 2004. Antigenic and genetic diversity of early European isolates of Infectious bursal disease virus prior to the emergence of the very virulent viruses: Early European epidemiology of Infectious bursal disease virus revisited? Arch. Virol. 149, 465-480. El-Batrawy, A., 1990. Studies on severe outbreaks of infectious bursal disease, Proc. 2nd Sci. Conf., Egypt. Vet. Poult. Assoc, pp. 239-252. Escaffre, O., Le Nouen, C., Amelot, M., Ambroggio, X., Ogden, K.M., Guionie, O., Toquin, D., Muller, H., Islam, M.R., Eterradossi, N., 2013. Both genome segments contribute to the pathogenicity of very virulent infectious bursal disease virus. Journal of virology 87, 2767-2780. Eterradossi, N., Arnauld, C., Tekaia, F., Toquin, D., Le Coq, H., Rivallan, G., Guittet, M., Domenech, J., Van den Berg, T., Skinner, M., 1999. Antigenic and genetic relationships between European very virulent infectious bursal disease viruses and an early West African isolate. Avian Pathol. 28, 36-46. Eterradossi, N., Arnauld, C., Toquin, D., Rivallan, G., 1998. Critical amino acid changes in VP2 variable domain are associated with typical and atypical antigenicity in very virulent infectious bursal disease viruses. Arch. Virol. 143, 1627-1636. Eterradossi, N., Gauthier, C., Reda, I., Comte, S., Rivallan, G., Toquin, D., de Boisséson, C., Lamandé, J., Jestin, V., Morin, Y., 2004. Extensive antigenic changes in an atypical isolate of very virulent infectious bursal disease virus and experimental clinical control of this virus with an antigenically classical live vaccine. Avian Pathol. 33, 423-431. Eterradossi, N., Picault, J., Drouin, P., Guittet, M., L'Hospitalier, R., Bennejean, G., 1992. Pathogenicity and preliminary antigenic characterization of six infectious bursal disease virus strains isolated in France from acute outbreaks. J. Vet. Med. Ser. B 39, 683-691. Eterradossi, N., Rivallan, G., Toquin, D., Guittet, M., 1997a. Limited antigenic variation among recent infectious bursal disease virus isolates from France. Arch. Virol. 142, 2079-2087.

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Eterradossi, N., Saif, Y.M., 2013. Chapter 7 : Infectious bursal disease. In “Diseases of Poultry”, XIIIth Edition, Edited by Swayne, D.E., Glisson, J.R., McDougald, L.R., Nolan, L.K., Suarez, D.L., Nair, V., John Wiley & Sons Inc., Ames, Iowa, USA, pp 219-246 Eterradossi, N., Toquin, D., Rivallan, G., Guittet, M., 1997b. Modified activity of a VP2-located neutralizing epitope on various vaccine, pathogenic and hypervirulent strains of infectious bursal disease virus. Arch. Virol. 142, 255-270. Hassan, M.K., Afify, M., Aly, M.M., 2002. Susceptibility of vaccinated and unvaccinated Egyptian chickens to very virulent infectious bursal disease virus. AVIAN PATHOL. 31, 149-156. Ismail, N.M., Saif, Y.M., Moorhead, P.D., 1988. Lack of pathogenicity of five serotype 2 infectious bursal disease viruses in chickens. Avian Dis 32, 757-759. Jackwood, D.J., Sommer-Wagner, S.E., 2011. Amino acids contributing to antigenic drift in the infectious bursal disease Birnavirus (IBDV). Virology 409, 33-37. Jackwood, D.J., Sommer-Wagner, S.E., Crossley, B.M., Stoute, S.T., Woolcock, P.R., Charlton, B.R., 2011. Identification and pathogenicity of a natural reassortant between a very virulent serotype 1 infectious bursal disease virus (IBDV) and a serotype 2 IBDV. Virology 420, 98-105. Kasanga, C., Yamaguchi, T., Munang’andu, H., Ohya, K., Fukushi, H., 2013. Genomic sequence of an infectious bursal disease virus isolate from Zambia: classical attenuated segment B reassortment in nature with existing very virulent segment A. Archives of virology 158, 685-689. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Molecular biology and evolution 33, 1870-1874. Le Nouen, C., Rivallan, G., Toquin, D., Darlu, P., Morin, Y., Beven, V., de Boisseson, C., Cazaban, C., Comte, S., Gardin, Y., 2006. Very virulent infectious bursal disease virus: reduced pathogenicity in a rare natural segment-B-reassorted isolate. J. Gen. Virol. 87, 209-216. Li, K., Courtillon, C., Guionie, O., Allée, C., Amelot, M., Qi, X., Gao, Y., Wang, X., Eterradossi, N., 2015. Genetic, antigenic and pathogenic characterization of four infectious bursal disease virus isolates from China suggests continued evolution of very virulent viruses. Infection, Genetics and Evolution 30, 120-127. Mawgod, S.A., Arafa, A.S., Hussein, H.A., 2014. Molecular genotyping of the infectious bursal disease virus (IBDV) isolated from Broiler Flocks in Egypt. International Journal of Veterinary Science and Medicine 2, 46-52. McFerran, J., McNulty, M., McKillop, E., Connor, T., McCracken, R., Collins, D., Allan, G., 1980. Isolation and serological studies with infectious bursal disease viruses from fowl, turkeys and ducks: demonstration of a second serotype. Avian Pathol. 9, 395-404. Metwally, A.M., Yousif, A.A., Shaheed, I.B., Mohammed, W.A., Samy, A.M., Reda, I.M., 2009. Reemergence of very virulent IBDV in Egypt. Int. J. Virol. 5, 1-17. Michel, L.O., Jackwood, D.J., 2017. Classification of infectious bursal disease virus into genogroups. Arch. Virol. 162, 3661-3670. Mohamed, M.A., Elzanaty, K.E., Bakhit, B.M., Safwat, M.M., 2014. Genetic Characterization of Infectious Bursal Disease Viruses Associated with Gumboro Outbreaks in Commercial Broilers from Asyut Province, Egypt. ISRN veterinary science 2014. Müller, H., Becht, H., 1982. Biosynthesis of virus-specific proteins in cells infected with infectious bursal disease virus and their significance as structural elements for infectious virus and incomplete particles. Journal of virology 44, 384-392. Müller, H., Schnitzler, D., Bernstein, F., Becht, H., Cornelissen, D., Lütticken, D., 1992. Infectious bursal disease of poultry: antigenic structure of the virus and control. Vet. Microbiol. 33, 175-183. Noor, M., Mahmud, M.S., Ghose, P.R., Roy, U., Nooruzzaman, M., Chowdhury, E.H., Das, P.M., Islam, M.R., Müller, H., 2014. Further evidence for the association of distinct amino acid residues with in vitro and in vivo growth of infectious bursal disease virus. Arch. Virol. 159, 701-709. OIE, 2008. Chapter 2.3.4. 2.3.12. INFECTIOUS BURSAL DISEASE (Gumboro disease). Available online at:http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.03.12_IBD.pdf.

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Figure 1 Consensus phylogenetic trees deduced from partial nucleotide sequences for segment A (A) and segment B (B) of Egyptian and reference IBDV strains using the Neigbor-Joining method. Relevant groups are highlighted by colour boxes. Gray dots indicate the sequences of isolates 99323 and 00154, collected in 1999. Sequences with a triangle indicate isolates collected in 2001. Sequences with a gray square indicate isolates collected in 2015. The red arrow shows reassortment for isolate 160019 (red squares). Bootstrap values > 75% are indicated. Genogroups for VP2 as proposed by Michel et al., 2017 are indicated. Genbank accession numbers are listed in Supplementary Table 1.

Journal Pre-proof Figure 2 Amino acid alignment of VP2 HVR for studied Egyptian isolates in comparison with reference sequences. Coloured boxes indicate exposed loops of VP2 P domain. Positions 253 and 284, that are important for cell-culture adaptation, are shaded in gray. Positions 220, 254 and 321, for which specific residues are present in Egyptian isolates, are shaded in orange.

Table 1. Samples analyzed in this study. NA: Not Available. Samples Name

Governorates

Collection Year

Age (days)

Flock Size

Vaccination age (vaccine)

7 ,14 and 17days 10 days 7 ,15 days

KY597845 KY597846 KY597847 [KY597863]

KY597833 KY597834 KY597835 [KY610529]

NA

Neg.

Neg.

KY597848 [KY597859] KY597849 KY597850 [KY597861] KY597851 KY597852

KY597836 [KY610530] KY597837 KY597838 [KY610531] KY597839 KY597840

NA

NA

KY597853 KY597854 KY597855 KY597856 KY597858 [KY597859] [KY597862]

KY597841 KY597842 KY597843 KY597844 KY597857 [KY610528] [KY610532]

Dakahlia Dakahlia Damietta

2015 2015 2015

21 18 43

9000 7000 10000

160020 160021

Dakahlia Damietta

2015 2015

20 33

8000 NA

160022 160023

Damietta Damietta

2015 2015

30 NA

12500 NA

7,15 and18 days NA

160024 160025 160026

Kafr El Sheikh Kafr El Sheikh Kafr El Sheikh

2015 2015 2015

17 28 22

60000 10000 11000

(IBD L) NA 8 , 14 and 18 days

02049F2 02049F12 02049H21 02049H22 02049F4 00154 Bursine

NA NA NA NA NA NA NA

2001 2001 2001 2001 2001 2000 NA

NA NA NA NA NA NA NA

NA NA NA NA NA NA NA

NA NA NA NA NA NA NA

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160017 160018 160019

Accession number Partial sequence [complete sequence] Segment B Segment A

Table 2. Unique amino acid substitutions in full VP5, polyprotein and VP1 for selected Egyptian isolates in comparison with reference sequences (between asterisks(*)) for a typical vvIBDV strain (89163, right panel) and cell-culture adapted IBDV strains (Bursine and Lukert strains, left panel). Viral Strain Protein Position *Bursine* *Lukert* 160019 00154 160021 160023 *89163*

VP5

Polyprotein

VP2

14 16 45 74 118 133 134 220 222 249 251 253 254

K D G I H R H Y S H S H G

K D G I H R H Y S H R Q G

K D G I H R Y Y L H S Q G

E A R F H W H F A Q S Q S

K A R F H W H F A Q S Q S

K A R F H W H F A Q S Q S

E A R F Y W H Y A Q S Q G

Journal Pre-proof I G A A A T I S S T L H I L Y N S D L L Q A

f

A N T S T I L N N A I L V L C K P H I Q P T

A D T S T I L N S A I L V L C K P H I Q P T

I G A A A T I S S T L H I I Y N S D L L Q A

I G A A A T I S S A L H I L Y N S D L L Q A

I G A A A T I S S A L H I L Y N S D L L Q A

VP1

*Bursine*

4 61 121 141 145 146 147 172 227 242 243 287 356 390 393 479 508 511 527 562 646 687 695 720 817 880 881

I V T V N E G Q K D F T M L E T R R N S G S K L A * -

*Lukert* I V P V N E G E R D F T V L E P R R S S G S K F V Q P

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Position

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Protein

Viral Strain 160019 00154 160021

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Table 2 (continued).

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pr

VP3

A D T S T I L N S A I L V L C K P H I L P T

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VP4

256 258 270 278 284 286 294 299 317 321 451 471 541 548 680 685 715 751 786 922 993 1005

I I P I T D N Q R E F A M M E T K S N P S P R L V * -

I I P I T D N Q R E F A M M D T K S N P S P R L V * -

I I P I T D N Q R E F A M M D T K S N P S P R L V * -

160023

*89163*

I I P I T D N Q R E F A M M D T K S N P S P R L V * -

V I P V T D N Q R E Y A M M D T K S N P S P R L V * -

Table 3. Antigenic characterization of selected Egyptian isolates by antigen -capture ELISA. Reactivity of each mAb is expressed as the percentage of binding compared with a polyclonal reagent. Shaded values indicate efficient binding of the considered mAb (binding >25%). For comparison purpose the antigenic profiles of

Journal Pre-proof typical classical strain (F52/70), typical vvIBDV strain (89163), Egyptian strain (99323) and atypical vvIBDV strain (91168) are presented with asterisks (*).

68 99 45 71 67 70 97 84 54 26 60 63

84 149 100 4 0 0 7 1 0 0 1 0

mAb 4 P BC

78 83 67 9 1 0 18 0 0 0 0 0

mAb 5 P DE

51 72 48 65 0 0 83 0 0 1 0 0

mAb 6

mAb 7 P HI

mAb 8

101 98 54 120 97 66 159 1 0 2 0 0

101 99 77 119 101 79 62 109 63 43 64 78

100 158 128 81 68 62 3 17 8 14 15 13

f

mAb 3

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pr

*F52/70* 02049F4 02049F12 *89163* 160023 02 049 H21 *91168* *99323* 00154 02 049 H22 02 049 F2 160021

mAb 1 Unknown

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Paratope localization Strain

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Antigenically atypical very virulent IBDV (vvIBDV) are detected in Egypt These viruses have persisted for 15 years in Egypt Antigenically atypical and typical vvIBDV co-circulate in Egypt

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  

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Highlights:

mAb 9 unknown

70 36 29 55 53 29 79 15 16 12 22 23

Figure 1

Figure 2