Persistent Circulation of Very virulent Infectious Bursal Disease virus (IBDV) in Egypt: Phylogenetic analysis, Pathogenicity and Immunogenicity of an immune-complex vaccine

Persistent Circulation of Very virulent Infectious Bursal Disease virus (IBDV)

in Egypt: Phylogenetic analysis, Pathogenicity and Immunogenicity of

an immune-complex vaccine

Mostafa Saleh^*, Zienab Mosaad^**, Ola Abdel Aziz^**, Ali Zanaty^**

^*Animal Health Research Institute, Mansoura branch

**Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Agriculture Research Center (ARC) Giza 12618, Egypt.

ABSTRACT

Infectious bursal disease (IBD) viruses continue to cause considerable economic losses in the Egyptian poultry industry. The purpose of this study was to investigate the molecular features of IBDV isolated in Egypt from 2021 to march 2022, and assess the pathogenicity, immunogenicity, and protection of the IBD immune-complex vaccinations (Transmune®-CEVA vaccine). Twenty-three field samples (bursa of Fabricius) were collected from broiler farms and a highly variable region encompassing VP2 gene was targeted for IBDV screening utilizing RT-PCR. Out of 23 tested farms, 19 were positive by RT-PCR.Six positive samples were chosen for viral isolation, sequence, and phylogenetic analysis. Phylogenetically, five of the strains under study belonged to the very virulent (vvIBDV) strains, with 95-98% resemblance to Giza 2008 belonging to Genogroup 3 of IBDV strain. The remaining strain were identified as a vaccination strain (Genotype 1) and  matched the winter field 2512 vaccine strain by a similarity percentage of 96%. One day old commercial chicks were vaccinated (Transmune®-CEVA vaccine) then challenged with selected very virulent strain (OP056767). The Fabricius Bursa was examined grossly and histologically. Furthermore, the Bursal Body Weight Ratio and Bursa Index were computed. The transmune IBD vaccination was able to elicit a high ELISA mean titer of 3179 at 32 days of age (4 day post challenge). Moreover, the greater raised mean ELISA titers of 9264 (38day) and 9354 (42 day) post vaccination, indicating that the challenge IBDV serves as booster immunization. Beside the Efficacy of transmune®-CEVA vaccine in reducing mortality in comparison to Pathogenicity group. The bursal body weight ratio and index demonstrate that the IBD vaccine was able to indicate an inflammatory response in the bursa of fibricia, resulting in a better immunological response and the safety of the Transmune®-CEVA vaccine. Finally, our findings show the dual circulation of both G1 and G3 strain in poultry flocks, and the immune-complex vaccination is still effective in protecting commercial broiler chicks against dominant circulating vvIBD strains.

INTRODUCTION

IBDV caused by a tiny, non-enveloped virus that belongs to the Birnaviridae family and has a bi-segmented ds-RNA genome (Kibenge et al. 1988). Segment A of the viral genome encodes a precursor polyprotein, which is autoclaved into the proteins VP2, VP4, and VP3 ((Müller and Becht 1982). The key immunogenic determinants are carried by VP2, which is the capsid protein (Letzel et al. 2007; Schnitzler et al. 1993). Also responsible for antigenic site which  neutralizing the antibodies (Becht et al. 1988). Within VP2 a small area known as the variable domain (Bayliss et al. 1990). Genomic segment B (2.8 kbp) encodes the viral RNA-dependent RNA polymerase (RdRp) VP1 (Mundt et al. 1995) as well as the nonstructural protein VP5 (Garriga et al. 2007). Both segments have a role in virus replication and virulence (Müller et al. 2003)

IBDV, like any other RNA virus, has a high polymerase mutation rate, which leads to genetic evolution and the generation of new viruses with unique features that contribute to antigenic diversity and virulence modification (Van den Berg et al. 2000). Virus neutralization (VN) tests revealed two IBDV serotypes (Ashraf et al. 2006). Pathogenic strains are classified as mild, intermediate, intermediate plus, classical, variant, and very virulent in serotype 1. Serotype 2 strains, on the other hand, are mostly non-pathogenic and are mostly isolated from turkeys (Van den Berg et al. 2000). Sequence studies between pathogenic and non-pathogenic strains revealed nucleotide alterations across the genome (Absalón et al. 2017; Brown and Skinner, 1996) which likely contribute to the virulence's mutagenic aspect (Escaffre et al. 2013).

In 1975, IBDV was discovered in Egyptian broiler chickens for the first time (Ayoub and Malek, 1976). The its first introduction of very virulent IBDV Since 1989 in vaccinated Egyptian flocks (EL-BATRAWI, 1990). Currently, circulating variant IBDV strains have been identified from flocks vaccinated with IBDV vaccinations (Abou El-Fetouh and Abdallah, 2018; El-Bagoury et al. 2018; Helal et al. 2012; Mawgod et al. 2014; Samy et al. 2020; Shehata et al. 2017). Despite the use of a high range of vaccinations, Egypt has been stricken by recurrent IBD episodes in the latest period (Mohamed et al. 2014; Shehata et al. 2017; Zanaty et al. 2022)

Despite strict sanitary precautions, the IBDV is very contagious, immunosuppressive, resistant, and it tends to persist in the environment. As a consequence, vaccination is seen as an important means of protecting young birds during their first few weeks of life (Eterradossi et al. 2008; Faragher et al. 1972). IBDV infection may worsen existing infections with other infectious agents and reduce the bird's capacity to react to vaccination because the virus inhibits humoral and cellular immune responses (Fan et al. 2020). Hyper immunization of breeders with inactivated vaccinations is used to reduce IBDV infection in chicks. Although passive immunity protects chickens well during their early weeks of life, lasting protection against IBD necessitates the use of live vaccines (Müller et al. 2012). Mild vaccinations are harmless, while intermediate and hot vaccines are significantly more effective in the case of strong maternal antibodies or against particularly virulent strains of IBDV, although they can cause moderate to severe lesions in the Fabricius Bursa (Camilotti et al. 2016).

 As a result, novel vaccines that combine safety and efficacy, such as immune complex and recombinant vaccines have been produced to address these issues. The recombinant vaccine employs a viral vector to contain and produce the immunogenic protein VP2 of IBDV, causing the development of particular antibodies even in the presence of passive immunity (Camilotti et al. 2016; Rage et al. 2020; Sze et al. 2016).The immune-complex vaccine, on the other hand, is novel in comparison to traditional live vaccines because the vaccine virus is coated with anti-IBD antibodies and its pathological effects are delayed for up to one week when administered to one-day-old chicks, during which the level of maternal antibodies is greatly reduced (Camilotti et al. 2016; Shazali, 2008).

This study aims to investigate the molecular features of the currently circulating IBDV isolated in Egypt (2021-2022), and to characterize the pathogenicity and immunogenicity of IBD immune-complex commercial vaccines (Transmune®-CEVA vaccine) as well as the degree of protection afforded by those vaccinations in chickens challenged with a highly-virulent strain of IBDV.

MATERIAL AND METHODS

Collection and preparation of IBDV field strain:

The chicken flocks suffered from high Morbidity andmortalities with depression, watery diarrhea, ruffled feathers, and dehydration. At necropsy, the cloacal bursa is swollen, edematous, yellowish, occasionally congested. Bursal of fibricia (10 bursae/farm) were taken aseptically from 23 broiler chicken farms located in different provinces of Egypt (Table 1). Bursa samples were homogenized, and the supernatant was collected and filtered in accordance to  (Yovel et al. 2008). Following the manufacturer's instructions, RNA from the prepared samples was extracted using a QiaAmp® Viral RNA Mini Kit (QIAGEN GmbH, Hilden, Germany). The RNAs were then confirmed for IBDV using AgPath-ID™ One-Step RT-PCR Reagents Kit (Applied Biosystems, USA) on the extracted RNA was performed to process the reverse transcriptase-polymerase chain reaction (RT-PCR). Forward and reverse primers were used to amplify a 620 bp included in HVR of the VP2 gene (Metwally et al. 2009). ProFlex PCR System thermal cycler (Applied Biosystems, California, and USA) was used to carry out the reaction.PCR Analysis was performed by gel electrophoresis 1.5% against 100 bp Plus DNA Ladder GeneRuler™ (Fermentas).

Virus isolation and Titration

The filtrated supernatant of positive Bursa samples was injected on the chorioallantoic membrane (CAM) in embryonated SPF eggs 10-11 day old embryos According to (Dufour-Zavala, 2008; Hirai et al. 1972). Then, they were incubated at 37°C with candling daily. The allantoic fluids were collected at 4-5 days post-inoculation (Hirai et al. 1972), PCR testing was applied to confirm isolation (Metwally et al. 2009).Then Titration of the selected dominant IBDV strain according to (Soubies et al. 2018), using Specific Pathogen Free eggs (SPF). (Van den Berg et al. 2004). The EID₅₀of local strain (EGY-IBDV-Domiedta-VV22-2022-VP2) was determined using the following formula according to (Muench, 1938).

Sequence and phylogenetic analysis of VP2 gene hyper variable region

A QiaAmp purification kit (Qiagen, Germany) was used to purify the positive PCR samples. BigDye Terminator V3.1 cycle sequencing kit (Perkin-Elmer, Foster city, CA, USA) was used to sequence the VP2 gene. The nucleotide sequences were determined using an Applied Biosystems 3500XL Genetic Analyzer (Applied Biosystems,Foster City, CA). To edit and assemble sequences, the Bio-edit application (version 7.2.5) was used (Hall et al. 2011). BLASTn was used on the NCBI website (www.ncbi.nlm.nih.gov/BLAST), to compare IBDV sequences to GenBank sequences.The sequences of the unique strains were deposited in the GenBank database (Table 1).