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Further Evidence for the Association of Distinct Amino Acid Residues with in Vitro and in Vivo Growth of Infectious Bursal Disease Virus

M. Noor
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh

H. Muller
Institute for Virology, Faculty of Veterinary Medicine,University of Leipzig, Leipzig, Germany,Present Address: Genolytic GmbH, Deutscher Platz 5, 04103 Leipzig, Germany

M. R. Islam
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh

P. M. Das
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh

E. H. Chowdhury
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh

M. Nooruzzaman
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh

U. Roy
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh

P. R. Ghose
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh

M. S. Mahmud
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh

Abstract/ Executive Summary:

A cell-culture-adapted reverse genetics strain of very virulent infectious bursal disease virus (IBDV) of chickens, designated as BD-3tcC, having four amino acid substitutions (Gln253His, Asp279Asn, Ala284Thr and Ser330Arg) in the capsid protein VP2 was tested for its genetic stability during serial passage in chickens and chicken embryo fibroblast (CEF) cell culture. Results of in vitro and in vivo experiments demonstrated that all four introduced mutations in BD-3tcC remained stable during serial passage in CEF cell culture, but during passage in chickens, amino acid residues at position 253 and 284 reverted from histidine to glutamine and threonine to alanine, respectively. In a parallel experiment, the same substitutions also occurred in a conventionally attenuated vaccine strain D-78 on serial passage in chickens. However, no reversion or substitution took place at positions 279 and 330 during in vivo passage of the mutant virus BD- 3tcC or vaccine virus D-78. The findings provide conclusive evidence that while IBDV requires histidine and threonine at positions 253 and 284 for cell culture adaptation, glutamine and alanine at these positions are selected preferentially during in vivo replication.

Keywords: Virulent, Cell Culture, Pathogenicity, IBDV

Location: Department of Pathology, Faculty of Veterinary Science,Bangladesh Agricultural University, Mymensingh, Bangladesh

Start Date:

End Date:

Program Area: Pest Management

Commodity: Diseases

Objectives:

To provide further conclusive evidence that distinct amino acid residues at position 253 and 284 are associated with in vitro and in vivo growth of IBDV, through an experimental approach using mutants generated by reverse genetics

Methodology:

Wild-type and recombinant IBDV strains

The wild-type vvIBDV (BD-3/99 or BD-3wt) from Bangladesh served as the parental strain of the recombinant IBDV strains used in this study. Construction of fulllength clones of the genome segments A and B of BD 3/99, site-directed mutagenesis, and rescue of IBDV reverse genetics strains BD-3tc and BD-3tcC having two (Gln253His and Ala284Thr) or four (Gln253His, Asp279Asn, Ala284Thr and Ser330Arg) amino acid substitutions in the VP2, respectively, have been described.

Pathogenicity of recombinant IBDV strains for commercial chickens

For the pathogenicity experiment, a total of 100 one-day-old Hisex brown layer chickens, obtained from a commercial source, were raised in relative isolation. At 5 weeks of age they were divided into four groups with 25 birds in each group and placed in four separate houses. Three groups of birds were inoculated with BD-3wt, BD- 3tc, and BD-3tcC, respectively, through the intraocular and intranasal route at a dose of 104 EID50/bird, while the fourth group served as an uninfected control. The birds were observed daily for the appearance of clinical signs and mortality. At day 0, 3, 7 and 14 post inoculation (p.i.), five birds from each group were selected randomly, killed, and subjected to routine post-mortem examination. The bursa/ body weight ratio was determined on each sampling occasion. A part of the BF of each sampled bird was fixed in 10% neutral buffered formalin for histopathological examination, and the other part was collected aseptically for the detection of viral RNA by RT-PCR. The formalinfixed tissues were processed, sectioned and stained with haematoxylin and eosin following standard histological techniques.

Serial passage of BD-3tcC in chickens and cell culture

The recombinant IBDV strain BD-3tcC was passaged serially five times in chickens in two separate experiments. In the first experiment (Experiment 2), three-week-old commercial layer chicks were used that had been raised in relative isolation since they were one day old. In the first passage, 60 µ1 (6X9 10³ EID₅₀) of the virus suspension (tissue culture supernatant) was administered through the intraocular and intranasal route in each of five chicks. At day 4 p.i., the chickens were killed, and the bursae were harvested aseptically and homogenized to make a 10 % (w/v) suspension in PBS. The methodology was the same as above except that chicks were inoculated at four weeks of age and amplification and sequencing of the VP2 hypervariable region of the virus was done only after the fifth passage, but from individual birds. The reverse genetics IBDV strain BD-3tcC was also passaged serially 12 times in CEF cell culture.

Preparation of a 3D diagram of the VP2 subunit

To prepare a 3D diagram of the VP2 subunit of IBDV, the protein sequence was submitted to the on-line structure homology modelling server SWISS MODEL (http://www.swissmodel.expasy.org). The result was received in pdb (protein data base) format and was viewed using RasWin software (http://www.rasmol.org).

Source of Information: Arch Virol, Springer-Verlag Wien 2013, Vol.: 158, Number 10, October 2013

Article Link (Online Journal): http://link.springer.com/article/10.1007%2Fs00705-013-1885-2#page-1

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

This study demonstrates that although the amino acid residues His and Thr at positions 253 and 284 remain stable during serial passage in tissue culture, they are not favored during in vivo replication of vvIBDV and classical virulent IBDV; hence they revert to Gln and Ala, respectively. The reason for this reversion still remains unknown. As this reversion does not take place during in vitro growth of the virus, it is not likely to be an absolute requirement for morphogenesis of virus particles. Instead, it might provide a special advantage for better growth of the virus in vivo. The amino acid residues at positions 253 and 284 are located in two exposed loops, and it has been shown that they participate directly in particle-to-particle interactions and, hence, paracrystalline array formation. It would be interesting to know if Gln and Ala residues at position 253 and 284 enhance or strengthen particle- to-particle interaction with the formation of a more rigid crystalline array that would be resistant to natural and acquired host defences.

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