Identification and characterization of a novel infectious bursal disease virus from outbreaks in Maharashtra Province of India

Abstract

Aim: The study was undertaken to isolate infectious bursal disease virus (IBDV) from clinical cases in broiler and cockerel flocks of Maharashtra state, India, and its molecular epidemiological investigation.

Materials and methods: The morbid bursal tissues were collected from flocks suspected for IBD. The samples were subjected for virus adaptation in primary chicken embryo fibroblast (CEF) cells followed by confirmation by reverse transcription polymerase chain reaction (RT-PCR) for partial VP₂ sequence and phylogenetic analysis.

Results: The isolation of IBDV from field samples took seven blind passages for adaptation in CEF. The cytopathic effects included rounding, aggregation, vacuolation, and detachment of the cells. The RT-PCR showed amplification of 627 bp amplicon specific to the primers for VP₂ gene fragment which confirmed successful adaptation and isolation of IBDV using CEF. The nucleotide and deduced amino acids based on phylogeny clustered the current isolate in a distinct clade with classical virulent and antigenic variants. It showed divergence from very virulent (vv) and vaccine strains of Indian origin. The isolate showed unique amino acid substitution at A329V as compared to all other IBDVs. The variation in key amino acids was reported at A222, I242, Q249, Q253, A256, T270, N279, T284, I286, L294, N299, and V329. It shared conserved amino acids at position A222, I242, and Q253 as reported in vvIBDV isolates. However, the amino acids reported at position T270, N279, T284, L294, and N299 are conserved in classic, antigenic variant and attenuated strains of IBDV. The amino acids at positions N279 and T284 indicated that the isolate has key amino acids for cell culture replication.

Conclusion: The IBDV field isolate does not reveal the full nucleotide sequence signature of vvIBDV as well as vaccine strains. Hence, we can conclude that it might not belong to vvIBDVs of Indian origin and the vaccine strain used in the region. This may be suggestive of the evolution of the IBDV in the field due to the coexistence of circulating field strains and live attenuated hot strains, resulting into morbidity and mortality, warranting the need for safer protective vaccines, and implementation of stringent biosecurity measures to minimize loss to farmers.

Keywords: VP2; adaptation; chicken embryo fibroblast; epidemiology; isolate infectious bursal disease virus.

Figure-1

Symptoms and lesions of infectious bursal disease-suspected flocks (a and b) ruffled feathers, depression, and droopy appearance, (c-e) hemorrhages on thigh and breast muscles, (f and g) enlargement of bursa and nephritis, (h-k) bursal lesions.

Figure-2

Primary chicken embryo fibroblast cells (a) uninfected, (b) 24 h post infection (PI), (c) 36 h PI, (d) 72 h PI at Passage No. 6, (e) uninfected, (f) infectious bursal disease virus-infected cells showing rounding, aggregation, vacuolation, and detachment 48 h PI at Passage No. 7.

Figure-3

Infectious bursal disease virus (IBDV) VP₂ gene amplicon 627 bp (Lane 2: Marker, Lane 4: VP₂ (IBDV, PS, Nagpur, India), Lane 5: IBDV-Positive control, Lane 6: Negative control).

Figure-4

Phylogenetic tree constructed with MEGA 7.0, illustrating relationship among infectious bursal disease virus (IBDV), PS, Nagpur, India and 33 global IBDV nucleotide sequences from different geological origins. The tree was generated using neighbor-joining algorithm based on pairwise nucleotide differences in VP₂ hypervariable region after constructing distance matrices using Kimura two-parameter model. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown below the branches.

Figure-5

Alignment of deduced amino acid sequences for the hypervariable region of VP2 gene of Indian very velogenic, classical, variant, and attenuated strains of infectious bursal disease virus (IBDVs) with IBDV, PS, Nagpur, India. Identity of the aligned amino acids is shown by dots and differences by single letter.

Figure-6

Phylogenetic tree constructed with MEGA7, illustrating relationship among infectious bursal disease virus (IBDV), PS, Nagpur, India and 30 global IBDVs based on deduced amino acid sequences from different geological origins. The tree was generated using neighbor-joining algorithm and evolutionary distances were computed using the Poisson correction method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown below the branches.