VP2 gene sequencing based Geno-grouping of infectious bursal disease viruses isolated from Gujarat and Maharashtra state (India)

Abstract

Infectious bursal disease (IBD), caused by infectious bursal disease virus (IBDV), has recently been reported in chickens vaccinated with classical or intermediate types of vaccines from various regions of India due to the emergence of novel very virulent strains of infectious bursal disease virus (vvIBDV). In the present study, suspected samples of IBD were collected from poultry flocks of districts of Gujarat and Nagpur (Maharashtra), identified using PCR and grouped as per traditional and new genogrouping pattern. Out of 54 bursa samples, 21 (38.89%) yielded the expected amplicon of 743 bp (701–1444 bp), and were found positive for IBDV. Among these 21 positive flocks, 11 (52.38%) were already vaccinated. Upon nucleotide sequencing of amplicon and its deduction into amino acids, it was found that all the sequences of present study were related to vvIBDV according to old classification pattern. Considering the new genogrouping pattern, nine and four sequences of this study fell within G3a and G3b lineage, respectively. These sequences revealed important differences at key amino acid positions with respect to classical (G1 genogroup), variant (G2 genogroup) type of IBDV and classical vaccines. Further divergence from prototypic vvIBDV strains was revealed as, D-N at 212 position (N = 9) and 279 position (N = 1). In sequences from Maharashtra (group 2 of G3a lineage), occurrence of V instead of P/T/A at 222 position was recorded as a novel and conspicuous substitution in the immunodominant peak A of VP2 hypervariable region. Additional changes at 270 (3 sequences) and 272 positions (4 sequences) could be attributed to reverse mutation or recombination with vaccine strains. In conclusion, both point mutation and genetic reassortment with intermediate type of vaccines were found to be responsible for generation of novel vvIBDV strains in this area which belonged to G3a and G3b genogroups.

Infectious bursal disease (IBD), also known as Gumboro disease, is caused by infectious bursal disease virus (IBDV) in young chickens. The disease is characterized by functional loss of the Bursa of Fabricius accompanied by severe immunosuppression, resulting in substantial mortality due to secondary infections [18]. Out of the two serotypes of IBDV (1 and 2), only serotype 1 is pathogenic and further classified as classical virulent IBDV, antigenic variant IBDV and very virulent IBDV (vvIBDV) [2]. Recently, these strains of IBDV have been classified into seven genogroups based on the marked changes in the amino acids in the hypervariable region (206–350 amino acid) of the VP2 protein (hVP2) [11]. These novel strains with additional virulent properties have evolved due to high mutation rate of RNA viruses and selection pressure generated by intensive vaccination, thereby inflicting higher morbidity and mortality in the susceptible flocks [9].

Consequently, despite adhering to vaccination regimen, several outbreaks of IBD have been recorded in the recent past in India [13, 14]. These outbreaks largely occurred due to emergence of vvIBDV [16] and hence necessitates for better characterization of IBDV strains by supplementation of Reverse transcriptase-Polymerase chain reaction (RT-PCR) based diagnosis, either with restriction fragment length polymorphism (RFLP)/ restriction enzyme digestion (RE digestion) or nucleic acid sequencing methods [5]. In India, sequencing data generated from different regions indicated two possible mechanisms for evolution of novel and more pathogenic strains. One is point mutations in classical IBDVs [15, 19] which could be traced back to emergence of vvIBDV strains, since 2001 [7, 17], and secondly a novel mechanism of recombination of field strains with attenuated vaccine strains [1, 12, 16]. Looking towards the importance of determining the prevalent strains of the area and possible mechanism of strains evolution, the present study has been focused on RT-PCR based diagnosis, sequencing and genotyping of IBDV isolates from IBD affected flocks of Gujarat and Maharashtra state.

During the study, a total of 54 flocks with the history of IBD were visited. From these flocks, 54 pooled samples of bursa of Fabricius (in each affected flock, bursae were pooled from two–three randomly selected recently dead birds) were aseptically collected and diagnosed through RT-PCR. The PCR reaction mixture was prepared as per manufacturer’s instructions of RT-PCR kit (QIAGEN One-step RT-PCR kit) and template RNA was added to make final volume of 25 µl. RNA isolated from vaccine virus was used as positive control. PCR amplification was carried out using gene specific primers described by Jackwood and Sommer [6] which encompasses nucleotide position from 701 to 1444 bp to yield amplicon of 743 bp. Results were visualized with appropriate ladder on 2% agarose gel and gel-documented. After purification and quantification of positive amplicons, representative samples based on location and mortality rates (N = 13) were sent for sequencing to Eurofins, Bangalore. The sequencing results were edited and analyzed using Editseq of the Lasergene 6.0 (DNAstar, USA), BioEdit version 7.05.3 and Sequencher™ 5.4.6 softwares. Finally, a fragment of 421 bp was used for analysis for the presence of specific mutations and subsequently aligned with available sequences in gene bank including various reference strains of geno-groups and lineages (Table 1), popular vaccine strains and representative strains of India and neighboring countries (Table 2). Phylogenetic tree was also constructed using neighbor-joining method with bootstrapped value 1000 of MEGA.

Table 1.

Comparison of amino acid sequences of present study with reference strains of IBDV at key positions

S. No	Sample ID	Sample details	Important mutations in VP2 gene of IBDV—Amino acid position
		Amino acid position	212	222	242	249	253	254	256	270	272	279	284	294	299	300	330
1.	G1-G1a-D00499.1	Classical, G1 ref	D	P	V	Q	Q	G	V	T	I	D	A	L	N	E	S
2.	G2-G2a-AF133904.1	Variant, G2 ref	∙	T	∙	K	∙	S	∙	A	∙	N	∙	∙	∙	∙	∙
3.	G3-G3a-NC004178.1	Very virulent (G3 ref)	∙	A	I	∙	∙	∙	I	A	∙	∙	∙	I	S	∙	∙
4.	G3-G3b/G2c- MF142539.1		N	A	I	∙	∙	∙	I	A	∙	∙	∙	I	S	∙	∙
5.	G3-G3c-MF142563.1		N	T	I	∙	∙	D	I	A	∙	N	∙	I	S	∙	∙
6.	G5 (DQ916210)	G5 ref		P	I	∙	∙	N	V	K	∙	N	∙				-
7.	G7 (AJ878908)	G7 ref		T		∙	∙		L	T	T	G	∙	I	S	∙	-
8.	Group-1 (G3a)	Field Isolates (This study)	N	A	I	∙	∙	∙	I	A	∙	∙	∙	I	S	∙	∙
9.	Group-2A (G3b)		∙	A	I	∙	∙	∙	I	A	T	∙	∙	I	S	∙	∙
10.	Group-2B (G3b)			A	I	∙	∙	∙	I	A	T	N	∙	I	S	∙	∙
11.	Group-3 (G3a)		N	V	I	∙	∙	∙	I	∙	∙	∙	∙	I	S	∙	∙

Table 2.

Comparison of amino acid sequences of present study with sequences of IBDV from India and neighboring countries

Sample ID	Sample details	212	216	222	240	242	249	253	254	256	270	272	279	284	294	299	300	Reference
Amino-acid Position
UK661	vvIBDV Reference	D	F	A	L	I	Q	Q	G	I	A	I	D	A	I	S	E	Khan et al. 2019
AF362776.1 old strain BD 3/99	vvIBDV Neighboring countries	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	A
KT884486.1 Henan		∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙
KY484079, vvIBD Pakistan		N	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙
IBDMZIND 1–7	Field isolates (India)	N	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	Rajkhowa et al. 2018
IBDMZIND 8		∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	T	N	∙	∙	∙	∙
IBDMZIND 9–10		∙	C	∙	F	∙	∙	∙	∙	∙	∙	∙	∙	∙	V	∙	A
IBDMZIND 10		∙	∙	∙	F	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙
Johrat PD5		∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	P
Chikodara, Napad, West Bengal		–	–	∙	∙	∙	∙	∙	∙	∙	∙	T	N	∙	∙	∙	∙	Patel et al. 2016
Guwahati 06–07, Hyderabad		–	–	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	V	∙	∙
CVASP1-5,7,14, 16,17,20–22		–	–	∙	F	∙	∙	∙	∙	∙	∙	∙	∙	∙	V	∙	A	Nandhakumar et al. 2020
CVASP6		–	–	∙	F	∙	∙	∙	∙	∙	∙	∙	∙	∙	V	N	A
CVASP8		–	–	P	∙	V	∙	H	∙	V	∙	∙	Y	T	L	N	∙
CVASP15,18		–	–	∙	∙	∙	∙	∙	∙	∙	∙	T	N	∙	∙	∙	∙
CVASP19		–	–	P	∙	V	∙	∙	∙	∙	T	∙	N	T	L	N	∙
IBDVNE1-4		–	–	∙	–	∙	–	∙	–	∙	–	–	∙	∙	V	∙	–	Morla et al. 2016
HY12		–	–	–	–	–	–	∙	–	∙	∙	T	N	∙	∙	∙	∙	Raja et al. 2018
MB11		–	–	–	–	–	–	H	–	V	T		N	T	L	N	∙
EDE14		–	–	–	–	–	–	∙	–	∙	∙	∙	∙	∙	V	N	∙
NKL14, PY12,RPM14, VCN14, BGE14		–	–	–	–	–	–	∙	–	∙	∙	∙	∙	∙	V	∙	A
PSNGP-MS-IND		∙	∙	∙	∙	∙	∙	∙	∙	A	T	∙	N	T	L	N	∙	Awandhker et al. 2018
Y18682 (RJ 1/94)		–	–	–	–	–	S	–	–	–	–	–	–	–	–	–	–	Kataria et al. 2001
Ventri-IBDV-Plus	Vaccine strains	–	–	∙	∙	∙	∙	∙	∙	∙	∙	T	N	∙	∙	∙	∙	Rajkhowa et al. 2018
Georgia Vaccine		–	–	P	∙	V	∙	H	∙	V	∙	∙	Y	T	L	N	∙	Nandhakumar et al. 2020
SKN-IBD Group-2A	Field isolates Present study	∙	–	∙	–	∙	∙	∙	∙	∙	∙	T	∙	∙	∙	∙	∙	Present Study
SKN-IBD Group-2B		∙	–	∙	–	∙	∙	∙	∙	∙	∙	∙	N	∙	∙	∙	∙
SKN-IBD Group-1		N	–	∙	–	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙
SKN-IBD Group-3		N	–	V	–	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙	∙

Out of total 54 tissue samples, 21 (38.89%) and 33 samples (61.11%) were found positive and negative, respectively through RT-PCR (Fig. 1). Among these 21 positive flocks, 11 (52.38%) had received vaccination (two flocks with classical Georgia strain and others with intermediate and/or intermediate plus vaccine strain), whereas 10 (47.62%) flocks were unvaccinated. It was found that all the sequences of present study were related to vvIBDV according to old classification pattern.

Fig. 1.

PCR amplification of VP2 gene of IBDV isolates. Lane 1:GelPilotMid range ladder (Cat. No.239135), Lane 2–10: Positive samples (743 bp amplicon), Lane 11: Negative control

Considering the new classification suggested by Michel and Jackwood [11], nine sequences of this study fell within G3a lineage and four sequences within G3b lineage (Table 1; Fig. 2). The nine isolates, falling in G3a lineage were having corresponding mortality less than 15% and could be further divisible into two groups, based on differences in amino acid positions. The groups were designated as group 1(N = 6) which consisted of isolates from Gujarat state and the group with isolates from Maharashtra state was designated as group 3 (N = 3). While corresponding isolates of G3b were designated as group 2 (N = 4), this group consisted of isolates from Gujarat and were showing mortality more than 15%. Though one isolates in group 2 (shown as 2B in tables) had an additional change at amino acid position 279.The further divergence of sequences was also found from prototypic vvIBDV(UK661), sequences retrieved from recently reported outbreaks from India, and intermediate and intermediate plus vaccines (Table 2).

Fig. 2.

Phylogenetic tree was constructed using various reference strains of geno-groups and lineage, popular vaccine strains and representative strains of neighboring countries using neighbor-joining method with bootstrapped value 1000 of MEGA

On comparison of amino acid positions with the classical virulent strain (or G1 of new classification) and variant IBDV (G2 of new classification) (Table1), at aa 222, A/V instead of P (classical) and T (variant) was found. At 242 and 265 position isoleucine (I) at the place of valine of classical /variant type was present. Whereas at 270 position, A to T mutation was noted in all the isolates, except in that of group 3. Comparison with classical IBDV vaccine (Georgia strain) revealed substitution as H253Q, A284T, L294 I, N299S and R330S. On comparison with newer type of attenuated vaccines (Intermediate or intermediate plus), it was observed that our sequences have similarities to them as A222, I242, Q253, I256, D279, A284, I294, S299 and S330 with some notable findings. In the isolates of group 2 (N = 4), D and T were located at 212 and 272, respectively and one isolate has N at 279 position which was similar to these vaccine strains but different from other isolates present study. The major exceptions were noted in group 3 (isolated from Maharashtra state), with presence of V and T at 222 and 272, respectively. Variation at the position 222 V was novel and yet unrecorded whereas T at 270 indicated reversal towards classical strain (Table 2).

Phylogenetic tree clearly indicated that the sequences of this study have branched away from classical virulent strains (D00499), variant virulent strain (AF133904.1), vaccines prepared from these strains (KF573194, Georgia vaccine) and classical field isolates still prevalent in south India (MK861038, MK861027 and KM064548). Four strains from the study (isolates number IBD-1,8,10,19 of group 2) showed close resemblance with reference strain of G3b (MF142539), intermediate plus (EF066500) and Ventri plus vaccine strain sequences (KJ547670). These sequences also have resemblance with recently reported sequences from southern (MK861037) and north east (KX342919) regions of India. The other nine sequences (Group 1 and 3) resembled to reference very virulent strains like UK 661 (NC004178.1) and possibly originated from progenitor IBD sequence of China [16] through initially reported vvIBD strains of India (AJ249520). These nine sequences have ancestral relationship with prototypic vvIBD strains from Tamil Nadu (EF066491). These sequences are still prevalent in neighboring nation Pakistan (KY484079) and have recently been reported from different parts of India. These nine sequences were further divisible into two clusters, one having three (isolate no- IBD39, 43, 49) and the second consisting of six sequences (isolate no -IBD 18, 20, 23, 28, 31, 35) (Fig. 2).

In the present work, 21/54 (38.9%) flocks were found infected with infectious bursal disease virus (IBDV) with RT-PCR. As out of 21 positive flocks, 11 were already vaccinated, the present work reaffirmed that field strains have evaded immunity generated by classical IBD vaccine strains or newer intermediate type of vaccines [12], which is similar to reports from other parts of India [12–14, 16] as well as its neighboring countries viz. Pakistan [3, 8], Bangladesh [4] and China [2]. Flock wise prevalence was well corroborated with the results of Nandhakumar et al. [13] from India, Magwood et al. [10] from Egypt and Zahoor et al. [20] from Pakistan, respectively. But, it was much lower than other reports from India [16] and Pakistan [3].

The nucleic acid sequencing and its translation to amino acid clearly indicated that this vaccine failure is attributed to very virulent IBDV (vvIBDV) and sequences differed considerably from classical IBDV, variant IBDV and classical vaccine strain e.g. Georgia strain. Further divergence was noted from prototypic vvIBDV strain UK661, which has previously been mentioned by workers from India [1, 7, 12–14, 16], Pakistan [3] and Bangladesh [4]. Changes obtained in isolates at 222 ( V instead of P/T/A) from Nagpur of Maharashtra province and 212 position ( N substituted D) from Gujarat (Group 1) were the highly conspicuous mutations because of their location in peak A of VP2 protein which may be the cause of failure of binding of antibodies at this site [14]. The mutation at 212 position is said to be originated from North China but less documented from India [7, 12]. The second group (Group 2) was found more divergent from prototypic vvIBD strain and sequences of this study as well. The changes noted here, N212D, T272I and N279D (in one isolate only) were similar with intermediate type vaccine strains (manufactured by several manufactures in Gujarat) (Table 2) and indicative of genetic reassortment with these vaccine strains as previously reported [14]. Additionally, Morla et al. [12] substantiated this notion with the fact that the low virulent intermediate vaccine strains may sometime show reversion to the virulent pathotype, which may be the cause of higher mortality observed in outbreaks because of these strains.

The earlier phenotypic classification of IBDV has now been modified and a new genogrouping has been proposed [11]. Out of seven groups proposed only genogroups 1 and 3 have been reported from South Asian countries in recent years [8]. All the isolates belonged to vvIBD strains of present work have therefore been placed in to genogroup 3 of this classification which indicates replacing of classical strains by more devastating very virulent strains. Absence of sequences corresponding to classical virulent (Genogroup 1) or classical vaccine strain, were in contrast to the findings from south India [13, 15] and Pakistan [8] where these strains are still prevalent. It was further evident that our isolates could be further placed either in G3a and G3b sub groups, of which G3b sub group showed identity with intermediate vaccine strains and sequences reported from India [12, 14, 16] with the major exception of presence of I294V mutation in one or more isolates.

Declarations

Conflicts of interest

All authors declare no conflict of interest.