Rapid selection in chickens of subpopulations within ArkDPI-derived infectious bronchitis virus vaccines

Figures & data

Table 1.  Primers for RT-PCR and sequencing

Name	Sequence	Orientation	Position in Ark vaccine S-coding sequence	Reference
NEWS1OLIGO5′	5′-TGA AAC TGA ACA AAA GAC-3′	Forward	–43 to –65	Jackwood et al. (1997)
S1OLIGO3′	5′-CAT AAC TAA CAT AAG GGC AA-3′	Reverse	1675 to 1656	Kwon et al. (1993)
S1R	5′-CAT CTG AAA AAT TGC CAG-3′	Reverse	742 to 725	Lanqing Li, personal communication
S2F	5′-TAG AGG TTT GTT AGC ATG-3′	Forward	696 to 713	Lanqing Li, personal communication
S2R	5′-CCT TGT ACA ACG AAG ATG TC-3′	Reverse		Lanqing Li
S3F	5′GTT TAT GTT ACT AAG AGC G-3′	Forward		Lanqing Li
S5F	5′-GGT ATT CTC ACT TCA CGT AAT G-3′	Forward	1540 to 1561
S7F	5′-TAC ATC ACT GCA CGA GAT ATG-3′	Forward	1968 to 2988
S3′	5′-GAS^a TTT GAA TCA TTA AAC AGA C-3′	Reverse	3522 to 3501	Modified from Callison et al. (1999)
S9R	5′-ATG TAC TCC GAC TGC TTA GC-3′	Reverse	2687 to 2668
S11F	5′-TTC AAG ACA TTT ACC AGC AAC-3′	Forward	2570 to 2590
S12R	5′-ATT TAC ACT TAC ATA ATT TGC TTG AC-3′	Reverse	3063 to 3038
H43	5′-GGA CAT GGT TGG CAT TTA C-3′	Forward	109 to 127
N326	5′-TGG ATG GTA AGA TCC ATA CAT ATA ATT-3′	Reverse	1002 to 976
Additional primers for DE072			Position in DE072 vaccine S-coding sequence
S4F	5′-TGG CAT ATT TTG TGA ATG G-3′	Forward	611 to 629
DE072S2	5′-CCA TAA GTT ACA TAT GGA CGA-3′	Reverse	1649 to 1629
^aS = G or C.

Table 2.  Positions in S genes of ArkDPI-derived IBV vaccines exhibiting heterogeneity in vaccine vial and/or differences between vaccine vial and vaccinated chicken

	S1																S2							Amino acid differences between vaccine in chickens and predominant vaccine sequence in vial
Nucleotide position	127	162	226	355	388	511	593	637	968	976	1009	1030 to 1032	1157	1192	1195	1228	2034	2068	2162	2188	3108	3110	3200
Codon number/amino acid position	43	54	76	119	130	171	198	213	323	326	337	344	386	398	399	410	678	690	721	730	1036	1037	1067
Vaccine A: vial
Predominant nucleotide	T	T	C	T	A	T	A	T	C	T	S	AAT	G	C	C	A	T	C	T	C	T	C	A
Minor nucleotide (% of total^a)	C (36%)	G (16%)	T (12%)	C (12%)	G (20%)	C (12%)	C (12%)	G (28%)	G (26%), T (17%)	A (26%)	–^b	ΔΔΔ^c (26%)	A (17%)	G (22%)	T (9%)	–	C (37%)	–	A (45%)	T (36%)	A (23%)	A (39%)	G (9%)
Predominant amino acid encoded	Y ^d	S	L	S	S	Y	K	S	T	Y	S	N	R	Q	H	K	S	P	M	L	F	A	D
Minor amino acid encoded	H	R	F	P	G	H	T	A	R, M	N	S	▵	H	E	Y	–	S	–	K	F	L	D	G
Vaccine A: Chickens (Harderian glands, trachea, or tears)
17 of 17 samples^e	H	S	L	S	G	Y	K	A	R	N	S	▵	R	Q	H	K	S (c ) ^f	P	K	F	L	D	G	11
Two of 5 Harderian glands three d.p.i.^g	H	S	F	P	S	H	T	A	R	N	S	Δ	R	Q	H	K	S (c)	P	K	F	ND^h	ND	ND	≥11
Vaccine B: vial
Predominant amino acid encoded	Y	S	L	S	S	Y	K	S	T	Y	S	N	R	Q	H	K	S (t)	P	M	L	F	A	D
Minor amino acid encoded (% of total^i)	–	–	–	–	–	–	–	–	R (43%)	–	–	–	–	E (44%)	–	–	–	–	–	–	–	–	–
Vaccine B: chickens (tears)
One of five chickens	Y	S	L	S	S	Y	K	S	R	Y	G , S	N	R	Q	H	K	S (t)	P	M	L	F	A	D	1
One of five chickens	Y	S	L	S	S	Y	K	S	R	Y	S	N	R	Q	H	K	S (t)	P	M	L	F	A	D	1
Two of five chickens	Y	S	L	S	S	Y	K	S	T	Y	S	N	R	E	H	K	S (t)	P	M	L	ND	ND	ND	1
One of five chickens	H	S	F	P	S	H	T	A	R	N	S	D	R	Q	H	K	S (c)	P	K	F	ND	ND	ND	≥11
Vaccine C: vial
Predominant amino acid encoded	H	S	L	S	S	Y	K	A	R	Y	S	N	R	Q	H	K	S (c)	P	M	L	F	D	D
Minor amino acid encoded (% of total)	Y (32%)	–	–	–	–	H (39%)	–	–	M (26%)	N (31%)	–	Δ (38%)	–	–	–	E (38%)	S (t) (25%)	S (30%)	K (47%)	–	–	–	–
Vaccine C: chickens (tears)
Four of four chickens	H	S	L	S	S	Y	K	A	R	N	S	Δ	R	Q	H	K	S (c)	S	K	F	F	D	D	5
Vaccine D: vial
Predominant amino acid encoded	Y	S	L	S	S	Y	K	S	R	Y	S	N	R	Q	H	K	S (t)	P	M	L	F	A	D
Vaccine D: chickens (tears)
Five of 5 chickens	H	S	L	S	S	Y	K	A	R	N	S	Δ	R	Q	H	K	S (c)	S	K	L/ F	L	D	D	9
One of 5 chickens 6 and 9 d.p.i.^j	Y	S	L	S	S	Y	K	A	M	Y	S	N	R	Q	H	K	S (t)	P	M	L	F	A	D	1
Vaccine consensus
Vaccine vial	Y	S	L	S	S	Y	K	S	R(T)	Y	S	N	R	Q	H	K	S (t)	P	M	L	F	A	D
Chickens (vaccines A, C, D)	H	S	L	S	S( G )	Y	K	A	R	N	S	Δ	R	Q	H	K	S (c)	S (P)	K	F ( F /L)	L (F)	D	D ( G )
Field isolates
ARkDPI p11^k	H	S	L	S	S	Y	K	A	R	N	S	Δ	R	Q	H	K	S (c)	P	K	L	F	D	D
Field consensus^l	H	S	F/L	S/P	S	H/Y	K/T	A	R	D/ N	S	N	R/H	Q	Y/H	K	S	P/T	M/N/L	L	L	D /N	D
d.p.i., days post inoculation. ^aIn the S1 subunit, based on incidence in 25 cDNA clones; in theS2 subunit, estimated based on chromatogram peak height. ^bNo minor codon readily visible. ^cDeletion. ^dRegular font = predominant vaccine amino acid, vaccine amino acid in chickens, ArkDPI p11 are same; italic = predominant vaccine amino acid differs from ArkDPI p11; bold italic = vaccine in chickens same as ArkDPI p11 and different from vaccine predominant amino acid; underlined = a minor amino acid (or predominant in some, but not all vaccines) not present in vaccine in most chickens; underlined bold = a minor amino acid in vaccine present in some chickens that does not match ArkDPI passage 11 or vaccine consensus. ^eS1 sequence determined for 17 samples; S2 sequence determined for seven samples. ^fAll codons at position 678 encode serine. The lowercase letter in parentheses indicates the nucleotide in the third position of the codon (c or t). ^gThese two samples contained mixed sequences, including the sequences shown in line above (found in all 17 samples). ^hND, not determined. ^iFor vaccines B and C, estimated based on chromatogram peak height. ^jThese samples contained mixed sequences, including the sequences shown in line above (found in all five chickens). ^kPasssage 11 in embryonated eggs. From GenBank accession numbers AF006244 and AF239272. In addition to the positions shown, DE072 passage 11 differs from all vaccine sequences at S1 codon 498 and several codons of S2 (see Table 5). ^lBased on IBV field isolate sequences in GenBank with >90% nucleotide sequence homology (S1 and S2 portions evaluated separately) with Ark vaccines in chickens; consensus indicates amino acid encoded by at least 90% of isolates.

Figure 1.  Sequence chromatogram sections showing heterogeneity of codon 43 in Ark vaccines A and C, lack of heterogeneity in codon 43 in Ark vaccines B and D, and codon 43 different from vaccine vial in chickens vaccinated with Ark vaccines A and D. 1a: vaccine A in vaccine vial. 1b: vaccine B in vaccine vial. 1c: vaccine C in vaccine vial. 1d: vaccine D in vaccine vial. 1e: vaccine A in trachea of individual chicken 3 days post-vaccination. 1f: vaccine D in tears of individual chicken 6 days post-vaccination. Arrowheads, heterogeneous nucleotide positions. Amino acids encoded are indicated in single-letter amino acid code. Amino acids in parentheses are encoded by the minor codon. Bold, amino acid encoded by vaccine subpopulation selected in chickens and by low passage parental ArkDPI isolate. The codon present in vaccine subpopulations selected in chickens is minor in vaccine A, major in vaccine C, and unapparent in vaccines B and D prior to passage in chickens.

Figure 2.  Sequence chromatogram sections showing heterogeneity of codon 213 in Ark vaccine A, lack of heterogeneity in codon 213 in Ark vaccines B, C, and D, and codon 213 different from vaccine vial in chickens vaccinated with Ark vaccines A and D. 2a: vaccine A in vaccine vial. 2b: vaccine B in vaccine vial. 2c: vaccine C in vaccine vial. 2d: vaccine D in vaccine vial. 2e: vaccine A in Harderian gland of individual chicken three days post-vaccination. 2f: vaccine D in tears of individual chicken 4 days post-vaccination. Arrowhead, heterogeneous nucleotide position. Amino acids encoded are indicated in single-letter amino acid code. Amino acids in parentheses are encoded by the minor codon. Bold, amino acid encoded by vaccine subpopulation selected in chickens and by low passage parental ArkDPI isolate. The codon present in vaccine subpopulations selected in chickens is minor in vaccine A, the only codon apparent in vaccine C, and unapparent in vaccines B and D prior to passage in chickens.

Table 3.  Heterogeneous positions of DE072 vaccine S1 sequences in vaccine vial and vaccinated chickens

Nucleotide position	95	134	258	876
Codon number/amino acid position	32	45	86	292
Vaccine vial
Predominant nucleotide	A	C	G	T
Minor nucleotide (% of total^a)	G (12)	T (19)	T (7)	G (40)
Predominant amino acid encoded	E^b	A	R	I
Minor amino acid encoded	G	V	S	M
Vaccine in chickens (tears)
Amino acid encoded in three of six chickens	E	A	R	I/M
Amino acid encoded in three of six chickens	E	V	S	M
DE/072/92 isolate (unattenuated)^c
Amino acid encoded	E	A	S	I
^aEstimated based on chromatogram peak heights. ^bBold font = same as unattenuated DE/072/92; italic font = different from unattenuated DE/072/92 and same as predominant vaccine amino acid; underlined = different from both unattenuated DE/072/92 and predominant vaccine amino acid. ^cGenBank accession number U77298. In addition to the positions shown, unattenuated DE/072/92 differs from vaccine codons at codons 120, 181, 466, and 519.

Table 4.  Vaccine S1 nucleotide sequences identified by amplification with primers specific for Ark vaccine selected by chickens

	Primers	S1
Nucleotide position		127	162	226	355	388	511	593	637	968	976
Codon number		43	54	76	119	130	171	198	213	323	326
Vaccine A
Vial	NEWS1OLIGO5′/S1OLIGO-3′	T(C)^a	T(G)	C(T)	T(C)	A(G)	T(C)	A(C)	T(G)	C(G,T)	T(A)
Vial	H43/N326	C	T	Y^b	Y	R^c	Y	M^d	G	G	A
Chickens (17 of 17 samples)	NEWS1OLIGO5′/S1OLIGO-3′	C	T	C	T	G	T	A	G	G	A
Chickens (two of five Harderian glands 3 d.p.i.)	NEWS1OLIGO5′/S1OLIGO-3′	C	T	T	C	A	C	C	G	G	A
Vaccine B
Vial	NEWS1OLIGO5′/S1OLIGO-3′	T	T	C	T	A	T	A	T	S	T
Vial	H43/N326	C	T	Y	Y	A(G)	Y	M	G	G	A
Chickens (one of five chickens)	NEWS1OLIGO5′/S1OLIGO-3′	C	T	T	C	A	C	C	G	G	A
Vaccine C
Vial	NEWS1OLIGO5′/S1OLIGO-3′	C(T)	T	C	T	A	Y	A	G	G(T)	T(A)
Vial	H43/N326	C	T	C	T	A	T((C))	A	G	G	A
Chickens (four of four chickens)	NEWS1OLIGO5′/S1OLIGO-3′	C	T	C	T	A	T	A	G	G	A
Vaccine D
Vial	NEWS1OLIGO5′/S1OLIGO-3′	T	T	C	T	A	T	A	T	G	T
Vial	H43/N326	C	T	C	T	A	T	A	G	G	A
Chickens (five of five chickens)	NEWS1OLIGO5′/S1OLIGO-3′	C	T	C	T	A	T	A	G	G	A
d.p.i., days post inoculation. ^aNucleotides in parentheses are minor nucleotides. ^bY = C and T (in approximately equal amounts). ^cR = G and A (in approximately equal amounts). ^dM = C and A (in approximately equal amounts).

Table 5.  Comparison of S2 amino acid sequences of Ark vaccines and Ark vaccine supopulations selected in chickens with those of various attenuation passage levels

	S2
Amino acid position in entire S	678	690	721	730	773	797	1036	1037	1067	1105	1111	1121
Attenuation passage
p11^a	S(c)^b,c	P	K	L	T	V	F	D	D	V	P	V
p25^d	S(c)	P	K	L	A	L	F	D	D	L	T	L
p30^e	S(c)	P	K	L	T	L	F	D	D	L	T	L
p40^f	S(c)	P	K	L	A	L	F	D	D	L	T	L
p55^g	S(c)	P	M	L	A	L	F	D	D	L	T	L
p75^h	S(t)	P	M	L	A	L	F	A	D	L	T	L
Vaccine in vial
Consensus	S(t)	P	M	L	A	L	F	A	D	L	T	L
Minor	S(c)	S	K	F	-	-	L	D	G	–	–	–
Vaccine selected in chickens
Vaccine A	S(c)	P	K	F	A	L	L	D	G	L	T	L
Vaccine C	S(c)	S	K	F	A	L	F	D	D	L	T	L
Vaccine D	S(c)	S	K	(F/L)	A	L	L	D	D	L	T	L
^aGenBank accession number AF239272. ^bBold font = same as ArkDPI passage 11; italic font = different from ArkDPI passage 11 and same as predominant vaccine amino acid; underlined = different from both ArkDPI passage 11 and predominant vaccine amino acid. ^cAll codons at position 678 encode serine. The lowercase letter in parentheses indicates the nucleotide in the third position of the codon (c or t). ^dGenBank accession number AF239269. ^eGenBank accession number AF239271. ^fGenBank accession number AF239270. ^gGenBank accession number AF239273. ^hGenBank accession number AF335555.

Callison , S.A. , Jackwood , M.W. and Hilt , D.A. 1999 . Infectious bronchitis virus S2 gene sequence variability may affect S1 subunit specific antibody binding . Virus Genes , 19 : 143 – 151 .

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