key: cord-0724933-xy3fnygf
authors: Cavanagh, D.; Davis, P. J.
title: Sequence analysis of strains of avian infectious bronchitis coronavirus isolated during the 1960s in the U.K.
date: 1993
journal: Arch Virol
DOI: 10.1007/bf01309675
sha: fb620d7a3b1769ef430cb8e143be05957a871e26
doc_id: 724933
cord_uid: xy3fnygf

Sequencing of parts of the spike, small membrane, and integral membrane protein genes of English isolates of avian infectious bronchitis virus (IBV) isolated in the 1960s revealed that they were not the direct ancestors of those isolated in the 1980s.

Smnmary. Sequencing of parts of the spike, small membrane, and integral membrane protein genes of English isolates of avian infectious bronchitis virus (IBV) isolated in the 1960s revealed that they were not the direct ancestors of those isolated in the 1980s.

In the late 1970s and early 1980s in the Netherlands and the U.K. pathogenic strains of IBV were isolated which were unrelated by virus neutralization (VN) tests to those of the Massachusetts and other serotypes which had been detected previously in the U.S.A. [7, 10, 12] . The question arose as to whether the 1970/ 80s European isolates were closely related to strains present in earlier years or represented a distinct group(s) of strains. Fortunately Dawson and Gough [11] had isolated IBV strains from many regions of England between the years 1965 and 1967. Since some IBV strains appear to be only distantly related on the basis of serum VN tests while actually having very similar (>~ 97% amino acid identity) S 1 proteins [6] it was decided to compare the 1960s isolates by nucleotide sequencing. The VN antibodies that have been the basis for comparison of IBV isolates are induced largely by the N-terminal S 1 subunit of the S protein [3, 14, 16] and S 1 genes of several of the 1970/80s isolates have been sequenced [1, 6, 13, 15, 17, 20] as have sM (small membrane; previously referred to as 3 c) and M genes [-2, 19] .

The following isolates, isolated in England between 1965 and 1967 and propagated in embryonated domestic fowl eggs [11] , were kindly provided by R. The end of the sM gene and the beginning of the M gene overlap and we have previously examined this region for many strains isolated in the 1980s in the U.K. and the Netherlands and for a large number of Massachusetts serotype strains isolated in the U.S.A. and Europe [2] . Therefore we examined this part of the genome in respect of 11 of the pre-1970 U.K. isolates and compared the sequences with the consensus sequence of 12 Massachusetts serotype strains ( Fig. 1) . Two of the 11 isolates (Allen/47 and 265/66) differed by only one nucleotide from the consensus sequence of the Massachusetts serotype, a similarity subsequently confirmed by sequencing of the S 1 gene. The remaining nine isolates differed substantially from Massachusetts serotype strains and eight of them formed a recognisable group on the basis of a six nucleotide deletion, effectively making the M protein two amino acids shorter at the extreme amino-terminus compared with Massachusetts strains. The remaining isolate (604/67) had a nine base deletion but was otherwise similar to the other eight strains. This particular six/nine nucleotide deletion was one that we had not encountered previously except for one 1980s isolate, UK/101/86 (Fig. 1) . The sequence of three other 1980s strains, representative of three different deletion types, is shown for comparison in Fig. 1 .

The sequence of S 1 of strain 918/67 was determined in its entirety and comprised 1577 nucleotides (plus a leader sequence of 54 bases) encoding a polypeptide of 526 amino acids, making this the longest IBV S t sequenced to- (Table 2 ), in keeping with the sM/M sequence determined for Allen/47 and 265/ 66 (Fig. 1) . The four other 1960s strains for which S 1 data was obtained resembled 918/67 more than M 41. However, variation among these 1960s isolates was 3-to 5-fold greater in regions I-IV than in the corresponding regions of a group of 12 Massachusetts serotype strains [4, 5] and 3-to 4-fold greater in regions I-III and 6-to 10-fold more in region IV than in a group of seven 1980s strains [6] . Thus, even when the M 41-like strains were excluded, the remaining 1960s isolates formed a rather heterogeneous group. One of the most variable S I regions (M 41 nucleotide sequence 270 to 394, excluding the signal sequence) contained an additional 15 contiguous bases for some of the non-Massachusetts-like 1960s strains (demonstrated for 225/67, 690/67, and 918/67), which largely accounted for the greater length of S 1 of these strains when compared with published sequences (data not shown). These bases were located between nucleotides 369 and 370 (amino acids 123 and 124) in the M4t sequence. Isolate 48/65 had nine rather than 15 additional bases at this position. These amino acids were within a region (site E) which, for many 1980s strains, induced VN antibodies [14] .

The data indicates that it is unlikely that the IBV strains isolated in the late 1970s and 1980s in the U.K. had evolved directly from those present during

Nucleotide variation in four regions of the S1 gene of UK IBV isolates and M 4I [4, 5, 9] . The more recent isolate 101/86 had a sM/M region which resembled that of most of the 1960s strains (Fig. 1 ) which led us to believe that it was directly related of them. However, S 1 data suggested otherwise (Table 2) . Rather, it would appear that the major IBV strains present in the U.K. during the 1980s either were absent in the 1960s, were present only at a low level or were possibly located in other areas from which virus isolations had not been attempted. It is possible that the later strains had evolved directly from the earlier ones, the process having been disguised by a high mutation rate, of the order of 20% in a decade or so. However, this seems unlikely. Analysis of Massachusetts serotype strains isolated over a half century period has indicated that rapid mutation is not necessarily intrinsic to IBV [-4, 6] . Some strains isolated between 1978 and 1986 in the Netherlands and the U.K. differed by only 2% or so of S 1 nucleotides, again counter-indicative of rapid mutation rates [1, 6, 13, 15, 17] . Rather it would appear that the strains dominant in the U.K. 25 years ago have declined and been replaced by others. Analysis, by VN tests, of 1980s strains indicated that a similar change in the U.K. IBV population was occurring during the 1980s [8] .

As the number of IBV isolates sequenced increases it emerges that there is a wide spectrum of IBV strains. Isolates with S sequence intermediate between one group and another blur the distinction between them, defying attempts to allocate an isolate to one IBV group or another. This is compounded by the fact that isolates which closely resemble each other in one gene may be very different in another, suggestive of recombination [2, 5, 18] . Our results emphasise that, on the one hand, IBV strains with 20% differences in S 1 can coexist over a relatively small area, while on the other that the IBV population in a given area is in a state of flux.

Comparison of the spike sequences of coronavirus IBV strains M 41 and 6/82 with that of IBV Beaudette

Evolution of avian coronavirus IBV: sequence of the matrix glycoprotein gene and intergenic regions of several serotypes

Induction of humoral neutralising and haemagglutination inhibiting antibody by the spike protein of avian infectious bronchitis virus

Amino acids within hypervariable region 1 of avian coronavirus IBV (Massachusetts serotype) spike glycoprotein are associated with neutralization epitopes

Sequence of IBV strains

Infectious bronchitis virus: evidence for recombination within the Massachusetts serotype

Location of the amino acid differences in the S 1 spike glycoprotein subunit of closely related serotypes of infectious bronchitis virus

The classification of new serotypes of infectious bronchitis virus isolated from poultry flocks in Britain between 1981 and 1983

Long term studies on serotyping European IBV strains

Taxonomic studies on strains of avian infectious bronchitis virus using neutralisation tests in tracheal organ cultures

Occurrence and significance of infectious bronchitis virus variant strains in egg and broiler production in the Netherlands

Antigenic variation in strains of avian infectious bronchitis virus

197~) Serological comparisons of strains of infectious bronchitis virus using plaque-purified isolants

Nucleotide sequence of the gene coding for the peplomer protein (= spike protein) of infectious bronchitis virus, strain D 274

Location of antigenic sites defined by neutralizing monoclonal antibodies on the S 1 avian infectious bronchitis virus glycopolypeptides

Nucleotide and amino acid sequence of the S 1 subunit of the spike glycoprotein of avian infectious bronchitis virus, strain D 3896

Antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions

Phylogeny of antigenic variants of avian coronavirus IBV

Sequence evidence for RNA recombination in field isolates of avian coronavirus infectious bronchitis virus

A polycistronic mRNA specified by the coronavirus infectious bronchitis virus

The peplomer protein sequence of the M41 strain of coronavirus IBV and its comparison with Beaudette strains

We gratefully acknowledge the provision of IBV strains by Dick Gough and Jane Cook and thank Rachel Purvis for technical assistance.

Authors' address: D. Cavanagh, Division of Molecular Biology, Institute for Animal Health, AFRC, Compton Laboratory, Newbury, Berkshire RG 16 ONN, U.K.Received July 15, 1992