key: cord-288309-6pw7t512
authors: Kusters, J. G.; Jager, E. J.; Niesters, H.G.M.; van der Zeijst, B.A.M.
title: Sequence evidence for RNA recombination in field isolates of avian coronavirus infectious bronchitis virus
date: 1990-12-31
journal: Vaccine
DOI: 10.1016/0264-410x(90)90018-h
sha: 
doc_id: 288309
cord_uid: 6pw7t512

Abstract Under laboratory conditions coronaviruses were shown to have a high frequency of recombination. In The Netherlands, vaccination against infectious bronchitis virus (IBV) is performed with vaccines that contain several life-attenuated virus strains. These highly effective vaccines may create ideal conditions for recombination, and could therefore be dangerous in the long term. This paper addresses the question of the frequency of recombination of avian coronavirus IBV in the field. A method was sought to detect and quantify recombination from sequence data. Nucleotide sequences of eight IBV isolates in a region of the genome suspected to contain recombination, were aligned and compared. Phylogenetic trees were constructed for different sections of this region. Differences in topology between these trees were observed, suggesting that in three out of eight strains in vivo RNA recombinant had occurred.

Infectious bronchitis virus (IBV) causes considerable damage in the poultry industry by infections of the respiratory tract and the reproductive organs 1. IBV is the prototype strain of the Coronaviridae, a family of positive stranded RNA viruses with a genome of about 28kb. This genome contains the information for three structural proteins and a number of enzymes involved in virus replication (Figure 2 ). Although vaccines offer protection, they are often made ineffective by the continuous emergence of new serotypes 2"3. Serotypespecific protective immunity is thought to be mediated by antibodies to a protein on the surface of the virion, the so-called peplomer protein *--7. The peplomer protein is synthesized as a precursor glycoprotein, which is cleaved into the subunits S1 and $2, derived from its N-and C-terminal half, respectively a-~°. Point mutations are obviously involved in the generation of new serotypes. However, previous studies 1~ suggested that RNA recombination is also involved in the generation of new antigenic variants. To address the question of the frequency of recombination in the generation of new field isolates the nucleotide sequences in five windows of homologous sequences of the genomes of eight IBV strains have been compared. To make this comparison the sequences of the $2 genes of D207 and D1466 were newly determined. Both strains are Dutch field isolates and are included in vaccines currently used in The Netherlands. The alignment of the sequences suggests that the Dutch field strain D20712, the British field strain 6/8213 and the Japanese field strain KB8523 t4 result from RNA recombination events.

The origin of the IBV strains as well as the procedures used for the cloning and sequencing of the peplomer genes have been published previously 3'11'15. Sequence data were processed using the programs of Microgenic (Beckman Instruments, Ref. 16) . Phylogenetic trees were derived from nucleotide difference-matrices using the program of Dr J. Felsenstein (University of Seattle) distributed as part of the PHYLIP package 2.617.

The $2 sequences

The newly determined D207 and D 1466 sequences are based on data from two or more independent cDNA clones. With strain D1466, two nucleotide differences between cDNA clones were observed. Neither of these differences resulted in an amino acid replacement. From the nucleotide sequences (submitted to the EMBL Genbank and DDBJ Nucleotide Sequence Databases), amino acid sequences have been deduced. In Figure 1 the amino acid sequences are listed together with those of all other known $2 sequences.

Assuming the conserved Arg-Arg-X-Arg-Arg-Ser sequence to be the cleavage site between S1 and $28, the (Table I) . However, 19 out of 20 cysteine residues and most glycosylation sites are completely conserved.

S K V V N RK L S K V T V -- -- R V z E N M V V -- ----F E K V Y S V -- P ASG E K V H S V -- -- P ASG FQ KQ S S H I GLA QDNA MIQ R D NSF -'F--A D ~ I••DLLFT••E••GLPTDDAYKN•TA•PLGFLKDLA•AREYNGLL•L•PI•TAEM•TLYT••L•A•MAFGGITAAGAIPFA N F A -- A -- V IC- s K F I S DK L G S QK S V V QK AM I S M41U,~Y~DA~ANA~DRL~TGRLs~Ls~LA~AK~AEH~R~s~RELAT~K~NE~vK~RY~F~GNGR/~LTIP~NAPNG F Y L Y T S D I YY S D PI YY K LE L YAK T KE T G G M Ibis M41U,H M42H '\ M428 KB8523 6/82 D207 D1466 96Q 97Q 980 99Q 1000 1010 1020 1030 IvFIHFsYTPD~FvNvT---A~vGF~VKPANA-~S~YAI~PANGRGIFI~vNG--SSYYITARDMYMPRA~TAGDI~TLTs~ANYvs V T E ~ ~ V ~ H T E N D T E -- N ~ ~ D L M L T E Y ----V 8D-'D'TE GL VT VE ---T S N G V K Q M41U,H M42H,S KB8523 6/82 D207 D1466 M41U M41H M42S,H KB8523 6•82 D207 D1466 104Q 1050 106Q 107Q 1080 1090 ll0O iii0 VNKT____.VITTFVDNDDFDFNDEL SKWWNDT___~ELPDFDKFNYT___VP ILDID S EIDR IQGVIQGLNDS LIDLEKLS ILKTYIKW E D i'E--Y 0 --I V N TYD K EE K -- T -- D -- E-- G T i-'r- r. ~ --rSRDF n q ~I v N__~SN ~. ----

The nucleotide and amino acid sequences were compared for possible recombination points. From this comparison we conclude that the Japanese field strain KB8523 is probably a recombinant. The $2 protein of this strain is almost identical to $2 of M41, except for the region corresponding to amino acids 929 to 1102 ( Figure I) where the sequence is remarkably similar to D1466. This suggests a recombination event with two crossovers in this region. The alignment of the $2 genes indicates a second potential recombinant: the British field isolate 6/82. Its S1 gene and most of the $2 gene are almost identical to those of strain D207. At the 3' end of the $2 sequence (at the Lys-1105 codon) the percentage of identical nucleotides switches from 99% upstream to 57% downstream. At exactly the same position the nucleotide sequence of 6/82 becomes highly similar to the D1466 sequence: from 73% identity upstream to 97% downstream. The nucleotide identity between D207 and Figure 1 D1466 is about the same on both sides of this putative crossover site: 73 and 67%, respectively. The above comparisons are both time consuming and subjective. A method was sought to test and quantify these results more rigorously. This was achieved by comparing phylogenetic trees for each of five sections (windows) of the $2 gene and its flanking sequences (see Figure 2a ). The $2 gene was divided into three sections.

Vaccine, Vol. 8, December 1990 607

In vivo recombination of IBV: J.G. Kusters et al. The first section (II in Figure 2b ) contained the 5'-part of the $2 gene up to the putative KB8523 crossover site (in the codon for Ile-929 in Figure 1) . A second $2 tree (III in Figure 2b ) was constructed for the part of the $2 gene between the KB8523 and 6/82 crossover sites (from Ile-929 to Lys-1105). The 3'-border of this region did not exactly coincide with the second KB8523 crossover site (in the Ser-ll02 instead of the Lys-ll05 codon) but changing this border had no significant effect on the topology. The third $2 tree (IV in Figure 2b ) was calculated for the region from the 6/82 crossover site (in the Lys-ll05 codon) until the stop codon on position 1182.

Sequences upstream and downstream from the $2 gene were also used to construct phylogenetic trees. The tree based on S1 gene sequences (I in Figure 2b ) has the same topology as an $2 tree based on region II in $2. KB8523 has shifted to the D1466 branch in a tree for region III of $2. Region IV of $2 yields a tree in which 6/82 has shifted from the D207 to the D1466 branch, while KB8523 is again near M41 and M42. Finally a tree (V) constructed from the E1 gene sequences shows that, compared to tree IV, also strain D207 has shifted towards the D1466 branch, and therefore may also be a recombinant.

With the murine coronavirus MHV a high frequency of recombination was found both in vitro and in mouse brain after infection with a ts mutant of MHV strain A59 and wild type JHM-virus 18'19 Phylogenetic trees constructed from five different windows of the genomes of eight IBV strains were used to detect crossover events. A clear advantage of this method is that, due to its simplicity, large numbers of homologous genes can easily be screened. The data presented in this paper suggest that genomic recombination not only occurs under laboratory conditions but also plays an important role in the generation of new virulent strains; at least three IBV strains, all isolated from outbreaks, are potential recombinants. Pre,)ious data ~1 already indicated that the Dutch field isolate V1397 is also the product of a recombination. Considering the limited number of sequences analysed so far, in vivo recombination of IBV seems to occur at a rather high frequency. This has implications for the use of IBV vaccines consisting of several serotypes of live attenuated IBV strains. These vaccines offer the best protection, but they may prove dangerous in the long term by the induction of new variants, not only by point mutations but also through recombination. In this respect vaccination protocols that combine IBV with other live attenuated viruses are equally suspect. They may create ideal conditions for non-homologous recombination which may result in the emergence of recombinant virus species with unwanted properties.

The biology and pathogenesis of coronaviruses

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

Molecular epidemiology of infectious bronchitis virus in the Netherlands

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

Coronavirus IBV; partial amino terminal sequencing of spike polypeptide $2 identifies the sequence Arg-Arg-Phe-Arg-Arg at the cleavage site of the spike precursor of IBV strains Beaudette and M41

Antigenic differentiation of avian bronchitis virus variant strains employing monoclonal antibodies

Monoclonal antibodies to the $1 spike and membrane proteins of avian infectious bronchitis virus strain Massachusetts M4t

Coronavirus IBV: virus retaining spike glycopolypeptide S2 but not $1 is unable to induce virus-neutralizing or haemagglutination-inhibiting antibody, or induce chicken tracheal protection

Eyidence for a coiled-coil structure in the spike protein of coronaviruses

Coronavirus proteins: biogenesis of avian bronchitis virus virion proteins

Phylogeny of antigenic variants of avian coronavirus IBV

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

Comparison of the spike precursor sequences of coronavirus IBV strains M41 and 6•82 with that of IBV Beaudette

Cloning and sequencing of genes encoding structural proteins of avian infectious bronchitis virus

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

A comprehensive sequence analysis program for the IBM personal computer

Confidence limits on phylogenies: an approach using the bootstrap

Highfrequency RNA recombination of murine coronaviruses

In vivo RNA-RNA recombination of coronavirus in mouse brain

Cloning and sequencing of the gene encoding the spike protein of coronavirus IBV

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

The authors are grateful to Ms G.A.W.M. Kremers and Ms K.A. Zwaagstra for experimental contributions and to Dr J.A. Lenstra for advice and help with computer programs. The use of the CAOS/CAMM computer