Infectious bronchitis virus: Evidence for ...

Avian Pathology

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Infectious bronchitis virus: Evidence for recombination within the Massachusetts serotype D. Cavanagh , P. J. Davis & J. K. A. Cook To cite this article: D. Cavanagh , P. J. Davis & J. K. A. Cook (1992) Infectious bronchitis virus: Evidence for recombination within the Massachusetts serotype, Avian Pathology, 21:3, 401-408, DOI: 10.1080/03079459208418858 To link to this article: http://dx.doi.org/10.1080/03079459208418858

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Date: 28 January 2016, At: 19:50

Avian Pathology (1992) 21, 401-408

Infectious bronchitis virus: evidence for recombination within the Massachusetts serotype D. CAVANAGH, P. J. DAVIS & J. K. A. COOK AFRC Institute for Animal Health, Houghton Laboratory, Houghton, Huntingdon, Cambridgeshire PE17 2DA, England

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SUMMARY The spike (S) glycoprotein gene (which encodes two subunits, S1 and S2), the membrane (M) glycoprotein gene and the gene which encodes the products of gene 3 are situated in the infectious bronchitis virus (IBV) genome in the order S1-S2-3-M. The S1 gene of four isolates of the Massachusetts (Mass) serotype, isolated between 1970 and 1984, each differed by only 2 to 3% from that of older Mass serotype isolates, including M41 and H120. Similarly, sequencing of the end of gene 3 and the beginning of the M gene showed that three of the isolates differed from the older Mass isolates by 2% or less. In contrast, the fourth Mass strain, Portugal/322/82, differed by 11% and 24% in gene 3, and the first part of gene M, respectively, suggesting that this strain was a recombinant. It was not possible to identify a putative recombination site but the finding that the S2 gene of Portugal/322/82 differed from other strains much less than did the S1 gene suggests that recombination might have occurred in S2 as a consequence of the high S2 nucleotide homology among IBV strains.

INTRODUCTION The first isolates of IBV were assigned to the Masschusetts (Mass) serotype and strains of this serotype were among the first to be used to make live vaccines, e.g. HI20 which have been used widely (King & Cavanagh, 1991). It is the SI subunit of the S protein which induces neutralizing antibody (Cavanagh et al., 1986a, 1988; Koch et ah, 1990), the basis of IBV serotyping, and it was not surprising to find that the SI protein of the Mass vaccines had 95% or greater amino acid identity with each other (Cavanagh et al., 1988). Partial sequencing also showed that these vaccine strains had gene 3 and M proteins which were about 99% identical (Cavanagh & Davis, 1988). During a subsequent study of more recently isolated strains of the Mass serotype it was observed that one of them, Portugal/322/82, had gene 3 and M sequences atypical of all the other Mass strains examined. This suggests that the strain is a recombinant and shows there can be marked genetic heterogeneity within the Mass serotype.

MATERIALS AND METHODS IBV strains Strains UK/VF873/70 (Darbyshire et al., 1979), UK/68/84 (Cook & Huggins, 1986),

Received 9 May 1991, Accepted 21 November 1991.

401

402

D. CAVANAGH ET AL.


and UK/183/66 (Dawson & Gough, 1971) were isolated in the UK in the years indicated by the last number. Isolate Netherlands (NL)/V1385/84 (Kusters et al., 1987) was kindly provided by Dr A. G. Burger, Doom, The Netherlands, and strains Portugal/322/82 and Portugal/325/83 were received from Dr M. Fevereiro, National Veterinary Investigation Laboratory, Lisbon, Portugal. The HI 20 vaccine strain, derived from an isolate obtained in 1956 (Bijlenga, 1956) and strain M41, isolated in 1941, have been described previously (Cavanagh et al., 1988). The viruses had been passaged a number of times in chick embryo tracheal organ cultures (OC) (Cook, 1984), including two and three passages at limiting dilution in the case of Portugal/322/82 and UK/68/84, respectively. Viruses were passaged once in embryonated eggs to produce the working stocks that were used for the preparation of IBV RNA. Serum neutralization test Antisera were produced by infecting groups of five specified-pathogen-free chickens intranasally with one virus strain and bleeding them after 28 days. The sera of birds within a group were pooled. The neutralization test was performed in OC using a constant amount of virus (2.0 log10 median ciliostatic doses [CD50]) and sequential doubling dilutions of serum (Darbyshire et al., 1979). RNA sequencing IBV RNA was purified and sequenced as described by Cavanagh & Davis (1988) except that (a) the reactions were performed in flexible assay plates (Falcon 3911, Becton Dickinson) rather than in tubes; (b) the reverse transcription was stopped by addition of only 2 fil of formamide dye mix; (c) the nucleic acids were denatured by heating the uncovered plate in an oven at 80°C for 6 min, which also reduced the volume; (d) each sample was analysed on only one 6% acrylamide, urea-containing gel containing a salt gradient of 0.54 to 4.0 xTBE buffer. TBE buffer contained 10.8 g Tris base, 5.5 g boric acid and 0.95 g EDTA.2H20 per litre. Electrophoresis was terminated when the bromophenol blue dye had run to the bottom of the gel. RESULTS Sequence of the SI spike glycopolypeptide The S genes of IBV Beaudette and M41 were the first to be cloned and sequenced (Binns et al., 1985, 1986; Niesters et al., 1986) and were shown to encode a precursor protein of 1165 amino acids, of which the first 18 form an amino-(N-)terminal signal sequence which is not present in the mature, virion-associated S. The S protein is cleaved at residues 519/520 in Mass strains to yield two glycopolypeptides, namely N-terminal SI and carboxy-(C-)terminal S2 (Cavanagh et al., 1986b). For this report we have sequenced the SI gene for four IBV isolates (UK/VF873/70, NL/VI385/84, Portugal/322/82 and Port/325/83) which were shown by neutralization tests using OC to be of the Mass serotype (Table 1). Where two-way cross-neutralization tests had been performed the analysis of Archetti & Horsfall (1950) confirmed that the strains had been correctly assigned to the Mass serotype (data not shown). As has been described previously (Darbyshire et al., 1979), there was low reciprocity with some pairs of strains. Although UK/VF873/70 was neutralized poorly by sera against M41 and HI20, antis-

IBV RECOMBINATION

403

erum to UK/VF873/70 neutralized M41 and H120 to a similar extent as it neutralized UK/VF873/70. The SI sequences of these strains have been compared with those of M41 and HI20 as representatives of older Mass serotype strains.


Table 1. Cross-neutralization between IBV strains Antiserum

M41

H120

UK VF873/70

NL V1385/84

Port 322/82

Port 325/83

M41 H120 UK/VF873/70 NL/V1385/84 Port/322/82 Port/325/83

575* 137 64 12

145 256 115 46 NT 288

36 36 80 20 NT 46

230 256 162 64 NT 256

104 50 64 22 NT 25

94 712 128 50

NTf 40

NT 224

•Reciprocal neutralization titre (homologous reaction underlined). t N T = not tested

Each of the four newly sequenced Mass isolates differed in SI from M41 by only 2 to 3% of nucleotides (data not shown). Figure 1 shows, correspondingly, that all four of the Mass isolates had very similar SI amino acid sequences to each other and to the previously analysed Mass strains. Three of the isolates, UK/VF873/70, NL/V1385/84 and Port/325/83, had slightly less identity with M41 (96 to 97%) than with H120, each differing from HI20 by three to six residues (Figure 1). Portugal/322/82 had 95% amino acid identity with M41 (Figure 1). Many of the differences between it and M41 were shared with the other Mass strains, although it was clearly distinct from all of them and from the vaccinal strains. In all cases, the majority of the differences from M41 were located in the first 25% of SI. Sequence of gene 3 and M Our previous sequencing of the end of gene 3 and the beginning of the M gene (Cavanagh & Davis, 1988) had shown that some non-Mass serotype IBV isolates had deletions, with respect to M41 and other Mass strains, at the end of gene 3 and at the beginning of the M gene, in addition to many other nucleotide differences. Three of the Mass strains in the current study (UK/VF873/70, NL/V1385/84 and Port/325/83) resembled M41 and HI20 and other Mass vaccinal strains (Cavanagh & Davis, 1988) very closely, having only three or four nucleotide differences (less than 2%) from M41 (data not shown). These strains were slightly more like HI20 than M41, correlating with SI data. The exception to this uniformity was Portugal/322/82 which had deletions, with respect to the other Mass strains, at the end of gene 3 and at the beginning of the M gene (Figure 2). For comparison we also sequenced gene 3 and the beginning of M of two non-Mass strains, UK/68/84 and UK/183/66. These had deletions which were smaller than that of Portugal/322/82. We have previously sequenced the overlap region of genes 3 and M for about 50 strains of IBV. Apart from Portugal/322/82 only one other isolate, received from Portugal at the same time as Portugal/322/82, had the particular deletions shown for Portugal/322/82 in Figure. In addition to deletions, the M gene of Portugal/322/82 differed from that of M41 at 15 of the first 54 nucleotides (28%, excluding deletions), which encodes the short N-terminal part of M which is exposed at the virus surface (Cavanagh et al., 1986c), and 15 of the 69 nucleotides (22%) which encode the first membrane-spanning part of M (data not shown).

404

D. CAVANAGH ETAL.

10 20 30 40 50 60 70 M41 ALYDSSSYVYYYQSAFRPPNGWHLHGGAYAWNISSESNNAGSSPGCIVGTIHGGRWNASSIAMTAPSS VF873/70 S T I V1385/84 S T I 325/83 S T I 322/82 LSD T Q H P H120 S T I


80 90 100 110 120 130 140 M41 GMAWSSSQFCTAHCNFSDTTVFVTHCYKYDGCPITGMRQKNFLRVSAMKNGQLFYNLTVSVAKYPTFKSF VF873/70 Y HG L QHS V1385/84 Y HV L QHS 325/83 Y HV L Q S 322/82 N GR Y I H L Q SI H120 Y HV QH 150 160 170 180 190 200 210 M41 QCVNNLTSVYLNGDLVYTSNETTDVTSAGVYFKAGGPITYKVMREVKALAYFVNGTAQDVILCDGSPRGL VF873/70 R V1385/84 R 325/83 R 322/82 H120 R 220 230 240 250 260 270 280 M41 LACQYNTGNFSDGFYPFINSSLVKQKFIVYRENSVNTTFTLHNFTFHNETGANPNPSGVQNIQTYQTQTA VF873/70 T V1385/84 T 325/83 T 322/82 I H120 T 290 300 310 320 330 340 350 M41 QSGYYNFNFSFLSSFVYKESNFMYGSYHPSCNFRLETINNGLWFNSLSVSIAYGPLQGGCKQSVFSGRAT VF873/70 V1385/84 325/83 322/82 H120 360 370 380 390 400 410 420 M41 CCYAYSYGGPSLCKGVYSGELDLNFECGLLVYVTKSGGSRIQTATEPPVITRHNYNNITLNTCVDYNIYG VF873/70 L H Q V1385/84 L H Q 325/83 L H Q 322/82 L H Q H120 L Q 430 440 450 460 470 480 490 M41 RTGQGFITNVTDSAVSYNYLADAGLAILDTSGSIDIFWQGEYGLTYYKVNPCEDVNQQFWSGGKLVGI VF873/70 S N V1385/84 S N 325/83 S S N 322/82 S S N H120 S N 500 510 M41 LTSRNETGSQLLENQFYIKITNGTRRFRR VF873/70 V1385/84 325/83 G 322/82 H120

Figure 1. Comparison of the amino acid sequence of the SI subunit (without the signal sequence) of the S glycoprotein of six Mass serotype IBV strains. The sequence of the S protein of the M41 strain (Binns et al., 1985) is shown in its entirety. Only differences from this sequence are shown for HI20 (Cavanagh et al., 1988; Kusters et al., 1989) and the four Mass isolates sequencedfor this report.

We then sequenced the whole of gene 3 for these strains; the sequences will appear in the EMBL, DDBJ and Genbank Nucleotide Sequence Databases under the accession numbers X59819 (Portugal/322/82), X59820 (UK/183/66) and X60712 (UK/68/84).

IBV RECOMBINATION

620 M41

630

640

405

650

660

670

AGAACGGUUGGAAUAAUAAAAAUCCAGCAAAUUUUCAAGAUGUCCAACGAGACAAAUUGU

322/82

AC

********************************AU

68/84

C

C

A*********AU GA

C

A****** UGAC GA

C

A AA


183/66

G

C

Figure 2. Comparison of nucleotides 610 to 670 of gene 3 of four isolates of IBV. The numbers relate to gene 3 of the M41 strain which had 678 nucleotides. Only differences from M41 are shown. The asterisks (*) show where nucleotides were missing from the other strains. The M protein coding sequence starts before the end of gene 3, i.e. genes 3 and M overlap. The AUG translation start codons for M are marked by arrow heads

Table 2. Two-way comparisons of the gene 3 nucleotide differences among four isolates of IBV Number1 (%) nucleotide differences Virus

322/82

Port/322/82 UK/68/84 UK/183/66 M41

78(13) 67(11) 74(12)

68/84

183/66

M41

78(13)

67(11) 45(7)

74(12) 61(10) 65(10)

45(7) 61(10)

65(10)

'The first 626 nucleotides of gene 3 were compared i.e. excluding the region near the end of gene 3 in which there were deletions with respect to the M41 sequence (see Figure 2).

The sequence of Portugal/322/82 differed from that of M41 evenly throughout gene 3, the nucleotides differing by 12%, similar to the extent to which each of these strains differed from the other isolates examined (Table 2). To be certain that Portugal/322/82 did have a S gene very similar to the other Mass isolates but greatly different 3 and M genes, another preparation of Portugal/322/82 RNA was made. Parts of the S and M genes were sequenced again; the data confirmed our original rinding.

Sequence of S2 of Portugal/322/82 The finding that the SI gene of Portugal/322/82 was extremely similar to that of M41 and all other Mass strains examined, but markedly different in gene 3 and that for M suggested that Portugal/322/82 might be a recombinant. Since the sequence of gene 3 of Portugal/322/82 differed from that of the other Mass strains throughout the gene it was unlikely that the proposed recombination event had occurred within gene 3. To ascertain if recombination might have occurred within the C-terminal S2 region of the S gene we sequenced the whole of S2 (1875 nucleotides) for Portugal/322/82. The S2 of Portugal/322/82 differed at only 44/1875 (2.3%) from that of M41, with 2.4% of amino acid differences (data not shown). It was not possible to identify unequivocally a putative recombination site.

406

D. CAVANAGH ET AL.


DISCUSSION Three of the four mass serotype isolates examined for this report were very similar, with respect to their S, gene 3 and M proteins, to the Mass vaccine strains that have previously been characterised (Cavanagh & Davis, 1988; Cavanagh et al, 1988; Kusters et al, 1989), but differed sufficiently for it to be concluded that they were not a result of the reisolation of vaccine strains. It is possible that they are mutants of vaccine strains. Alternatively, they may be genuine field isolates which have changed very little since the time of isolation of the isolates that were subsequently used to make vaccines. It is not possible to distinguish between these two possibilities. However, in the case of Portugal/322/82 there is no doubt that this strain is not a simple mutant of Mass vaccine strains; it differs substantially in genes 3 and M. In addition, while the SI sequence was very similar to that of the other Mass strains, there were several differences unique to Portugal/322/82 (Figure 1, position 44 to 78). The finding that a strain isolated in 1982 differed in SI, a gene noted for its variability (Binns et al., 1986; Kusters et al, 1989), by only 3% of nucleotides when compared with the Mass vaccine strain H120, derived from an isolate obtained in 1956, indicates that S genes can survive relatively unchanged for at least several decades. The most interesting finding was that the gene 3 and M sequences of Portugal/322/82 were substantially different from all the other nine Mass strains (this report and Cavanagh et al., 1988) with which it has been compared. This raises the question as to how Portugal/322/82 has evolved. One possibility is that, by chance, the gene 3 and M sequences have mutated by about 12% in gene 3 and up to 24% for the beginning of the M gene while SI and S2 have changed only 2 to 3%. This seems unlikely. That part of M which is exposed at the virus surface is known to be very variable (Cavanagh & Davis, 1988) and it might be misleading to make deductions concerning the evolution of IBV strains based only on sequence data of that part of M. However, although the amino acid sequence of the first membrane-spanning part of M is highly conserved, the nucleotide variation within this region is as high as in the sequence encoding the exposed part of the M protein (Cavanagh & Davis, 1988). The SI protein can vary by up to 49% (Kusters et al., 1989), much more than gene 3 or M. Thus, comparison of the nucleotide sequences throughout genes S and 3 and the first part of the M gene is likely to give an unbiased indication of the evolution of this part of the genome of Portugal/322/82 and suggests that the notion that different rates of random mutation alone in these genes would be sufficient to explain the sequence of Portugal/322/82 was not correct. An alternative hypothesis to explain the lack of correlation between the data for gene S and genes 3 and M for Portugal/322/82 is that this isolate is a recombinant which arose during mixed infection in the field. Recombination by the coronavirus murine hepatitis virus has been demonstrated experimentally, both in vitro and in vivo ((Lai et al., 1985; Keck et al., 1988). The possession by two Dutch strains of IBV, D207 and D1466, of highly similar M and three genes (Cavanagh & Davis, 1988), but very different SI (49%) (Kusters et al, 1989) and S2 (27%) (Kusters et al, 1990) proteins further supports the view that recombination has occurred among IBV strains in vivo, in the field. The data on Portugal/322/82 (genes, SI, S2 3 and M) and the observations of Kusters et al, (1990) for S2 of several European IBV isolates, when considered alongside sequence comparisons of SI of many isolates (Binns et al, 1986; Kusters et al, 1989) indicates that the S2 region of the S gene had greater potential as a site of recombination than either SI or gene 3. This is probably because high frequency recombination will depend in part on the presence of highly homologous nucleotide sequences between the parental strains;

IBV RECOMBINATION

407


nucleotide sequence similarity between most strains is higher in S2 than in either SI or gene 3 (Kusters et al., 1990). In the specific case of Portugal/322/82 it is unlikely that the recombination event occurred within SI, where the sequence throughout was very similar to other Mass strains, or gene 3 in which there was an evenly distributed variation of up to 13%. It is more likely that recombination occurred within S2. The relatively small proportion of S2 differences between Mass and non-Mass strains meant that it was not possible to define accurately the recombination position. While the evidence for recombination of IBV strains in the field is only circumstantial it is clear that isolates of the same serotype can differ substantially in some genes. Clearly, serological analyses which measure properties only of the S protein, e.g. neutralization tests, may result in misleading conclusions with regard to the relatedness of strains. ACKNOWLEDGEMENTS We thank Rachel Purvis and Marjorie Ellis for excellent technical assistance, and Richard Gough (Central Veterinary Laboratory, Weybridge) for IBV isolate UK/183/66. This work was supported by the Ministry of Agriculture, Fisheries and Food. REFERENCES ARCHETTI, I. & HORSFALL, F.L. (1950). Persistent antigenic variation of influenza A viruses after incomplete neutralization in ovo with heterologous immune serum. Journal of Experimental Medicine, 92, 441-462. BIJLENGA, G. (1956). Het infectieuze bronchitis virus bij kuikens in Nederland aangetoond met behulp van ei-enting, dierexpermenteel onderzoek en serumneutralisatieproeven. Tijdschrift voor diergeneeskunde, 81, 43-54. BINNS, M.M., BOURSNELL, M.E.G., CAVANAGH, D., PAPPIN, D.J.C. & BROWN, T.D.K. (1985). Cloning and sequencing of the gene encoding the spike protein of the coronavirus IBV. Journal of General Virology, 66, 719-726. BINNS, M.M., BOURSNELL, M.E.G., TOMLEY, F.M. & BROWN, T.D.K. (1986). Comparison of the spike precursor sequences of coronavirus IBV strains M41 and 6/82 with that of IBV Beaudette. Journal of General Virology, 67, 2825-2831. CAVANAGH, D. & DAVIS, P.J. (1988). Evolution of avian coronavirus IBV: sequence of the matrix glycoprotein gene and intergenic region of several serotypes. Journal of General Virology, 69, 621-629. CAVANAGH, D., DAVIS, P.J., DARBYSHIRE, J.H. & PETERS, R.W. (1986a). Coronavirus IBV: virus retaining spike glycopolypeptide S2 but not S1 is unable to induce virus-neutralising or haemagglutination inhibiting antibody, or induce chicken tracheal protection. Journal of General Virology, 67, 1435-1442. CAVANAGH, D., DAVIS, P.J., PAPPIN, D.J.C., BINNS, M.M., BOURSNELL, M.E.G. & BROWN, T.D.K. (1986b). Coronavirus

IBV: partial amino terminal sequencing of spike polypeptide S2 identifies the sequence Arg-Arg-Phe-Arg-Arg at the cleavage site of the spike precursor propolypeptide of IBV strains Beaudette and M41. Virus Research, 4, 133-143. CAVANAGH, D., DAVIS, P.J. & PAPPIN, D.J.C. (1986c). Coronavirus IBV glycopolypeptides: locational studies using proteases and saponin, a membrane permeabilizer. Virus Research, 4, 145-156. CAVANAGH, D., DAVIS, P.J. & MOCKETT, A.P.A. (1988). Amino acids within hypervariable region 1 of avian coronavirus IBV (Massachusetts serotype) spike glycoprotein are associated with neutralisation epitopes. Virus Research, 11, 141-150. COOK, J.K.A. (1984). The classification of new serotypes of infectious bronchitis virus isolated from poultry flocks in Britain between 1981 and 1983. Avian Pathology, 13, 733-741. COOK, J.K.A. & HUGGINS, M.B. (1986). Newly isolated serotypes of infectious bronchitis virus: their role in disease. Avian Pathology, 15, 129-138. ' • . . DARBYSHIRE, J.H., ROWELL, J.G., COOK, J.K.A. & PETERS, R.W. (1979). Taxonomic studies of strains of avian infectious bronchitis virus using neutralisation tests in tracheal organ cultures. Archives of Virology, 61, 227-238. DAWSON, P.S. & GOUGH, R.E. (1971). Antigenic variation in strains of avian infectious bronchitis virus. Archiv fur de gesamte Virusforschung, 34, 32-39. KECK, J.G., MATSUSHIMA, G.K., MAKINO, S., FLEMING, J.O., VANNIER, D.M., STOHLMAN, S.A. & LAI, M.M.C. (1988).

In vivo RNA-RNA recombination of coronavirus in mouse brain. Journal of Virology, 62, 1810-1813. KING, D.J. & CAVANAGH, D. (1991). Infectious Bronchitis. In: B. W. CALNEK, H. J. BARNES, C. W. BEARD, W. M. REID

and H. W. YODER (Eds) Diseases of Poultry, Ninth Edition pp. 471-484 (Ames, Iowa State University Press). KOCH, G., HARTOG, L., GRANT, A. & VAN ROOZELAAR, D.J. (1990). Antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions. Journal of General Virology, 71, 1929-1935. KUSTERS, J.G., NIESTERS, H.G.M., BLEUMINK-PLUYM, N.M.C., DAVELAAR, F.G., HORZINEK, M.C. & VAN DER ZEIJST,

B.A.M. (1987). Molecular epidemiology of infectious bronchitis virus in the Netherlands. Journal of General Virology, 68, 343-352.

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KUSTERS, J.G., NIESTERS, H.G.M., LENSTRAR07, J.A., HORZINEK, M.C. & VAN DER ZEIJST, B.A.M. (1989). Phylogeny of

antigenic variants of avian coronavirus IBV. Virology, 169, 217-221. KUSTERS, J.G., JAGER, E.J., NIESTERS, H.G.M. & VON DER ZEIJST, B.A.M. (1990). Sequence evidence for RNA recombination in field isolates of avian coronavirus infectious bronchitis virus. Vaccine, 8, 605-608. LAI, M.M.C., BARIC, R.S., MARINO, S., KECK, J.G., EGBERT, J., LEIBOWITZ, J.L. & STOHLMAN, S.A. (1985).

Recombination between nonsegmented RNA genomes of murine coronaviruses. Journal of Virology, 56, 449-456. NIESTERS, H.G.M., LENSTRA, J.A., SPAAN, W.J.M., ZIJDERVELD, A.J., BLEUMINK-PLUYM, N.M.C., HONG, F., VAN

SCHARRENBURG, G.J.M., HORZINEK, M.C. & VAN DER ZEIJST, B.A.M. (1986). The peplomer protein sequence of the M41 strain of coronavirus IBV and its comparison with Beaudette strains. Virus Research, 5, 253-263.

RESUME


Virus de la bronchite infectieuse: démonstration de recombinaison à l'intérieur du sérotype Massachusetts Le gène de la glycoprotéine du spicule (S), qui code pour deux sous-unités: S1 et S2, le gène de la glycoprotéine de membrane (M) et le gène qui code les produits de gène 3, sont situés dans le génome du virus de la bronchite infectieuse (IBV) dans l'ordre S1, S2, 3 et M. Le gène S1 de quatre souches appartement au sérotype Massachusetts (Mass) isolées entre 1970 et 1984, diffère chacun seulement de 2 à 3% par rapport aux souches plus anciennes de sérotype Mass, incluant M41 et H120. De la même manière, le séquençage de la fin du gène 3 et le commencement du gène M ont montré que trois des souches isolées différaient des anciennes souches Mass par 2% au moins. Par contre, la quatrième souche Mass, Portugal/322/82, différait par 11 et 24% pour le gène 3 et la première partie du gène M, respectivement, indiquant que cette souche était un recombinant. Il n'a pas été possible d'identifier un site éventuel de recombinaison mais le fait que le gène S2 de Portugal/322/82 différait des autres souches, moins que ne le faisait le gène S1, suggère que la recombinaison ait pu intervenir en S2 comme conséquence d'une grande homologie du nucléotide S2 parmi les souches d'IBV.

ZUSAMMENFASSUNG Virus der infektiösen Bronchitis: Nachweis einer Rekombination innerhalb des Serotyps Massachusetts Das Spike (S)-Glykoprotein-Gen (das die beiden Untereinheiten S1 und S2 kodiert), das Membran (M)Glykoprotein-Gen und das Gen, das die Produkte des Gens 3 kodiert, liegen im Genom des Virus der infektiösen Bronchitis (IBV) in der Reihenfolge S1-S2-3-M. Das S1-Gen von vier Zwischen 1970 und 1984 isolierten Stämmen vom Serotyp Massachusetts (Mass) unterschied sich jeweils um nur 2 bis 3% von dem älterer Mass-Isolate, einschließlich der Stämme M41 und H120. Dem ähnlich, ergab die Sequenzierung des Endes von Gen 3 und des Anfangs des M-Gens, daß sich drei der Isolate um 2% oder weniger von den älteren Mass-Isolaten unterschieden. Im Gegensatz dazu unterschied sich der vierte Mass-Stamm, Portugal/322/82, um 11% beim Gen 3 und um 24% beim Anfangsteil des M-Gens, was daraufschließen ließ, daß es sich bei diesem Stamm um eine Rekombinante handelte. Es war nicht möglich, eine mutmaßliche Rekombinationsstelle festzustellen, aber der Befund, daß sich das S2-Gen des Stammes Potugal/322/82 viel weniger als das Sl-Gen von anderen Stämmen unterschied, läßt darauf schließen, das die Rekombination infolge der großen S2Nukleotidhomologie unter IBV-Stämmen bei S2 erfolgt war.

RESUMEN Virus de la bronquitis infecciosa: evidencias de recombinación en el serotipo Massachusetts El gen de la glicoproteína de la espícula (S) que codifica para dos subunidades, la S1 y la S2, el gen de la glicoproteína de membrana (M) y el gen que codifica para los productos del gen 3 están situados en el genoma del virus de la bronquitis infecciosa (IBV) en el orden S1-S2-3-M. El gen Sl procedente de cuatro aislamientos del serotipo Massachusetts (Mass) aislados entre 1970 y 1984 difirió sólo entre un 2 y 3% de aislamientos más antiguos del mismo serotipo, como el M41 y el H120. De forma similar, la secuenciación del final del gen 3 y el comienzo del gen M mostró que los tres aislamientos diferían de los aislamientos más antiguos de Mass en un 2% o menos. Por el contrario, una cuarta cepa de Mass, la Portugal/322/82 difirió un 11 y un 24% en el gen 3 y la primera parte del gen M, respectivamente, sugiriendo que esta cepa era recombinante. No fue posible identificar un lugar de recombinación putativo pero el hallazgo de que el gen S2 de Portugal/322/82 difería del de ostras cepas mucho menos que el gen Sl sugiere que pudiera haber ocurrido una recombinación en S2 como consecuencia de una elevada homología de nucleótidos S2 entre las cepas de IBV.