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Short Communication |
1 INRA-ENVT, UMR1225 – Interactions Hosts-Pathogens (IHAP), Ecole Nationale Vétérinaire, 31076 Toulouse cedex 03, France
2 Laboratoire Départemental Vétérinaire LVD09, 09007 Foix cedex, France
Correspondence
Gilles Meyer
g.meyer{at}envt.fr
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ABSTRACT |
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MAIN TEXT |
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). The antigenic and genetic diversity among independent isolates of HRSV has been extensively documented and the existence of two major groups (A and B) has been clearly established, as well as additional variability within each individual group (reviewed by Sullender, 2000; Cane, 2001; Zlateva et al., 2004, 2005). The most extensive differences were found on the attachment glycoprotein G, which differed by up to 45 % in its amino acid sequence between groups A and B (Johnson et al., 1987; Mufson et al., 1985). The extent of antigenic variation observed with BRSV is considerably less than for HRSV and the different subgroups identified may only represent variants of a single major antigenic group (Furze et al., 1994; Schrijver et al., 1996; Prozzi et al., 1997). While BRSV nucleotide sequence variation of the variable G gene did not exceed 11 % between independent isolates (Larsen et al., 2000; Valarcher et al., 2000), this genetic variability may have biological implications such as escape from previous vaccine immunity, as suggested by a comparative phylogenetic study on the G, F and N genes (Valarcher et al., 2000).
Genetic variation is a hallmark of RNA viral pathogens. For RNA viruses, mutation rates operating during RNA replication have been estimated to be in the range of 10–3 to 10–5 substitutions per nucleotide per round of copying (Drake, 1993; Drake & Holland, 1999). The biochemical basis of the limited replication fidelity is the absence or low efficiency of 3'
5' exonuclease proofreading activity in viral RNA-dependent RNA polymerases, together with lack of post-replicative mismatch repair mechanisms (Domingo & Holland, 1997). As a consequence, populations of RNA viruses evolve as dynamic distributions of closely related mutant genomes that exist in equilibrium around a theoretical consensus sequence. Such mutants would provide evidence of quasispecies dynamics, implying the presence of a variant reservoir for viral adaptation. Despite the biological significance of this population structure, no such analyses of mutant spectra have been reported for BRSV and HRSV to date. In the present work, we demonstrate that BRSV populations evolve, in vitro and in vivo, as complex and dynamic mutant swarms, despite apparent genetic stability.
The replicative environment provided by cell culture conditions may exert a selective pressure on replicating RNA viral populations (Domingo et al., 2001b). To test the genetic stability of BRSV upon virus isolation and propagation in cell culture, we analysed the consensus nucleotide sequence of the highly variable glycoprotein G coding region of a natural population of BRSV at different stages of its evolutionary history (Fig. 1). BRSV W2-00131 was isolated by bronchoalveolar lavage (BAL) of a calf with respiratory distress syndrome (BAL-T) and further propagated in bovine turbinate cells (BT; ATCC CRL-1390) (Valarcher et al., 2001). To prove that multiple variants are produced de novo upon replication of a single BRSV genome, biological cloning was performed as follows: serial virus dilutions (order of 2) were used to infect BT cells in fresh agar minimum essential medium (Valarcher et al., 2001). After 4 days, one individual syncytium at the lowest dilution was picked under microscopy and amplified for one round in BT cells. Two additional steps of cloning were performed using the same method. Finally a single syncytium, 3C, was picked and propagated in BT cells. Populations 3Cp3, 3Cp9 and 3Cp10 were isolated after 3, 9 and 10 passages of the clone 3C in BT cells (Fig. 1). In addition, population 3Cp9 was used in experimental infection. For this, calves were reared in biocontainment facilities from birth to euthanasia, as prescribed by the guidelines of the EU Council on Animal Care (86/609/CEE). Calves were infected at 4–5 months of age with 107 p.f.u. virus by aerosolization (UltraNeb 99 Nebulizer; DevilBiss). BAL-E and BAL-G populations were then isolated by BAL at 7 and 9 days post-infection from calves E and G respectively, as already described (Valarcher et al., 2001).
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). Five independent PCRs, from two reverse transcription steps, were performed on the entire BRSV G gene with primers G1 and G2 (positions 4682–4703 and 5501–5475, GenBank accession no. NC_001989
[GenBank]
) for 3Cp and BAL-T, or on the partial G gene with primers VG1 and VG4 (positions 4835–4855 and 5376–5356, accession no. NC_001989
[GenBank]
) for BAL-E and BAL-G. DNA amplification was performed using the high-fidelity ThermalAce DNA polymerase (Invitrogen) in a GeneAmp PCR system 9700 (Applied Biosystems) for 35 cycles (95 °C for 30 s, 58 °C for 30 s, 72 °C for 45 s). RT-PCR controls were performed on RNA diluted 1/10 to ensure that stock RNAs were not at a low concentration. Sequence determination of the glycoprotein G coding region revealed that the consensus viral genome found in BAL of calf T remained dominant following virus isolation and was genetically stable over 10 serial infections in BT cells. Likewise, no modification of the G consensus sequence of BRSV W2-00131 populations was observed following virus replication in calves.
The genetic stability exhibited by the glycoprotein G led us to examine the mutant spectrum of the BRSV populations derived from this field isolate. For each population, RT-PCR products generated above were cloned in the pCR4 Blunt TOPO plasmid (Zero blunt TOPO PCR cloning kit; Invitrogen). Twenty-six to 29 molecular clones were sequenced and analysed (SeqMan 4; DNASTAR) for each population (five to six positive Escherichia coli clones for each of the five independent PCRs). The mutation frequency of 3Cp populations ranged from 6.8 to 9.9x10–4 substitutions per nucleotide (Table 1). A significant heterogeneity was also observed within BRSV populations replicating in the animal host with mutation frequencies that ranged from 7.5 to 10.1x10–4 substitutions per nucleotide. These mutation frequencies are similar to those observed for highly variable RNA viruses, such as vesicular stomatitis virus and poliovirus (Domingo & Holland, 1994). However, conservation among independent isolates often parallels conservation within mutant spectra (Domingo et al., 2001a). Consequently, mutation frequencies obtained in this study could be overestimated, since the G gene was the most highly variable. Confirmation would require sequencing of other BRSV genomic regions.
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), the final error rate of the system was calculated to be 0.9x10–4 mutations per nucleotide. ANOVA and a multiple comparison test, with the Bonferroni correction, revealed significant differences when each BRSV population frequency was compared with the system error rate. This indicated that the mutation frequencies observed cannot be the result of misincorporations during RT-PCR amplification of BRSV RNA.
The genetic heterogeneity derived from clone 3C suggests a constant generation of mutant genomes in the course of BRSV replication. To provide further evidence of the error-prone replication of BRSV genome, we analysed the mutant spectrum of a recombinant BRSV (rBRSV) population, derived by transfection of a single infectious cDNA clone of ATue 51908 strain (Buchholz et al., 1999) and kindly provided by Dr K. K. Conzelmann (Federal Research Centre for Virus Diseases of Animals, Tübingen, Germany) after two passages in Madin–Darby bovine kidney cells. Five additional passages of rBRSV in BT cells were performed before analysis. The rBRSV mutation frequency of 7.7x10–4 substitutions per nucleotide for the glycoprotein G coding region is in the range of previous mutation frequencies for other isolates (Table 1
). These results clearly indicated that multiple variants are produced de novo upon replication of a single viral genome. Recently, genetic variation of the consensus sequence of HRSV strains likely derived from one single clone was also demonstrated with mutation rates of 2.5–3.0x10–3 substitutions per nucleotide per year (Trento et al., 2006).
Several arguments support the fact that the great majority of mutations found were present in the BRSV W2-00131 RNA populations. A large dominance of non-synonymous (Nsyn) over synonymous mutations was observed in all BRSV mutant spectra. These Nsyn mutations accumulate in all parts of the G protein ectodomain, preferentially but not significantly in the two mucin-like regions (Fig. 2). Variability within the G immunodominant central domain of the mutant spectrum was also observed in consensus sequences of published BRSV isolates (Prozzi et al., 1997; Valarcher et al., 2000). However, it contrasts with observations made from HRSV sequences, which indicate that Nsyn mutations were specifically located in the mucin-like regions within subgroups (Cane et al., 1991). In our study, Nsyn mutations preferentially mapped in antigenic regions of BRSV G glycoprotein located in the C-terminal part of the protein (Rueda et al., 1991, 1995; Sullender, 1995; Melero et al., 1997) and in defined domains of the mucin-like and the central conserved regions (Lerch et al., 1990; Langedijk et al., 1996; Prozzi et al., 1997; Walsh et al., 1998). Selection of HRSV immune escape mutants was shown to be associated with such a single amino acid substitution in these antigenic domains (Garcia Barreno et al., 1990; Rueda et al., 1991, 1995; Martinez et al., 1997). However, for BRSV, Woelk & Holmes (2001) failed to detect positive selection in the G gene, suggesting that some of the variability observed in the G protein was not a consequence of selection by the immune response. In this study, no major selective pressure was present, as corroborated by the identity of the G consensus sequences of all BRSV populations. Consequently, the high level of amino acid variation that we found in the mutant spectrum more probably reflects reduced constraints for variation of glycoprotein G in the ectodomain. The fact that many of these amino acid changes were encountered in the consensus sequence of independent isolates (Valarcher et al., 2000) also indicates that these mutations are well tolerated and may not adversely affect protein function. Several mechanisms observed for glycoprotein G variability also illustrated the capacity of this protein to accommodate multiple sequence changes (Melero et al., 1997). These mechanisms include amino acid substitutions but also insertions/deletions, changes in the stop codon usage and frameshift mutations. Some of these situations were found in molecular clones of the BRSV populations studied, suggesting that they were present in the mutant spectrum. Two BRSV populations (BAL-T and 3Cp10) contained one clone with a mutation in the stop codon (TAG 258 glutamic acid), leading to a protein 6 aa longer. Differences in the length of the BRSV and HRSV G glycoproteins were demonstrated to result from use of such alternative stop codons (Garcia Barreno et al., 1990; Rueda et al., 1991; Zlateva et al., 2005). Remarkably, the number of deletions, found in all but one population, was high in this study (Table 1). Examination of the sequence context indicated that 85 % of the deletions included one or two nucleotides within short homopolymeric-A and -C tracts. Two poly-A tracts (nt 607–610 and 622–626 of the G gene) correspond to poly-A runs which were described for HRSV to be very prone to polymerase errors, resulting in frameshifts because of the insertion or deletion of adenosine residues (Cane et al., 1993). Surprisingly, we also found two clones in populations 3Cp10 and BAL-T with a C deletion in one homopolymeric poly-C run (nt 467–471). This deletion introduced a frameshift with a putative protein containing 155 aa of the N-terminal part of the G glycoprotein followed by 55 aa of new sequence. Viability of this clone was not demonstrated in this study but, for HRSV, escape mutants with frameshift mutations in the consensus sequence generated by deletions in poly-A runs were clearly selected for with antibody 63G (Garcia-Barreno et al., 1990). Some limited use of alternative frames and termination codons has also been found among HRSV isolates from patients (Sullender et al., 1991; Cane & Pringle, 1995).
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, 2000). This does not exclude potential changes in the consensus sequence of other parts of the BRSV genome. Results indicated the occurrence in BRSV populations of a dominant genomic sequence, which ranges from 48 to 68 % of the genome, together with several variants. Except for one, all mutations were unique, suggesting that despite mutant genomes arising at a high rate, each specific variant remained at a low frequency. The dominant and consensus sequences were identical in all populations analysed and the values of Shannon entropy ranged from 0.38 to 0.52, with no significant differences among populations tested. Consequently, under those circumstances, the mutant spectrum is probably nearly optimal, at the population equilibrium, suggesting that BRSV is well adapted to its biological environment. In this view, the 89 % or higher consensus sequence identities of G glycoprotein found among independent BRSV isolates (Valarcher et al., 2000) would be the result of negative selection on many newly arising mutants and convergence of consensus and average sequences (Eigen & Biebricher, 1988; Domingo et al., 2000). On the other hand, the 11 % BRSV genetic variability was probably shaped by positive selective pressures of the replicative environment. Positive selection on the G glycoprotein was clearly demonstrated to be associated with mutations in the G gene (Melero et al., 1997; Sullender, 2000; Woelk & Holmes, 2001; Huang & Anderson, 2003). In conclusion, this study shows the heterogeneous nature of BRSV genome populations and the low fidelity of their replication, despite genetic stability. Continuous generation of mutant virus is currently regarded as a key adaptative strategy of RNA viruses. Studies are under way to define the biological implications of BRSV viral heterogeneity.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 26 October 2006;
accepted 14 December 2006.
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, passages in BT; 
, BRSV biological cloning by three-step end-point dilutions; 
, populations selected for genetic studies.