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Journal of Virology, March 2009, p. 2397-2403, Vol. 83, No. 5
0022-538X/09/$08.00+0     doi:10.1128/JVI.02189-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Genome Comparison of a Nonpathogenic Myxoma Virus Field Strain with Its Ancestor, the Virulent Lausanne Strain {triangledown} ,{dagger}

Mónica Morales,1,§ Miguel A. Ramírez,1,{ddagger} María J. Cano,1 Mario Párraga,1 Joaquín Castilla,1,|| Luis I. Pérez-Ordoyo,2 Juan M. Torres,1 and Juan Bárcena1*

Centro de Investigación en Sanidad Animal (CISA-INIA), Valdeolmos, 28130 Madrid, Spain,1 Departamento de I+D, Laboratorios Syva S. A. Avda, Párroco Pablo Díez, 49-57, 24010 León, Spain2

Received 16 October 2008/ Accepted 4 December 2008


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ABSTRACT
 
One of the best-studied examples of host-virus coevolution is the release of myxoma virus (MV) for biological control of European rabbits in Australia and Europe. To investigate the genetic basis of MV adaptation to its new host, we sequenced the genome of 6918, an attenuated Spanish field strain, and compared it with that of Lausanne, the strain originally released in Europe in 1952. Although isolated 43 years apart, the genomes were highly conserved (99.95% identical). Only 32 of the 159 MV predicted proteins revealed amino acid changes. Four genes (M009L, M036L, M135R, and M148R) in 6918 were disrupted by frameshift mutations.


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TEXT
 
Myxoma virus (MV), the causative agent of myxomatosis, belongs to the Leporipoxvirus genus of the Poxviridae family (9). Two distinct types of MV have been identified: South American MV, which circulates in Sylvilagus brasiliensis, and Californian MV, which circulates in Sylvilagus bachmani. Each virus is highly adapted to its host, causing a benign cutaneous fibroma at the site of inoculation. Both types of MV infect the European rabbit (Oryctolagus cuniculus), causing myxomatosis. The Californian strain MSW is more virulent for European rabbits than South American strains such as SLS or Lausanne (54). Another leporipoxvirus, Shope fibroma virus (SFV), is found in eastern North America in Sylvilagus floridanus. SFV protects European rabbits against myxomatosis (24), and it is routinely used as a vaccine.

One of the best-studied examples of host-virus coevolution is the use of MV for biological control of European rabbits (22, 23, 29). It is particularly unusual because the precise time the virus was released is known, and the original viruses are available for comparison with current strains. MV (the SLS strain) was deliberately released in Australia in 1950 and soon after (1952) in France (the Lausanne strain), whence it rapidly spread across Europe, and it has become endemic since then. For almost 60 years, a complex coevolution of host and virus has occurred, characterized by the emergence of attenuated viral strains and rabbits selected for resistance to MV (11, 12, 30).

The MV Lausanne strain and SFV have been completely sequenced (13, 61). MV encodes 171 genes, versus 165 encoded by SFV. The genetic information is highly conserved between the two viruses. Recently, preliminary sequencing of the MSW strain indicated that the major genomic differences with the Lausanne strain localize at the left terminal end of the MSW genome (31). In MSW, the terminal inverted repeats (TIRs) are extended, causing the duplication of five complete open reading frames (ORFs), which are present as a single copy near the right TIR in the Lausanne strain (9). To date, little molecular analysis concerning the adaptation of MV to its new host has been performed. Studies involving Australian field strains found small differences with reference to the SLS and Lausanne strains (49, 50), suggesting that adaptation (and the concomitant attenuation) of MV is not associated with major genetic changes such as large deletions. This finding is in contrast to what has been reported for attenuated poxviruses obtained by extensive cell culture passaging, which usually present substantial genomic deletions or rearrangements (5, 25, 36, 47, 48).

Strain 6918 is a naturally attenuated MV isolated in Spain in 1995 (7). It is therefore a descendant of the Lausanne strain recovered after 43 years of continuous evolution in the field. It has been used for the development of a "transmissible vaccine" intended to protect wild-rabbit populations against both myxomatosis and rabbit hemorrhagic disease virus (RHDV) in Spain, where the European rabbit plays a key role in the Mediterranean ecosystems (18). For this purpose, a recombinant virus, 6918VP60-T2, was constructed by inserting the capsid gene of RHDV into the genome of strain 6918 (4, 6, 7, 56, 57). The genomes of 6918 and 6918VP60-T2 have been sequenced. Here we report the results of our comparison of the genomic sequences of Lausanne and 6918. To our knowledge, this is the first comparative genomic analysis involving two poxvirus field strains linked by a clearly recorded lineage, one being fully virulent and the other virtually nonpathogenic. The results provide relevant insights into the mechanisms of MV attenuation that occurred as a consequence of the adaptation of the virus to its new host.

Genome sequencing. Viral genomic DNA was extracted from infected RK-13 cells as described previously (20). The DNA was used to generate 26 overlapping PCR amplicons of 6 to 7 kbp (Expand long template PCR system, Roche) spanning the MV genome. Amplicons were used for sequencing reactions, using ABI 3730xl automated DNA sequencers (PE Biosystems). Primers were designed to anneal at approximately every 500 bases across the templates. The final consensus DNA sequence represented an average fourfold redundancy. Sequence data were assembled by using the CAP3 program. All nucleotide changes with respect to Lausanne observed in 6918 were verified in a subsequent set of sequencing reactions. For comparative genomic analysis, we used software tools from the Viral Bioinformatics Resource Center (VBRC) (34, 35). Pairwise alignments were performed, using CLC Free Workbench 4.6 software (CLC bio).

Update on the genomic sequence of the Lausanne strain. The sequence analysis performed enabled the detection of what seem to be two sequencing errors in the published sequence of the Lausanne strain, affecting two conserved ORFs. ORF M069L encodes a protein phosphatase with dual tyrosine/serine specificity (13). According to the published genomic sequence, the predicted protein is 178-amino-acids (aa) long (13). However, a previous publication reported this ORF to be 172-aa long (45). The difference between the two sequences is a T deletion next to a TT stretch (nucleotides 66102 to 66103 of the Lausanne genome) in the sequence reported by Cameron et al. (13) with respect to that reported by Mossman et al. (45). The sequence of 6918 M069L encodes a predicted protein of 172 aa. This protein length is in better agreement with that of the corresponding orthologs across the different chordopoxvirus genera (data not shown). Furthermore, we sequenced the corresponding region from the Lausanne virus available at our lab and confirmed a predicted protein of 172 aa.

ORF M020L encodes a serine/threonine protein kinase (13). ORF M020L from 6918 encodes a predicted protein of 446 aa, one more than M020L from Lausanne. The difference between the two sequences is the insertion of a CTC codon (leucine) at position 52 in M020L from 6918. This additional leucine is conserved in the SFV ortholog (61). Furthermore, a blast search against the VBRC database indicated that this leucine residue is strictly conserved among orthologs of the different chordopoxvirus genera (data not shown). The corresponding region from the Lausanne isolate available at our lab was sequenced, and the result confirmed the presence of the additional leucine.

According to the above-mentioned findings, the full-length genome of the Lausanne strain might in fact be 4 nucleotides longer than the published sequence. We used this corrected version of the genome for the sequence comparison reported in this study.

Comparison of Lausanne and 6918 genome sequences. The genome of 6918 was determined to be 161,766-bp long, only 11 bp shorter than that of Lausanne (161,777 bp). All the 159 ORFs previously assigned for Lausanne (of which 12 are present in diploid in the TIRs, giving a total of 171 genes) were present in 6918, although four ORFs were severely disrupted (Table 1).


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  		TABLE 1. Comparison of ORFs of the MV Lausanne and 6918 strains

Comparison of the two strains on a nucleotide-by-nucleotide basis revealed only 73 differences, involving 82 nucleotides across a pairwise sequence alignment of 161,779 nucleotide positions (see the alignment in Fig. S1 and see Table S1 in the supplemental material), indicating that the two genomes are 99.95% identical. The differences consisted of 67 base substitutions, four deletions, and two insertions. Seventy-one of the changes were located within coding regions, and only two (two deletions of 1-bp each) were located at intergenic regions. The maximum number of changes per ORF observed was four independent mutations in ORF M036L.

Sixty-five of the 67 base substitutions were transitions, and only 2 were transversions. Twenty-two base substitutions caused synonymous codon changes, and the other 45 caused nonsynonymous codon changes. Of these, 21 resulted in conserved amino acid changes, 15 originated semiconserved changes, and 9 caused nonconserved changes.

Of the 159 different MV ORFs, 112 were identical in both strains and 15 presented only synonymous substitutions. Therefore, 127 of the 159 predicted viral proteins (79.9%) were identical in both strains. Twenty-eight ORFs (17.6%) presented nonsynonymous substitutions, of which 16 exhibited only conserved amino acid changes, 8 contained semiconserved changes, and 4 were affected by a single nonconserved amino acid change. Finally, four ORFs (2,5%), M009L, M036L, M135R, and M148R, were disrupted by frameshift mutations.

The distribution of the differences observed (Table 2) agreed with the general observation that the central portion of poxvirus genomes specify mainly conserved proteins essential for virus replication, whereas terminal regions encode for more-divergent proteins, including those modulating host range and virulence (43). In the MV genome, the central 124.5 kb (M012L to M142R) include the set of 90 genes which are present in all chordopoxviruses sequenced so far (26, 35, 60), while the terminal flanking 37.2 kb, 14.1 kb at the left (M000.5L to M011L) and 23.1 kb at the right (M143R to M000.5R), are enriched for genes specific to the Leporipoxvirus genus (13). The terminus-flanking regions, representing 23% of the genome, contained 41% of the total nucleotide changes and 40% of the single-nucleotide substitutions (Table 2). Interestingly, the mutational bias toward the terminus-flanking regions was even higher in the case of the nonsynonymous substitutions and nonconserved amino acid changes (Table 2). Similar results have been obtained in previous studies comparing poxvirus strains isolated at different times or at different geographic locations (1, 17, 21, 28, 41, 46, 51).


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  		TABLE 2. Distribution of nucleotide and amino acid changes between Lausanne and 6918 strains

Insertions and deletions. Figure 1 depicts the four deletions and two insertions detected in 6918 with respect to Lausanne. Two deletions of 1 bp each were located at the intergenic regions between ORFs M138L and M139R and ORFs M153R and M154L (Fig. 1A and B). The other two deletions and two insertions were located at ORFs M009L, M036L, M135R, and M148R. ORF M009L was affected by a 10-bp deletion within a sequence containing two 6-bp direct repeats (CATCGA) (Fig. 1C). This finding indicated that the deletion was originated by a homologous recombination event between the direct repeats of the type previously described (15, 19, 32, 50, 52, 62). The rest of the deletions and insertions involved slippery sequences consisting of stretches of 5 to 8 identical nucleotides (Fig. 1D to F).


Figure 1
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  		FIG. 1. Nucleotide insertions and deletions found in the genome sequence of the 6918 strain with respect to that of the Lausanne strain (Laus). The nucleotide changes are shown in boldface lowercase letters. A 6-bp direct repeat is underlined. The resultant amino acid changes in ORFs are indicated by shaded boldface italics.

Disrupted genes. The analysis performed suggested that the attenuated phenotype of 6918 potentially maps to the four disrupted genes.

The gene M135R (encoding a protein of 178 aa) was affected by a frameshift mutation (Table 1 and Fig. 1E), causing the gene to split into two putative ORFs, M135aR (encoding a protein of 40 aa) and M135bR (encoding a protein of 135 aa). M135R protein is a homolog to the protein encoded by the B19R gene of the vaccinia virus (VV) Copenhagen strain (B18R in the Western Reserve strain) (8). This VV protein is a soluble alpha/beta interferon (IFN-{alpha}/β) receptor that prevents IFN-{alpha}/β from triggering a host antiviral response (3, 16, 55). Recently, M135R has been shown to be a novel cell surface virulence factor of MV (10), and in contrast to its predicted properties, it does not interact with IFN-{alpha}/β. Pathogenesis studies with a targeted M135 knockout construct (vMyx135KO) demonstrated that the deletion of M135R severely attenuates MV pathogenesis, indicating that M135R is a critical virulence factor for myxomatosis (10). Interestingly, M135R is one of the seven MV genes absent in SFV (13, 61). It is noteworthy that the clinical symptoms induced by vMyx135KO (10) closely resemble those previously described for 6918 or the derived recombinant virus 6918VP60-T2 (6, 7, 56, 57), strongly suggesting that disruption of M135R is an important determinant of 6918 attenuation.

The gene M148R (encoding a protein of 675 aa) was affected by a frameshift mutation (Table 1 and Fig. 1F) originating two putative ORFs, M148aR (encoding a protein of 464 aa) and M148bR (encoding a protein of 171 aa). Although the functions of M148R remain to be elucidated, the M148R protein forms part of the ankyrin repeat-containing family (13). Ankyrin repeats are involved in protein-protein interactions (37, 42). Poxvirus ankyrin repeat proteins have been associated with host range functions, and they may inhibit virus-induced apoptosis (27, 44). Studies comparing genome sequences of virulent and cell culture-attenuated vaccine strains of different poxviruses have revealed that members of the ankyrin repeat family tend to be disproportionately affected by passage-specific mutations (5, 25, 32, 39, 47, 59). MV contains four members of the ankyrin repeat family, M005R/L, M148R, M149R, and M150R. SFV has counterparts for three of these ankyrin proteins but lacks a complete homolog of M150R (61), while MSW, the most virulent leporipoxvirus, has a partially duplicated M150R gene (31). The deletion of M005R/L or M150R from wild-type MV results in an almost complete loss of virulence (14, 44). M148R is the largest MV ankyrin-containing protein (77.4 kDa). It contains nine ankyrin repeats and is the only one for which transmembrane regions are predicted (13). It also contains a predicted F-box domain (46), which is present in the majority of poxvirus ankyrin repeat proteins (40). Overall, it seems likely that the disruption in the M148R gene plays a role in the attenuation of 6918.

The gene M009L (encoding a protein of 509 aa) was affected by a deletion causing a frameshift mutation (Table 1 and Fig. 1C) originating two putative ORFs, M009aL (encoding a protein of 416 aa) and M009bL (encoding a protein of 112 aa). M009L forms part of the kelch-like protein family (13), which has members in several poxvirus genera (2, 13, 33, 53, 58, 59). MV contains five members of the kelch-like protein family (M006L/R, M008L/R, M009L, M014L, and M140R), the functions of which remain to be elucidated. M009L is nonessential for virus replication in cell culture (38). Remarkably, the M009L gene is disrupted in the MSW strain (31), whereas it is partially duplicated in SFV (61). These data argue against M009L being a critical virulence factor in MV. Consequently, it is unclear if the truncation of M009L contributes to the attenuated phenotype of 6918.

Finally, the M036L gene (encoding a protein of 680 aa) was affected by a deletion causing a frameshift mutation (Table 1 and Fig. 1D) originating two putative ORFs, M036aL (encoding a protein of 535 aa) and M036bL (encoding a protein of 139 aa). The function of M036L is unknown. The only indication previously reported comes from an epidemiology study conducted in Australia (50). It was shown that certain field isolates contained a 98-bp deletion within ORF M036L. This mutation appeared to have no effect on virulence in laboratory rabbits (50). Consequently, it is unlikely that the truncation of M036L contributes to 6918 attenuation.

Genome sequence of 6918VP60-T2. Comparison of the 6918 and 6918VP60-T2 genomes (data not shown) indicated that the only difference corresponded to the presence of the RHDV-derived sequence inserted between ORFs M061R and M062R in 6918VP60-T2, as expected (6). Therefore, no uncontrolled recombination event had occurred during the generation of 6918VP60-T2, as has been reported for other recombinant MVs (38). Furthermore, no nucleotide changes had accumulated during the selection of the recombinant virus. This result was in agreement with our previous observations, indicating that both viruses exhibited indistinguishable biological features (6, 7, 56, 57).

It was of interest to analyze the stability of the most relevant genetic differences reported in this study after serial passages of the recombinant virus. For this analysis, 6918VP60-T2 was subjected to five serial passages in RK-13 cells and in inoculated rabbits. Subsequently, the sequence around 48 nucleotide changes, including the four deletions, the two insertions, and 42 nonsynonymous substitutions, was determined for the virus obtained from the last passage. The results (data not shown) demonstrated that the 48 nucleotide changes monitored persisted after passages in either cell cultures or rabbits. This finding was in agreement with previous analysis on the biological and genetic stabilities of 6918VP60-T2 (6, 56), further indicating that the attenuated nature of 6918VP60-T2 seems to be a stable trait. The molecular characterization of 6918 and 6918VP60-T2 is an important step toward the use of the recombinant virus as a vaccine against myxomatosis and RHD for wild-rabbit populations.

Nucleotide sequence accession numbers. Genomic sequences of 6918 and 6918VP60-T2 are available through GenBank (accession numbers EU552530 and EU552531) and two curated poxvirus sites, www.poxvirus.org/ and www.biovirus.org/.


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ACKNOWLEDGMENTS
 
This work was supported by a collaborative grant from INIA, Fundación para el Estudio y Defensa de la Naturaleza y la Caza, Fundación Biodiversidad, and Laboratorios Syva, S. A. (CC03-034).


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FOOTNOTES
 
* Corresponding author. Mailing address: Centro de Investigación en Sanidad Animal (CISA-INIA), Valdeolmos, 28130 Madrid, Spain. Phone: 34 91 620 23 00. Fax: 34 91 620 22 47. E-mail: barcena{at}inia.es Back

{triangledown} Published ahead of print on 17 December 2008. Back

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/. Back

§ Present address: Grupo de Biología y Toxicología Ambiental, Departamento de Física Matemática y de Fluidos, Facultad de Ciencias, Universidad Nacional de Educación a Distancia (UNED), C/ Senda del Rey 9, 28040 Madrid, Spain. Back

{ddagger} Present address: Departamento de Reproducción Animal, INIA, Ctra, de La Coruña Km 5.9, 28040 Madrid, Spain. Back

|| Present address: Scripps Research Institute, 5353 Parkside Drive, Jupiter, FL 33458. Back


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Journal of Virology, March 2009, p. 2397-2403, Vol. 83, No. 5
0022-538X/09/$08.00+0     doi:10.1128/JVI.02189-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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