Genetic Characterization of a Recombinant Myxoma Virus in the Iberian Hare (Lepus granatensis)

Abstract

Myxomatosis is a lethal disease in wild European and domestic rabbits (Oryctolagus cuniculus), which is caused by a Myxoma virus (MYXV) infection-a leporipoxvirus that is found naturally in some Sylvilagus rabbit species in South America and California. The introduction of MYXV into feral European rabbit populations of Australia and Europe, in the early 1950s, demonstrated the best-documented field example of host-virus coevolution, following a cross-species transmission. Recently, a new cross-species jump of MYXV has been suggested in both Great Britain and Spain, where European brown hares (Lepus europaeus) and Iberian hares (Lepus granatensis) were found dead with lesions consistent with those observed in myxomatosis. To investigate the possibility of a new cross-species transmission event by MYXV, tissue samples collected from a wild Iberian hare found dead in Spain (Toledo region) were analyzed and deep sequenced. Our results reported a new MYXV isolate (MYXV Toledo) in the tissues of this species. The genome of this new virus was found to encode three disruptive genes (M009L, M036L, and M152R) and a novel ~2.8 kb recombinant region, which resulted from an insertion of four novel poxviral genes towards the 3' end of the negative strand of its genome. From the open reading frames inserted into the MYXV Toledo virus, a new orthologue of a poxvirus host range gene family member was identified, which was related to the MYXV gene M064R. Overall, we confirmed the identity of a new MYXV isolate in Iberian hares, which, we hypothesized, was able to more effectively counteract the host defenses in hares and start an infectious process in this new host.

Keywords: Lepus granatensis; Myxoma virus; poxvirus; recombinant virus.

Figure 1

Iberian hare with myxomatosis-compatible lesions. (A) Blepharitis and conjunctivitis with seropurulent discharge. (B) Myxomas at the base of the left ear (arrows). (C) Severe acanthosis of the eyelid skin, with hyperkeratosis. (D) Ballooning degeneration of the epidermal cells, and intracytoplasmic eosinophilic inclusion bodies in the eyelid skin (arrowheads).

Figure 2

Representation of the aligned genome organization of both RFV-Kas (AF170722), MYXV-Lau (AF170726), and Myxoma virus-Toledo (MYXV-Tol) (MK836424). Blue ORF illustrations represent truncated genes; purple shows the location of M060R, M061R, M062R, M063R, M064R, and M065R genes in both MYXV virus; orange shows the M009L gene (intact in RFV-Kas, MYXV-Lau, and disrupted in MYXV-Tol) and shades of blue represent the new gene “cassette” identified in MYXV-Tol, which is highly likely derived from a recombinant event with an unsampled poxvirus.

Figure 3

Illustration of the “four gene cassette” sequences identified in the genomes of the representative capripoxviruses, cervidpoxviruses, suipoxviruses, yatapoxviruses, and three unclassified poxviruses (BeAn 58058 virus, cotia virus and eptesipoxvirus) that share similarity to the one found encoded on the negative strand of MYXV-Tol genome sequence.

Figure 4

Pairwise nucleotide identity matrix (upper image) and maximum-likelihood phylogenetic tree (model GTR+G+I) showing the relationships of the MYXV-Tol recombinant “four gene cassette” to similar sequences in the genomes of capripoxviruses, cervidpoxviruses, suipoxviruses, yatapoxviruses, and three unclassified poxviruses (BeAn 58058 virus, cotia virus, and eptesipoxvirus). Branches with aLRT support of 0.95 are indicated by black circles whereas branches exhibiting 0.9–0.95 and 0.8–0.9 aLRT supports are indicated by grey and white circles, respectively.

Figure 5

Pairwise amino acid identity matrix (upper image) and maximum-likelihood phylogenetic tree (model JTT+G), showing the relationships of the rPox-virion protein (highlighted in red) found in the recombinant region of MYXV-Tol and its homologous proteins, found in representative sequences (NCBI RefSeq) of poxvirus. Branches with aLRT support of 0.95 are indicated by black circles whereas branches exhibiting 0.9–0.95 and 0.8–0.9 are indicated by grey and white circles, respectively.

Figure 6

Pairwise amino acid identity matrix (upper image) and maximum-likelihood phylogenetic tree (model WAG+G+F), showing the relationships of the rPox-thymidine kinase (highlighted in red) found in the recombinant region of MYXV-Tol and its homologous proteins found in representative sequences (NCBI RefSeq) of poxvirus. Branches with aLRT support 0.95 are indicated with black circles, whereas branches exhibiting 0.9–0.95 and 0.8–0.9 are indicated with grey and white circles, respectively.

Figure 7

Pairwise amino acid identity matrix (upper image) and maximum-likelihood phylogenetic tree (JTT+G+F) showing the relationships of the rPox-host range protein (highlighted in red) found in the recombinant region of MYXV-Tol and its homologous proteins found in representative sequences (NCBI RefSeq) of poxvirus. Branches with aLRT support 0.95 are indicated by black circles whereas branches exhibiting 0.9–0.95 and 0.8–0.9 are indicated by grey and white circles, respectively.

Figure 8

Pairwise amino acid identity matrix (upper image) and maximum-likelihood phylogenetic tree (model JTT+G+F) showing the relationships of the rPox-poly(A) Pol subunit (highlighted in red), found in the recombinant region of MYXV-Tol, and its homologous proteins, found in the representative sequences (NCBI RefSeq) of poxvirus. Branches with aLRT support 0.95 are indicated by black circles whereas branches exhibiting 0.9–0.95 and 0.8–0.9 are indicated by grey and white circles, respectively.