Genetic characterization of a reptilian calicivirus (Cro1)

Abstract

Background: Vesiviruses in the family Caliciviridae infect a broad range of animal hosts including mammals, birds, fish, amphibians and reptiles. The vesivirus Cro1 strains were isolated from diseased snakes in the San Diego zoo in 1978 and reported as the first caliciviruses found in reptiles. The goal of this study was to characterize the Cro1 strain 780032I that was isolated in cell culture from a rock rattlesnake (Crotalus lepidus) in the original outbreak.

Results: We re-amplified the original virus stock in Vero cells, and determined its full-length genome sequence. The Cro1 genome is 8296 nucleotides (nt) in length and has a typical vesivirus organization, with three open reading frames (ORF), ORF1 (5643 nt), ORF2 (2121 nt), and ORF3 (348 nt) encoding a nonstructural polyprotein, the major capsid protein precursor, and a minor structural protein, respectively. Phylogenetic analysis of the full-length genome sequence revealed that the Cro1 virus clustered most closely with the VESV species of the genus Vesivirus, but was genetically distinct (82-83% identities with closest strains).

Conclusions: This is the first description of a full-length genome sequence from a reptile calicivirus (Cro1). The availability of the Cro1 genome sequence should facilitate investigation of the molecular mechanisms involved in Cro1 virus evolution and host range.

Figure 1

In vitro growth characterization of the 780032I strain isolated from Crotalus lepidus.A) The growth of the 780032I strain results in lysis of infected Vero cells and in the subsequent formation of plaques in a cell monolayer overlayed with 1% agarose containing growth medium. Plaques can be visualized with crystal violet staining after cells are fixed with formaldehyde. B) The virus titers of the 780032I strain produced by the Vero cells. The cell monolayers were infected with MOI=0.01 and virus titer was measured at different time points by plaque assay. The titers shown are expressed as the mean from two independent experiments performed in duplicate.

Figure 2

Schematic representation of the 780032I strain genome organization. The 780032I genome is comprised of three major ORFs. ORF1 encodes a nonstructural polyprotein, ORF2 – a precursor of the virus capsid protein, VP1, and ORF3 – a minor capsid protein, VP2. Mature virus proteins are shown as rectangular boxes with calculated protein molecular masses indicated below. The putative borders of the virus mature nonstructural proteins are indicated with predicted cleavage sites and arrows. The E¹⁵²/S site that is likely cleaved by the virus-encoded protease during capsid protein maturation is shown for the ORF2-encoded precursor. The 780032I predicted cleavage sites in the ORF1 were based on an alignment of VESV-like ORF1 polyprotein sequences with the established map of FCV [23].

Figure 3

Schematic representation of the vesivirus genome organization and the sequence conservation along the alignment of the full-length genome sequences. The Plotcon program was used for the sequence comparison of either the VESV-like viruses only (black color line) or all vesiviruses (grey color line). Similarity plots were generated with a sliding window of 200 nt. Each plotted point was an average of the position similarities within the window, and the position similarities were calculated using the algorithm described in plotcon ( http://emboss.open-bio.org/rel/rel6/apps/plotcon.html).

Figure 4

Phylogenetic relationship of the 780032I strain isolated from reptile with other vesiviruses. Phylogenetic trees for the alignments of the vesivirus full-length genome (A), ORF1 (B) and subgenomic RNA (C) sequences were inferred using Bayesian method (MrBayes 3.1.2) and parameters described in Materials and methods section. Bayesian clade probability values are shown next to the nodes.

Figure 5

Amino acid sequence variability of the VESV-like virus VP1 proteins.. A) Multiple sequence alignment of the VP1 P2 regions of the VESV-like viruses Asterisks indicate positions where differences in amino acid sequences between strains v810 and 780032I are observed. Structural elements such as β−strands and α−helices found in the structure of SMSV4 VP1 are depicted above the alignment as hollow box arrows and coils, respectively. B) Ribbon and surface representation of the SMSV4 VP1 protein and its virus capsid assembly inferred using the Chimera program. Color coding of the alignment, ribbon structure and virus capsid were generated using the ConSurf software and is based on variability scale from 0 to 100%, with the most variable residues colored dark purple.