- Open Access
Genetic characterization of a reptilian calicivirus (Cro1)
© Sandoval-Jaime et al.; licensee BioMed Central Ltd. 2012
Received: 15 August 2012
Accepted: 22 November 2012
Published: 29 November 2012
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.
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).
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.
The family Caliciviridae is a large group of small, non-enveloped RNA viruses that includes important human and animal pathogens . The family is comprised of five genera: Lagovirus, Vesivirus, Nebovirus, Sapovirus and Norovirus and two new genera have been proposed . Despite marked genetic and antigenic diversity, caliciviruses share several common features. All have icosahedral virions with a protein shell containing 180 copies of a major capsid protein, VP1 . The virions carry a positive-sense single-stranded RNA genome of approximately 6.4 to 8.5 kb in length. The 3’-end of the calicivirus RNA is polyadenylated, and the 5’-end is covalently linked to a small protein encoded by the virus genome, VPg . Calicivirus RNA genomes share similar organization; they are comprised either of two or three ORFs. The large ORF1 encodes the virus nonstructural proteins and is expressed from the genomic RNA template. In the genomes of sapo-, lago-, and neboviruses, the nonstructural region is fused to the gene encoding virus capsid protein, VP1. In the genomes of vesi- and noroviruses, the capsid protein is encoded by a separate ORF2, located towards the 3’-end of the virus genome. For all caliciviruses, the capsid proteins are produced from an abundant subgenomic RNA synthesized during virus replication. The same RNA serves as bicistronic template for the expression of a minor capsid protein, VP2. The ORF encoding VP2 is near the 3’-end of the virus genome and is conserved among caliciviruses .
The genus Vesivirus currently contains two approved species, Vesicular exanthema of swine virus (VESV) and Feline calicivirus (FCV), and a diverse group of unassigned, phylogenetically-related viruses . There are several well-recognized animal pathogens among vesiviruses that have been associated with a variety of disease conditions. These include diarrheal disease in dogs , respiratory illness, vesicular lesions, and epidemic hemorrhagic fever in cats [8, 9], and vesicular lesions in several other host species including swine, pinnipeds and humans [10–12]. The prototype virus of the genus Vesivirus, VESV, was originally isolated from pigs with clinical signs compatible with those caused by infection with foot-and-mouth disease virus (FMDV) . In 1972, a virus with morphological and biochemical characteristics indistinguishable from those of VESV was isolated on San Miguel Island, California from sea lions and named San Miguel Sea lion virus (SMSV) [13, 14]. When the experimental infection of pigs with SMSV resulted in a vesicular disease clinically mimicking FMDV and VESV infection, marine animals including ocean fish were retrospectively implicated to be the original source of VESV outbreaks [13, 14]. Since then, VESV, SMSV, and other related caliciviruses have frequently been designated as the “marine vesiviruses.” In contrast to FCV, which are considered to have restricted host specificity to cats of the family Felidae, the marine vesiviruses have been described as having an unusually broad host range [15–18].
In 1978–79, sixteen new vesivirus strains were isolated from four poikilothermic species (Aruba Island rattlesnake, Crotalus unicolor, Rock rattlesnake, Crotalus lepidus, Eyelash viper Bothrops schlegeli, Bell’s horned frog Ceretophyrs ornate) in a California zoological collection. The sixteen viruses were antigenically related and were not neutralized by the available VESV-like reference sera . The new viruses were proposed as members of a new reptilian caliciviruses (RCV) Crotalus 1 (Cro1) serotype . Sequence analysis of a 453 nt region of the Cro1 polymerase gene provided additional evidence for a new vesivirus group . The Cro1 serotype did not appear to be restricted geographically or temporally, or limited to reptile and amphibian hosts. In 1986–7, vesiviruses neutralized by the Cro1 typing serum were isolated from samples collected from three different marine mammals species (Eumetopias jubatus, Zalophus californianus californianus, and Callorhinus ursinus) along the coast of Oregon and California states .
In this study, we determined the full-length genome sequence of Cro1 strain 780032I, isolated from the intestine of a Rock rattlesnake (Crotalus lepidus) housed in the San Diego Zoo in 1978. Comparison of the genome sequence of 780032I with those available in GenBank database shows that this Cro1 virus represents a genetically distinct vesivirus strain within the species VESV.
Results and discussion
The vesivirus ORF1 encodes a nonstructural polyprotein that undergoes co-translational proteolytic processing during virus replication . A cascade of proteolytic events mediated by the virus-encoded proteinase gives rise to the virus mature nonstructural proteins and their intermediate forms. The Cro1 ORF1 encodes an 1880 amino acid nonstructural polyprotein with a predicted molecular mass of 209 kDa. Motif scanning [24, 25] and comparative sequence analysis of the Cro1 polyprotein revealed the presence of the characteristic NTPase (590GppgcGKT597), 3C-like proteinase (1303GDCG1306) and 3D-like RNA-dependent RNA polymerase (1653GLPSG1657 and 1701YGDD1704) catalytic motifs in an order conserved among caliciviruses. Alignment of the Cro1 ORF1 polyprotein sequence with that of another vesivirus, FCV, that had an experimentally established cleavage map  allowed prediction of the putative cleavage sites and, correspondingly, the sizes of mature nonstructural proteins. The predicted Cro1 polyprotein cleavage sites were conserved among VESV-related vesiviruses and, with the exception of cleavage site between NS3 and NS4 proteins, all carried a glutamic residue in the P1 position and alanine, serine or glycine residues at the position P1’ (Figure 2). Of interest, the NS3-NS4 scissile bond was predicted between glutamine and alanine residues. The order and sizes of the proteins defined by these cleavage sites were as follows: 16.2 kDa NS1 – 32.2 kDa NS2 – 39.6 kDa NS3NTPase – 31.2 kDa NS4 – 13.5 kDa NS5VPg – 76.5 kDa NS6-7ProPol.
The vesivirus ORF2 encodes a precursor of the virus capsid protein that is processed by cleavage during capsid protein maturation. Processing of the FCV capsid precursor at an E124-A125 dipeptide by virus NS6-7ProPol results in removal of an 124 aa-long N-terminal leader sequence (LC) and release of the 59–60 kDa mature form of the virus capsid protein, VP1 . The Cro1 ORF2 sequence is predicted to encode a precursor protein with a molecular mass of ~78 kDa. Alignment with the FCV capsid precursor sequence showed the presence of a putative scissile bond between 152E and 153S (Figure 2). It is likely that, similar to FCV, this cleavage site defines the border between the virus capsid protein leader sequence and mature VP1. Of interest, the site is conserved in the corresponding sequences of the recently characterized v810 and v1415 vesivirus strains isolated from Steller sea lions . The expression of the v810 and v1415 ORF2 sequences that were N-terminally truncated at this site resulted in production and self-assembly of virus-like particles morphologically and antigenically similar to virions .
The small ORF3 located at the 3’-end of the vesivirus genome encodes a minor component of virus capsids, the VP2 protein . The protein is expressed from the virus bicistronic subgenomic RNA and has been shown to be essential for the formation of infectious vesivirus virions . The substitution of a C for a T at nucleotide 8115 converted a stop codon (TAG) to a glutamine codon (CAG) resulting in a five codon-extension of the ORF3 compared to other VESV-related vesiviruses (data not shown). The 3’-end of the calicivirus RNA genome has been shown to play a crucial role in the initiation of virus replication [31, 32]. Nevertheless, modifications introduced into the genomes of feline calicivirus and murine norovirus showed that this region could tolerate a number of sequence changes while supporting virus replication [30, 32].
Percent nucleotide and amino acid identity of Cro1 strain 780032I with other versiviruses
The profiles of the genome plots generated in a comparison of VESV-, FCV- and CaCV-like vesivirus sequences as well as that for VESV-like sequences alone were similar. Consistent with the generally higher level of genetic divergence existing between vesiviruses from different groups, the similarity indexes calculated for the all-vesivirus sequence alignments were significantly lower. Interestingly, the comparative analysis showed that the NS1 and part of the NS4 sequence were not conserved when compared to the corresponding sequences of the FCV-like and CaCV-like viruses (Figure 3). Furthermore, predicted vesivirus NS1 gene sequences varied in length from 138 to 534 nt between these groups of viruses. For the Cro1, the nucleotide identity of the NS1 gene (predicted to be 148 aa long) was 29.7-38.5% with the corresponding sequences of CaCV-like viruses, and 51.6-52.2% with those of FCV-like viruses. The level of similarity for the NS1 deduced amino acid sequences was calculated to be 19.6-22.2% and 8.8% between Cro1 and CaCV-like and between Cro1 and FCV-like viruses, respectively. The function of the calicivirus NS1 protein is unknown and cannot be predicted based on the protein sequence since it does not show significant homology with any established functional sequence motifs. Moreover, the level of similarity of NS1 amino acid sequences between different calicivirus genera does not exceed that of random sequences, precluding identification of conserved sequence motifs.
The observed level of the NS4 sequence identity was significantly lower when the Cro1 sequence was aligned to those of FCV- and CaCV-like viruses. For the latter two, it ranged from 55.2 (Cro1 vs FCV) to 56.4% (Cro1 vs CaCV). In contrast, the lowest level of sequence identity among VESV-like virus NS4 genes was 86.4%. The similarity profile showed that the most variable sequences of the NS4 gene were located near the 5’-end (Figure 3). Similar to NS1, the NS4 gene is not conserved among viruses from different Caliciviridae genera and represents the second most variable region in the nonstructural ORF of their genomes. Nevertheless, all calicivirus NS4 proteins share a conserved structural feature, which is the presence of a hydrophobic domain. Of interest, a cluster of hydrophobic amino acid residues is located near the C-terminus of the Cro1 NS4 (MacVector’s Protein Analysis Toolbox), with amino acids 249–271 predicted to form a membrane-associated helix (TMpred server). Consistent with the putative role of this domain in membrane interactions, biochemical studies showed that transiently expressed NS4 behaved as an integral membrane protein . In addition, different forms of the NS4 were found to localize to the membrane-associated virus replication complexes in calicivirus infected cells [36, 37]. The significant sequence diversity suggests that NS4 might play an important role in determining the specificity of protein-membrane interactions in the host cell. For example, the subcellular localization of the norovirus NS4 is determined by an ER export signal motif (MERES) conserved only among the noroviruses . Moreover, the presence of the MERES motif was shown to be critical to the NS4 antagonist role in ER/Golgi trafficking [38, 39]. Of interest, computational analysis showed that the MERES motif was not present in the Cro1 sequence. In addition, scanning of the Cro1 NS4 sequence with software designed to identify putative signal and subcellular localization motifs (see Materials and Methods) found no known targeting sequences. The presence of such signals in the Cro1 NS4 protein remains to be established.
Another region of marked sequence variation was observed downstream from the ORF1-ORF2 junction. Plotcon analysis of the vesivirus sequences revealed a low level of nucleotide identity among the virus LC genes, with the lowest identity (30.8%) between the Cro1 and FCV65 viruses. Accordingly, a lower level of similarity was found for the compared deduced amino acid sequences of this protein, 30.1-30.7% for FCV-Cro1 and 39.4-43.1% for CaCV-Cro1 pairs. The function of this protein remains unknown; however, cleavage of the LC from the capsid precursor molecule was crucial for production of infectious virus particles .
Consistent with genetic distance analysis, the genomic sequence of the Cro1 780032I strain clustered together with those of the VESV-like viruses, making it a member of the marine vesivirus group (Figure 4). Within this cluster, the inferred phylogenetic relationships between RaV, SMSV1, VESV A48, PAN1, WCV and Steller sea lion v810 and v1415 strains were found to be similar to those reported earlier [17, 27, 40]. Of interest, when the phylogenetic tree was inferred for the virus subgenomic RNA sequences, the 780032I strain clustered together with the v810 strain. Similar clustering was observed when a set of the capsid sequences was extended to include two additional sequences available in GenBank, SMSV4 and SMSV17 (accession numbers M87482 and U52005, respectively). In the extended capsid tree, SMSV4 formed a separate group with VESV A48, while SMSV17 clustered with the 780032I, v810 and v1415 strains. However, the latter cluster showed a lack of strong statistical support, with the posterior probability value reaching only 0.68 (data not shown). Of note, the ORF1 and subgenomic sequences of the SMSV1 strain showed inconsistent grouping with those of RaV and WCV. The observed incongruence of the phylogenetic clustering of these viruses suggested a possible recombinant origin of the SMSV1 strain. Similarity plot analysis of the SMSV1 and other vesivirus genome sequences demonstrated an increased level of nucleotide sequence identity between the subgenomic regions of SMSV1 and RaV/WCV strains with a predicted putative recombination site near the junction of the SMSV1 ORF1 and ORF2 sequences (data not shown). However, our preliminary analysis could not identify the second parental strain or its probable lineage (data not shown). Confirmation of a possible recombination event will require further investigation.
Our finding that Cro1 strain 780032I (isolated from Rock rattlesnake) shares strong sequence identity with the VESV-like marine vesiviruses is consistent with reports that the Cro1 serotype has become established in both aquatic and terrestrial hosts [16, 19]. The original tissue samples from reptiles in the San Diego Zoo Cro1 outbreak of 1978 are no longer available for analysis, so it is impossible to investigate the outbreak retrospectively. Although the source of this Cro1 virus remains unknown, the continued genetic characterization of known viral isolates in concert with the increasing use of deep sequencing techniques for virus discovery may help track the origin and spread of this and other caliciviruses in nature.
Materials and methods
Sample isolation and virus amplification
Reptile tissue samples (small intestine, liver, kidney) were collected in 1978 from hatchling and breeding snakes in the San Diego Zoo. Snakes were experiencing high mortality rates thought to be associated with enteritis and hepatitis of an unknown cause . The tissue samples were homogenized and clarified by low-speed centrifugation. To amplify the virus, the corresponding supernatants were filtered through 0.22 μm polysulfone filters, and the resulting filtrates were added to Vero cell monolayers maintained in Eagle’s minimal essential medium that was supplemented with penicillin (200 units/ml), streptomycin (100 μg/ml), L-glutamine (2 mM) and 10% heat-inactivated fetal bovine serum. The inoculated cells were monitored until the appearance of visible CPE. When CPE in monolayers exceeded 90%, virus stocks were generated by collection of growth media and low speed clarification of supernatants. After viruses were plaque-purified three times, the amplification procedure was repeated and the clarified supernatants were aliquoted and stored at −80°C.
RNA extraction and RT-PCR sequencing
Viral RNA was extracted from virus stock samples with the RNeasy Mini Kit (Qiagen). The extracted RNA was used as a template for reverse transcription (RT) and PCR amplification of cDNA fragments. The RT-PCR reactions were performed using One-Step RT-PCR Kit (Invitrogen, Carlsbad, CA) and vesivirus genome-specific primer pairs. Briefly, following the initial RT reaction, 30 min at 45°C, a denaturation step at 94°C was performed for 2 min, followed by 40 cycles of 15 s at 94°C, 30 s at 50°C, and 3 min at 68°C. Amplified cDNA fragments were resolved by electrophoresis in 1.0% agarose gels containing ethidium bromide. The corresponding DNA bands were visualized with UV light and excised from the gel. The DNA was extracted with QIAquick Gel Extraction Kit (Qiagen) and subjected to nucleotide sequencing using the Big Dye Terminator v3.1 Cycle Sequencing Ready Reaction Kit and an automated sequencer, ABI 3100 (Applied Biosystems, Carlsbad, CA). Sequences of the primers that were employed for cDNA fragment amplification are given in Additional file 1: Table S1 and sequences of the primers used for genomic sequencing are available upon request. The sequences of the 5’-end regions of the virus genome were determined using 5’/3 RACE Kit 2nd Generation (Roche). The corresponding cDNA fragments were synthesized and amplified using 5’-end sequence-specific and anchor (5’-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTV-3’) primers according to protocol provided by the manufacturer. Following agarose-gel purification, fragments were subjected to direct sequence analysis as described above. Virus genome sequences were assembled using the Sequencher 4.9 program (Genecodes, Ann Arbor, MI).
Computer sequence analysis
Derived Cro1 nucleotide and amino acid sequences were analyzed, aligned and compared with vesivirus sequences available from the GenBank database using MacVector (MacVector, Inc., Cary, NC), EMBOSS  and Mega5  software packages. The GenBank accession numbers for complete vesivirus genome sequences were: Canine calicivirus (CaCV), AB070225; Vesicular exanthema of swine virus (VESV A48), U76874; San Miguel Sea Lion virus-1 (SMSV1), AF181081; Primate calicivirus 1 (Pan1), AF091736; Walrus calicivirus (WCV), AF321298; Rabbit vesivirus (RaV), AJ866991; Feline calicivirus F9 strain (FCVF9), M86379; Feline calicivirus F4 strain (FCVF4), D31836; Feline calicivirus F65 strain (FCVF65), AF109465; Feline calicivirus Urbana strain (FCVURB), L40021; Steller sea lion vesivirus-v810 (v810), EF193004; Steller sea lion vesivirus-v1415 (v1415), EF195384; Calicivirus isolate Allston 2008/US (ACV8), GQ475302; Calicivirus isolate Geel 2008/Belgium (GCV8), GQ475303; Calicivirus isolate Allston 2009/US (ACV9), GQ475301). The genome sequence of the 780032I strain was submitted to GenBank and assigned accession number JX047864.
A plot of average similarity for each set of the aligned vesivirus sequences was generated using the Plotcon program from the EMBOSS software package (http://emboss.open-bio.org/rel/rel6/apps/plotcon.html).
Scanning for the protein domains and motifs in the predicted protein sequences were performed using InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan). Signal peptide and subcellular localization motifs predictions were performed using iPSORT (http://hc.ims.u-tokyo.ac.jp/iPSORT), PSORTII (http://psort.nibb.ac.jp/form2.html), SOSUIsignal (http://bp.nuap.nagoya-u.ac.jp/sosui/sosuisignal/sosuisignal_submit.html), SIG-Pred (http://bmbpcu36.leeds.ac.uk/prot_analysis/Signal.html), Golgi Predictor (http://ccb.imb.uq.edu.au/golgi/golgi_predictor.shtml), PTS1 predictor (http://mendel.imp.ac.at/mendeljsp/sat/pts1/PTS1predictor.jsp), Predotar (http://urgi.versailles.inra.fr/predotar/predotar.html), and SignalIP (http://www.cbs.dtu.dk/services/SignalP). Prediction of the membrane associated domains was performed using TMpred Server (http://www.ch.embnet.org/software/TMPRED_form.html).
Bayesian inference of phylogeny was carried out with Mr.Bayes 3.1.2 software . The analysis was performed using Markov chain Monte Carlo sampling under a general time-reversible model of nucleotide substitution with a gamma distribution of rates and a proportion of invariant sites. The search was run for one million generations with tree sampling occurring every 100th generation. Bayesian posterior probabilities and tree topologies were calculated from the consensus of collected tree samples after excluding the first 25% trees as burn-in. The analyses were performed two times for every set of the sequences. The resulting trees were visualized with FigTree1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/).
Generation of multiple-cycle growth curve
Vero cell monolayers in six-well plates were infected with the 780032I strain at MOI=0.01, and virus titers were measured at different time points post infection. Briefly, virus stocks were diluted in a supplemented medium (see above), added to cells and allowed to adsorb for one hour at 37 °C. After the incubation, the inocula were removed, the infected cells were washed and fresh growth medium was added to each well. At 0, 8, 16, and 24 hpi, the plates were frozen and stored at −80 °C. All collected samples were freeze-thawed three times, and cell debris was removed by low speed centrifugation. Aliquots of the clarified supernatants were used to determine virus titers at each time point by plaque assay .
This research was supported by the Intramural Research Program of the NIH, NIAID.
We would like to thank Dr. David Matson at the Eastern Virginia Medical School, Norfolk, Virginia for providing virus samples.
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