Comparative genomic analysis of the family Iridoviridae: re-annotating and defining the core set of iridovirus genes

One objective of this project was to demonstrate the application of comparative genomics to annotating viral genomes, particularly those that have been poorly characterized experimentally. In an earlier study, we utilized comparative genomics to identify previously unannotated small viral ORFs in the Poxviridae [17]. Here, we focused our analysis on the Iridoviridae family, which represents a challenge in genome annotation since there is little experimental evidence available to confirm gene expression. Another problem is that iridovirus promoter elements have not been well characterized, and thus cannot be used as a reliable criterion for assigning ORFs. These combined factors made previous iridovirus gene annotation a somewhat arbitrary process, resulting in closely related iridovirus species with dramatic differences in their genomic annotations. Therefore, we decided to analyse all members of this family using a standardized comparative genomics approach, using the fact that ORFs that are conserved in more than one divergent species are likely to be functional genes.

Using this approach, a substantial number of ORFs were either added to, or deleted from members of the Megalocytivirus genus (Table 2). OSGIV and RBIV share 99% DNA sequence identity, and thus are probably different strains of the same virus; however, previous annotation described only 82 out of 118 total annotated ORFs shared by the two genomes [18, 19]. After our re-analysis, the RBIV and OSGIV genomes had an identical complement of annotated genes. Furthermore, this re-annotated ISKNV genome contained 110 ORFs orthologous with both RBIV and OSGIV (compared to 71 in the old annotation.) (Table 2) [18, 20].

In the process of re-examining these genomes, we annotated a number of genes containing apparent frameshift mutations between species. In RBIV we annotated ten genes with potential frameshift mutations, while OSGIV had four such genes (Table 2). All of the genes containing potential frameshift mutations had orthologs in the other two members of the Megalocytivirus genus (Table 2). In some cases, these mutations may be the result of natural mutations within the viruses; however, it is also possible that these apparent frameshift mutations are actually sequencing errors. For both RBIV and OSGIV, PCR primers based on the ISKNV sequence were used to amplify genomic fragments, which were subsequently sequenced [18, 19]. It is possible that errors were introduced during the PCR process, leading to apparent frameshifts in the reported sequence. It is interesting to note that the genomic sequence of ISKNV (sequenced using subcloned fragments rather than PCR products) [20], had significantly fewer annotation changes made during our re-analysis. Though we have not experimentally proven that the frameshift mutations in OSGIV and RBIV are the result of sequencing errors, it would be useful to focus future sequencing efforts on these regions, to determine if the reported sequences are indeed correct.

After re-annotating the Megalocytivirus genus, we applied the same comparative genomic analysis to the Ranavirus genus. The genus contains five sequenced members divided into two groups, each with a high degree of sequence conservation and a co-linear arrangement of genes. The first group is comprised of frog virus 3 (FV3), tiger frog virus (TFV), and Ambystoma tigrinum virus (ATV). The second group contains Singapore grouper iridovirus (SGIV) and grouper iridovirus (GIV).

The first step in the re-annotation of the Ranavirus genus was a comparative genomic analysis of FV3, TFV, and ATV. This resulted in an increase in the number of conserved annotated genes from 76 to 87 (Table 3). Subsequent re-analysis of the second Ranavirus group, containing SGIV and GIV, resulted in an increase from 131 to 138 conserved annotated ORFs (Table 4). It should be noted that two of the newly annotated ORFs, SGIV 0.5L and GIV 120.5L, appear to "wrap around", beginning at one end of the genome with the remainder of the ORF located at the opposite end [21, 22]. These apparent "split ORFs" are actually the result of the circularly permutated iridovirus genome being represented as a linear genomic sequence, when the arbitrarily chosen start point happens to fall in the middle of an ORF [23].