Detection, classification, genetic diversity and molecular evolution of algal viruses based on DNA polymerase genes

Chen, Feng, 1964-
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A polymerase chain reaction (PCR) technique with three highly degenerate primers (group-specific primers AVS1/AVS2 and nested primer POL) was developed to detect and amplify DNA polymerase genes (DNA pol) from 13 virus isolates which infect three genera of distantly related microalgae, Chlorella strains NC64A and Pbi, Micromonas pusilla and Chrysochromulina spp. Amplified DNA pol fragments were cloned and sequenced, and the genetic relatedness among algal viruses examined. Phylogenetic trees based on DNA pol sequences and hybridization of viral genomic DNA showed similar branching patterns. Genetic relatedness calculated from the hybridization and sequencing data showed a good concordance (r=0.90), indicating that DNA pol can be used to determine genetic relatedness and infer phylogenetic relationships among these viruses, and potentially among other organisms. It was found that nucleotide sequences of DNA pol share higher similarities among algal viruses than with other organisms, and viruses infecting the same species had higher sequence similarities than those infecting different species. Sequence analysis of inferred amino acid sequences of DNA pol from 24 double-stranded DNA viruses indicated that viruses which infect these microalgae form a distinct phyletic group, and are more closely related to herpes viruses than to other dsDNA viruses. The branching patterns of the phylogenetic tree corresponded well with groupings based on the International Committee on Taxonomy of Viruses. The study suggests that these algal viruses share a common ancestor and belong to the Phycodnaviridae. Nested PCR was also used to amplify DNA pol genes from natural virus communities concentrated by ultrafiltration and centrifugation, from the Gulf of Mexico. DNA pol genes from algal viruses were detected in 5 of 6 stations. The PCR products from an offshore station (station B11) were cloned, analyzed by restriction fragment length polymorphism (RFLP) and representative clones sequenced to examine the genetic diversity of algal viruses in nature. The five RFLP patterns or operational taxonomy units (OTUs) that were observed ranged from 9 to 34% of the 33 clones in the library. Based on sequence analysis four OTUs were closely related to the Micromonas viruses, whereas one OTU was an unknown algal virus