Phylogenetic analysis of members of the Phycodnaviridae virus family using amplified fragments of the Major Capsid Protein gene

Department of Biology, University of Bergen, P.O. Box 7800, N-5020 Bergen, Norway

^* To whom correspondence should be addressed. Email: jens.larsen{at}bio.uib.no.

	Abstract

Algae viruses are considered ecological important by affecting host population dynamics and nutrient flow in aquatic foodwebs. Members of the family Phycodnaviridae are in addition interesting due to their extraordinary genome size. Few algal viruses in the Phycodnaviridae family have been sequenced, and those that are have few genes in common and low gene homology. It has hence been difficult to design general PCR primers that allow further studies of their ecology and diversity. In this study we screened the nine type I core genes of the Nuclear Cytoplasmatic Large DNA Viruses (NCLDV) for sequences suitable for designing a general set of primers. Sequence comparison between members of the Phycodnaviridae family, including three partly sequenced viruses infecting the Prymnesiophyte Pyramimonas orientalis, and the Haptophytes Phaeocystis pouchetii and Chrysochromulina ericina (Pyramimonas orientalis Virus 01B (PoV-01B), Phaeocystis phouchetii Virus 01 (PpV-01), and Chrysochromulina ericina Virus 01B (CeV-01B) respectively), revealed eight conserved regions in the Major Capsid Protein (mcp). Two of these regions also showed conservation at the nucleotide level and this allowed us to design degenerate PCR primers. The primers produced 347-518 bp amplicons when applied on lysate from algal viruses kept in culture and natural viral communities. The aim of this work was then to use the mcp as a proxy to infer phylogenetic relationships and genetic diversity among members of the phycodnaviridae family, and to determine the occurrence and diversity of this gene in natural viral communities. The results support the current legitimate genera in the Phycodnaviridae based on algae host species. However, while placing the Mimivirus in close proximity to the PBCV-1 type species of Phycodnaviridae along with the three new viruses assigned to the family (PoV-01B, PpV-01 and CeV-01B), the results also indicate that the Coccolithoviruses and Phaeoviruses are more diverged from this group. Phylogenetic analysis of amplicons from virus assemblages from Norwegian coastal waters as well as isolated algae viruses, revealed a cluster of viruses infecting members of the Prymnesio- and Prasinophyte algae families. Other distinct clusters were also identified, containing amplicons from this study as well as sequences retrived from the Sargasso Sea metagenome. This shows that closely related sequences of this family are present at geographical distant locations within the marine environment.