Global analysis of Chlorella variabilis NC64A mRNA profiles during the early phase of Paramecium bursaria chlorella virus-1 infection.

The PBCV-1/Chlorella variabilis NC64A system is a model for studies on interactions between viruses and algae. Here we present the first global analyses of algal host transcripts during the early stages of infection, prior to virus replication. During the course of the experiment stretching over 1 hour, about a third of the host genes displayed significant changes in normalized mRNA abundance that either increased or decreased compared to uninfected levels. The population of genes with significant transcriptional changes gradually increased until stabilizing at 40 minutes post infection. Functional categories including cytoplasmic ribosomal proteins, jasmonic acid biosynthesis and anaphase promoting complex/cyclosomes had a significant excess in upregulated genes, whereas spliceosomal snRNP complexes and the shikimate pathway had significantly more down-regulated genes, suggesting that these pathways were activated or shut-down in response to the virus infection. Lastly, we examined the expression of C. varibilis RNA polymerase subunits, as PBCV-1 transcription depends on host RNA polymerases. Two subunits were up-regulated, RPB10 and RPC34, suggesting that they may function to support virus transcription. These results highlight genes and pathways, as well as overall trends, for further refinement of our understanding of the changes that take place during the early stages of viral infection.

Deep RNA sequencing reveals hidden features and dynamics of early gene transcription in Paramecium bursaria chlorella virus 1.

Paramecium bursaria chlorella virus 1 (PBCV-1) is the prototype of the genus Chlorovirus (family Phycodnaviridae) that infects the unicellular, eukaryotic green alga Chlorella variabilis NC64A. The 331-kb PBCV-1 genome contains 416 major open reading frames. A mRNA-seq approach was used to analyze PBCV-1 transcriptomes at 6 progressive times during the first hour of infection. The alignment of 17 million reads to the PBCV-1 genome allowed the construction of single-base transcriptome maps. Significant transcription was detected for a subset of 50 viral genes as soon as 7 min after infection. By 20 min post infection (p.i.), transcripts were detected for most PBCV-1 genes and transcript levels continued to increase globally up to 60 min p.i., at which time 41% or the poly (A+)-containing RNAs in the infected cells mapped to the PBCV-1 genome. For some viral genes, the number of transcripts in the latter time points (20 to 60 min p.i.) was much higher than that of the most highly expressed host genes. RNA-seq data revealed putative polyadenylation signal sequences in PBCV-1 genes that were identical to the polyadenylation signal AAUAAA of green algae. Several transcripts have an RNA fragment excised. However, the frequency of excision and the resulting putative shortened protein products suggest that most of these excision events have no functional role but are probably the result of the activity of misled splicesomes.

Evaluation of higher plant virus resistance genes in the green alga, Chlorella variabilis NC64A, during the early phase of infection with Paramecium bursaria chlorella virus-1.

With growing industrial interest in algae plus their critical roles in aquatic systems, the need to understand the effects of algal pathogens is increasing. We examined a model algal host-virus system, Chlorella variabilis NC64A and virus, PBCV-1. C. variabilis encodes 375 homologs to genes involved in RNA silencing and in response to virus infection in higher plants. Illumina RNA-Seq data showed that 325 of these homologs were expressed in healthy and early PBCV-1 infected (≤60min) cells. For each of the RNA silencing genes to which homologs were found, mRNA transcripts were detected in healthy and infected cells. C. variabilis, like higher plants, may employ certain RNA silencing pathways to defend itself against virus infection. To our knowledge this is the first examination of RNA silencing genes in algae beyond core proteins, and the first analysis of their transcription during virus infection.

High abundances of cyanomyoviruses in marine ecosystems demonstrate ecological relevance.

The distribution of cyanomyoviruses was estimated using a quantitative PCR (qPCR) approach that targeted the g20 gene as a proxy for phage. Samples were collected spatially during a > 3000 km transect through the Sargasso Sea and temporally during a gyre-constrained phytoplankton bloom within the southern Pacific Ocean. Cyanomyovirus abundances were lower in the Sargasso Sea than in the southern Pacific Ocean, ranging from 2.75 × 10(3) to 5.15 × 10(4) mL(-1) and correlating with the abundance of their potential hosts (Prochlorococcus and Synechococcus). Cyanomyovirus abundance in the southern Pacific Ocean (east of New Zealand) followed Synechococcus host populations in the system: this included a decrease in g20 gene copies (from 4.3 × 10(5) to 9.6 × 10(3) mL(-1) ) following the demise of a Synechococcus bloom. When compared with direct counts of viruses, observations suggest that the cyanomyoviruses comprised 0.5 to >25% of the total virus community. We estimated daily lysis rates of 0.2-46% of the standing stock of Synechococcus in the Pacific Ocean compared with c. < 1.0% in the Sargasso Sea. In total, our observations confirm this family of viruses is abundant in marine systems and that they are an important source of cyanobacterial mortality.

Viral and bacterial abundance and production in the Western Pacific Ocean and the relation to other oceanic realms.

We completed a transect through the Western Pacific Warm Pool to examine how environmental variables may influence viral and bacterial abundance and production rates in this globally important oceanic region. Of the variables analyzed, viral abundance and production had the most significant relationship to bacterial cell abundance: viral parameters were not significantly correlated to the measured environmental variables, including temperature. Bacterial production rates were significantly correlated to temperature in open ocean waters, but not in waters close to land masses. Analyses of 16S rRNA gene by pyrosequencing indicated only minor changes in eubacterial community structure across the transect, with α-proteobacteria dominating all sampled populations. Diversity within the prokaryotic community did not correlate directly with viral abundance or activity. Comparisons to two other ocean-scale transects (> 8000 km of open ocean in total) in the Atlantic Ocean indicated that correlations between viral and bacterial abundance and production relative to environmental variables are regime dependent. In particular, correlations to temperature showed remarkable differences across the three transects. Collectively, our observations suggest that seemingly similar oceanic regions may have very different microbial community responses to environmental variables. Our observations and analyses demonstrate that ocean-scale generalizations may not apply in the case of viral ecology.

Application of the major capsid protein as a marker of the phylogenetic diversity of Emiliania huxleyi viruses.

Studies of the Phycodnaviridae have traditionally relied on the DNA polymerase (pol) gene as a biomarker. However, recent investigations have suggested that the major capsid protein (MCP) gene may be a reliable phylogenetic biomarker. We used MCP gene amplicons gathered across the North Atlantic to assess the diversity of Emiliania huxleyi-infecting Phycodnaviridae. Nucleotide sequences were examined across >6000 km of open ocean, with comparisons between concentrates of the virus-size fraction of seawater and of lysates generated by exposing host strains to these same virus concentrates. Analyses revealed that many sequences were only sampled once, while several were over-represented. Analyses also revealed nucleotide sequences distinct from previous coastal isolates. Examination of lysed cultures revealed a new richness in phylogeny, as MCP sequences previously unrepresented within the existing collection of E. huxleyi viruses (EhV) were associated with viruses lysing cultures. Sequences were compared with previously described EhV MCP sequences from the North Sea and a Norwegian Fjord, as well as from the Gulf of Maine. Principal component analysis indicates that location-specific distinctions exist despite the presence of sequences common across these environments. Overall, this investigation provides new sequence data and an assessment on the use of the MCP gene.

ISOLATION OF A NON-PHAGE-LIKE LYTIC VIRUS INFECTING AUREOCOCCUS ANOPHAGEFFERENS(1).

We have been working to characterize viruses that infect the HAB-forming pelagophyte Aureococcus anophagefferens Hargraves et Sieburth. Field samples were collected during brown-tide events in 2002 and tested for the presence of lytic agents. Here, we describe a recently isolated, lytic virus-like particle (VLP) that is morphologically similar to particles observed in thin sections of infected A. anophagefferens cells from natural samples. TEM and SEM have revealed VLPs consistent with the morphological characteristics of previously described Phycodnaviridae. Large icosahedral particles (∼140 nm) of similar shape and morphology dominate cell lysates and are accompanied by smaller phage-like particles and heterotrophic prokaryotes that appear to be incurable from our cultures. To determine which of these particles interacts with the Aureococcus cells, we preserved cultures during the early stage of infection so that SEM could be used to visualize those particles that attach to the surface of naïve cultures. SEM revealed that 63% of the large icosahedral-shaped particles attached to A. anophagefferens cells after only 30 min of exposure, while no significant frequency of attachment to the alga was observed for the phage-like particles. The results of these observations are in contrast to previous studies, where phage-like particles were reported to infect cells. When considered in conjunction with field observations, the results suggest that this newly isolated virus represents the dominant virus-morphotype associated with bloom collapse and termination.