Isolation and Characterization of a Virus Infecting the Freshwater Algae Chrysochromulina Parva

Virology 486 (2015) 105–115

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Virology

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Isolation and characterization of a virus infecting the freshwater algae Chrysochromulina parva

S.F. Mirza a,1, M.A. Staniewski b,1, C.M. Short a, A.M. Long b, Y.V. Chaban a, S.M. Short a,b,n a Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada L5L 1C6 b Department of Ecology and Evolutionary Biology, University of Toronto, 25 Willcocks Street, Toronto, Ontario, Canada M5S 3B2 article info abstract

Article history: Water samples from Lake Ontario, Canada were tested for lytic activity against the freshwater haptophyte Received 4 August 2015 algae Chrysochromulina parva.Afilterable lytic agent was isolated and identified as a virus via trans- Returned to author for revisions mission electron microscopy and molecular methods. The virus, CpV-BQ1, is icosahedral, ca. 145 nm in 26 August 2015 diameter, assembled within the cytoplasm, and has a genome size of ca. 485 kb. Sequences obtained Accepted 8 September 2015 through PCR-amplification of DNA polymerase (polB) genes clustered among sequences from the family Available online 30 September 2015 Phycodnaviridae, whereas major capsid protein (MCP) sequences clustered among sequences from either Keywords: the Phycodnaviridae or Mimiviridae. Based on quantitative molecular assays, C. parva's abundance in Lake Algal virus Ontario was relatively stable, yet CpV-BQ1's abundance was variable suggesting complex virus-host NCLDV dynamics. This study demonstrates that CpV-BQ1 is a member of the proposed order Megavirales with Prymnesiovirus characteristics of both phycodnaviruses and mimiviruses indicating that, in addition to its complex Phycodnaviridae Mimiviridae ecological dynamics, it also has a complex evolutionary history. Haptophyta & 2015 Elsevier Inc. All rights reserved.

Introduction These newly isolated haptophyte viruses, like most algal virus isolates, have dsDNA genomes and are most closely related to Nagasaki and Bratbak (2010) recently noted that research on species within the Nucleocytoplasmic Large DNA Viruses (NCLDV), algal and protistan viruses has passed beyond an initial phase of a group first recognized by Iyer et al. (2001). The NCLDV is a discovery and each year many articles documenting their role in monophyletic group that contains the families Phycodnaviridae the environment, their biology and biochemistry, and their ecol- and Mimiviridae (Koonin and Yutin, 2010), and has recently been ogy are published. This astute comment was based on the fact that proposed as a new virus order, the “Megavirales” (Colson et al., more than 40 algal virus species have been isolated infecting at 2013). All dsDNA algal viruses are formally classified as genera in least 29 species of freshwater and marine algae belonging to 11 the Phycodnaviridae, or, for those that have not yet been classified, different classes (Brussaard and Martínez, 2008; Nagasaki and are most closely related to taxa within the Phycodnaviridae or Bratbak, 2010) within the eukaryote kingdoms Plantae and Chro- Mimiviridae. In fact, recognition that several types of algal viruses mista sensu Cavalier-Smith (2010). Nevertheless, this number of are most closely related to mimiviruses had led to a proposed virus taxa pales in comparison to the number of potential hosts expansion of the Mimiviridae (Yutin et al., 2013). Moreover, recent since recent conservative estimates suggest that more than 72,000 genome sequencing of a virus (AaV) that infects the brown-tide species of algae exist worldwide (Guiry, 2012). Thus, efforts to forming algae Aureococcus anophagefferens has highlighted the isolate novel algal viruses remain an important endeavor in complexity and challenges associated with classifying NCLDV aquatic virology, yet reports of newly discovered viruses have been because this particular virus encodes genes that are distinctly sporadic. Most recently, several viruses that infect multiple species mimivirus-like, yet it also encodes phycodnavirus-like genes of marine haptophyte phytoplankton were isolated (Johannessen (Moniruzzaman et al., 2014). Clearly, the rapid pace of discovery of et al., 2015), challenging past observations that most algal viruses new NCLDV species in recent years, especially from aquatic are specific for individual species, or even strains of algae (Short, environments, is driving ongoing revision of NCLDV classification 2012). schemes, and many issues related to the taxonomy of this remarkable group of giant viruses are not yet resolved. PCR-based studies of algal viruses in a variety of temperate n Corresponding author at: Department of Biology, University of Toronto freshwater environments have revealed the presence of DNA Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada L5L 1C6. E-mail address: [email protected] (S.M. Short). polymerase (polB) and major capsid protein (MCP) gene fragments 1 Authors contributed equally. related to both phycodnaviruses or mimiviruses demonstrating http://dx.doi.org/10.1016/j.virol.2015.09.005 0042-6822/& 2015 Elsevier Inc. All rights reserved. 106 S.F. Mirza et al. / Virology 486 (2015) 105–115 that these groups are diverse and ubiquitous in these environ- abundances of the lytic virus and its host, C. parva, with the aim to ments (Clasen and Suttle, 2009; Gimenes et al., 2012; Rozon and initiate ecological studies of this newly discovered algal virus. Short, 2013; Short et al., 2011; Short and Short, 2008; Zhong and Jacquet, 2014). Surprisingly, with the exception of a single gene fragment detected in a small pond in the city of Mississauga, Results Ontario, Canada, all PCR-amplified algal virus polB sequences have clustered among the cultivated Prasinovirus and Chlorovirus genera C. parva lysis of Phycodnaviridae that infect green algae (chlorophytes). Although this one exceptional gene fragment (clone UP.18sep09.01, GenBank Following the initial inoculation of C. parva cultures with accession # HM776037) was amplified from only a single pond 0.45 mm-filtered environmental water, several rounds of culture water sample, observation of this gene sequence provided the first inoculation with filtered lysates were performed to fulfill Koch's evidence that freshwater algal virus diversity extends beyond postulates and ensure that the observed lytic activity against C. viruses of chlorophytes (Short et al., 2011). Nevertheless, so far the parva was stable. Following these initial lysis experiments, a final only algal viruses that have been isolated and cultivated from 0.20 mm-filtered lysate was added to cultures which caused a freshwater environments infect the Chlorella-like algae Chlorella steady decline in C. parva concentration after 48 h. By 155 h post variabilis, Chlorella heliozoae, and Micratinium conductrix (Jean- lysate inoculation, cells were no longer detectable whereas control niard et al., 2013); the hosts of other freshwater algal viruses cultures grew normally to an abundance approaching 106 cells detected via molecular methods remain unknown. mL1 (Fig. 1). TEM thin-section images of samples collected Phylogenetic analysis of the unusual freshwater algal virus polB between 72 and 96 h after lysate addition confirmed infection and fragment, UP.18sep09.01, suggested that it was amplified from a lysis of C. parva cells by virus particles (Fig. 2). novel Prymnesiovirus since it is most closely related to polB sequences from marine viruses that infect prymnesiophyte (hap- Virus morphology and growth characteristics tophyte) algal species like Haptolina brevifila, basionym: Chryso- chromulina brevifilum (Edvardsen et al., 2011), and Phaeocystis TEM thin-section micrographs of samples collected at 72 and globosa (Short et al., 2011). To avoid confusion, it should be noted 96 h following lysate inoculation revealed the presence of that P. globosa is infected by at least two different types of NCLDV; numerous virus particles with hexagonal cross-sections within the group I PgVs have large genomes (460 kb) and are closely rela- cytoplasm of infected cells (Fig. 2B) as well as cells that were ted to mimiviruses, whereas group II PgVs have smaller genomes partially disintegrated (Fig. 2D). Though readily identifiable in (170 kb) and appear to be most closely related to phycodna- uninfected cells (Fig. 2A), an intact nucleus was never observed in viruses (Baudoux et al., 2006; Brussaard et al., 2004; Santini et al., any cell that contained these particles; at the magnification used 2013). The polB sequence detected in the Mississauga pond sam- to scan fields of view (3500x), there were roughly 20 cells per field ple, UP.18sep09.01, clustered among members of the Phycodna- and more than 200 fields of view were visualized. The particles viridae and is most closely related to group II PgVs. varied in size from 140 to 155 nm in diameter, with an average Past phycological records from nearby freshwater environ- diameter of 145 nm (Fig. 2C). As apparent in many intact infected ments such as Lake Ontario and the other Great Lakes (e.g., cells, virus particle assembly (capsid formation, nucleic acid Munawar and Munawar, 1982, 1986, 1996) were used to determine packaging, etc.) appears to occur around a clearly delineated potential hosts for the virus encoding the polB fragment region of the cytoplasm, and particles at various stages of forma- UP.18sep09.01. As noted in these phycological reports, the most tion can be readily observed around the periphery of this cyto- commonly observed haptophyte algae (classified as Chrysophy- plasmic region (Fig. 3). For example, partially formed capsids, ceae according to previous algal systematic conventions) in the closed capsids with an unstained core, and closed capsids with Great Lakes include species such as Chromulina sp., Chrysomonas darkly stained cores can be observed around a lightly-stained, sp., Chrysochromulina parva, Dinobryon sociale and Ochromonas sp. roughly spherical region of the cytoplasm (Fig. 3). Notably, C. parva was the most commonly observed prymnesio- An estimate of minimum burst size of infected cells was phyte as it contributed more than 1% of total algal biomass in the obtained by counting particles in TEM micrographs. At their most spring, summer, and fall in 1970, 1972, 1977, 1979 and 1983 in Lake numerous, 96 readily identifiable virus particles were observed in Ontario (Munawar and Munawar, 1996). Since C. parva is a fresh- a single cell (Fig. 2B). For an experimentally-derived estimate of water congener of the marine species C. brevifilum, and C. brevifi- burst size based on virus particle counts from epifluorescence lum is the host of the virus that encodes the closest polB relative of UP.18.sep09.01, C. parva was used for laboratory experiments to test natural water samples for lytic activity. High abundances of gene fragments presumably encoded by haptophyte (prymnesiophyte)-infecting viruses have been detected in water samples from the Bay of Quinte, an embayment of Lake Ontario (Rozon and Short, 2013), and therefore were used to challenge cultures of C. parva. This study describes the isolation and partial characterization of a lytic virus infecting C. parva. To determine the nature and taxonomic affiliation of the isolated virus, microscopic and molecular methods were used. Specifically, transmission electron microscopy (TEM) of infected C. parva cultures was used to examine the morphological properties of the virus and infected cells. Molecular studies included PCR amplification of polB and MCP gene fragments for phylogenetic analyzes, and pulsed-field Fig. 1. Growth curves of C. parva. Average C. parva abundances as determined by flow-cytometry were plotted against time. Sterile medium was added to ‘control’ gel electrophoresis for genome size determination. Finally, quan- cultures while 0.20 μm-filtered lysate was added to ‘lysate-added’ cultures. Error titative PCR (qPCR) assays were developed to estimate the natural bars represent standard deviations from triplicate flasks. S.F. Mirza et al. / Virology 486 (2015) 105–115 107

Fig. 2. Transmission electron micrographs of C. parva and its virus. Thin-section micrographs of C. parva cells show: (A) an uninfected cell at 96 h post lysate inoculation, (B) a cell filled with virus particles at 96 h post lysate inoculation, (C) a close up image of the virus particles in panel B with scale bars, and (D) a lysed cell at 72 h post lysate inoculation. Examples of various ultrastructural features are abbreviated as follows: n (nucleus), c (chloroplast), cv (contractile vacuole), f (flagellum), g (Golgi apparatus), m (mitochondrion), s (scales), v (virus particles). microscopy, virus particle concentrations at 48 and 72 h post PCR amplification, cloning and sequencing of polB gene frag- lysate addition were estimated at 3.70 106 and 2.73 107 parti- ments from filtered lysate yielded 10 sequences (GenBank acces- cles mL1, respectively. The abundance of C. parva at these times, sion #s KT318525-34), all of which were 499% identical with estimated from flow cytometry, was 7.75 105 and 4.00 105 cells respect to nucleotides. When a representative of these polB frag- mL1. Thus, the virus particle and cell count-based estimate of ments was compared to sequences in GenBank, it was most similar burst size was approximately 63 virus particles produced per cell. (83% identity over 680 nucleotides) to a putative Prymnesiovirus In an experiment to test if the virus particles were sensitive to sequence, UP.18sep09.01, that was previously observed in a chloroform, treatment of filtered viral lysate with chloroform freshwater pond (Short et al., 2011). The most similar polB resulted in a complete loss of infectivity with cells growing at a sequences from cultivated viruses were from viruses that infect rate similar to controls with chloroform-treated medium (Fig. S1). the haptophyte algae C. brevifilum (e.g., CbV-PW1) and P. globosa Although the cultures with chloroform-treated medium or lysate (e.g., PgV-03T), with 73% identity over 718 nucleotides and 72% did not grow as rapidly as cells in untreated medium, cell abun- identity over 679 nucleotides, respectively (Fig. 4). fi fi dances in these cultures continued to increase throughout the Ampli cation of MCP gene fragments from ltered lysate ulti- experiment. Meanwhile, in control cultures with untreated lysate mately yielded 31 sequences (GenBank accession #s KT318535-66) C. parva abundances decreased steadily after the second day of the consisting of 6 singleton sequences that were not more than 97% experiment, and cells were no longer detectable after 5 days. identical to any other, and 3 groups of sequences, or OTUs, each consisting of 5, 9, and 11 sequences more than 97% identical to each other. Phylogenetic analysis of the MCP fragments revealed Molecular characterization one OTU of 11 sequences clustering among sequences annotated as “major capsid protein”, “capsid protein”,or“hypothetical protein” PFGE analysis of DNA extracted from agarose plugs containing from the Phycodnaviridae, while all other OTUs and singleton concentrated material from pooled 0.2 mm-filtered lysates revealed sequences clustered among major capsid proteins, capsid proteins, two distinct bands, one approximately 485 kb in size as well as a or hypothetical proteins from the mimivirus-like viruses including much smaller band o48.5 kb that migrated further than the those that infect other haptophyte algae such as P. globosa (PgV) smallest band in the ladder (Fig. S2). For all PCRs performed to and Chrysochromulina ericina (CeV) (Fig. 5). amplify gene fragments from C. parva (actin) and its virus (polB Sequences were also obtained from three clones of PCR- and MCP), negative control lanes were clear, and bands of the amplified actin gene fragments from C. parva. With respect to expected sizes were observed for all experimental reactions their nucleotide sequences, these three fragments were 499% (i.e., 700 bp for polB, 400 and 500 bp for MCP, 800 bp for actin). identical to each other, and all nucleotide differences were 108 S.F. Mirza et al. / Virology 486 (2015) 105–115

500 nm

100 nm

Fig. 3. Transmission electron micrographs of cytoplasmic virus factories. Representative cells that show the cytoplasmic location for virus assembly were imaged from samples collected at either 72 h (panels A, C, and D) or 78 h (panel B) post lysate inoculation. The open arrowheads indicate partially assembled virions, while the closed arrowhead in panel B shows a capsid that appears to be partially packaged, and the closed arrowhead in panel C shows an apparently mature virion with a dense, darkly stained core surrounded by a lighter icosahedral capsid. restricted to the PCR primer regions. Based on BLAST searches in present at only low copy numbers at all three sites in July ranging GenBank, the C. parva actin sequence (accession # KT318535) was from 3 to 12 copies mL1; virus polB genes were not even detected most closely related to actin sequences from the haptophytes Emi- at the Belleville and Napanee sites on July 5, or Hay Bay on July 19. liania huxleyi CCMP1516 (e.g. accession # XM 005770879.1), and On the other hand, in August virus polB gene copy abundance Chrysochromulina sp. NIES-1333 (accession # DQ980418.1), (Fig. S3) increased dramatically, ranging from 58 to 69 copies mL1 at with 93% identity over 780 nucleotides for both. Belleville station, 601 to 5935 copies mL1 at Napanee and 1199 to 5057 copies mL1 at Hay Bay. Environmental abundance assays

Quantitative PCR assays targeting C. parva (actin) and CpV-BQ1 (polB) gene fragments were designed to estimate the environ- Discussion mental abundances of both this lytic virus and its host in an attempt to examine host–virus dynamics. For all qPCRs triplicate Altogether, algal growth curves, transmission electron micro- no-template control reactions resulted in no detectable amplifi- scopy, and molecular analysis of culture lysate provided incon- cation, and sample triplicate reactions had Ct standard deviations trovertible evidence that C. parva, a freshwater haptophyte, can be o fi fi 2 0.96 Ct. Ampli cation ef ciencies and r values from standards, infected by a new type of virus that is a member of the recently i.e., serially-diluted, cloned fragments of target DNA, were all proposed virus order, Megavirales. Following the conventions of the – fi 2 within acceptable ranges (93.6 109.0% ef ciency, and r values International Committee on Taxonomy of Viruses and incorporating from 0.995–0.999). For the purpose of analysis, and to ensure into its name the location from which it was isolated, the Bay of plate-to-plate consistency since not all samples were analyzed Quinte, this newly isolated virus will henceforth be referred to as within a single set of qPCR reactions, a single threshold fluores- Chrysochromulina parva virus-BQ1, or CpV-BQ1. Surprisingly, CpV- cence value and standard curve equation was applied to the ana- BQ1 is the first new type of freshwater algal virus isolated since the lysis of each series of reactions; the equations were Y¼3.406 * log(X)þ40.34 for C. parva actin, and Y¼3.281 * log(X)þ38.74 for Chlorovirus ATCV-1 was discovered more than a decade ago (Bubeck fi fi C. parva virus polB. and P tzner, 2005). Moreover, CpV-BQ1 is the rst non-chlorophyte C. parva actin gene-copy abundance in the Bay of Quinte was algal virus isolated from any freshwater environment. This report similar at all three sampling sites (Belleville, Napanee and Hay provides the first glimpse into the biology and ecology of a virus Bay) throughout July and August 2011, varying between 3 and 28 that can serve as a new model for studies of algal virus ecology and gene copies mL1 (Fig. 6). Similarly, C. parva virus polB genes were the evolutionary relationships among the NCLDV. S.F. Mirza et al. / Virology 486 (2015) 105–115 109

Fig. 4. Unrooted maximum likelihood phylogeny of NCLDV polB sequences. Sequence comparisons were based on inferred amino acid sequences. The dashed branch lines indicate sequences from viruses that are formally classiﬁed as, or are putative phycodnaviruses, while the solid branch lines indicate viruses belonging to the Mimiviridae as well as other unclassiﬁed NCLDV. GenBank accession numbers are listed beside virus common names or abbreviations, and the scale bar shows the number of substitutions per site.

C. parva growth and viral lysis be obtained. Nonetheless, estimates from epifluorescence microscopy and TEM micrographs of infected cells provided similar A particular challenge of growing C. parva in culture is its burst sizes of fewer than 100 particles produced per cell; it should relatively slow growth rate. Throughout several years of semi- be noted that the burst size estimate from changes in virus particle continuous cultivation, C. parva growth rates have never exceeded and cell abundances are conservative because the calculation did 1 0.2 d (i.e., doubling times of 5 days), and modifications to the not account for cell growth (i.e., division) during the experiment. cultivation regime such as gentle stirring, increased temperature, These low estimates place CpV-BQ1's burst sizes at the low end of or increased light have all been detrimental. For the lysis experi- the spectrum when compared to other NCLDV algal viruses that ment depicted in Fig. 1, the overall growth rate of cells in the typically range from 100 s to 1000 s of particles produced per lytic 1 control cultures was 0.18 d despite higher growth rates during event (reviewed in Brussaard and Martínez, 2008; Short, 2012). fi the rst day of the experiment. Nonetheless, lysis proceeded as Given the relatively slow growth rate of C. parva in culture as well fl fi expected in the experimental asks with added ltered lysate as observations that host physiological status can influence burst where, after the sixth day of the experiment, no viable cells could size (Bratbak et al., 1998), the low burst sizes observed during this be detected. On several occasions lysed cultures were left to study may reflect the actual biology and ecology of this virus-host incubate for up to 6 months and no regrowth was ever detected system. even when fresh medium was added. Furthermore, it appears that C. parva is susceptible to infection at any stage of growth as other lysis experiments have demonstrated that cultures can be lysed Virus morphology even when viruses are added after cultures reach stationary phase (data not shown). Thus, unlike other algal virus hosts that can be Thin section images of intact cells (Fig. 2A) revealed well- fi resistant to infection (e.g., Frada et al., 2008; Kimura and Tomaru, de ned organelles and morphological features characteristic of C. 2014; Thomas et al., 2011; Tomaru et al., 2009), there is no evi- parva such as a large central vacuole that is diagnostic for this dence that C. parva can resist infection by CpV-BQ1. species (Parke et al., 1962). Hence, it is apparent that the pre- Samples collected at 72, 78, and 92 h post inoculation with servation and preparation of thin sections did not lead to artifacts filtered lysate demonstrated that one-step growth of the virus was that could have distorted virus and host morphologies. Cross not achieved in this experiment; viruses were added at an MOI of sections through CpV-BQ1 virions revealed 145 nm diameter, approximately 5–10 based on titters estimated from similar lysis icosahedral particles (e.g., Fig. 2C) that are morphologically experiments. Cells with no signs of infection (e.g. Fig. 2A) as well indistinguishable from many other algal viruses within the Phy- as cells that had lysed (e.g. Fig. 2D) could be observed at all of codnaviridae. Moreover, its sensitivity to chloroform could suggest these time points. Unfortunately, this means that details such as that the CpV-BQ1 virion, like many other NCLDV such as mimi- the length of the virus's lytic cycle or the duration of the eclipse viruses, chloroviruses, and certain prymnesioviruses and prasino- phase could not be determined during this study. Moreover, this viruses (e.g. Brussaard and Martínez, 2008; Martinez et al., 2015; also meant that only crude estimates of CpV-BQ1's burst size could Mutsafi et al., 2014; Van Etten et al., 2002), may have a lipid 110 S.F. Mirza et al. / Virology 486 (2015) 105–115

Fig. 5. Unrooted maximum likelihood phylogeny of NCLDV major capsid protein (MCP) sequences. Sequence comparisons were based on inferred amino acid sequences. The dashed branch lines indicate sequences from viruses that are formally classified as, or are putative, phycodnaviruses, while the solid branch lines indicate viruses belonging to the Mimiviridae as well as other unclassified NCLDV. The symbols preceding virus names or abbreviations are provided to highlight multiple capsid protein sequences annotated from a single virus genome. CpV-BQ1 sequences are shown in bold followed by parentheses with the number of redundant sequences within an OTU (where appropriate) and GenBank Accession numbers. Similarly, for all other sequences the GenBank accession numbers are listed beside virus common names or abbreviations. The scale bar shows the number of substitutions per site. component. Overall, based on the morphology of the virion, CpV- that no intact nuclei were observed in any cell with obvious signs BQ1 appears to be a member of the NCLDV. of infection (i.e., visible intracellular particles or capsid-like Cultures inoculated with CpV-BQ1 completely lyse, and structures). This suggests that the degradation of the nucleus numerous partially disintegrated cells that had released viruses occurs in the early stages of infection, and may indicate that, like into the surrounding medium were observed in TEM micrographs Mimiviruses and Poxviruses (Mutsafi et al., 2014), CpV-BQ1 (e.g., Fig. 2D). Hence, it is apparent that the release of CpV-BQ1 replicates entirely in the cytoplasm. However, evidence for or virions occurs as a result of cell lysis rather than by budding. At the against an entirely cytoplasmic replication cycle is limited so it is other end of the viral life cycle, it was not clear from this study premature to draw any firm conclusions about CpV-BQ1's repli- how the virus enters cells. While some algal viruses such as the cation. Clearly, additional microscopic investigations will be nee- chloroviruses inject only their genome into their hosts leaving ded to address these basic questions about CpV-BQ1's cellular empty capsids on the cell surface (Yamada et al., 2006), other algal entry and replication. viruses such as Emiliania huxleyi virus 86 (Mackinder et al., 2009) On the other hand, it is clear from numerous TEM images that as well as many other NCLDV like Mimivirus enter their hosts as virus assembly takes place within the cytoplasm, a characteristic intact particles via phagocytosis (Mutsafi et al., 2014). Although typical for NCLDV. Many infected cells revealed the same cellular many cells were observed at various stages of infection including morphology: intact capsids assembling around a distinct region of apparently uninfected cells (Fig. 2A), cells with only a few visible the cytoplasm suggesting the formation of a viroplasm, or virus intracellular virus particles (not shown), cells with numerous factory (Fig. 3). Because partially formed capsids and empty cap- intracellular particles (Fig. 2B), and lysed cells (Fig. 2D), no virus sids were generally observed closer to the center of the viroplasm particles were observed on the cell surface, or within phagocytic relative to darkly stained and presumably mature virions, the vacuoles. Hence, the mechanism by which the CpV-BQ1 genome viroplasm appears to be the site of virus assembly and viruses gains entry into the host cell remains unknown. It is also notable move away from the core of the virus factory as they mature. S.F. Mirza et al. / Virology 486 (2015) 105–115 111

despite arguments that microbial transitions between marine and freshwater environments are rare (Logares et al., 2009). Surpris- ingly, the CpV-BQ1 polB sequence was only 83% identical to the environmental sequence, UP.18sep09.01, that originally stimulated these efforts to isolate a novel freshwater prymnesiovirus. Hence, there is compelling evidence for the existence of other types of freshwater prymnesioviruses that infect C. parva or hosts that have not yet been identified. Whereas only a single polB sequence was obtained from CpV- BQ1 lysates and phylogenetic analysis of that sequence suggests that CpV-BQ1 should be classified as a member of the Phycodna- viridae, results from the analysis of CpV-BQ1's MCP sequences were less conclusive. Multiple sequences were obtained via amplification with universal primers that amplify MCP gene fragments from a variety of algal viruses including phycodnaviruses and others that are, based on their polB sequences, more closely related to mimiviruses (Larsen et al., 2008). Moreover, while most CpV-BQ1 MCP sequences clustered among mimiviruses and mimivirus-like algal viruses such as the group I PgVs, CeV, and PoV, one CpV-BQ1 OTU (CpV-BQ1.bL1.02) clustered among MCP genes from the Phycodnaviridae genera Chlorovirus and Prasinovirus (Fig. 5). There are several possible explanations for the observation of multiple MCP sequences amplified from CpV-BQ1 lysate. First, it is possible that CpV-BQ1 is not a clonal virus so the different MCP gene fragments could have been amplified from different strains that coexisted in the lysate. That said, only one polB sequence was obtained from the same CpV-BQ1 lysate sample that was used for MCP amplification, and it is unlikely that different strains of CpV would encode identical polB sequences especially considering that closely related strains of other algal viruses such as phycodnaviruses that infect the prasi- nophytes Ostreococcus, Bathyococcus, and Micromonas (e.g., Bellec et al., 2014, 2009), and the prymnesiophyte Chrysochromulina brevifilum (Chen and Suttle, 1996) encode similar but non-identical polB genes. Alternatively, it is plausible that CpV-BQ1 encodes multiple capsid proteins that are paralogs, as for example has been observed in the chlorovirus PBCV-1 (Dunigan et al., 2012). The

Fig. 6. Average environmental abundances of C. parva (actin) and CpV-BQ1 (polB) facts that the PCR primers that were used to amplify these gene gene fragments. Gene abundances are shown for samples collected on the dates fragments were highly degenerate, and that all of the algal viruses indicated at three sites in the Bay of Quinte: Belleville, Napanee, and Hay Bay. ND that have been fully sequenced encode multiple sequences anno- indicates that no gene copies were detected in a particular sample, and error bars tated as putative major capsid proteins, or capsid proteins lends represent standard deviations from triplicate qPCR reactions. credibility to this argument; for the phylogenetic tree shown in Fig. 5, CpV-BQ1 capsid protein sequences were compared to the Although the virus factories observed in other NCLDV such as top 250 BLAST hits in GenBank, and all non-redundant sequences Mimivirus often appear as more densely-stained, clearly defined from the same NCLDV were included in the phylogeny. A final regions within the cytoplasm (Zauberman et al., 2008) compared possible explanation for the presence of multiple MCP sequences to the virus factories evident in the micrographs shown in Fig. 3, may be due to PCR or sequencing artefacts. Although the program CpV-BQ1's morphology of virus assembly occurring within a virus Bellerophon (Huber et al., 2004) did not identify any of the fi factory is typical for many NCLDV (Mutsa et al., 2014). sequences as chimeric, other artifacts cannot be ruled out since several singleton sequences were obtained that were closely Molecular characterization related and, due to low amplification yields and faint products from the first round of PCR, two rounds of PCR were required to Along with the morphological characteristics noted in the obtain enough PCR products for successful ligation and transfor- preceding section, the molecular characteristics of CpV-BQ1 also mation. It is certainly possible that the high number of PCR cycles indicate that it is a bona-fide member of the NCLDV, or Mega- could introduce errors, however, it is very unlikely that this would virales. The polB sequence obtained from CpV-BQ1 via PCR produce such distinct OTUs as those that clustered with very dif- amplification using algal-virus-specific primers clustered within ferent types of NCLDV. Presently, it cannot be resolved if CpV-BQ1 the Phycodnaviridae, one of the formally recognized families encodes multiple capsid proteins, or if it actually represents a within the Megavirales (Colson et al., 2013). In fact, the CpV-BQ1 consortium of closely related virus strains. Additional end-point polB sequence was most closely related to sequences from prym- dilution experiments and sequencing, or whole genome sequen- nesioviruses such as PgV-03T and CbV-PW1 that infect marine cing will eventually resolve these questions about the source of haptophyte algal species (Fig. 4). This observation bolsters the the multiple MCP sequences obtained in this study. argument for host–virus coevolution among this group of viruses Whatever the case, it is important to note that PCR amplifica- (Brussaard et al., 2004) suggesting that this co-evolutionary his- tion from a CpV-BQ1 lysate generated at least one OTU of redun- tory transcends the boundaries of marine and freshwater algae dant MCP sequences that clustered with the Phycodnaviridae as 112 S.F. Mirza et al. / Virology 486 (2015) 105–115 well as other OTUs and singleton sequences that clustered with In the few samples that were available for this analysis, C. parva the Mimiviridae (Fig. 5). This observation, combined with the genes were present at only low abundances (i.e. o100 copies per relatively large size of CpV-BQ1's genome (485 kb) that places it at mL 1), yet were relatively consistent across the sample sites and the high end of genomes within the Phycodnaviridae, or the low throughout the two months that were sampled. On the other end of the Mimiviridae, may indicate that CpV-BQ1 represents an hand, the abundance of CpV-BQ1 polB genes was much more evolutionary link between these families within the emerging variable with low or even undetectable numbers during the month virus order Megavirales. While this is an interesting though of July, but considerably higher numbers in August (Fig. 6). Inter- potentially controversial conclusion, it is not without precedence. estingly, the patterns of abundance observed for CpV-BQ1 were For example, Monier et al. (2008) presented evidence of a close similar to patterns observed for other virus genes previously phylogenetic relationship between the polB sequences of three quantified in the same samples (Rozon and Short, 2013). For algal viruses and that of Mimivirus, stating that there appears to be example, CpV-BQ1 gene abundances in the Bay of Quinte were 1 to a continuum between the viruses with respect to the sequence of 2 orders of magnitude lower in samples from the head of the bay this enzyme. They too suggested that the large genome sizes of the (Belleville) compared to samples collected closer to the mouth of three algal viruses may serve as another indicator of their close the embayment (Napanee and Hay Bay). Further, like CpV-BQ1, the relationship with Mimivirus. Further, recent genome sequencing of abundance of a virus gene identified as a mimivirus-like prym- the Aureococcus anophagefferens virus (AaV) demonstrated that it nesiovirus (Mimivirus-Prym 399), fluctuated from below detection 3 1 encodes genes which are exclusive to the Phycodnaviridae as well to ca. 10 copies mL during the months of July and August as others that are associated with the Mimiviridae, providing (Rozon and Short, 2013). Altogether, these data suggest that there compelling evidence that these families share a common ancestor is a complex community of prymnesiophyte viruses and prym- (Moniruzzaman et al., 2014). nesiophytes (i.e. haptophytes) in the Bay of Quinte. Furthermore, fl An interesting tangential observation related to estimating the much more dramatic uctuations in CpV-BQ1 gene abun- CpV-BQ1's genome size was the presence of the smaller o48.5 kb dances compared to C. parva hints at the ecological complexity of – DNA fragment in the PFGE gel (Fig. S2). Since this DNA fragment this host parasite system. It is possible that the observed migrated further than the smallest size standard, its size could not dynamics were due to the interactions of multiple strains of fi be accurately determined; extrapolation from the size standard phytoplankton and viruses as was rst observed by Waterbury and suggested that it was between 24 and 36 kb. Currently, the source Valois (1993), and later modeled by Rodriguez-Brito et al. (2010). and identity of this smaller fragment is unknown, but there are at Moreover, it is also possible that there are multiple species of hosts least two possibilities. First, C. parva was not grown axenically so for CpV-BQ1 that cannot be detected using our actin-based qPCR heterotrophic bacteria in the cultures could have supported the assay, especially considering the recent isolation of marine haptophyte viruses with multiple hosts (Johannessen et al., 2015). A growth of bacteriophage. Because most phage genomes range much more thorough, focused study of the dynamics of C. parva from 20 to 50 kb (Hatfull, 2008), it is plausible that the small and CpV-BQ1 in natural environments is necessary to fully fragment in the PFGE gel represents a phage genome. Alter- understand their apparently complex interactions. The isolation of natively, some NCLDV such as Mimivirus, CroV, and PgV-16T are this virus, and the availability of this new virus-host couple for known to be parasitized by the virophages Sputnik, Mavirus, and laboratory experimentation will undoubtedly provide opportu- PgVV, respectively (La Scola et al., 2008; Fischer and Suttle, 2011; nities for research on the role of viruses in algal ecology and Santini et al., 2013). Further, a related virophage, OLV, was also aquatic primary production. observed in environmental viral metagenomes (Yau et al., 2011). All of these virophages genomes were within the range of 18 (e.g. Sputnik) to 26 kb (e.g. OLV) and provide another possible source Conclusions for the small fragment in the PFGE gel. It is noteworthy that no phage or virophage particles were observed in the TEM micro- This study provides the first evidence for freshwater viruses graphs, but the relatively slow speed of the centrifugation used to that infect eukaryotic algae other than Chlorophytes. Because the prepare samples for sectioning may have excluded such particles. virus isolate described herein, CpV-BQ1, has only been partially Thus, the source of the smaller DNA fragment in the PFGE could characterized and its genome has not yet been sequenced, it not be determined, but is worth further investigation. Whatever cannot be classified with absolute certainty. Nonetheless, the the case, the morphological and molecular features of CpV-BQ1 characteristics of CpV-BQ1 that were determined including its that were observed in this study demonstrate that it certainly capsid size and morphology, cytoplasmic site for replication, large belongs to the NCLDV or Megavirales. genome size, and gene phylogenies allow it to be tentatively classified within the proposed order Megavirales. Although it is Virus-host ecology reasonable to speculate that, based on its polB sequence, CpV-BQ1 should be further classified within the Phycodnaviridae, its rela- Quantitative PCR analysis of C. parva and CpV-BQ1 genes in tively large genome size and several of its MCP sequences suggest Lake Ontario provided preliminary data related to the ecological mimivirus ancestry. Perhaps viruses within the sister families dynamics of this host–virus system. This data, while interesting, Phycodnaviridae and Mimiviridae represent an evolutionary con- must be interpreted cautiously. First, at the time of this study the tinuum with ongoing genetic exchange between individual taxa relationship between C. parva cell abundance and actin gene confounding current classification schemes. No matter the taxo- copies has not been resolved so gene copies should only be con- nomic identity of CpV-BQ1, this first glimpse into its ecology sidered a relative indicator of C. parva abundance. A similar caveat suggests virus–host co-existence, or perhaps additional undis- must also be considered for the relationship between CpV-BQ1 covered hosts. While multiple host species would indicate a pro- infectious titers and polB gene abundance. Second, there were only found ecological role for CpV-BQ1, the fact that its known host, C. a few samples available for simultaneous extraction of viral and parva, is an abundant and ubiquitous primary producer in many cellular nucleic acids so virus and host gene abundances were temperate freshwater environments also suggests that CpV has a monitored at a relatively crude temporal scale. These issues should major role in freshwater food webs. Ultimately, the cultivation of be relatively straightforward to resolve with future studies focused this virus–host pair has enabled new opportunities for research on on algal virus dynamics in natural environments. algal virus ecology and evolution. S.F. Mirza et al. / Virology 486 (2015) 105–115 113

Material and methods acid stain (Life Technologies), and SlowFade Gold Antifade, (Life Technologies). Epifluorescence microscopy was performed using Virus isolation and imaging an Eclipse 50i microscope (Nikon), 100 oil-emulsion objective, and blue light excitation. The observed increase in virus particle C. parva (strain CCMP 291) was obtained from the National abundance was divided by the absolute value of the corresponding Center for Marine Algae and Microbiota (East Boothbay, Maine, decrease in cell abundance to provide an estimate of burst-size. USA), and grown at a constant temperature of 15 °C, with a 12 h light-dark cycle at approximately 23 mEm2 s1 in DY-V medium Molecular characterization (Andersen, 2005). The original natural water sample that caused lysis of C. parva cultures was a 0–3 m depth-integrated sample The virus's genome size was estimated using pulsed-field gel collected from the Bay of Quinte near Belleville, ON, Canada (44° electrophoresis (PFGE) following the approach of Sandaa et al. 09.2200 N, 77° 20.7330 W) on Aug 30, 2011. After filtration through (2001) with minor changes. 1080 mL of 0.20 mm syringe-filtered a 47 mm diameter, 0.45 mm pore-size PVDF Durapores membrane lysate was obtained and was concentrated via ultracentrifugation filter (EMD Millipore), addition of 0.5 mL of this filtered water using a SW32Ti rotor as previously described (Short et al., 2011). sample caused four, 4 mL C. parva cultures to clear relative to After centrifugation, the pelleted material was resuspended in control cultures. The cleared cultures (i.e. lysates) were combined 10 mM Tris-Cl (pH 8.5), pooled, and stored at 4 °C. Agarose plugs and filtered through a 0.45 mm pore-size syringe filter (Millexs-HV were formed by gently mixing equal volumes of concentrated Durapores PVDF, EMD Millipore), and 2 mL of the filtered lysate lysate and molten 1.5% Megabase agarose (Bio-Rad) at 50 °C. Fol- was inoculated into duplicate 100 mL C. parva cultures. These lowing incubation for 30 min at 37 °C in 30 U of RNase-free DNase cultures also cleared within 6 days. This process was repeated I (Qiagen), the plugs were digested in 250 mM EDTA, 1% SDS, and three times and in every case the C. parva cultures lysed. The final 1 mg/mL proteinase K overnight in the dark at 30 °C, and were lysate was filtered through a 0.20 mm syringe filter (DISMIC-25cs washed four times in TE buffer (10 mM Tris, 1 mM EDTA). Virus cellulose acetate, Advantec-MFS). All filtrates/lysates were stored plugs as well as a concatenated λ DNA size standard (CHEF 48.5– at 4 °C. 1000 kb, Bio-Rad) were loaded in a 1% Certified Megabase agarose To monitor the lysis of C. parva and generate samples for gel (Bio-Rad) in 1 TBE (45 mM Tris–borate and 1 mM EDTA, pH transmission electron microscopy (TEM), 15 mL of 0.20 mm-filtered 8.0), electrophoresis was conducted following the parameters in lysate was added to 160 mL mid-log phase cultures. 1.9 mL sam- Sandaa et al. (2001), the gel was stained with 1 SYBR-Gold ples were collected from each flask at various time points from nucleic acid stain (Life Technologies) in 1 TBE, and then imaged. 0 to 155 h. Each sample was fixed with Grade I, 50% glutar- PCRs were performed using primers that target Phycodnavir- aldehyde (Sigma-Aldrich, final concentration 2.5%) followed by idae polB (Chen and Suttle, 1995) and major capsid protein (MCP) gentle mixing, incubation at 4 °C for 30 min, and flash freezing in (Larsen et al., 2008) gene fragments to obtain sequence informa- liquid nitrogen. These samples were stored at 80 °C, until they tion from filtered lysate, whereas primers that target eukaryotic were thawed on ice for cell counts on a Cell Lab Quanta™ SC actin genes (Yoon et al., 2008) were used to obtain sequence (Beckman Coulter) flow cytometer triggering on red fluorescence information from C. parva DNA. For PCRs of virus genes, the tem- at an event rate between 150 and 350 s1. plate was 0.45 μm-filtered, ultracentrifuge-concentrated lysate (as From the same cultures used to monitor cell lysis (see above), described above for PFGE) that was freeze-thaw treated as pre- 4 mL samples were collected and pooled from the lysate-treated viously described (Short and Short, 2008). Template for C. parva flasks at 72, 78 and 96 h post inoculation. These samples were PCR was DNA extracted using a CTAB (Cetyltrimethyl ammonium fixed with 50% Grade I glutaraldehyde (Sigma-Aldrich) to a final bromide)-based approach. First, C. parva cells were centrifuged at concentration of 1% and then incubated at 4 °C for 15 min. Fixed 3220xg for 30 min at 24 °C, and pellets were resuspended in samples were concentrated via centrifugation at 6000 g for residual liquid after decanting the supernatant. Resuspended cells 10 min at 4 °C, the supernatant was discarded, 1 mL of Karnovsky- were then combined with 0.25 g of sterilized 212–300 and 425– style fixative (3.2% paraformaldehyde, 2.5% glutaraldehyde in 600 mm diameter glass beads and 500 μL of CTAB buffer consisting 0.1 M monobasic and 0.1 M dibasic sodium phosphate salts, pH of 0.1 M Tris (pH 8), 1.4 M NaCl, 0.02 M EDTA (pH 8), as well as 7.2) was added, and samples were stored at 4 °C. Dehydration, 0.02 g CTAB, 0.04 g PVP and 5 μL β-mercaptoethanol per 1 mL of infiltration, polymerization, thin-sectioning and staining were buffer. The cells were rapidly agitated in a Mini-Beadbeat-96 performed at the University of Toronto, Faculty of Medicine, (BioSpec Products) cell disrupter at maximum speed for 6 min, Microscopy Imaging Laboratory. A Hitachi H7000 transmission were incubated at 55 °C for 1 h, and then mixed with 500 μLof electron microscope was used for imaging at an accelerating vol- 24:1 chloroform:isoamyl alcohol. After centrifugation for 10 min at tage of 75 kV. 21,100xg, the aqueous phase was combined with 0.08 volumes of 7.5 M ammonium acetate (pH 5.5) and 0.54 volumes of chilled Burst-size estimation (20 °C) isopropanol. Samples were left at 20 °C overnight and then were centrifuged for 3 min at 21,100xg. The supernatant was Viral burst-size was estimated by counting virus particles in aspirated, and DNA pellets were washed with 700 μL of chilled thin-section TEM images (see above), and by direct virus counts 70% ethanol, followed by 700 μL chilled 95% ethanol. Precipitated using epifluorescence microscopy. Briefly, 0.45 mm-filtered lysate DNA was resuspended with 200 μL of 10 mM Tris-Cl, pH 8.5. was added at a multiplicity of infection (MOI) of approximately 10 The volume of all PCRs was 50 μL consisting of 5 μL of tem- s (via most probable number estimation of infectious particles and plate, 1.5 mM MgCl2,200μM each dNTP, 1 U of Platinum Taq flow cytometry estimates of cell abundance) to 50 mL mid-log DNA polymerase (Life Technologies) and 5 μL of manufacturer phase C. parva cultures. At 48 and 72 h following lysate addition, supplied 10 reaction buffer. The amounts of forward and reverse cell abundances were determined via flow-cytometry. Simulta- primer added to the reactions were: 20 pmol each of actinf and neously, 500 mL samples were collected, incubated in the dark for actinr for actin, 20 and 30 pmol of AVS-1 and AVS-2 for polB, 20 min with Grade I glutaraldehyde (Sigma-Aldrich) at a final respectively, and 25 pmol each of mcp-Fwd and mcp-Rev for MCP. concentration of 0.5%, flash frozen in liquid nitrogen and then Gel electrophoresis and thermal cycling conditions for the polB stored at 80 °C. Virus particles were enumerated following the and MCP primers were as previously described (Rozon and Short, approach of Suttle and Fuhrman (2010) using SYBR-Gold nucleic 2013). Thermal cycling conditions for the actin-PCR were the same 114 S.F. Mirza et al. / Virology 486 (2015) 105–115

as those used for the AVS-PCR except the annealing and extension nuclease-free H2Oand2μL of template DNA. Sequences of the for- step durations were 90 s and 2 min, respectively. To increase the ward and reverse primers, and TaqMans probe targeting C. parva actin yield of PCR products from the MCP primers, a second round of genes were CAC GAT CAT GAA GTG CGA, GAG GTG ATC TCC TTG GAC, PCR was performed using first round PCR products purified with a and TGT ACT CCA ACA TCG TCC TCT CGG, respectively. Sequences of QIAquick PCR purification kit (Qiagen) as template. the forward and reverse primers, and TaqMans probe targeting C. PCR products from actin and AVS reactions were purified using parva virus polB genes were GTG CTG AAG AGT GGT ATA, GTG GCA a QIAquick PCR purification kit (Qiagen), whereas products from GAT ATT CTA TTC G, TTA TGG CGA CAC CGA CTC TAT, respectively. second round MCP-PCR were excised and gel purified using a Basedonthevolumesoftemplateusedineachreactionandthe QIAquick gel extraction kit (Qiagen). Purified PCR products were volumes of material extracted (i.e., volume filtered for phytoplankton, cloned using pGEM-T Vector System I as described in Short and or volume centrifuged for viruses), gene-copies per reaction were fi s α™ Short (2008), except Library Ef ciency DH5 Competent Cells converted to gene-copies per mL of environmental water. (Life Technologies) were used for transformations. Plasmid DNA was purified using a QIAprep Spin Miniprep Kit (Qiagen). Auto- mated sequencing of the insert fragments was performed by The Appendix A. Supplementary material Centre for Applied Genomics at the Hospital for Sick Children (Toronto, Canada). Sequences were grouped into OTUs (operations Supplementary data associated with this article can be found in taxonomic units) at a 97% nucleotide identity cut-off. the online version at http://dx.doi.org/10.1016/j.virol.2015.09.005. Representative sequences from each OTU of polB and MCP were used for phylogenetic analyzes using inferred amino acid sequences. Sequences were aligned with various NCLDV sequences Acknowledgments using MUSCLE (Edgar, 2004) in MEGA6 (Tamura et al., 2013); intein regions in various polB sequences were deleted prior to alignment. Based on tests of best protein models in MEGA6, both The authors would like to express their sincere thanks to Ste- maximum likelihood phylogenies were inferred using the LG ven Doyle at the University of Toronto, Faculty of Medicine, model (Le and Gascuel, 2008) with rates among sites gamma Microscopy Imaging Laboratory for technical assistance with distributed allowing for invariant sites. Positions containing gaps Transmission Electron Microscopy. This work was supported in and/or missing data were eliminated from the analysis; the final part by the Canadian Foundation for Innovation Leaders Oppor- data sets for the polB and MCP phylogenies were 209 and 74 amino tunity Fund (17254) and NSERC Discovery (35508-2011) grants acid positions, respectively. Branch support values for both phy- awarded to S.M.S, as well as NSERC USRA scholarships awarded to logenies were obtained from 500 bootstrap replicates. S.F.M. and Y.V.C., and NSERC PGS-D and OGS scholarships awarded to M.A.S. Chloroform sensitivity

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