The thesis defense outlined Cristian Quispe's research expanding knowledge of Chloroviruses and their symbiotic Chlorella hosts. Key findings included: 1) A 3-year study of Chlorovirus populations in a lake showed seasonal patterns and site-dependent diversity; 2) A new Chlorovirus genus, OSy, was found to exclusively infect Syngen cells; 3) OSyNE-5 can initiate infection of related algal species but only replicates in Syngen; 4) Metabolic studies revealed symbiotic Chlorella have specialized nutrient requirements compared to free-living species, such as preference for organic nitrogen sources.

1. 12/1/15
1
Expansion of the Chlorovirus Genus by Studies on Virus
Natural History and the Chlorella Host Metabolism
Thesis Defense
Cristian Quispe
Ph.D. Candidate
Dr. James Van Etten
Advisor
November 30th, 2015
Outline
1. Introduction
2. Chlorovirus natural history
3. Expansion of the Chlorovirus genus
4. Metabolism of the Chlorovirus symbiotic Chlorella hosts
5. Significance

2. 12/1/15
2
v Relative viral abundance
• Virus number typically exceeds that of cellular organisms by
at least an order of magnitude
– Ratio virus particles : cells
10 : 1
• 1 ml of sea water ~ 106-109 virus particles
1. Introduction
Viruses are the most abundant entities in the biosphere
November 30th, 2015
Nucleocytoplasmic Large DNA Viruses are the largest of all
known viruses

3. 12/1/15
3
Nucleocytoplasmic Large DNA Viruses (NCLDVs)
Koonin, et. al., 2015
Plaque assay development
Generalities
v Family Phycodnaviridae
v Genus Chlorovirus
Viruses infecting freshwater algae species
v Ancient evolutionary history
v Genome structure
• dsDNA
• genome size: 290-370 kb
• ~400 protein-encoding genes
• 5 to 16 tRNAs
Chlorovirus features

4. 12/1/15
4
o Model system: produce large quantities of viruses
o ∼40% of genes resemble proteins of unknown functions
o Unique characteristics
• Glycosylation independent of the host
• Internal membrane
• Restriction endonucleases
o Unexpected human health risk
• Effect on cognitive behavior in animals
Why we study Chloroviruses
Model
v Limitations
§ Inability to genetically manipulate the host or the virus

5. 12/1/15
5
Plaque assay development
PBCV-1 replication cycle
Paramecium bursaria chlorella virus 1 (PBCV-1) is the Chlorovirus prototype
Plaque assay development
Plaque assays

6. 12/1/15
6
Three-year Survey of Abundance, Prevalence
and Genetic Diversity of Chlorovirus Populations in
a Small Urban Lake
2. Chlorovirus natural history
Systematic study: Urban small lake in Lincoln, NE
Holmes Lake

7. 12/1/15
7
Experimental design
Plaque assay
Chlorovirus abundance and diversity

8. 12/1/15
8
Number of viruses infecting the NC64A host
Chloroviruses are widely dispersed, and local environmental
conditions enrich for certain viral types
Spring
Summer
Fall
Winter
v Chloroviruses were ubiquitous throughout the year
§ They were both host- and site-dependent
v Chloroviruses follow a seasonal pattern
• Highest titers between the 2nd week of April to the 1st week of July and
the 2nd week of October to the 2nd week of December
Site 2
Site 1

9. 12/1/15
9
Choosing a Sample
Places with more biodiversity have the highest titers
Virus concentrations can vary considerably within a small geographical region
v None of the symbiotic Chlorella strains grew in the collected water alone,
unless exogenous organic nitrogen source was added
Chloroviruses replicate in unknown host in nature

10. 12/1/15
10
Conclusions
Ø Three out of the four Chlorovirus types were present throughout the
year
§ Viruses infecting the Syngen host were the most abundant
§ Viruses infecting the Pbi host were not detected
Ø Places with less disturbance and more biodiversity have the highest
titers
Ø Two main viral peaks are consistent within the three years:
§ Transition between winter and spring
§ Transition between fall and winter
Hypothetical model for Chlorovirus distribution
Site 2
Site 1

11. 12/1/15
11
Characterization of a New Chlorovirus Type with Permissive
and Non-Permissive Features on Phylogenetically Related
Algae Strains
3. Expansion of the Chlorovirus genus
PBCV-1 replicates on three symbiotic algae strains
v Paramecium bursaria chlorella virus 1 (PBCV-1) is the prototype Chlorovirus
v NC64A, Syngen 2-3, and OK1-ZK are similar, but not identical Chlorella strains
Van Etten et al., 1983; Proschold et al., 2011

12. 12/1/15
12
Difference in number of plaques from indigenous water
v PFUs on Syngen lawns were significantly higher than the PFUs
on NC64A and OK1-ZK
Chlorovirus that might replicate in Syngen cells but not in NC64A
and OK1-ZK

13. 12/1/15
13
New genus OSy
OSyNE-5 is the prototype virus for the group
v New Chloroviruses that exclusively infect Syngen cells
v Syngen cells are host for two distinct virus populations
OSy-viruses are ubiquitous in nature
v Most abundant virus type in Nebraska freshwater
• 75% of the isolates only formed plaques on Syngen cells
(PBCV-1)

14. 12/1/15
14
OSyNE-5 has similar features to other Chloroviruses
Negative staining
Major capsid protein (MCP ) profile
v Two MCPs that migrated slightly slower than the single PBCV-1 MCP
v Same icosahedral morphology with a diameter of around 180–190 nm
OSyNE-5 and PBCV-1 exhibited high colinearity/synteny
Jeanniard et al. BMC Genomics 2013, 14:158
v Genome colinearity and phylogenetic affinity is limited to
chloroviruses infecting the same host
v The ability of both viruses to replicate in Syngen cells could explain
their high genomic colinearity

15. 12/1/15
15
OSyNE-5 has strong phylogenic affinity with viruses infection the
NC64A host (PBCV-1)
v OSyNE-5 core proteins have high (average of 89%) amino
acid identity with their PBCV-1 orthologous
Genomic comparison of OSyNE-5 and PBCV-1
* Virus-specific regions

16. 12/1/15
16
OSyNE-5 attaches and initiates infection not only in Syngen 2-3 but
also on NC64A and OK1-ZK cells
Flow cytometry
Fluorescent microscopy
v OSyNE-5 significantly rise the population of cells with increase
SYBR® Gold fluorescent intensity
v When 0.1% of the non-permissive cell population is infected by
OSyNE-5 the 99.9% can still divide and survive
NC64A and OK1-ZK cells are killed after OSyNE-5 infection but
there is no virus replication

17. 12/1/15
17
Conclusions: New Chlorovirus
v A ubiquitous virus type in Nebraska
v Most closely related at the genomic and phylogenetic level to
viruses infecting the NC64A host
v New genus have permissive and non-permissive features in
related algal species
§ can initiate early infection events in NC64A and OK1-ZK
cells but are unable to complete virus replication
Plaque assay development
Syngen 2-3 cells: Permissive
OSy-viruses
Syngen 2-3 cells
v When OSyNE-5 attaches and initiates infection
§ In permissive cells: continues with the normal virus
replication cycle

18. 12/1/15
18
Plaque assay development
NC64A and OK1-ZK cells: Non-permissive
NC64A cells
OK1-ZK cells
OSy-viruses
v When OSyNE-5 attaches and initiates infection
§ In non-permissive cells: can not replicate but cells are killed
after virus attachment. Infectious particles were not recovered
? ?
?
Comparative Metabolism, Genomics, and
Transcriptomics Distinguish Symbiotic from Free-
living Chlorella algae
4. Metabolism of the Chlorovirus symbiotic Chlorella hosts

19. 12/1/15
19
Model
Model

20. 12/1/15
20
Symbiotic algae possess fastidious nutrient requirements, and in
the isolation and axenic growth of symbiotic algae
v Physiological evaluation on alternative nitrogen and carbon
sources
BBM
MBBM
Symbiotic algae possess fastidious nutrient requirements
NC64A cannot efficiently assimilate NH4 as sole N sources
v Both strains are unable to utilize nitrate (NO3)
v NC64A strains grow better on low ammonium (NH4 )
concentrations (1 mM)

21. 12/1/15
21
NC64A is unable to utilize NO3
v NC64A grow better with galactose while free-living grow was
significantly compromised when galactose was supplemented
Asparagine, serine, and proline are better assimilated by NC64A
v Organic N sources are better assimilated by NC64A over the
free-living

22. 12/1/15
22
Galactose is the prefer carbon source in NC64A
v Galactose improve NC64A growth but it is inhibitory for the
free-living
Amino acid transporters are overrepresented in the NC64A
genome
.

23. 12/1/15
23
v NC64A is able to better assimilate unusual organic N sources likely by the
overrepresentation and expression of amino acid transporters in its genome
A. thaliana
C. variabilis NC64A
Some amino acid transporters are constitutively expressed in
axenic and during symbiosis C. variabilis growth
Supplementary Figure 1High-throughput nutritional analysis
v The only documented symbiotic-virus susceptible Chlorella species, which
can be cultured axenically

24. 12/1/15
24
Numerical scale
Hierarchical clustering analysis
v Tree clusters the strains and treatments according to similarities
in their growth patterns
v 64 media combinations (n=567)

25. 12/1/15
25
17
Figure 5A
Symbiotic algae can not efficiently assimilate NO3 or NH4 as sole
N sources
v Most symbiotic strains grow better on low concentrations of NH4
compared to free-living
40Asn, Ser and Pro metabolism might be important for symbiotic
growth
v Asn, Ser, and Pro appear to be symbiont-specific in that they were
used poorly, if at all by free-living Chlorella

26. 12/1/15
26
Conclusions
v All symbiotic strains exhibit similar metabolic phenotypes although
they are polyphyletic and arise as protozoan symbionts from different
origins
v Symbiotic algae are better able to assimilate organic N sources
v They also assimilate better galactose, an unusual C source
v Significant increase in the number of amino acid transporters proteins in
the genome of C. variabilis NC64A
v Some amino acid transporters are expressed in axenic and
endosymbiotic growth
5. Significance
Chlorovirus natural history
§ First systematic and long-term analysis of infections Chlorovirus
populations in freshwater
Expansion of the Chlorovirus genus
§ Permissive and non-permissive concepts in the Chloroviruses
Metabolism of the Chlorovirus symbiotic Chlorella hosts
§ Increase the tools to study host:pathogen interactions

27. 12/1/15
27
Acknowledgments
Current Lab members
Garry Duncan
David Dunigan
Irina Agarkova
Rebecca Richardson
Zeina Al-Ameeli
Ahmed Esmael
Past members
Les Lane
James Gurnon
Eric Noel
Kathleen Higgins
Undergraduates
Anya Seng
Olivia Sonderman
Michelle McQuinn
Brenna Rasmussen
Garrett Weber
James Van Etten
Graduate committee
Kenneth Nickerson
Amit Mitra
Wayne Riekhof
Luwen Zhang
Nebraska Center for Virology
UNL core facility
School of Biological Sciences
Family and friends
Questions?