research fellow project
TRANSCRIPT
![Page 1: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/1.jpg)
An investigation into the existence of An investigation into the existence of CyHV-3 encoded microRNAsCyHV-3 encoded microRNAs
Owen DonohoeOwen DonohoeResearch FellowResearch FellowMarine InstituteMarine Institute
GalwayGalway
![Page 2: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/2.jpg)
Cyprinid Herpesvirus-3 (CyHV-3)Cyprinid Herpesvirus-3 (CyHV-3)Classification: Member of Herpesvirales order Part of new family of HVs - Alloherpesviridae
Susceptible Cyprinid Fish: Common Carp (Cyprinus carpio carpio) Koi (Cyprinus carpio koi) Carp: 14% of freshwater aquaculture
Effects: Devastating effects on fish populations Morbidity ~100%, Mortality ~70-80%
![Page 3: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/3.jpg)
CyHV-3 LatencyCyHV-3 LatencyHerpesviruses have the ability to establish a latent infection.
DormantNo replicationImmune Evasion
CyHV-3 may be also capable of establishing a latent infection
Outside permissive temperature range ~16-25oC hosts survive as latent carriers
Asymptomatic long term infection
May reactivate
![Page 4: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/4.jpg)
Diagnostic Target Diagnostic Target Low levels of CyHV-3 DNA found in healthy long term survivorsLow levels of CyHV-3 DNA found in healthy long term survivors
BrainBrainKidneyKidneyGillGillGI TractGI TractLeucocytesLeucocytes
DNA is at low levels and sometimes difficult to detectDNA is at low levels and sometimes difficult to detect
![Page 5: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/5.jpg)
Latently Infected CellLatently Infected Cell
Latency Associated Transcripts as Latency Associated Transcripts as alternative diagnostic targetsalternative diagnostic targets
Viral genome maintained as non-integrated episome.Viral genome maintained as non-integrated episome.
Virus relies on specific subset of genes Virus relies on specific subset of genes
Viral Genome Viral Genome
Latently Associated Latently Associated TranscriptsTranscripts
Non-coding RNAs known as Non-coding RNAs known as Latency Associated TranscriptsLatency Associated Transcripts
Viral RNA from actively transcribed genes should be much more Viral RNA from actively transcribed genes should be much more abundant than viral genomeabundant than viral genome
Viral RNA transcripts: Viral RNA transcripts: Much moreMuch more ideal diagnostic target ideal diagnostic target
![Page 6: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/6.jpg)
Aims of this studyAims of this study
Potential use of Potential use of HIGH LEVELHIGH LEVEL
Latency Associated Transcripts Latency Associated Transcripts
in diagnosis of latent infectionsin diagnosis of latent infections
Identify CyHV-3 Latency Associated TranscriptsIdentify CyHV-3 Latency Associated Transcripts
![Page 7: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/7.jpg)
MicroRNAs (MiRNAs) as Latency MicroRNAs (MiRNAs) as Latency Associated transcripts: Associated transcripts:
MicroRNAs (miRNAs) are quite prominent during Herpesvirus latencyMicroRNAs (miRNAs) are quite prominent during Herpesvirus latency
Larger Primary TranscriptsLarger Primary Transcripts
(Primary-miRNAs)(Primary-miRNAs)
Folds into Imperfect Hairpin-like structureFolds into Imperfect Hairpin-like structure
(Pre-miRNAs)(Pre-miRNAs)miRNA component always present on the stem of hairpin structuremiRNA component always present on the stem of hairpin structure
miRNAs ~22 ntmiRNAs ~22 nt
![Page 8: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/8.jpg)
Cleavage – miRNA releasedCleavage – miRNA released
Mature miRNA Mature miRNA DuplexDuplex3’3’
3’3’
Involved in Involved in gene gene specific specific silencingsilencing
RISCRISCRISCRISC
ΔG
RNARNA
Base pairs with specific mRNA transcripts preventing Base pairs with specific mRNA transcripts preventing translation or causing them to be degradedtranslation or causing them to be degraded
Gene silencedGene silenced
MicroRNAs (miRNAs) are quite prominent during latencyMicroRNAs (miRNAs) are quite prominent during latency
MicroRNAs (MiRNAs) as Latency MicroRNAs (MiRNAs) as Latency Associated transcripts:Associated transcripts:
Incorporated into Incorporated into
RRNA NA IInduced nduced SSilencing ilencing CComplexomplex
Evading the immune responseEvading the immune responsePromotion of host cell survivalPromotion of host cell survivalControl of lytic cycleControl of lytic cycle
EssentialEssential for long term for long term maintenance of latencymaintenance of latency
DROSHA
EXP-5
Important regulators of genes during Lytic and Latent infectionsImportant regulators of genes during Lytic and Latent infections
![Page 9: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/9.jpg)
Scor
e
Position in genome
303 of predicted hairpins 303 of predicted hairpins
showed similar characteristics showed similar characteristics
to known viral pre-miRNAs to known viral pre-miRNAs
Stability (WC)Stability (WC)
Structure (Score)Structure (Score)
VMir: Precursor-miRNA PredictionsVMir: Precursor-miRNA Predictions
155 Classified as 155 Classified as “Real” using MiPred“Real” using MiPred
Sequence conservation is rare among viral miRNAsSequence conservation is rare among viral miRNAs
![Page 10: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/10.jpg)
Common Carp Brain Cells (CCBs) Common Carp Brain Cells (CCBs) infected with CyHV-3infected with CyHV-3
Viral replication, causing widespread CPEViral replication, causing widespread CPE
Identification of CyHV-3 miRNAsIdentification of CyHV-3 miRNAs
RNARNA (17-25nt)(17-25nt)
In-vitro In-vitro lytic infectionlytic infection
Deep SequencingDeep Sequencing
Latent infectionsLatent infectionsIn vivo In vivo not suitablenot suitable
No No in vitro in vitro modelmodel
Most latency associated miRNAs also present Most latency associated miRNAs also present during lytic infectionsduring lytic infections
![Page 11: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/11.jpg)
Deep Sequencing Results H361 infectionDeep Sequencing Results H361 infection
12,016,247 transcripts sequenced12,016,247 transcripts sequenced
Illumina Genome AnalyzerIllumina Genome Analyzer
212,399 (~1.8%) aligned212,399 (~1.8%) aligned
to the CyHV-3 genometo the CyHV-3 genome
30,217 Unique reads30,217 Unique reads
1.1% High Abundance small 1.1% High Abundance small RNAs from non-coding regions RNAs from non-coding regions
161,537 (76%) of viral transcripts161,537 (76%) of viral transcripts
![Page 12: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/12.jpg)
Rea
d co
unt
Position in genome
15432 15603
567
2711
ΔG = -78.00
3’3’3’3’
5’ Arm 5’ Arm
3’ Arm 3’ Arm
2 highly abundant CyHV-3 small RNAs mapping to the genome in very close proximity to each other2 highly abundant CyHV-3 small RNAs mapping to the genome in very close proximity to each other
Good example:Good example: Conforms with the basic characteristic of miRNAs Conforms with the basic characteristic of miRNAs
![Page 13: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/13.jpg)
Rea
d co
unt
Position in genome
301
159561 159696
21142114
30118 60
Additional transcripts derived from precursor immediately adjacent to the proposed miRNAsmicroRNA-offset-RNAs microRNAsMore abundant than 99% of other CyHV-3 transcripts in sample
![Page 14: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/14.jpg)
301301
21142114
1818
Identification of Pre-miRNA candidatesIdentification of Pre-miRNA candidates
Minor-formMinor-form
Major-formMajor-form
H361 InfectionH361 Infection
12161216
1039610396
8585
31713171
N076 InfectionN076 Infection Minor-formMinor-form
Major-formMajor-form
6060
microRNA-offset-RNAs
![Page 15: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/15.jpg)
21 Pre-miRNA candidates21 Pre-miRNA candidates
![Page 16: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/16.jpg)
In-depth analysis of pre-miRNA candidatesIn-depth analysis of pre-miRNA candidates
MiRNA 5’ end stabilityMiRNA 5’ end stability MiPred and CSHMM pre-miRNA classificationMiPred and CSHMM pre-miRNA classification Minimum Free Energy Minimum Free Energy Presence of 1-3 nt 3’ overhangsPresence of 1-3 nt 3’ overhangs Assessment of small RNA alignment signatureAssessment of small RNA alignment signature IsomiR 3’ End heterogeneityIsomiR 3’ End heterogeneity Discrete enriched lociDiscrete enriched loci
Consistency with model of miRNA biogenesisConsistency with model of miRNA biogenesis
![Page 17: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/17.jpg)
Terminal LoopTerminal Loop
Original RNA transcriptOriginal RNA transcript
Sequencing dataSequencing data
5’ miRNA5’ miRNA
3’ miRNA3’ miRNA
3’ moRNA3’ moRNA
5’ moRNA5’ moRNA
5’ miRNA5’ miRNA
3’ miRNA3’ miRNA
5’ moRNA5’ moRNA
3’ moRNA3’ moRNA
Original pre-miRNA transcriptOriginal pre-miRNA transcriptSequencing DataSequencing Data
MiRNA-like alignment signaturesMiRNA-like alignment signatures
Release of 2-4 smaller mature transcriptsRelease of 2-4 smaller mature transcripts
2-4 “stacks” representing 2-4 “stacks” representing processed mature transcriptsprocessed mature transcripts
![Page 18: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/18.jpg)
MD11776MD11776
![Page 19: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/19.jpg)
MR5057MR5057
![Page 20: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/20.jpg)
6 High probability Pre-miRNA candidates6 High probability Pre-miRNA candidates
MiRDeep
Mireap5 were identified using 2 automated methods
All primarily identified due to the high abundance of the proposed miRNAs
![Page 21: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/21.jpg)
Deep Sequencing suffers from Deep Sequencing suffers from enzymatic biasenzymatic bias
3’ Adaptor ligation3’ Adaptor ligation
5’ Adaptor ligation5’ Adaptor ligation
RTRT
PCR PCR
Bridge AmplificationBridge Amplification
Terminator SequencingTerminator Sequencing
Sample PreparationSample Preparation
SequencingSequencing
Over-representation / Under-RepresentationOver-representation / Under-Representation
![Page 22: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/22.jpg)
6 High probability Pre-miRNA candidates6 High probability Pre-miRNA candidates
Are some of these just Are some of these just over-representedover-represented transcripts? transcripts?
Are some of these Are some of these under-representedunder-represented transcripts? transcripts?
![Page 23: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/23.jpg)
DNA Array hybridizationDNA Array hybridization
30,000 Probes
Targeting
Supposedly highly abundant transcripts
MiRNA-like transcripts of lower abundance
Predicted miRNAs from earlier genomic analysis
Re-analysed H361 sample used in the sequencing experiment
![Page 24: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/24.jpg)
Array Hybridization ResultsArray Hybridization Results
Most of the miRNA candidates from deep sequencing were detected
Most were shown to be still among the most abundant CyHV-3 transcripts in the sample
Descending order of abundance from deep sequencing
Suggests that they were not over represented in deep sequencing due to enzymatic bias
No significant signals from negative control sample
Average Percentile Rank of 80.3
![Page 25: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/25.jpg)
Array Hybridization ResultsArray Hybridization Results
Confirmed that low probability miRNA candidates from deep sequencing are not miRNAs
Reinforced conclusions regarding most likely miRNA candidates
No new potential miRNA candidates identified
Probes targeting genuine miRNAs should not hybridize to anything in the 25-35nt size range, whereas probes targeting degradation products will
![Page 26: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/26.jpg)
6 High probability pre-miRNA 6 High probability pre-miRNA candidatescandidates
RNA from H361 infection analyzed using RNA from H361 infection analyzed using Deep SequencingDeep Sequencing
Array HybridizationArray Hybridization
![Page 27: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/27.jpg)
1.E-10
1.E-09
1.E-08
1.E-07
MR5057
MD1177
6
MD1111
5'
MD1111
3'
MR5075
5'
MR5075
3'
MD9812
MD1141
0
Target
N0 V
alue
s (L
og)
1.E+00
1.E+01
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
Transcript
Rea
d C
ount
s (L
og)
RT-qPCRRT-qPCR
Deep Sequencing Reads Deep Sequencing Reads Over/Under Over/Under
RepresentationRepresentation
PCR likely to be PCR likely to be more accuratemore accurate
No Change No Change hierarchy of hierarchy of expression levelsexpression levels
MiRNAs from MiRNAs from MD1111 were MD1111 were significantly over-significantly over-represented represented
Actually present Actually present at much lower at much lower levels relative to levels relative to other miRNAsother miRNAs
![Page 28: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/28.jpg)
Time Post InfectionTime Post InfectionCyHV-3 DNA
0.00001
100
200
300
400
500
600
NegativeCells
0 1 2 3 4 5 6 7 8 9
d.p.i.
Line
ar S
cale
Exp
ress
ion
Valu
es
CyHV-3 DNA
CyHV-3 Expression over course of CyHV-3 Expression over course of in vitroin vitro infection infection
![Page 29: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/29.jpg)
MR5057
1.00E-05
5.00E+00
1.00E+01
1.50E+01
2.00E+01
2.50E+01
3.00E+01
3.50E+01
NegativeCells
0 1 2 3 4 5 6 7 8 9
d.p.i.
N0
Valu
e
MR5057
MD11776
1.00E-05
2.00E+00
4.00E+00
6.00E+00
8.00E+00
1.00E+01
1.20E+01
1.40E+01
NegativeCells
0 1 2 3 4 5 6 7 8 9
d.p.i.
N 0 V
alue
MD11776
MD11410
1.00E-05
2.00E-01
4.00E-01
6.00E-01
8.00E-01
1.00E+00
1.20E+00
1.40E+00
1.60E+00
1.80E+00
2.00E+00
NegativeCells
0 1 2 3 4 5 6 7 8 9
d.p.i.
N0
Valu
e
MD11410
MD9812
1.00E-05
2.00E-01
4.00E-01
6.00E-01
8.00E-01
1.00E+00
1.20E+00
1.40E+00
1.60E+00
1.80E+00
2.00E+00
NegativeCells
0 1 2 3 4 5 6 7 8 9
d.p.i.
N0
Valu
e
MD9812
MD1111 3'
1.00E-05
2.00E-01
4.00E-01
6.00E-01
8.00E-01
1.00E+00
1.20E+00
1.40E+00
1.60E+00
1.80E+00
2.00E+00
NegativeCells
0 1 2 3 4 5 6 7 8 9
d.p.i.
N0
Valu
e
MD1111 3'
MR5075 3'
1.00E-05
2.00E-02
4.00E-02
6.00E-02
8.00E-02
1.00E-01
1.20E-01
1.40E-01
1.60E-01
1.80E-01
NegativeCells
0 1 2 3 4 5 6 7 8 9
d.p.i.
N0 V
alue MR5075 3'
1 d.p.i.1 d.p.i.
1 d.p.i.1 d.p.i.
1 d.p.i.1 d.p.i.
2 d.p.i.2 d.p.i.
2 d.p.i.2 d.p.i.
2 d.p.i.2 d.p.i.
![Page 30: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/30.jpg)
1.00E-04
1.00E-03
1.00E-02
1.00E-01
1.00E+00
1.00E+01
1.00E+02
NegativeCells
0 1 2 3 4 5 6 7 8 9
d.p.i.
N0
(Log
)
MR5057MD11776MD1111 5'MD1111 3'MR5075 5'MR5075 3'MD9812 MD11410
Steady-state increase in miRNA expression levelsSteady-state increase in miRNA expression levels
Linked to increased levels of viral transcription as more cells become infectedLinked to increased levels of viral transcription as more cells become infected
Similar profile to that observed in deep sequencing H361 infectionSimilar profile to that observed in deep sequencing H361 infection
Same profile repeatedly observed : May be more representative of the typicalSame profile repeatedly observed : May be more representative of the typical
profile during lytic infections profile during lytic infections in vitroin vitro
MiRNAs from MR5075 lower relative to other miRNAs MiRNAs from MR5075 lower relative to other miRNAs
![Page 31: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/31.jpg)
1.00E -05
2.00E -01
4.00E -01
6.00E -01
8.00E -01
1.00E+00
1.20E+00
1.40E+00
1.60E+00
1.80E+00
2.00E+00
NegativeCells
0 1 2 3 4 5 6 7 8 9
d.p.i.
N 0 V
alue
MD1111 5'MD1111 3'
1.00E-05
2.00E-02
4.00E-02
6.00E-02
8.00E-02
1.00E-01
1.20E-01
1.40E-01
1.60E-01
1.80E-01
NegativeCells
0 1 2 3 4 5 6 7 8 9
d.p.i.
N0 V
alue MR5075 5'
MR5075 3'
MD1111MD1111
MR5057MR5057
Major-formMajor-form
Major-formMajor-form
![Page 32: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/32.jpg)
Expression of CyHV-3 miRNAs Expression of CyHV-3 miRNAs in vivoin vivo
3 CyHV-3 miRNAs convincingly detected lytically infected fish
One CyHV-3 miRNA detected in suspected latent carrier
![Page 33: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/33.jpg)
1.00E-12
2.01E-10
4.01E-10
6.01E-10
8.01E-10
1.00E-09
1.20E-09
Sample
Line
ar S
cale
Exp
ress
ion
Valu
es
MR5057MD11776MR5075
Expression of CyHV-3 miRNAs Expression of CyHV-3 miRNAs in vivoin vivo
LyticSuspected Latent Carrier
![Page 34: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/34.jpg)
Detecting 3’ miRNA from precursor MR5057 by Detecting 3’ miRNA from precursor MR5057 by Northern BlottingNorthern Blotting
22nt
56nt
Mature miRNA
Pre-miRNA
Non-Infected Infected
100nt100nt
75nt75nt
50nt50nt
40nt40nt
30nt30nt
20nt20nt
Non-Infected Infected
Host miRNA
Let-7a (22nt)
Loading Control
Conclusive evidence to support the existence of this miRNA
![Page 35: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/35.jpg)
Seed
Seed
Identification of homologues in other CyHVsIdentification of homologues in other CyHVs
Pattern indicative of the presence of functionally important miRNA
![Page 36: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/36.jpg)
Predicted as pre-miRNA by VMir in CyHV-2
Classified as “Real” pre-miRNA by MiPred and CSHMM
May be a functional homologue of MD11776
Identification of homologues in other CyHVsIdentification of homologues in other CyHVs
![Page 37: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/37.jpg)
mRNAmRNA
3’ UTR3’ UTR
AAAAAAAAAA
LowLow ΔGG
Stop CodonStop Codon
AA
Function of these miRNAsFunction of these miRNAs
Target Site Target Site AccessibilityAccessibilityTargetScan : Looks for sites TargetScan : Looks for sites that possess some or all of these characteristicsthat possess some or all of these characteristics
Scores sites appropriately Scores sites appropriately
some bases 13-17 also base pairsome bases 13-17 also base pair Bases 2-8 Bases 2-8
(Seed Region)(Seed Region)
Significant “AU” contentSignificant “AU” content
NN
![Page 38: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/38.jpg)
Target site AccessibilityTarget site Accessibility
RISC
RISC
![Page 39: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/39.jpg)
PITAPITA
Free Energy potentially lost by Free Energy potentially lost by
un-folding of RNA around target Site un-folding of RNA around target Site
Free Energy potentially gained by Free Energy potentially gained by
binding of miRNA to target sitebinding of miRNA to target site
If difference between the two is negative, miRNA binding is most If difference between the two is negative, miRNA binding is most
thermodynamically favourable outcomethermodynamically favourable outcome
![Page 40: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/40.jpg)
5’ miRNA Target: 3’ UTR of ORF117
3’ miRNA Target: 3’ UTR of ORF122 and ORF123
MR5057
3’ miRNA: Correct temporal progression of viral gene expression during lytic infections
dUTPase
![Page 41: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/41.jpg)
MR5057
MD11111
MD11776
MR5075
MD9812
MD11410
MD11704
7 genes likely to be genuine CyHV-3 encoded Pre-miRNAs
ConclusionConclusion
Remaining 5 high probability pre-miRNAs all display the expected characteristics
![Page 42: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/42.jpg)
Further Research Further Research Identification of CyHV-3 encoded miRNAs & moRNAs opens up new lines of inquiry
Functions of CyHV-3 miRNAs (viral and host gene regulation)Functions of CyHV-3 moRNAs CyHV-3 miRNAs as diagnostic biomarkers of latent infectionsMiRNA genes in other members of the Alloherpesviridae family
These findings may help further our understanding of the biology CyHV-3 and other members of the Alloherpesviridae family
![Page 43: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/43.jpg)
Dr. Dermot Walls (Academic Supervisor, DCU)
Dr. Kathy Henshilwood (Supervisor, Marine Institute)
Dr. Keith Way (Cefas, UK)
Funding Bodies: Marine Institute, Defra
Thank you for your attention
![Page 44: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/44.jpg)
![Page 45: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/45.jpg)
![Page 46: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/46.jpg)
![Page 47: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/47.jpg)
![Page 48: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/48.jpg)
![Page 49: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/49.jpg)
![Page 50: Research Fellow Project](https://reader037.vdocuments.site/reader037/viewer/2022102703/589d57ea1a28abef688b533f/html5/thumbnails/50.jpg)