Chikungunya virus (CHIKV), a mosquito-borne alphavirus of the Togaviridae family, has recently emerged in the Americas from two originating continents, Asia and Africa. Historically, CHIKV circulated as at least four lineages worldwide with both enzootic and epidemic transmission cycles. To understand the recent re-emergence patterns, and the current status of the CHIKV spread, an updated phylogeny of the virus is needed. Here, we performed phylogenetic and comparative genomics screens of CHIKV genomes, taking advantage of the public availability of many recently sequenced isolates. Based on these new data and analyses, we derive a revised phylogeny from full open reading frame nucleotide sequences. Within this phylogeny, we propose the presence of several distinct lineages in Africa which were once considered a single lineage. In parallel, thermodynamic modeling of CHIKV untranslated regions (UTRs) reveals a cascade of evolutionarily conserved RNA structures, in particular in the 3' UTR. We provide evidence for the duplication of non-coding RNAs (ncRNAs) in recently emerged Carribean CHIKV isolates and propose the existence of lineage-specific duplicated ncRNAs in different CHIKV lineages.

1. Evolutionary traits in Chikungunya
virus untranslated regions
Michael T. Wolfinger
4th VDS Retreat, Mondsee
26 June 2019
Research Group Bioinformatics and Computational Biology
& Department of Theoretical Chemistry
University of Vienna

2. !2

3. Chikungunya Virus (CHIKV)
•Family Togaviridae / genus Alphavirus; mosquito-borne (Aedes spp.)
•Single-stranded (+) sense RNA virus
•“Chikungunya” (Makonde language): “Disease that bend up the joints”
•Chikungunya fever: febrile illness, arthralgia, rash, rarely causes hemorrhagic complications
•Enzootic in tropical and subtropical regions of Africa
•First outbreak described 1952 in Tanzania
•No vaccine available
WeaverandForrester,Antivir.Res.(2015)
!3
WeaverandForrester,Antivir.Res.(2015)

4. Alphavirus Genome Organization
• Non-segmented, single-stranded, (+)-sense RNA genomes of 11-12kB length
• Capped and polyadenylated
• AV genomes appear to host cells as mRNA for immediate translation upon entry into the cytoplasm
Viralzone/ExPASy
!4
https://viralzone.expasy.org/

5. Part I: Phylogeography
!5

6. CHIKV Epidemic Spread
Frickmannetal.,Viruses(2019)
!6
often associated with temporary outbreak events [9], leading to temporally increased infection risks in
contrast to arbovirus infections such as dengue fever, with continuously increasing case numbers [7,8].
Viruses 2019, 11, x FOR PEER REVIEW 2 of 8
in contrast to arbovirus infections such as dengue fever, with continuously increasing case numbers
[7,8].
Figure 1. Countries or regions with chikungunya virus (CHIKV) endemicity and/or outbreaks
mentioned in this review in the order of appearance (black dots and numerals 1–11). 1. Réunion; 2.
Figure 1. Countries or regions with chikungunya virus (CHIKV) endemicity and/or outbreaks mentioned

7. Updated CHIKV Phylogeny
• 590 CHIKV genomes
• iq-tree / SH-aLRT
!7
KY703952.1
KY703924.1
KY703926.1
KY703974.1
KY703891.1
KY703986.1
KY703982.1
KY703971.1
KY703907.1
KY703914.1
KY703892.1
KY703889.1
KY703995.1
KY703943.1KY703944.1KY703890.1KY703965.1KY703905.1KY703988.1KY703950.1KY703997.1KY703966.1KY703933.1KY703951.1KY703923.1KY703983.1KY703987.1KY703925.1KY703990.1KY703992.1KY703975.1KY703960.1KY703999.1KY703921.1
KY703968.1
KY703972.1
KY704002.1
KY703978.1
KY680375.1
KY703955.1
KY703948.1
KY703919.1
KY703959.1
KY703996.1
KY703935.1
KY703920.1
MF773560.1
KY680379.1
KY435459.1
KY435480.1
KY435456.1
KY435470.1
KY680399.1
KR559488.1
KR559475.1
KY703956.1
KY703898.1
KY680352.1
KY680361.1
KY680398.1
KY680384.1
KY703922.1
KY703908.1
KY680389.1
KR559476.1
KY703928.1
KY703909.1
KY703918.1
KY703901.1
KY703984.1
KY703947.1
KY680359.1
KT192707.1
KY680363.1
KY680355.1
KY703970.1
KY703994.1
KY703934.1
KY703899.1
KR559484.1
KR046232.1
KY703903.1
KY703902.1
KY703897.1
KY703927.1
KR559472.1
KY415985.1
KY415982.1
KY415978.1
KY415981.1
KY415979.1
KY415980.1
KY415983.1
KX702402.1
KY415984.1
KR559478.1
KX702401.1
KY680402.1
KY680356.1
KY680372.1
KY704001.1
KY435471.1
KY680382.1
KY435461.1
KY680386.1
KY680413.1
MG967666.1
KY680405.1
KY435455.1
KY703993.1
KY703940.1
KY703991.1
KY703894.1
KY703893.1
KY703976.1
KY703958.1
KY703937.1
KY703900.1
KY703973.1
KY703946.1
KY703954.1
KY703915.1
KY703938.1
KY703989.1
KY703979.1
KY703957.1
KY703942.1
KY703916.1
KY703985.1
KY703931.1
KY703888.1
KY680367.1
KR046230.1
KY703930.1
KY703895.1
KY703911.1
KY703980.1
KY703998.1
KY703967.1
KR559471.1
KR046229.1
KY680393.1
KY703917.1
KY703910.1
KY703962.1
KY703896.1
KR559481.1
KY703961.1
KY703936.1
KY703977.1
KY703913.1
MG921596.1
KY703945.1
KP851709.1
KY703906.1
KY680366.1
KY435476.1
KY680347.1
KY703904.1
KR046231.1
KY680364.1
KX262994.1KY680373.1KY680388.1KY435472.1KY435469.1KY435475.1KY435454.1KY704000.1KY703932.1KY703963.1KY703969.1KY703949.1KY680370.1KX262992.1KP164572.1KY435458.1KR046228.1KY435474.1KY575573.1KR559492.1
KY435481.1
KR559473.1
KY435483.1
KY435484.1
KR559494.1
KR559495.1
KR559485.1
KY435482.1
KY435462.1
KR559497.1
KR559491.1
KX496989.1
KY435465.1
KY435464.1
KY435477.1
KY680396.1
KY435473.1
KY435460.1
KY680376.1
KX262991.1
KY680407.1
KR559489.1
KP164567.1
KY435479.1
KY435457.1
KY435468.1
KR046227.1
KP851710.1
KY435467.1
KY435485.1
KY435478.1
KR559490.1
KR046234.1
KR046233.1
KR559496.1
KY435463.1
KY435486.1
KJ451624.1
KT327165.1
KT327166.1
KT327163.1
KT327164.1
KT327167.1
M
G
208125.1
KY680412.1
KY680368.1
KY272969.1
KY680397.1
KY272962.1
KY680414.1
KY680411.1
KY680362.1
KY680377.1
MF001512.1
KP164571.1
KY680378.1
KY680395.1
KY272963.1
KY680351.1
KU355832.1
KY680349.1
KY272970.1
KY272968.1
MF001519.1
MF001509.1
KY272965.1
KY435466.1
KY680403.1
KY680409.1
KR559482.1
KR559480.1
MF001506.1
KY680350.1
KY680348.1
MF001508.1
KY680380.1
MF001516.1
MF001518.1
KR264950.1
KR264949.1
KY575572.1
KY680404.1
KY680381.1
MF001517.1
KY680394.1
MF001514.1
KY680410.1
KR559486.1
KR559479.1
KY680390.1
KY680354.1
KY272967.1
MF001515.1
MF001505.1
KY680400.1
KY575566.1
MF001507.1
KY680387.1
KY680374.1
MF001510.1
KY680357.1
KY680358.1
KY680360.1
KY575569.1
KY680369.1
MF001513.1
KY272966.1
KY272961.1
KY575565.1
MF001511.1
KY680391.1
KY680353.1
KY680408.1
KY680385.1KY680401.1
KR559470.1
KY680406.1
KR559477.1
KR559498.1
KY680383.1
KY680371.1
KY272964.1
KY680392.1
KR264951.1
KR559474.1
KR559487.1
KR559483.1
MF773564.1
MF773563.1
KJ689453.1
KJ689452.1
KJ451623.1
KJ451622.1
MF773559.1
KR559493.1
MF773562.1
KT308162.1
KT308159.1
KT308160.1
KC488650.1
KF318729.1
KT308163.1
KT308161.1
MF773561.1
FJ807897.1
MF773565.1
KM923920.1
KM923917.1
KM923919.1
KM923918.1
KX168429.1
EU703761.1
EU703759.1
EU703760.1
KX097988.1
KX097982.1
KY883764.1
KM673291.1
MG664851.1
HM045790.1
HM045800.1
HM045791.1
HM045797.1
HM045802.1
HM045796.1
HM045787.1
KX262987.1
KX262988.1
HM045789.1
HM045808.1
HM045814.1
EF452493.1
HM045810.1
HM045803.1
EF027140.1
HM
045813.1
HM
045788.1
EF027141.1
M
F076575.1
M
F076573.1
M
F076574.1
M
F076572.1
JQ
861257.1
JQ
861255.1
JQ
861258.1
JQ
861253.1
JQ
861256.1
JQ
861254.1
M
F076570.1
M
F076569.1
M
F076568.1
M
F076577.1
M
F076576.1
M
F076571.1
JQ861260.1
JQ861259.1
KJ579186.1
KJ579184.1
KJ579185.1
KJ579187.1
GU301779.1
KT324224.1
FJ445430.1
KT324228.1
FJ445431.1
KX009167.1
KX009171.1
MF773567.1
KX262997.1
GU301781.1
MF773569.1
KC862329.1
FJ445463.1
FJ445433.1
FJ445432.1
FJ445445.1
KT324225.1
FJ445502.1
FJ445443.1
FJ445484.1
HQ846359.1
HQ846358.1
KF151175.1
KF151174.1
KF590565.1
KF590567.1
GU908223.1KF590566.1KF590564.1KT324226.1KX009168.1HQ846357.1HQ846356.1JX088705.1KX009170.1GU199353.1GU199352.1KT324227.1KP164869.1GU301780.1MF773568.1KX009169.1FJ807899.1GQ428213.1KT336782.1KT336781.1GQ428214.1GQ428212.1
FJ000069.1
KX262989.1
KP003811.1
EU244823.1
KX262993.1
KP003810.1
FJ513657.1
GU013530.1
FJ513629.1
FJ513628.1
GU013528.1
FJ513679.1
FJ513637.1
GU013529.1
FJ513635.1
HM045801.1
GU199350.1
GU189061.1
FJ445428.1
HM045799.1
GQ428211.1
MF774616.1
MF774615.1
MF774617.1
MF774619.1
MF774618.1
MF740874.1
MF774613.1
MF773566.1
MG049915.1
KY057363.1
KY751908.1
KX619426.1
MG925665.1
MF499120.1
MG137428.1
KT336780.1
KT336779.1
KT336778.1
JQ065890.1
MG664850.1
JQ065891.1
KJ679578.1
JQ065892.1
KX619423.1
KX619422.1
KX619425.1
KX619424.1
KX881784.1
JN558836.1
JN558835.1
JN558834.1
GQ428210.1
FJ513654.1
FJ513645.1
FJ513673.1
FJ445426.1
FJ513632.1
KY575570.1
GU199351.1
FJ513675.1
FJ000067.1
KT336777.1
FJ807898.1
EU372006.1
GQ428215.1
EF027134.1
KJ941050.1
HM045794.1
KY575568.1
KY575567.1
FJ000068.1
FJ000062.1
FJ000064.1
FJ000063.1
FJ445427.1
KC614648.1
JF274082.1
EU564335.1
FJ807896.1
FJ445511.1
FJ445510.1
FJ000065.1
FJ000066.1
EF027137.1
EF210157.1
EF027138.1
EF027136.1
EF027135.1
KY575571.1
KP003808.1
KP003807.1
KP003809.1
EU037962.1
EU564334.1
EF012359.1
KT449801.1
DQ443544.1
KP702297.1
KF283986.1
MG649985.1
MG649973.1
MG649971.1
MG649974.1
MG649972.1
MG649984.1
MG649983.1
MG649981.1
KY124329.1
MG649982.1
MG649978.1
MG649977.1
MG649976.1
MG649970.1
MG649975.1
KY124328.1
MG649980.1
MG649979.1
KY055011.1
KY704952.1
KY704942.1
KU940225.1
KP164569.1
KP164568.1
KP164570.1
MG000876.1
KY704955.1
MG000875.1KY704954.1KX228391.1HM045823.1KY575574.1HM045784.1HM045812.1EF027139.1KY038947.1HM045793.1KY038946.1HM045822.1KP003813.1KP003812.1KX262996.1KJ679577.1HM045821.1HM045806.1HM045811.1HM045805.1HM045792.1
HM045809.1
HM045819.1
HM045818.1
HM045820.1
HM045817.1
KX262995.1
AY726732.1
KX262986.1
HM045786.1
HM045804.1
HM045816.1
HM045785.1
HM045815.1
North
South
Central
Americas
Caribbean
East
Middle
South
West
East
South
South-East
West
Europe
Oceania
Asia Other
Africa
Phylogenetic Tree Leaves
Middle African /
South American Lineage
Middle African /
South American Lineage -
Middle African only
African/Asian Lineages
American Lineage
Asian Urban Lineage
Western African Lineage
Indian Ocean Lineage
Eastern African Lineage

8. CHIKV Phylogeography AUL
!8

9. CHIKV Phylogeography ECSA-IOL
!9

10. CHIKV Phylogeography ECSA-MASA
!10

11. Part II: Conserved ncRNAs
!11

12. Conserved RNAs in Viral 3’UTR: ZIKV
!12
5' 3'
ΨDBSL1 SL2 DB 3'-SL
EXCURSION

13. Conserved RNAs in Viral 3’UTR: ZIKV
!13
5' 3'
ΨDBSL1 SL2 DB 3'-SL
• Accumulation of short flavivirus RNA (sfRNA) upon infection
• Stable decay intermediates produced by partial exoribonuclease degradation
• Xrn1 is efficiently stalled at conserved xrRNA structures
EXCURSION

14. Conserved RNAs in Viral 3’UTR
!14
• Accumulation of short flavivirus RNA (sfRNA) upon infection
• Stable decay intermediates produced by partial exoribonuclease degradation
• Xrn1 is efficiently stalled at conserved xrRNA structures
5' 3'
3'
5' 3'CDS
SL1 SL2 DB1 DB2 3'-SLSLA SLB
EXCURSION

15. Conserved RNAs in Viral 3’UTR
!15
• Accumulation of short flavivirus RNA (sfRNA) upon infection
• Stable decay intermediates produced by partial exoribonuclease degradation
• Xrn1 is efficiently stalled at conserved xrRNA structures
5' 3'
3'
5' 3'CDS
SL1 SL2 DB1 DB2 3'-SLSLA SLB
sfRNA mediates
pathogenicity !
EXCURSION

16. Conserved RNAs in Viral 3’UTR
!16
• Accumulation of short flavivirus RNA (sfRNA) upon infection
• Stable decay intermediates produced by partial exoribonuclease degradation
• Xrn1 is efficiently stalled at conserved xrRNA structures
5' 3'
3'
5' 3'CDS
SL1 SL2 DB1 DB2 3'-SLSLA SLB
sfRNA mediates
pathogenicity !
EXCURSION
How to find them?

17. Conserved RNAs in Viral 3’UTR
!17
• Accumulation of short flavivirus RNA (sfRNA) upon infection
• Stable decay intermediates produced by partial exoribonuclease degradation
• Xrn1 is efficiently stalled at conserved xrRNA structures
5' 3'
3'
5' 3'CDS
SL1 SL2 DB1 DB2 3'-SLSLA SLB
sfRNA mediates
pathogenicity !
How to find them?
viruses
Article
Functional RNA Structures in the 30UTR of
Tick-Borne, Insect-Specific and No-Known-Vector
Flaviviruses
Roman Ochsenreiter 1 , Ivo L. Hofacker 1,2 and Michael T. Wolfinger 1,2,*
1 Department of Theoretical Chemistry, University of Vienna, Währingerstraße 17, 1090 Vienna, Austria;
romanoch@tbi.univie.ac.at (R.O.); ivo@tbi.univie.ac.at (I.L.H.)
2 Research Group BCB, Faculty of Computer Science, University of Vienna, Währingerstraße 29,
1090 Vienna, Austria
* Correspondence: michael.wolfinger@univie.ac.at
Received: 1 March 2019; Accepted: 20 March 2019; Published: 24 March 2019
Abstract: Untranslated regions (UTRs) of flaviviruses contain a large number of RNA structural
EXCURSION
https://doi.org/10.3390/v11030298

18. • High mutation rate in RNA viruses due to error-prone RdRP
• For base pair (i,j): GC/CG/AU/UA/GU/UG
• Consistent mutation: different standard combinations
• Compensatory mutation: both positions are mutated
• Presence of both strongly supports predicted base pair (i,j)
!18
RNA Covariation as Evolutionary Trait
((((((((...((((((((..............))))))))....))......((((......))))))))))
USUV.10 CCACGGCUCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
USUV.11 UCACGGCCCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
.........10........20........30........40........50........60........70..
C
C
A
C
G
G
CC
C
AA
GCGAACAG
A
C
GGU
G
A
U
G
C
G A _
A
C U G U U C G U
G
G A A
G G
A
C
U A
G
A
G
G
U
U A
G
A
G
G
A
G
A
C
C
C
C
G
U
G
G
f
types of pairs:
1 2 3 4 5 6
allowed pairs:
incompatible
pairs in column
0
1
2
A-U
U-A
G-C
C-G
G-U
U-G

19. • High mutation rate in RNA viruses due to error-prone RdRP
• For base pair (i,j): GC/CG/AU/UA/GU/UG
• Consistent mutation: different standard combinations
• Compensatory mutation: both positions are mutated
• Presence of both strongly supports predicted base pair (i,j)
!19
RNA Covariation as Evolutionary Trait
((((((((...((((((((..............))))))))....))......((((......))))))))))
USUV.10 CCACGGCUCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
USUV.11 UCACGGCCCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
.........10........20........30........40........50........60........70..
C
C
A
C
G
G
CC
C
AA
GCGAACAG
A
C
GGU
G
A
U
G
C
G A _
A
C U G U U C G U
G
G A A
G G
A
C
U A
G
A
G
G
U
U A
G
A
G
G
A
G
A
C
C
C
C
G
U
G
G
LIV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
SGEV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
TBEV T.xrRNA1 T.SL6 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
OHFV T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
LGTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
DTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
POWV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
KSIV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like
~ 300 nt
b
G
R
Y
R
C
G
G
C
A
G
C
A C
R
C
Y
Y
R
G
Y
G
A
C G G G A
A R
U
G
G
UCG
C
UCCCG
A
C
G
Y
A
R
R
Y
Y
5´
R
G
G
R
Y
R
Y
G
G
C
A
R
C
R
C
R
C
A Y
G R
GG
R
A
R
G
R
Y
G
C
C
C
R
R
R
Y
Y
Y
Y
5´ C
Y
Y
C
R
A
A
G G
Y
A
R
C
R
R R G5´
G
G
C
G
GY
U
C
U U
U
C
U
C
C C
R
A
G
C
Y
C
Y
Y
A
G
A
C
A
C
A G A
U
A
G
U
C
U
R
A C
A
G
RR
R
G
G
R
G
Y
U
Y
G
A
A
A
5´
R
R
R
C
C
C
C
G
R
R
Y
Y
G
G
A G
G
R
G
G
G
A
R
R
R
R
R
RA
A
A
U
U G G
C
A
R
Y
Y
Y
Y
Y
Y
U
C R G
G
A
U
UU
Y
Y
Y
Y
5´
T.xrRNA2 SL6 Y-shaped (Y1) Flavi_CRET.xrRNA1
5' 3'
.((((..((((((((...........))))))))......))))....
SGEV|261-306 CCCCCC-GCACCAUGACAAGGCCGAACAUGGUGCACCAAAGGGGAG-G 46
TBEV|532-577 CCCCCU-GCAUCAUGAUAAGGCCGAACAUGGUGCAUGAAAGGGGAG-G 46
LIV|268-312 CCCCCC-GCCCCAUGACAAGGCCGAACAUGGAGCAUUAAAGG-GAG-G 45
LGTV|339-381 CCCCUU-GCGUCCAGAGAAGGCCGAACUGGGCGU---UAUAAGGAG-G 43
OHFV|179-222 CCCCCC-GCACCAUG-GAAGGCCAAACAUGGUGCAUG-AAGGGAAA-G 44
KFDV|164-204 CCAAAA-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
ALKV|165-205 CCAAAG-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
DTV|248-294 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCAUUCUAGAG 47
POWV|249-293 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCGUU-UAG-G 45
TYUV|341-386 CUUUC--CAUGAGAGGAGACGGUCAACUCUCAUGGAACAAGAAGACCG 46
NEGV|129-174 CCCCCCCUGGCCAGAAAAAGGGGGGGCAAACAGGCC--AGGGGUGAAG 46
KSIV|233-270 CUGAC---CAUCCCU-CAAGGCCGAGUGGGAUGC------GUAUGAAG 38
.........10........20........30........40.......
Seq. Conservation:
*
*
c
d f
types of pairs:
1 2 3 4 5 6
SL6
e (((((..((((((((((....)))))))).))...((((((((...........)))))))).........)).)))
POWV|291-361 AGGAG--CCCCCGAGCAUAA---CUCGGGAGGAGGGAGGAAGA-AAAUUGGCAAUCUUCCUCGGGAUUUUUCCGCCU 71
DTV|290-354 UAGAG--CCCCCGGGCAUAA---CUCGGGAGGAGGGGGGAAGA-CAAUUGGCAAUCUUCCCCGGGAUU------UUU 65
LGTV|377-449 GGAGG--CCCCCAGGGGGAAACCCCUGGGAGGAGGGAAGAGAG-AAAUUGGCAACUCUCUUCAGGAUAUUUCC-UCC 73
TBEV|573-645 GGAGG--CCCCCGGAAGCACGCUUCCGGGAGGAGGGAAGAGAG-AAAUUGGCAGCUCUCUUCAGGAUUUUUCC-UCC 73
Y1
allowed pairs:
incompatible
pairs in column
0
1
2
A-U
U-A
G-C
C-G
G-U
U-G

20. !20
RNA Covariation as Evolutionary Trait
((((((((...((((((((..............))))))))....))......((((......))))))))))
USUV.10 CCACGGCUCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
USUV.11 UCACGGCCCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
SLEV.12 CGGCGGCCCAAACCAUGGAG----UGCGUGA---CCAUGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGCUG 66
.........10........20........30........40........50........60........70..
C
C
A
C
G
G
CC
C
AA
GCGAACAG
A
C
GGU
G
A
U
G
C
G A _
A
C U G U U C G U
G
G A A
G G
A
C
U A
G
A
G
G
U
U A
G
A
G
G
A
G
A
C
C
C
C
G
U
G
G
U
GC
G
• High mutation rate in RNA viruses due to error-prone RdRP
• For base pair (i,j): GC/CG/AU/UA/GU/UG
• Consistent mutation: different standard combinations
• Compensatory mutation: both positions are mutated
• Presence of both strongly supports predicted base pair (i,j)
LIV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
SGEV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
TBEV T.xrRNA1 T.SL6 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
OHFV T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
LGTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
DTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
POWV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
KSIV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like
~ 300 nt
b
G
R
Y
R
C
G
G
C
A
G
C
A C
R
C
Y
Y
R
G
Y
G
A
C G G G A
A R
U
G
G
UCG
C
UCCCG
A
C
G
Y
A
R
R
Y
Y
5´
R
G
G
R
Y
R
Y
G
G
C
A
R
C
R
C
R
C
A Y
G R
GG
R
A
R
G
R
Y
G
C
C
C
R
R
R
Y
Y
Y
Y
5´ C
Y
Y
C
R
A
A
G G
Y
A
R
C
R
R R G5´
G
G
C
G
GY
U
C
U U
U
C
U
C
C C
R
A
G
C
Y
C
Y
Y
A
G
A
C
A
C
A G A
U
A
G
U
C
U
R
A C
A
G
RR
R
G
G
R
G
Y
U
Y
G
A
A
A
5´
R
R
R
C
C
C
C
G
R
R
Y
Y
G
G
A G
G
R
G
G
G
A
R
R
R
R
R
RA
A
A
U
U G G
C
A
R
Y
Y
Y
Y
Y
Y
U
C R G
G
A
U
UU
Y
Y
Y
Y
5´
T.xrRNA2 SL6 Y-shaped (Y1) Flavi_CRET.xrRNA1
5' 3'
.((((..((((((((...........))))))))......))))....
SGEV|261-306 CCCCCC-GCACCAUGACAAGGCCGAACAUGGUGCACCAAAGGGGAG-G 46
TBEV|532-577 CCCCCU-GCAUCAUGAUAAGGCCGAACAUGGUGCAUGAAAGGGGAG-G 46
LIV|268-312 CCCCCC-GCCCCAUGACAAGGCCGAACAUGGAGCAUUAAAGG-GAG-G 45
LGTV|339-381 CCCCUU-GCGUCCAGAGAAGGCCGAACUGGGCGU---UAUAAGGAG-G 43
OHFV|179-222 CCCCCC-GCACCAUG-GAAGGCCAAACAUGGUGCAUG-AAGGGAAA-G 44
KFDV|164-204 CCAAAA-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
ALKV|165-205 CCAAAG-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
DTV|248-294 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCAUUCUAGAG 47
POWV|249-293 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCGUU-UAG-G 45
TYUV|341-386 CUUUC--CAUGAGAGGAGACGGUCAACUCUCAUGGAACAAGAAGACCG 46
NEGV|129-174 CCCCCCCUGGCCAGAAAAAGGGGGGGCAAACAGGCC--AGGGGUGAAG 46
KSIV|233-270 CUGAC---CAUCCCU-CAAGGCCGAGUGGGAUGC------GUAUGAAG 38
.........10........20........30........40.......
Seq. Conservation:
*
*
c
d f
types of pairs:
1 2 3 4 5 6
SL6
e (((((..((((((((((....)))))))).))...((((((((...........)))))))).........)).)))
POWV|291-361 AGGAG--CCCCCGAGCAUAA---CUCGGGAGGAGGGAGGAAGA-AAAUUGGCAAUCUUCCUCGGGAUUUUUCCGCCU 71
DTV|290-354 UAGAG--CCCCCGGGCAUAA---CUCGGGAGGAGGGGGGAAGA-CAAUUGGCAAUCUUCCCCGGGAUU------UUU 65
LGTV|377-449 GGAGG--CCCCCAGGGGGAAACCCCUGGGAGGAGGGAAGAGAG-AAAUUGGCAACUCUCUUCAGGAUAUUUCC-UCC 73
TBEV|573-645 GGAGG--CCCCCGGAAGCACGCUUCCGGGAGGAGGGAAGAGAG-AAAUUGGCAGCUCUCUUCAGGAUUUUUCC-UCC 73
Y1
allowed pairs:
incompatible
pairs in column
0
1
2
A-U
U-A
G-C
C-G
G-U
U-G

21. !21
RNA Covariation as Evolutionary Trait
C
C
A
C
G
G
CC
C
AA
ACCAACAA
A
_
___
G
A
C
G
C
G A _
_
_ C C A U C G C
G
G A A
G G
A
C
U A
G
A
G
G
U
U A
G
A
G
G
A
G
A
C
C
C
C
G
C
G
G
C
A_
A
((((((((...((((((((..............))))))))....))......((((......))))))))))
USUV.10 CCACGGCUCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
USUV.11 UCACGGCCCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
SLEV.12 CGGCGGCCCAAACCAUGGAG----UGCGUGA---CCAUGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGCUG 66
SLEV.13 CAGCGGCCCAAACCAUGGAG----UGCGUGA---CCAUGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGCUG 66
.........10........20........30........40........50........60........70..
• High mutation rate in RNA viruses due to error-prone RdRP
• For base pair (i,j): GC/CG/AU/UA/GU/UG
• Consistent mutation: different standard combinations
• Compensatory mutation: both positions are mutated
• Presence of both strongly supports predicted base pair (i,j)
LIV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
SGEV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
TBEV T.xrRNA1 T.SL6 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
OHFV T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
LGTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
DTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
POWV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
KSIV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like
~ 300 nt
b
G
R
Y
R
C
G
G
C
A
G
C
A C
R
C
Y
Y
R
G
Y
G
A
C G G G A
A R
U
G
G
UCG
C
UCCCG
A
C
G
Y
A
R
R
Y
Y
5´
R
G
G
R
Y
R
Y
G
G
C
A
R
C
R
C
R
C
A Y
G R
GG
R
A
R
G
R
Y
G
C
C
C
R
R
R
Y
Y
Y
Y
5´ C
Y
Y
C
R
A
A
G G
Y
A
R
C
R
R R G5´
G
G
C
G
GY
U
C
U U
U
C
U
C
C C
R
A
G
C
Y
C
Y
Y
A
G
A
C
A
C
A G A
U
A
G
U
C
U
R
A C
A
G
RR
R
G
G
R
G
Y
U
Y
G
A
A
A
5´
R
R
R
C
C
C
C
G
R
R
Y
Y
G
G
A G
G
R
G
G
G
A
R
R
R
R
R
RA
A
A
U
U G G
C
A
R
Y
Y
Y
Y
Y
Y
U
C R G
G
A
U
UU
Y
Y
Y
Y
5´
T.xrRNA2 SL6 Y-shaped (Y1) Flavi_CRET.xrRNA1
5' 3'
.((((..((((((((...........))))))))......))))....
SGEV|261-306 CCCCCC-GCACCAUGACAAGGCCGAACAUGGUGCACCAAAGGGGAG-G 46
TBEV|532-577 CCCCCU-GCAUCAUGAUAAGGCCGAACAUGGUGCAUGAAAGGGGAG-G 46
LIV|268-312 CCCCCC-GCCCCAUGACAAGGCCGAACAUGGAGCAUUAAAGG-GAG-G 45
LGTV|339-381 CCCCUU-GCGUCCAGAGAAGGCCGAACUGGGCGU---UAUAAGGAG-G 43
OHFV|179-222 CCCCCC-GCACCAUG-GAAGGCCAAACAUGGUGCAUG-AAGGGAAA-G 44
KFDV|164-204 CCAAAA-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
ALKV|165-205 CCAAAG-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
DTV|248-294 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCAUUCUAGAG 47
POWV|249-293 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCGUU-UAG-G 45
TYUV|341-386 CUUUC--CAUGAGAGGAGACGGUCAACUCUCAUGGAACAAGAAGACCG 46
NEGV|129-174 CCCCCCCUGGCCAGAAAAAGGGGGGGCAAACAGGCC--AGGGGUGAAG 46
KSIV|233-270 CUGAC---CAUCCCU-CAAGGCCGAGUGGGAUGC------GUAUGAAG 38
.........10........20........30........40.......
Seq. Conservation:
*
*
c
d f
types of pairs:
1 2 3 4 5 6
SL6
e (((((..((((((((((....)))))))).))...((((((((...........)))))))).........)).)))
POWV|291-361 AGGAG--CCCCCGAGCAUAA---CUCGGGAGGAGGGAGGAAGA-AAAUUGGCAAUCUUCCUCGGGAUUUUUCCGCCU 71
DTV|290-354 UAGAG--CCCCCGGGCAUAA---CUCGGGAGGAGGGGGGAAGA-CAAUUGGCAAUCUUCCCCGGGAUU------UUU 65
LGTV|377-449 GGAGG--CCCCCAGGGGGAAACCCCUGGGAGGAGGGAAGAGAG-AAAUUGGCAACUCUCUUCAGGAUAUUUCC-UCC 73
TBEV|573-645 GGAGG--CCCCCGGAAGCACGCUUCCGGGAGGAGGGAAGAGAG-AAAUUGGCAGCUCUCUUCAGGAUUUUUCC-UCC 73
Y1
allowed pairs:
incompatible
pairs in column
0
1
2
A-U
U-A
G-C
C-G
G-U
U-G

22. !22
RNA Covariation as Evolutionary Trait
C
C
A
C
G
G
CC
C
AA
GCCAUGAG
A
_
G_U
G
A
U
G
U
G A _
A
C U C A U G G C
G
G A A
G G
A
C
U A
G
A
G
G
U
U A
G
A
G
G
A
G
A
C
C
C
C
G
U
G
G
C
GC
G
((((((((...((((((((..............))))))))....))......((((......))))))))))
ALFV.09 CCACGGCCCGGGCCAUGAGU-GAUGAUGUUA-ACUCAUGGC-GAAGGACUAGAGGUUAGAGGAGACCCCGUGG 70
USUV.10 CCACGGCUCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
USUV.11 UCACGGCCCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
SLEV.12 CGGCGGCCCAAACCAUGGAG----UGCGUGA---CCAUGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGCUG 66
SLEV.13 CAGCGGCCCAAACCAUGGAG----UGCGUGA---CCAUGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGCUG 66
.........10........20........30........40........50........60........70..
• High mutation rate in RNA viruses due to error-prone RdRP
• For base pair (i,j): GC/CG/AU/UA/GU/UG
• Consistent mutation: different standard combinations
• Compensatory mutation: both positions are mutated
• Presence of both strongly supports predicted base pair (i,j)
LIV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
SGEV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
TBEV T.xrRNA1 T.SL6 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
OHFV T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
LGTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
DTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
POWV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
KSIV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like
~ 300 nt
b
G
R
Y
R
C
G
G
C
A
G
C
A C
R
C
Y
Y
R
G
Y
G
A
C G G G A
A R
U
G
G
UCG
C
UCCCG
A
C
G
Y
A
R
R
Y
Y
5´
R
G
G
R
Y
R
Y
G
G
C
A
R
C
R
C
R
C
A Y
G R
GG
R
A
R
G
R
Y
G
C
C
C
R
R
R
Y
Y
Y
Y
5´ C
Y
Y
C
R
A
A
G G
Y
A
R
C
R
R R G5´
G
G
C
G
GY
U
C
U U
U
C
U
C
C C
R
A
G
C
Y
C
Y
Y
A
G
A
C
A
C
A G A
U
A
G
U
C
U
R
A C
A
G
RR
R
G
G
R
G
Y
U
Y
G
A
A
A
5´
R
R
R
C
C
C
C
G
R
R
Y
Y
G
G
A G
G
R
G
G
G
A
R
R
R
R
R
RA
A
A
U
U G G
C
A
R
Y
Y
Y
Y
Y
Y
U
C R G
G
A
U
UU
Y
Y
Y
Y
5´
T.xrRNA2 SL6 Y-shaped (Y1) Flavi_CRET.xrRNA1
5' 3'
.((((..((((((((...........))))))))......))))....
SGEV|261-306 CCCCCC-GCACCAUGACAAGGCCGAACAUGGUGCACCAAAGGGGAG-G 46
TBEV|532-577 CCCCCU-GCAUCAUGAUAAGGCCGAACAUGGUGCAUGAAAGGGGAG-G 46
LIV|268-312 CCCCCC-GCCCCAUGACAAGGCCGAACAUGGAGCAUUAAAGG-GAG-G 45
LGTV|339-381 CCCCUU-GCGUCCAGAGAAGGCCGAACUGGGCGU---UAUAAGGAG-G 43
OHFV|179-222 CCCCCC-GCACCAUG-GAAGGCCAAACAUGGUGCAUG-AAGGGAAA-G 44
KFDV|164-204 CCAAAA-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
ALKV|165-205 CCAAAG-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
DTV|248-294 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCAUUCUAGAG 47
POWV|249-293 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCGUU-UAG-G 45
TYUV|341-386 CUUUC--CAUGAGAGGAGACGGUCAACUCUCAUGGAACAAGAAGACCG 46
NEGV|129-174 CCCCCCCUGGCCAGAAAAAGGGGGGGCAAACAGGCC--AGGGGUGAAG 46
KSIV|233-270 CUGAC---CAUCCCU-CAAGGCCGAGUGGGAUGC------GUAUGAAG 38
.........10........20........30........40.......
Seq. Conservation:
*
*
c
d f
types of pairs:
1 2 3 4 5 6
SL6
e (((((..((((((((((....)))))))).))...((((((((...........)))))))).........)).)))
POWV|291-361 AGGAG--CCCCCGAGCAUAA---CUCGGGAGGAGGGAGGAAGA-AAAUUGGCAAUCUUCCUCGGGAUUUUUCCGCCU 71
DTV|290-354 UAGAG--CCCCCGGGCAUAA---CUCGGGAGGAGGGGGGAAGA-CAAUUGGCAAUCUUCCCCGGGAUU------UUU 65
LGTV|377-449 GGAGG--CCCCCAGGGGGAAACCCCUGGGAGGAGGGAAGAGAG-AAAUUGGCAACUCUCUUCAGGAUAUUUCC-UCC 73
TBEV|573-645 GGAGG--CCCCCGGAAGCACGCUUCCGGGAGGAGGGAAGAGAG-AAAUUGGCAGCUCUCUUCAGGAUUUUUCC-UCC 73
Y1
allowed pairs:
incompatible
pairs in column
0
1
2
A-U
U-A
G-C
C-G
G-U
U-G

23. !23
RNA Covariation as Evolutionary Trait
C
C
A
C
G
G
CC
C
AA
GCCAUGAG
G
_
GGU
G
A
U
G
C
G A _
A
C U C A U G G C
G
G A A
G G
A
C
U A
G
A
G
G
U
U A
G
A
G
G
A
G
A
C
C
C
C
G
C
G
G
C
GU
A
C
G
((((((((...((((((((..............))))))))....))......((((......))))))))))
MVEV.08 CCGCAGCCCGGGCCGGGAGGAGGUGAUGCGA-AC-CCCGGC-GAAGGACUAGAGGUUAGAGGAGACCCUGCGG 70
ALFV.09 CCACGGCCCGGGCCAUGAGU-GAUGAUGUUA-ACUCAUGGC-GAAGGACUAGAGGUUAGAGGAGACCCCGUGG 70
USUV.10 CCACGGCUCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
USUV.11 UCACGGCCCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
SLEV.12 CGGCGGCCCAAACCAUGGAG----UGCGUGA---CCAUGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGCUG 66
SLEV.13 CAGCGGCCCAAACCAUGGAG----UGCGUGA---CCAUGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGCUG 66
.........10........20........30........40........50........60........70..
• High mutation rate in RNA viruses due to error-prone RdRP
• For base pair (i,j): GC/CG/AU/UA/GU/UG
• Consistent mutation: different standard combinations
• Compensatory mutation: both positions are mutated
• Presence of both strongly supports predicted base pair (i,j)
LIV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
SGEV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
TBEV T.xrRNA1 T.SL6 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
OHFV T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
LGTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
DTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
POWV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
KSIV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like
~ 300 nt
b
G
R
Y
R
C
G
G
C
A
G
C
A C
R
C
Y
Y
R
G
Y
G
A
C G G G A
A R
U
G
G
UCG
C
UCCCG
A
C
G
Y
A
R
R
Y
Y
5´
R
G
G
R
Y
R
Y
G
G
C
A
R
C
R
C
R
C
A Y
G R
GG
R
A
R
G
R
Y
G
C
C
C
R
R
R
Y
Y
Y
Y
5´ C
Y
Y
C
R
A
A
G G
Y
A
R
C
R
R R G5´
G
G
C
G
GY
U
C
U U
U
C
U
C
C C
R
A
G
C
Y
C
Y
Y
A
G
A
C
A
C
A G A
U
A
G
U
C
U
R
A C
A
G
RR
R
G
G
R
G
Y
U
Y
G
A
A
A
5´
R
R
R
C
C
C
C
G
R
R
Y
Y
G
G
A G
G
R
G
G
G
A
R
R
R
R
R
RA
A
A
U
U G G
C
A
R
Y
Y
Y
Y
Y
Y
U
C R G
G
A
U
UU
Y
Y
Y
Y
5´
T.xrRNA2 SL6 Y-shaped (Y1) Flavi_CRET.xrRNA1
5' 3'
.((((..((((((((...........))))))))......))))....
SGEV|261-306 CCCCCC-GCACCAUGACAAGGCCGAACAUGGUGCACCAAAGGGGAG-G 46
TBEV|532-577 CCCCCU-GCAUCAUGAUAAGGCCGAACAUGGUGCAUGAAAGGGGAG-G 46
LIV|268-312 CCCCCC-GCCCCAUGACAAGGCCGAACAUGGAGCAUUAAAGG-GAG-G 45
LGTV|339-381 CCCCUU-GCGUCCAGAGAAGGCCGAACUGGGCGU---UAUAAGGAG-G 43
OHFV|179-222 CCCCCC-GCACCAUG-GAAGGCCAAACAUGGUGCAUG-AAGGGAAA-G 44
KFDV|164-204 CCAAAA-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
ALKV|165-205 CCAAAG-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
DTV|248-294 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCAUUCUAGAG 47
POWV|249-293 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCGUU-UAG-G 45
TYUV|341-386 CUUUC--CAUGAGAGGAGACGGUCAACUCUCAUGGAACAAGAAGACCG 46
NEGV|129-174 CCCCCCCUGGCCAGAAAAAGGGGGGGCAAACAGGCC--AGGGGUGAAG 46
KSIV|233-270 CUGAC---CAUCCCU-CAAGGCCGAGUGGGAUGC------GUAUGAAG 38
.........10........20........30........40.......
Seq. Conservation:
*
*
c
d f
types of pairs:
1 2 3 4 5 6
SL6
e (((((..((((((((((....)))))))).))...((((((((...........)))))))).........)).)))
POWV|291-361 AGGAG--CCCCCGAGCAUAA---CUCGGGAGGAGGGAGGAAGA-AAAUUGGCAAUCUUCCUCGGGAUUUUUCCGCCU 71
DTV|290-354 UAGAG--CCCCCGGGCAUAA---CUCGGGAGGAGGGGGGAAGA-CAAUUGGCAAUCUUCCCCGGGAUU------UUU 65
LGTV|377-449 GGAGG--CCCCCAGGGGGAAACCCCUGGGAGGAGGGAAGAGAG-AAAUUGGCAACUCUCUUCAGGAUAUUUCC-UCC 73
TBEV|573-645 GGAGG--CCCCCGGAAGCACGCUUCCGGGAGGAGGGAAGAGAG-AAAUUGGCAGCUCUCUUCAGGAUUUUUCC-UCC 73
Y1
allowed pairs:
incompatible
pairs in column
0
1
2
A-U
U-A
G-C
C-G
G-U
U-G

24. !24
RNA Covariation as Evolutionary Trait
C
C
G
C
G
G
CC
C
AA
GCCAUGAG
G
_
GGU
G
A
U
G
U
G A _
A
C C C A U G G C
G
G A A
G G
A
C
U A
G
A
G
G
U
U A
G
A
G
G
A
G
A
C
C
C
C
G
C
G
G
C
GC
A
C
G
((((((((...((((((((..............))))))))....))......((((......))))))))))
WNV.07 ACGCGGCCCAAAUCCUGGUGA-U-GGUGUUA-UGCCAGGGUGGAAGGACUAGAGGUUAGAGGAGACCCCGCGU 70
MVEV.08 CCGCAGCCCGGGCCGGGAGGAGGUGAUGCGA-AC-CCCGGC-GAAGGACUAGAGGUUAGAGGAGACCCUGCGG 70
ALFV.09 CCACGGCCCGGGCCAUGAGU-GAUGAUGUUA-ACUCAUGGC-GAAGGACUAGAGGUUAGAGGAGACCCCGUGG 70
USUV.10 CCACGGCUCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
USUV.11 UCACGGCCCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
SLEV.12 CGGCGGCCCAAACCAUGGAG----UGCGUGA---CCAUGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGCUG 66
SLEV.13 CAGCGGCCCAAACCAUGGAG----UGCGUGA---CCAUGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGCUG 66
.........10........20........30........40........50........60........70..
• High mutation rate in RNA viruses due to error-prone RdRP
• For base pair (i,j): GC/CG/AU/UA/GU/UG
• Consistent mutation: different standard combinations
• Compensatory mutation: both positions are mutated
• Presence of both strongly supports predicted base pair (i,j)
LIV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
SGEV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
TBEV T.xrRNA1 T.SL6 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
OHFV T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
LGTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
DTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
POWV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
KSIV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like
~ 300 nt
b
G
R
Y
R
C
G
G
C
A
G
C
A C
R
C
Y
Y
R
G
Y
G
A
C G G G A
A R
U
G
G
UCG
C
UCCCG
A
C
G
Y
A
R
R
Y
Y
5´
R
G
G
R
Y
R
Y
G
G
C
A
R
C
R
C
R
C
A Y
G R
GG
R
A
R
G
R
Y
G
C
C
C
R
R
R
Y
Y
Y
Y
5´ C
Y
Y
C
R
A
A
G G
Y
A
R
C
R
R R G5´
G
G
C
G
GY
U
C
U U
U
C
U
C
C C
R
A
G
C
Y
C
Y
Y
A
G
A
C
A
C
A G A
U
A
G
U
C
U
R
A C
A
G
RR
R
G
G
R
G
Y
U
Y
G
A
A
A
5´
R
R
R
C
C
C
C
G
R
R
Y
Y
G
G
A G
G
R
G
G
G
A
R
R
R
R
R
RA
A
A
U
U G G
C
A
R
Y
Y
Y
Y
Y
Y
U
C R G
G
A
U
UU
Y
Y
Y
Y
5´
T.xrRNA2 SL6 Y-shaped (Y1) Flavi_CRET.xrRNA1
5' 3'
.((((..((((((((...........))))))))......))))....
SGEV|261-306 CCCCCC-GCACCAUGACAAGGCCGAACAUGGUGCACCAAAGGGGAG-G 46
TBEV|532-577 CCCCCU-GCAUCAUGAUAAGGCCGAACAUGGUGCAUGAAAGGGGAG-G 46
LIV|268-312 CCCCCC-GCCCCAUGACAAGGCCGAACAUGGAGCAUUAAAGG-GAG-G 45
LGTV|339-381 CCCCUU-GCGUCCAGAGAAGGCCGAACUGGGCGU---UAUAAGGAG-G 43
OHFV|179-222 CCCCCC-GCACCAUG-GAAGGCCAAACAUGGUGCAUG-AAGGGAAA-G 44
KFDV|164-204 CCAAAA-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
ALKV|165-205 CCAAAG-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
DTV|248-294 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCAUUCUAGAG 47
POWV|249-293 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCGUU-UAG-G 45
TYUV|341-386 CUUUC--CAUGAGAGGAGACGGUCAACUCUCAUGGAACAAGAAGACCG 46
NEGV|129-174 CCCCCCCUGGCCAGAAAAAGGGGGGGCAAACAGGCC--AGGGGUGAAG 46
KSIV|233-270 CUGAC---CAUCCCU-CAAGGCCGAGUGGGAUGC------GUAUGAAG 38
.........10........20........30........40.......
Seq. Conservation:
*
*
c
d f
types of pairs:
1 2 3 4 5 6
SL6
e (((((..((((((((((....)))))))).))...((((((((...........)))))))).........)).)))
POWV|291-361 AGGAG--CCCCCGAGCAUAA---CUCGGGAGGAGGGAGGAAGA-AAAUUGGCAAUCUUCCUCGGGAUUUUUCCGCCU 71
DTV|290-354 UAGAG--CCCCCGGGCAUAA---CUCGGGAGGAGGGGGGAAGA-CAAUUGGCAAUCUUCCCCGGGAUU------UUU 65
LGTV|377-449 GGAGG--CCCCCAGGGGGAAACCCCUGGGAGGAGGGAAGAGAG-AAAUUGGCAACUCUCUUCAGGAUAUUUCC-UCC 73
TBEV|573-645 GGAGG--CCCCCGGAAGCACGCUUCCGGGAGGAGGGAAGAGAG-AAAUUGGCAGCUCUCUUCAGGAUUUUUCC-UCC 73
Y1
allowed pairs:
incompatible
pairs in column
0
1
2
A-U
U-A
G-C
C-G
G-U
U-G

25. !25
RNA Covariation as Evolutionary Trait
C
C
A
C
G
G
CC
C
AA
ACCAUGAG
G
_
_G_
G
A
U
G
C
G A _
A
C C C A U G G C
G
G A A
G G
A
C
U A
G
A
G
G
U
U A
G
A
G
G
A
G
A
C
C
C
C
G
C
G
G
C
AC
A
C
G
((((((((...((((((((..............))))))))....))......((((......))))))))))
JEV.06 CCACGGCCCAAACCUCAUCUA-G-GAUGCAAUAGAUGAGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGUGG 71
WNV.07 ACGCGGCCCAAAUCCUGGUGA-U-GGUGUUA-UGCCAGGGUGGAAGGACUAGAGGUUAGAGGAGACCCCGCGU 70
MVEV.08 CCGCAGCCCGGGCCGGGAGGAGGUGAUGCGA-AC-CCCGGC-GAAGGACUAGAGGUUAGAGGAGACCCUGCGG 70
ALFV.09 CCACGGCCCGGGCCAUGAGU-GAUGAUGUUA-ACUCAUGGC-GAAGGACUAGAGGUUAGAGGAGACCCCGUGG 70
USUV.10 CCACGGCUCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
USUV.11 UCACGGCCCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
SLEV.12 CGGCGGCCCAAACCAUGGAG----UGCGUGA---CCAUGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGCUG 66
SLEV.13 CAGCGGCCCAAACCAUGGAG----UGCGUGA---CCAUGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGCUG 66
.........10........20........30........40........50........60........70..
• High mutation rate in RNA viruses due to error-prone RdRP
• For base pair (i,j): GC/CG/AU/UA/GU/UG
• Consistent mutation: different standard combinations
• Compensatory mutation: both positions are mutated
• Presence of both strongly supports predicted base pair (i,j)
LIV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
SGEV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
TBEV T.xrRNA1 T.SL6 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
OHFV T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
LGTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
DTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
POWV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
KSIV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like
~ 300 nt
b
G
R
Y
R
C
G
G
C
A
G
C
A C
R
C
Y
Y
R
G
Y
G
A
C G G G A
A R
U
G
G
UCG
C
UCCCG
A
C
G
Y
A
R
R
Y
Y
5´
R
G
G
R
Y
R
Y
G
G
C
A
R
C
R
C
R
C
A Y
G R
GG
R
A
R
G
R
Y
G
C
C
C
R
R
R
Y
Y
Y
Y
5´ C
Y
Y
C
R
A
A
G G
Y
A
R
C
R
R R G5´
G
G
C
G
GY
U
C
U U
U
C
U
C
C C
R
A
G
C
Y
C
Y
Y
A
G
A
C
A
C
A G A
U
A
G
U
C
U
R
A C
A
G
RR
R
G
G
R
G
Y
U
Y
G
A
A
A
5´
R
R
R
C
C
C
C
G
R
R
Y
Y
G
G
A G
G
R
G
G
G
A
R
R
R
R
R
RA
A
A
U
U G G
C
A
R
Y
Y
Y
Y
Y
Y
U
C R G
G
A
U
UU
Y
Y
Y
Y
5´
T.xrRNA2 SL6 Y-shaped (Y1) Flavi_CRET.xrRNA1
5' 3'
.((((..((((((((...........))))))))......))))....
SGEV|261-306 CCCCCC-GCACCAUGACAAGGCCGAACAUGGUGCACCAAAGGGGAG-G 46
TBEV|532-577 CCCCCU-GCAUCAUGAUAAGGCCGAACAUGGUGCAUGAAAGGGGAG-G 46
LIV|268-312 CCCCCC-GCCCCAUGACAAGGCCGAACAUGGAGCAUUAAAGG-GAG-G 45
LGTV|339-381 CCCCUU-GCGUCCAGAGAAGGCCGAACUGGGCGU---UAUAAGGAG-G 43
OHFV|179-222 CCCCCC-GCACCAUG-GAAGGCCAAACAUGGUGCAUG-AAGGGAAA-G 44
KFDV|164-204 CCAAAA-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
ALKV|165-205 CCAAAG-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
DTV|248-294 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCAUUCUAGAG 47
POWV|249-293 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCGUU-UAG-G 45
TYUV|341-386 CUUUC--CAUGAGAGGAGACGGUCAACUCUCAUGGAACAAGAAGACCG 46
NEGV|129-174 CCCCCCCUGGCCAGAAAAAGGGGGGGCAAACAGGCC--AGGGGUGAAG 46
KSIV|233-270 CUGAC---CAUCCCU-CAAGGCCGAGUGGGAUGC------GUAUGAAG 38
.........10........20........30........40.......
Seq. Conservation:
*
*
c
d f
types of pairs:
1 2 3 4 5 6
SL6
e (((((..((((((((((....)))))))).))...((((((((...........)))))))).........)).)))
POWV|291-361 AGGAG--CCCCCGAGCAUAA---CUCGGGAGGAGGGAGGAAGA-AAAUUGGCAAUCUUCCUCGGGAUUUUUCCGCCU 71
DTV|290-354 UAGAG--CCCCCGGGCAUAA---CUCGGGAGGAGGGGGGAAGA-CAAUUGGCAAUCUUCCCCGGGAUU------UUU 65
LGTV|377-449 GGAGG--CCCCCAGGGGGAAACCCCUGGGAGGAGGGAAGAGAG-AAAUUGGCAACUCUCUUCAGGAUAUUUCC-UCC 73
TBEV|573-645 GGAGG--CCCCCGGAAGCACGCUUCCGGGAGGAGGGAAGAGAG-AAAUUGGCAGCUCUCUUCAGGAUUUUUCC-UCC 73
Y1
allowed pairs:
incompatible
pairs in column
0
1
2
A-U
U-A
G-C
C-G
G-U
U-G

26. !26
RNA Covariation as Evolutionary Trait
C
C
A
C
G
G
CC
C
AA
GCCAUGAG
G
A
_G_
G
A
U
G
C
G A _
A
C C C A U G G C
G
G A A
G G
A
C
U A
G
A
G
G
U
U A
G
A
G
G
A
G
A
C
C
C
C
G
U
G
G
C
GC
A
C
G
((((((((...((((((((..............))))))))....))......((((......))))))))))
JEV.05 CCACGGCCCAAGUCUCGUCCA-G-GAUGCAAUGGACGAGAUGUAAGGACUAGAGGUUAGAGGAGACCCCGUGG 71
JEV.06 CCACGGCCCAAACCUCAUCUA-G-GAUGCAAUAGAUGAGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGUGG 71
WNV.07 ACGCGGCCCAAAUCCUGGUGA-U-GGUGUUA-UGCCAGGGUGGAAGGACUAGAGGUUAGAGGAGACCCCGCGU 70
MVEV.08 CCGCAGCCCGGGCCGGGAGGAGGUGAUGCGA-AC-CCCGGC-GAAGGACUAGAGGUUAGAGGAGACCCUGCGG 70
ALFV.09 CCACGGCCCGGGCCAUGAGU-GAUGAUGUUA-ACUCAUGGC-GAAGGACUAGAGGUUAGAGGAGACCCCGUGG 70
USUV.10 CCACGGCUCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
USUV.11 UCACGGCCCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
SLEV.12 CGGCGGCCCAAACCAUGGAG----UGCGUGA---CCAUGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGCUG 66
SLEV.13 CAGCGGCCCAAACCAUGGAG----UGCGUGA---CCAUGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGCUG 66
.........10........20........30........40........50........60........70..
• High mutation rate in RNA viruses due to error-prone RdRP
• For base pair (i,j): GC/CG/AU/UA/GU/UG
• Consistent mutation: different standard combinations
• Compensatory mutation: both positions are mutated
• Presence of both strongly supports predicted base pair (i,j)
LIV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
SGEV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
TBEV T.xrRNA1 T.SL6 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
OHFV T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
LGTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
DTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
POWV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
KSIV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like
~ 300 nt
b
G
R
Y
R
C
G
G
C
A
G
C
A C
R
C
Y
Y
R
G
Y
G
A
C G G G A
A R
U
G
G
UCG
C
UCCCG
A
C
G
Y
A
R
R
Y
Y
5´
R
G
G
R
Y
R
Y
G
G
C
A
R
C
R
C
R
C
A Y
G R
GG
R
A
R
G
R
Y
G
C
C
C
R
R
R
Y
Y
Y
Y
5´ C
Y
Y
C
R
A
A
G G
Y
A
R
C
R
R R G5´
G
G
C
G
GY
U
C
U U
U
C
U
C
C C
R
A
G
C
Y
C
Y
Y
A
G
A
C
A
C
A G A
U
A
G
U
C
U
R
A C
A
G
RR
R
G
G
R
G
Y
U
Y
G
A
A
A
5´
R
R
R
C
C
C
C
G
R
R
Y
Y
G
G
A G
G
R
G
G
G
A
R
R
R
R
R
RA
A
A
U
U G G
C
A
R
Y
Y
Y
Y
Y
Y
U
C R G
G
A
U
UU
Y
Y
Y
Y
5´
T.xrRNA2 SL6 Y-shaped (Y1) Flavi_CRET.xrRNA1
5' 3'
.((((..((((((((...........))))))))......))))....
SGEV|261-306 CCCCCC-GCACCAUGACAAGGCCGAACAUGGUGCACCAAAGGGGAG-G 46
TBEV|532-577 CCCCCU-GCAUCAUGAUAAGGCCGAACAUGGUGCAUGAAAGGGGAG-G 46
LIV|268-312 CCCCCC-GCCCCAUGACAAGGCCGAACAUGGAGCAUUAAAGG-GAG-G 45
LGTV|339-381 CCCCUU-GCGUCCAGAGAAGGCCGAACUGGGCGU---UAUAAGGAG-G 43
OHFV|179-222 CCCCCC-GCACCAUG-GAAGGCCAAACAUGGUGCAUG-AAGGGAAA-G 44
KFDV|164-204 CCAAAA-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
ALKV|165-205 CCAAAG-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
DTV|248-294 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCAUUCUAGAG 47
POWV|249-293 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCGUU-UAG-G 45
TYUV|341-386 CUUUC--CAUGAGAGGAGACGGUCAACUCUCAUGGAACAAGAAGACCG 46
NEGV|129-174 CCCCCCCUGGCCAGAAAAAGGGGGGGCAAACAGGCC--AGGGGUGAAG 46
KSIV|233-270 CUGAC---CAUCCCU-CAAGGCCGAGUGGGAUGC------GUAUGAAG 38
.........10........20........30........40.......
Seq. Conservation:
*
*
c
d f
types of pairs:
1 2 3 4 5 6
SL6
e (((((..((((((((((....)))))))).))...((((((((...........)))))))).........)).)))
POWV|291-361 AGGAG--CCCCCGAGCAUAA---CUCGGGAGGAGGGAGGAAGA-AAAUUGGCAAUCUUCCUCGGGAUUUUUCCGCCU 71
DTV|290-354 UAGAG--CCCCCGGGCAUAA---CUCGGGAGGAGGGGGGAAGA-CAAUUGGCAAUCUUCCCCGGGAUU------UUU 65
LGTV|377-449 GGAGG--CCCCCAGGGGGAAACCCCUGGGAGGAGGGAAGAGAG-AAAUUGGCAACUCUCUUCAGGAUAUUUCC-UCC 73
TBEV|573-645 GGAGG--CCCCCGGAAGCACGCUUCCGGGAGGAGGGAAGAGAG-AAAUUGGCAGCUCUCUUCAGGAUUUUUCC-UCC 73
Y1
allowed pairs:
incompatible
pairs in column
0
1
2
A-U
U-A
G-C
C-G
G-U
U-G

27. !27
RNA Covariation as Evolutionary Trait
C
C
A
C
G
G
CC
C
AA
GCCAUGAG
G
A
_G_
G
A
U
G
C
G A _
A
C A C A U G G C
G
G A A
G G
A
C
U A
G
A
G
G
U
U A
G
A
G
G
A
G
A
C
C
C
C
G
U
G
G
C
GA
A
C
G
((((((((...((((((((..............))))))))....))......((((......))))))))))
JEV.04 CCACGGCCCAAGCUUCGUCUA-G-GAUGCAAUAGACGAGGUGUAAGGACUAGAGGUUAGAGGAGACCCCGUGG 71
JEV.05 CCACGGCCCAAGUCUCGUCCA-G-GAUGCAAUGGACGAGAUGUAAGGACUAGAGGUUAGAGGAGACCCCGUGG 71
JEV.06 CCACGGCCCAAACCUCAUCUA-G-GAUGCAAUAGAUGAGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGUGG 71
WNV.07 ACGCGGCCCAAAUCCUGGUGA-U-GGUGUUA-UGCCAGGGUGGAAGGACUAGAGGUUAGAGGAGACCCCGCGU 70
MVEV.08 CCGCAGCCCGGGCCGGGAGGAGGUGAUGCGA-AC-CCCGGC-GAAGGACUAGAGGUUAGAGGAGACCCUGCGG 70
ALFV.09 CCACGGCCCGGGCCAUGAGU-GAUGAUGUUA-ACUCAUGGC-GAAGGACUAGAGGUUAGAGGAGACCCCGUGG 70
USUV.10 CCACGGCUCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
USUV.11 UCACGGCCCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
SLEV.12 CGGCGGCCCAAACCAUGGAG----UGCGUGA---CCAUGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGCUG 66
SLEV.13 CAGCGGCCCAAACCAUGGAG----UGCGUGA---CCAUGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGCUG 66
.........10........20........30........40........50........60........70..
• High mutation rate in RNA viruses due to error-prone RdRP
• For base pair (i,j): GC/CG/AU/UA/GU/UG
• Consistent mutation: different standard combinations
• Compensatory mutation: both positions are mutated
• Presence of both strongly supports predicted base pair (i,j)
LIV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
SGEV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
TBEV T.xrRNA1 T.SL6 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
OHFV T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
LGTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
DTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
POWV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
KSIV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like
~ 300 nt
b
G
R
Y
R
C
G
G
C
A
G
C
A C
R
C
Y
Y
R
G
Y
G
A
C G G G A
A R
U
G
G
UCG
C
UCCCG
A
C
G
Y
A
R
R
Y
Y
5´
R
G
G
R
Y
R
Y
G
G
C
A
R
C
R
C
R
C
A Y
G R
GG
R
A
R
G
R
Y
G
C
C
C
R
R
R
Y
Y
Y
Y
5´ C
Y
Y
C
R
A
A
G G
Y
A
R
C
R
R R G5´
G
G
C
G
GY
U
C
U U
U
C
U
C
C C
R
A
G
C
Y
C
Y
Y
A
G
A
C
A
C
A G A
U
A
G
U
C
U
R
A C
A
G
RR
R
G
G
R
G
Y
U
Y
G
A
A
A
5´
R
R
R
C
C
C
C
G
R
R
Y
Y
G
G
A G
G
R
G
G
G
A
R
R
R
R
R
RA
A
A
U
U G G
C
A
R
Y
Y
Y
Y
Y
Y
U
C R G
G
A
U
UU
Y
Y
Y
Y
5´
T.xrRNA2 SL6 Y-shaped (Y1) Flavi_CRET.xrRNA1
5' 3'
.((((..((((((((...........))))))))......))))....
SGEV|261-306 CCCCCC-GCACCAUGACAAGGCCGAACAUGGUGCACCAAAGGGGAG-G 46
TBEV|532-577 CCCCCU-GCAUCAUGAUAAGGCCGAACAUGGUGCAUGAAAGGGGAG-G 46
LIV|268-312 CCCCCC-GCCCCAUGACAAGGCCGAACAUGGAGCAUUAAAGG-GAG-G 45
LGTV|339-381 CCCCUU-GCGUCCAGAGAAGGCCGAACUGGGCGU---UAUAAGGAG-G 43
OHFV|179-222 CCCCCC-GCACCAUG-GAAGGCCAAACAUGGUGCAUG-AAGGGAAA-G 44
KFDV|164-204 CCAAAA-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
ALKV|165-205 CCAAAG-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
DTV|248-294 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCAUUCUAGAG 47
POWV|249-293 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCGUU-UAG-G 45
TYUV|341-386 CUUUC--CAUGAGAGGAGACGGUCAACUCUCAUGGAACAAGAAGACCG 46
NEGV|129-174 CCCCCCCUGGCCAGAAAAAGGGGGGGCAAACAGGCC--AGGGGUGAAG 46
KSIV|233-270 CUGAC---CAUCCCU-CAAGGCCGAGUGGGAUGC------GUAUGAAG 38
.........10........20........30........40.......
Seq. Conservation:
*
*
c
d f
types of pairs:
1 2 3 4 5 6
SL6
e (((((..((((((((((....)))))))).))...((((((((...........)))))))).........)).)))
POWV|291-361 AGGAG--CCCCCGAGCAUAA---CUCGGGAGGAGGGAGGAAGA-AAAUUGGCAAUCUUCCUCGGGAUUUUUCCGCCU 71
DTV|290-354 UAGAG--CCCCCGGGCAUAA---CUCGGGAGGAGGGGGGAAGA-CAAUUGGCAAUCUUCCCCGGGAUU------UUU 65
LGTV|377-449 GGAGG--CCCCCAGGGGGAAACCCCUGGGAGGAGGGAAGAGAG-AAAUUGGCAACUCUCUUCAGGAUAUUUCC-UCC 73
TBEV|573-645 GGAGG--CCCCCGGAAGCACGCUUCCGGGAGGAGGGAAGAGAG-AAAUUGGCAGCUCUCUUCAGGAUUUUUCC-UCC 73
Y1
allowed pairs:
incompatible
pairs in column
0
1
2
A-U
U-A
G-C
C-G
G-U
U-G

28. !28
RNA Covariation as Evolutionary Trait
C
C
A
C
G
G
CC
C
AA
GCCAUGAG
G
A
_G_
G
A
U
G
C
G A _
A
C A C A U G G C
G
U A A
G G
A
C
U A
G
A
G
G
U
U A
G
A
G
G
A
G
A
C
C
C
C
G
U
G
G
C
GA
A
C
G
((((((((...((((((((..............))))))))....))......((((......))))))))))
APCV.03 CCGCGGCCCAACCAGUUCAGACU-GAUGCUA--UGAACUGGGUAAGGACUAGAGGUUAGAGGAGACCCCGCGG 70
JEV.04 CCACGGCCCAAGCUUCGUCUA-G-GAUGCAAUAGACGAGGUGUAAGGACUAGAGGUUAGAGGAGACCCCGUGG 71
JEV.05 CCACGGCCCAAGUCUCGUCCA-G-GAUGCAAUGGACGAGAUGUAAGGACUAGAGGUUAGAGGAGACCCCGUGG 71
JEV.06 CCACGGCCCAAACCUCAUCUA-G-GAUGCAAUAGAUGAGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGUGG 71
WNV.07 ACGCGGCCCAAAUCCUGGUGA-U-GGUGUUA-UGCCAGGGUGGAAGGACUAGAGGUUAGAGGAGACCCCGCGU 70
MVEV.08 CCGCAGCCCGGGCCGGGAGGAGGUGAUGCGA-AC-CCCGGC-GAAGGACUAGAGGUUAGAGGAGACCCUGCGG 70
ALFV.09 CCACGGCCCGGGCCAUGAGU-GAUGAUGUUA-ACUCAUGGC-GAAGGACUAGAGGUUAGAGGAGACCCCGUGG 70
USUV.10 CCACGGCUCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
USUV.11 UCACGGCCCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
SLEV.12 CGGCGGCCCAAACCAUGGAG----UGCGUGA---CCAUGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGCUG 66
SLEV.13 CAGCGGCCCAAACCAUGGAG----UGCGUGA---CCAUGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGCUG 66
.........10........20........30........40........50........60........70..
• High mutation rate in RNA viruses due to error-prone RdRP
• For base pair (i,j): GC/CG/AU/UA/GU/UG
• Consistent mutation: different standard combinations
• Compensatory mutation: both positions are mutated
• Presence of both strongly supports predicted base pair (i,j)
LIV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
SGEV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
TBEV T.xrRNA1 T.SL6 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
OHFV T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
LGTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
DTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
POWV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
KSIV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like
~ 300 nt
b
G
R
Y
R
C
G
G
C
A
G
C
A C
R
C
Y
Y
R
G
Y
G
A
C G G G A
A R
U
G
G
UCG
C
UCCCG
A
C
G
Y
A
R
R
Y
Y
5´
R
G
G
R
Y
R
Y
G
G
C
A
R
C
R
C
R
C
A Y
G R
GG
R
A
R
G
R
Y
G
C
C
C
R
R
R
Y
Y
Y
Y
5´ C
Y
Y
C
R
A
A
G G
Y
A
R
C
R
R R G5´
G
G
C
G
GY
U
C
U U
U
C
U
C
C C
R
A
G
C
Y
C
Y
Y
A
G
A
C
A
C
A G A
U
A
G
U
C
U
R
A C
A
G
RR
R
G
G
R
G
Y
U
Y
G
A
A
A
5´
R
R
R
C
C
C
C
G
R
R
Y
Y
G
G
A G
G
R
G
G
G
A
R
R
R
R
R
RA
A
A
U
U G G
C
A
R
Y
Y
Y
Y
Y
Y
U
C R G
G
A
U
UU
Y
Y
Y
Y
5´
T.xrRNA2 SL6 Y-shaped (Y1) Flavi_CRET.xrRNA1
5' 3'
.((((..((((((((...........))))))))......))))....
SGEV|261-306 CCCCCC-GCACCAUGACAAGGCCGAACAUGGUGCACCAAAGGGGAG-G 46
TBEV|532-577 CCCCCU-GCAUCAUGAUAAGGCCGAACAUGGUGCAUGAAAGGGGAG-G 46
LIV|268-312 CCCCCC-GCCCCAUGACAAGGCCGAACAUGGAGCAUUAAAGG-GAG-G 45
LGTV|339-381 CCCCUU-GCGUCCAGAGAAGGCCGAACUGGGCGU---UAUAAGGAG-G 43
OHFV|179-222 CCCCCC-GCACCAUG-GAAGGCCAAACAUGGUGCAUG-AAGGGAAA-G 44
KFDV|164-204 CCAAAA-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
ALKV|165-205 CCAAAG-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
DTV|248-294 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCAUUCUAGAG 47
POWV|249-293 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCGUU-UAG-G 45
TYUV|341-386 CUUUC--CAUGAGAGGAGACGGUCAACUCUCAUGGAACAAGAAGACCG 46
NEGV|129-174 CCCCCCCUGGCCAGAAAAAGGGGGGGCAAACAGGCC--AGGGGUGAAG 46
KSIV|233-270 CUGAC---CAUCCCU-CAAGGCCGAGUGGGAUGC------GUAUGAAG 38
.........10........20........30........40.......
Seq. Conservation:
*
*
c
d f
types of pairs:
1 2 3 4 5 6
SL6
e (((((..((((((((((....)))))))).))...((((((((...........)))))))).........)).)))
POWV|291-361 AGGAG--CCCCCGAGCAUAA---CUCGGGAGGAGGGAGGAAGA-AAAUUGGCAAUCUUCCUCGGGAUUUUUCCGCCU 71
DTV|290-354 UAGAG--CCCCCGGGCAUAA---CUCGGGAGGAGGGGGGAAGA-CAAUUGGCAAUCUUCCCCGGGAUU------UUU 65
LGTV|377-449 GGAGG--CCCCCAGGGGGAAACCCCUGGGAGGAGGGAAGAGAG-AAAUUGGCAACUCUCUUCAGGAUAUUUCC-UCC 73
TBEV|573-645 GGAGG--CCCCCGGAAGCACGCUUCCGGGAGGAGGGAAGAGAG-AAAUUGGCAGCUCUCUUCAGGAUUUUUCC-UCC 73
Y1
allowed pairs:
incompatible
pairs in column
0
1
2
A-U
U-A
G-C
C-G
G-U
U-G

29. !29
RNA Covariation as Evolutionary Trait
C
C
A
C
G
G
CC
C
AA
GCCAUGAC
G
A
_G_
G
A
U
G
C
G A _
A
C C C A U G G U
G
U A A
G G
A
C
U A
G
A
G
G
U
U A
G
A
G
G
A
G
A
C
C
C
C
G
C
G
G
U
GC
A
C
C
((((((((...((((((((..............))))))))....))......((((......))))))))))
KUNV.02 ACGCGGCCCUAGCUCUGGCAA--UGGUGUUA--ACCAGAGUGAAAGGACUAGAGGUUAGAGGAGACCCCGCGU 69
APCV.03 CCGCGGCCCAACCAGUUCAGACU-GAUGCUA--UGAACUGGGUAAGGACUAGAGGUUAGAGGAGACCCCGCGG 70
JEV.04 CCACGGCCCAAGCUUCGUCUA-G-GAUGCAAUAGACGAGGUGUAAGGACUAGAGGUUAGAGGAGACCCCGUGG 71
JEV.05 CCACGGCCCAAGUCUCGUCCA-G-GAUGCAAUGGACGAGAUGUAAGGACUAGAGGUUAGAGGAGACCCCGUGG 71
JEV.06 CCACGGCCCAAACCUCAUCUA-G-GAUGCAAUAGAUGAGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGUGG 71
WNV.07 ACGCGGCCCAAAUCCUGGUGA-U-GGUGUUA-UGCCAGGGUGGAAGGACUAGAGGUUAGAGGAGACCCCGCGU 70
MVEV.08 CCGCAGCCCGGGCCGGGAGGAGGUGAUGCGA-AC-CCCGGC-GAAGGACUAGAGGUUAGAGGAGACCCUGCGG 70
ALFV.09 CCACGGCCCGGGCCAUGAGU-GAUGAUGUUA-ACUCAUGGC-GAAGGACUAGAGGUUAGAGGAGACCCCGUGG 70
USUV.10 CCACGGCUCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
USUV.11 UCACGGCCCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
SLEV.12 CGGCGGCCCAAACCAUGGAG----UGCGUGA---CCAUGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGCUG 66
SLEV.13 CAGCGGCCCAAACCAUGGAG----UGCGUGA---CCAUGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGCUG 66
.........10........20........30........40........50........60........70..
• High mutation rate in RNA viruses due to error-prone RdRP
• For base pair (i,j): GC/CG/AU/UA/GU/UG
• Consistent mutation: different standard combinations
• Compensatory mutation: both positions are mutated
• Presence of both strongly supports predicted base pair (i,j)
LIV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
SGEV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
TBEV T.xrRNA1 T.SL6 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
OHFV T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
LGTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
DTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
POWV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
KSIV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like
~ 300 nt
b
G
R
Y
R
C
G
G
C
A
G
C
A C
R
C
Y
Y
R
G
Y
G
A
C G G G A
A R
U
G
G
UCG
C
UCCCG
A
C
G
Y
A
R
R
Y
Y
5´
R
G
G
R
Y
R
Y
G
G
C
A
R
C
R
C
R
C
A Y
G R
GG
R
A
R
G
R
Y
G
C
C
C
R
R
R
Y
Y
Y
Y
5´ C
Y
Y
C
R
A
A
G G
Y
A
R
C
R
R R G5´
G
G
C
G
GY
U
C
U U
U
C
U
C
C C
R
A
G
C
Y
C
Y
Y
A
G
A
C
A
C
A G A
U
A
G
U
C
U
R
A C
A
G
RR
R
G
G
R
G
Y
U
Y
G
A
A
A
5´
R
R
R
C
C
C
C
G
R
R
Y
Y
G
G
A G
G
R
G
G
G
A
R
R
R
R
R
RA
A
A
U
U G G
C
A
R
Y
Y
Y
Y
Y
Y
U
C R G
G
A
U
UU
Y
Y
Y
Y
5´
T.xrRNA2 SL6 Y-shaped (Y1) Flavi_CRET.xrRNA1
5' 3'
.((((..((((((((...........))))))))......))))....
SGEV|261-306 CCCCCC-GCACCAUGACAAGGCCGAACAUGGUGCACCAAAGGGGAG-G 46
TBEV|532-577 CCCCCU-GCAUCAUGAUAAGGCCGAACAUGGUGCAUGAAAGGGGAG-G 46
LIV|268-312 CCCCCC-GCCCCAUGACAAGGCCGAACAUGGAGCAUUAAAGG-GAG-G 45
LGTV|339-381 CCCCUU-GCGUCCAGAGAAGGCCGAACUGGGCGU---UAUAAGGAG-G 43
OHFV|179-222 CCCCCC-GCACCAUG-GAAGGCCAAACAUGGUGCAUG-AAGGGAAA-G 44
KFDV|164-204 CCAAAA-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
ALKV|165-205 CCAAAG-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
DTV|248-294 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCAUUCUAGAG 47
POWV|249-293 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCGUU-UAG-G 45
TYUV|341-386 CUUUC--CAUGAGAGGAGACGGUCAACUCUCAUGGAACAAGAAGACCG 46
NEGV|129-174 CCCCCCCUGGCCAGAAAAAGGGGGGGCAAACAGGCC--AGGGGUGAAG 46
KSIV|233-270 CUGAC---CAUCCCU-CAAGGCCGAGUGGGAUGC------GUAUGAAG 38
.........10........20........30........40.......
Seq. Conservation:
*
*
c
d f
types of pairs:
1 2 3 4 5 6
SL6
e (((((..((((((((((....)))))))).))...((((((((...........)))))))).........)).)))
POWV|291-361 AGGAG--CCCCCGAGCAUAA---CUCGGGAGGAGGGAGGAAGA-AAAUUGGCAAUCUUCCUCGGGAUUUUUCCGCCU 71
DTV|290-354 UAGAG--CCCCCGGGCAUAA---CUCGGGAGGAGGGGGGAAGA-CAAUUGGCAAUCUUCCCCGGGAUU------UUU 65
LGTV|377-449 GGAGG--CCCCCAGGGGGAAACCCCUGGGAGGAGGGAAGAGAG-AAAUUGGCAACUCUCUUCAGGAUAUUUCC-UCC 73
TBEV|573-645 GGAGG--CCCCCGGAAGCACGCUUCCGGGAGGAGGGAAGAGAG-AAAUUGGCAGCUCUCUUCAGGAUUUUUCC-UCC 73
Y1
allowed pairs:
incompatible
pairs in column
0
1
2
A-U
U-A
G-C
C-G
G-U
U-G

30. !30
RNA Covariation as Evolutionary Trait
((((((((...((((((((..............))))))))....))......((((......))))))))))
WNV.01 GCGCAGCCAUAACCUUGGUGA--AGGUGUUA--GCCAAGGAGAAGGGACUAGAGGUUAGCAGAGAUCCUGCGC 69
KUNV.02 ACGCGGCCCUAGCUCUGGCAA--UGGUGUUA--ACCAGAGUGAAAGGACUAGAGGUUAGAGGAGACCCCGCGU 69
APCV.03 CCGCGGCCCAACCAGUUCAGACU-GAUGCUA--UGAACUGGGUAAGGACUAGAGGUUAGAGGAGACCCCGCGG 70
JEV.04 CCACGGCCCAAGCUUCGUCUA-G-GAUGCAAUAGACGAGGUGUAAGGACUAGAGGUUAGAGGAGACCCCGUGG 71
JEV.05 CCACGGCCCAAGUCUCGUCCA-G-GAUGCAAUGGACGAGAUGUAAGGACUAGAGGUUAGAGGAGACCCCGUGG 71
JEV.06 CCACGGCCCAAACCUCAUCUA-G-GAUGCAAUAGAUGAGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGUGG 71
WNV.07 ACGCGGCCCAAAUCCUGGUGA-U-GGUGUUA-UGCCAGGGUGGAAGGACUAGAGGUUAGAGGAGACCCCGCGU 70
MVEV.08 CCGCAGCCCGGGCCGGGAGGAGGUGAUGCGA-AC-CCCGGC-GAAGGACUAGAGGUUAGAGGAGACCCUGCGG 70
ALFV.09 CCACGGCCCGGGCCAUGAGU-GAUGAUGUUA-ACUCAUGGC-GAAGGACUAGAGGUUAGAGGAGACCCCGUGG 70
USUV.10 CCACGGCUCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
USUV.11 UCACGGCCCAAGCGAACAGACGGUGAUGCGA-ACUGUUCGUGGAAGGACUAGAGGUUAGAGGAGACCCCGUGG 72
SLEV.12 CGGCGGCCCAAACCAUGGAG----UGCGUGA---CCAUGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGCUG 66
SLEV.13 CAGCGGCCCAAACCAUGGAG----UGCGUGA---CCAUGGCGUAAGGACUAGAGGUUAGAGGAGACCCCGCUG 66
.........10........20........30........40........50........60........70..
C
C
G
C
G
G
CC
C
AA
GCCAUGGC
G
A
_G_
G
A
U
G
C
G A _
A
G C C A U G G U
G
U A A
G G
A
C
U A
G
A
G
G
U
U A
G
A
G
G
A
G
A
C
C
C
C
G
C
G
G
U
GC
G
G
C
• High mutation rate in RNA viruses due to error-prone RdRP
• For base pair (i,j): GC/CG/AU/UA/GU/UG
• Consistent mutation: different standard combinations
• Compensatory mutation: both positions are mutated
• Presence of both strongly supports predicted base pair (i,j)
LIV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
SGEV T.xrRNA1 T.xrRNA2 T.SL6 T.xrRNA2 Flavi_CRE
TBEV T.xrRNA1 T.SL6 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
OHFV T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
LGTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
DTV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
POWV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like Flavi_CRE
KSIV T.xrRNA1 T.xrRNA2 T.SL6 xrRNA-like
~ 300 nt
b
G
R
Y
R
C
G
G
C
A
G
C
A C
R
C
Y
Y
R
G
Y
G
A
C G G G A
A R
U
G
G
UCG
C
UCCCG
A
C
G
Y
A
R
R
Y
Y
5´
R
G
G
R
Y
R
Y
G
G
C
A
R
C
R
C
R
C
A Y
G R
GG
R
A
R
G
R
Y
G
C
C
C
R
R
R
Y
Y
Y
Y
5´ C
Y
Y
C
R
A
A
G G
Y
A
R
C
R
R R G5´
G
G
C
G
GY
U
C
U U
U
C
U
C
C C
R
A
G
C
Y
C
Y
Y
A
G
A
C
A
C
A G A
U
A
G
U
C
U
R
A C
A
G
RR
R
G
G
R
G
Y
U
Y
G
A
A
A
5´
R
R
R
C
C
C
C
G
R
R
Y
Y
G
G
A G
G
R
G
G
G
A
R
R
R
R
R
RA
A
A
U
U G G
C
A
R
Y
Y
Y
Y
Y
Y
U
C R G
G
A
U
UU
Y
Y
Y
Y
5´
T.xrRNA2 SL6 Y-shaped (Y1) Flavi_CRET.xrRNA1
5' 3'
.((((..((((((((...........))))))))......))))....
SGEV|261-306 CCCCCC-GCACCAUGACAAGGCCGAACAUGGUGCACCAAAGGGGAG-G 46
TBEV|532-577 CCCCCU-GCAUCAUGAUAAGGCCGAACAUGGUGCAUGAAAGGGGAG-G 46
LIV|268-312 CCCCCC-GCCCCAUGACAAGGCCGAACAUGGAGCAUUAAAGG-GAG-G 45
LGTV|339-381 CCCCUU-GCGUCCAGAGAAGGCCGAACUGGGCGU---UAUAAGGAG-G 43
OHFV|179-222 CCCCCC-GCACCAUG-GAAGGCCAAACAUGGUGCAUG-AAGGGAAA-G 44
KFDV|164-204 CCAAAA-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
ALKV|165-205 CCAAAG-CCUCCCAGAGAAGGCCGAACUGGGAGGCC-----AUGAA-G 41
DTV|248-294 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCAUUCUAGAG 47
POWV|249-293 CCAAAAGGCCUCCUG-GAAGGCUCACCAGGAGUUAGGCCGUU-UAG-G 45
TYUV|341-386 CUUUC--CAUGAGAGGAGACGGUCAACUCUCAUGGAACAAGAAGACCG 46
NEGV|129-174 CCCCCCCUGGCCAGAAAAAGGGGGGGCAAACAGGCC--AGGGGUGAAG 46
KSIV|233-270 CUGAC---CAUCCCU-CAAGGCCGAGUGGGAUGC------GUAUGAAG 38
.........10........20........30........40.......
Seq. Conservation:
*
*
c
d f
types of pairs:
1 2 3 4 5 6
SL6
e (((((..((((((((((....)))))))).))...((((((((...........)))))))).........)).)))
POWV|291-361 AGGAG--CCCCCGAGCAUAA---CUCGGGAGGAGGGAGGAAGA-AAAUUGGCAAUCUUCCUCGGGAUUUUUCCGCCU 71
DTV|290-354 UAGAG--CCCCCGGGCAUAA---CUCGGGAGGAGGGGGGAAGA-CAAUUGGCAAUCUUCCCCGGGAUU------UUU 65
LGTV|377-449 GGAGG--CCCCCAGGGGGAAACCCCUGGGAGGAGGGAAGAGAG-AAAUUGGCAACUCUCUUCAGGAUAUUUCC-UCC 73
TBEV|573-645 GGAGG--CCCCCGGAAGCACGCUUCCGGGAGGAGGGAAGAGAG-AAAUUGGCAGCUCUCUUCAGGAUUUUUCC-UCC 73
Y1
allowed pairs:
incompatible
pairs in column
0
1
2
A-U
U-A
G-C
C-G
G-U
U-G

31. CHIKV 3’UTR: Sequence Repeats
!31
IG. 2. Schematic representation of repeated sequence elements (RSE) and lengths of 39-NTRs of 35 alphaviruses (adapted from Strauss and
uss, 1994). Both the lengths of the repeats and the overall lengths of the 39-NTRs have been drawn to scale. The downward-pointing arrow
Pfeffer et al., Virology (1998)
Repeat Sequence Elements

32. CHIKV 3’UTR: Sequence Repeats
!32
IG. 2. Schematic representation of repeated sequence elements (RSE) and lengths of 39-NTRs of 35 alphaviruses (adapted from Strauss and
uss, 1994). Both the lengths of the repeats and the overall lengths of the 39-NTRs have been drawn to scale. The downward-pointing arrow
Chen et al., PLoS Pathog. (2013)
Lineage-specific Direct Repeats (DRs)
Pfeffer et al., Virology (1998)
Repeat Sequence Elements (RSEs)

33. CHIKV 3’UTR: Sequence Repeats
!33
Chen et al., PLoS Pathog.(2013)
Lineage-specific Direct Repeats (DRs)
Pfeffer et al., Virology (1998)
Repeat Sequence Elements (RSEs)
Do they form RNA structures ?

34. CHIKV 3’UTR
!34
WA
ECSA-IOL
ECSA-MASA
AUL-Am
AUL-As
AUL-Cbn
• 110 full length CHIKV 3’UTRs
• Variable length (510nt — 930nt)
• Structural alignments + Covariance models
• Thermodynamic modelling based on ViennaRNA Package

35. CHIKV 3’UTR: Conserved RNA Structures
!35
WA
ECSA-IOL
ECSA-MASA
AUL-Am
AUL-As
AUL-Cbn
CSE
CSE
CSE
CSE
CSE
CSE
G A G A U G U U A U U U U G U U U U U A A U A U U U C
G A G A U G U U A U U U U G U U U U U A A U A U U U C
GAGAUGUUAUUUUGUUUUUAAUAUUUC
GAGAUGUUAUUUUGUUUUUAAUAUUUC
G
A
G
A
U
G
U
U
A
U
U
U
U G U
U
U
U
U
A
A
U
A
U
U
U
C
UG
UA
AU
UG
(((((((((.........)))))))))
KF318729.1/688-714 GAGAUGUUAUUUUGUUUUUAAUAUUUC 27
KT327163.2/862-888 GAGAUGUUAUUUUGUUUUUAAUAUUUC 27
JF274082.1/472-498 GAGAUGUUAUUUUGUUUUUAAUAUUUC 27
KY038946.1/472-498 GAGAUGUUAUUUUGUUUUUAAUAUUUC 27
AY726732.1/540-566 GAGAUGUUAUUUUGUUUUUAAUAUUUC 27
MF001517.1/685-707 GAGAUGUUAUUUUGUUUUUAA----UC 23
.........10........20......
Conserved Sequence Element (CSE)

36. !36
WA
ECSA-IOL
ECSA-MASA
AUL-Am
AUL-As
AUL_Cbn
Y-a CSE
Y-a CSE
Y-a CSE
Y-a Y-b CSE
Y-a Y-b CSE
Y-a Y-b CSE
CHIKV 3’UTR: Conserved RNA Structures
C G G A A G A G A U G U A G G U A C U U A A G C U U C U U A A A A G C A G C C G A A C U C A C U U U G A G A U G U A G G C A U A G C A U A C C G A A C U C U U C C A
C G G A A G A G A U G U A G G U A C U U A A G C U U C U U A A A A G C A G C C G A A C U C A C U U U G A G A U G U A G G C A U A G C A U A C C G A A C U C U U C C A
CGGAAGAGAUGUAGGUACUUAAGCUUCUUAAAAGCAGCCGAACUCACUUUGAGAUGUAGGCAUAGCAUACCGAACUCUUCCA
CGGAAGAGAUGUAGGUACUUAAGCUUCUUAAAAGCAGCCGAACUCACUUUGAGAUGUAGGCAUAGCAUACCGAACUCUUCCA
C G
G
A
A
G
A
G
A
U
G
U
A G
G
U
A
C
U
U
A
A
G
C
U
U
C
U
U A
A
A
A
G
C A
G
C
C
G
A
A C
U
C
A
C U
U
U
G
A
G
A
U
G
UAG
G
C
A
U
A
G
C
AUA
C
C
G
A
A
C
U
C
U
U
C
C ACG
A
U
.(((((((.....(((......((((.....)))).(((...((((...)))).....))).......)))...))))))).
KY038946.1/363-444 CGGAAGAGAUGUAGGUACUUAAGCUUCCUAAAAGCAGCCGAACUCACUUUGAGAUGUAGGCAUAGCAUACCGAACUCUUCCA 82
KF318729.1/395-476 CGGAAGAGAUGUAGGUACUUAAGCUUCUUAAAAGCAGCCGAACUCGCUUUGAGAUGUAGGCGUAGCACACCGAACUCUUCCA 82
MF001517.1/392-473 CGGAAGAGAUGUAGGUACUUAAGCUUCUUAAAAGCAGCCGAACUCGCUUUGAGAUGUAGGCGUAGCACACCGAACUCUUCCA 82
KT327163.2/569-650 CGGAAGAGAUGUAGGUACUUAAGCUUCUUAAAAGCAGCCGAACUCGCUUUGAGAUGUAGGCGUAGCACACCGAACUCUUCCA 82
KF318729.1/580-660 UGGAAGAGACGUAGGUAC-UAAGCUUCUUAAAAGCAGCCGAACUCACUUUGAGAUGUAGGCAUAGCAUACCGAACUCUUCCA 81
MF001517.1/577-657 UGGAAGAGACGUAGGUAC-UAAGCUUCUUAAAAGCAGCCGAACUCACUUUGAGAUGUAGGCAUAGCAUACCGAACUCUUCCA 81
KT327163.2/754-834 UGGAAGAGACGUAGGUAC-UAAGCUUCUUAAAAGCAGCCGAACUCACUUUGAGAUGUAGGCAUAGCAUACCGAACUCUUCCA 81
JF274082.1/363-444 CGGAAGAGAUGUAGGCACUUAAGCUUCCUAAAAGCAGCCGAACUCACUUUGAGAAGUAGGCAUAGCAUACCGAACUCUUCCA 82
AY726732.1/431-512 GAGAAGAGACGUAGGUAUUUAAGCUUCCUAAAAGCAGCCGAACUCACUUUGAGACGUAGGCAUAGCAUACCGAACUCUUCCA 82
.........10........20........30........40........50........60........70........80.
Y-shaped Element (SL-Y)

37. !37
CHIKV 3’UTR: Conserved RNA Structures
Stem-loop a (SL-a)
WA
ECSA-IOL
ECSA-MASA
AUL-Am
AUL-As
AUL-Cbn
Y-a CSE
SL-a Y-a CSE
SL-a Y-a CSE
SL-a Y-a Y-b CSE
SL-a Y-a Y-b CSE
SL-a Y-a Y-b CSE
U G A C A A C U A G G U A U G A A G G C A U A C G C G U C C C U A A A G A G A C A C A C C G C A U A U A G C U A G G A A U C
U G A C A A C U A G G U A U G A A G G C A U A C G C G U C C C U A A A G A G A C A C A C C G C A U A U A G C U A G G A A U C
UGACAACUAGGUAUGAAGGCAUACGCGUCCCUAAAGAGACACACCGCAUAUAGCUAGGAAUC
UGACAACUAGGUAUGAAGGCAUACGCGUCCCUAAAGAGACACACCGCAUAUAGCUAGGAAUC
U G
A
C
A
A
C
U
A
G
G
U
A
U
G
A
A
G
G
C
A
U
A
C G
C G
U
C
C C
U A
A
A
G
AG
A
C
AC
A
C
C
G
C
A
U
A
U
A
G
C
U
A
G
G
A
A
U
C
GC
UA
CG
.((...((((.((((..((.....(.(((.(....).))).).))...)))).))))...))
KF318729.1/3-64 UGACAACUAGGUAUGAAGGCAUACGCGUCCCUAAAGAGACACACCGCAUAUAGCUAGGAAUC 62
MF001517.1/3-64 UGACAACUAGGUAUGAAGGCAUACGCGUCCCUAAAGAGACACACCGCAUAUAGCUAGGAAUC 62
KT327163.2/3-64 UGACAACUAGGUAUGAAGGCAUACGCGUCCCUAAAGAGACACACCGCAUAUAGCUAGGAAUC 62
JF274082.1/3-64 UGACAAUUAAGUAUGAAGGUAUAUGUGUCCCCUAAGAGACACACUGUACAUAGCAAAUAAUC 62
KY038946.1/3-64 CGACAACUAAGUAUGAAGGUAUAUGUGUCCCCUAAGAGACACACUGUACAUAGCUAAUAAUC 62
.........10........20........30........40........50........60.

38. !38
CHIKV 3’UTR: Conserved RNA Structures
Stem-loop b (SL-b)
A G G U A U A U G U G U C C C C U A A G A G A C A C A C C A U A U A U A G C U
A G G U A U A U G U G U C C C C U A A G A G A C A C A C C A U A U A U A G C U
AGGUAUAUGUGUCCCCUAAGAGACACACCAUAUAUAGCU
AGGUAUAUGUGUCCCCUAAGAGACACACCAUAUAUAGCU
A G G
U
A
U
A
U
G
U
G
U
CC
CC
U
A A
G A
G
A
C
A
C A
C
C
A
U
A
U
A
U A G C U
AU
..(((((((((((.(....).)))))...))))))....
KF318729.1/171-209 AGGUAUAUGUGUCCCCUAAGAGACACACCAUAUAUAGCU 39
MF001517.1/168-206 AGGUAUAUGUGUCCCCUAAGAGACACACCAUAUAUAGCU 39
KT327163.2/168-206 AGGUAUAUGUGUCCCCUAAGAGACACACCAUAUAUAGCU 39
KT327163.2/345-383 AGGUAUAUGUGUCCCCUAAGAGACACACCAUAUAUAGCU 39
JF274082.1/149-187 AGGUAUACGUGUCCCCUAAGAGACACAUUGUAUGUAGGU 39
KY038946.1/149-187 AGGUAUACGUGUCCCCUAAGAGACACAUUGUAUGUAGGU 39
AY726732.1/184-222 AGGUAUAAGUGUCCCCUAAGAGACACAUUGUAUGUAGGU 39
AY726732.1/54-92 AGGUAUAUGUGCCCCUUAAGAGGCACAAUAUAUAUAGCU 39
.........10........20........30........
WA
ECSA-IOL
ECSA-MASA
AUL-Am
AUL-As
AUL-Cbn
SL-b SL-b Y-a CSE
SL-a SL-b Y-a CSE
SL-a SL-b Y-a CSE
SL-a SL-b Y-a Y-b CSE
SL-a SL-b Y-a Y-b CSE
SL-a SL-b SL-b Y-a Y-b CSE

39. !39
CHIKV 3’UTR: Conserved RNA Elements
Unstructured repeat (UR)
5′
1
A
C
U
2
U
A
3
U
4
A
5
G
6
A
7
U
8
C
9
U
A
10
A
11
A
12
G
13
G
14
G
15
C
16
C
U
17
A
G
18
U
19
U
C
A
20
U
G
A
21
U
C
22
A
23
A
24
C
25
C
26
C
27
C
28
U29
G30
A31
A32U33
A34
A
G
35
U
36
A
37
A
38
C
39
A
40
A
41
A
42
A
43
C
U
44
A
45
C
U
46
G
A
47
A
48
A
49
A
50
A
51
C
U
52
C
53
A
54
C
A
55
C
U
56
A
57
A
58
A
59
A
60
A
61
C
U
62
U
C
63
A
64
C
U
65
A
66
A
67
A
68
A
69
A
U
70
C
71
G
A
72
A
G
73
A
74
A
75
A
76
A
3′
WA
ECSA-IOL
ECSA-MASA
AUL-Am
AUL-As
AUL-Cbn
SL-b UR-1 SL-b UR-2 UR-3 Y-a CSE
SL-a UR-1 SL-b UR-2 UR-3 Y-a CSE
SL-a UR-1 SL-b UR-2 UR-3 Y-a CSE
SL-a UR-1 SL-b UR-2 UR-3 Y-a UR-4 Y-b CSE
SL-a UR-1 SL-b UR-2 UR-3 Y-a UR-4 Y-b CSE
SL-a UR-1 SL-b UR-2 SL-b UR-3 UR-4 Y-a UR-5 Y-b CSE

40. !40
CHIKV-Cbn Accessibility
WA
ECSA-IOL
ECSA-MASA
AUL-Am
AUL-As
AUL-Cbn
SL-b UR-1 SL-b UR-2 UR-3 Y-a CSE
SL-a UR-1 SL-b UR-2 UR-3 Y-a CSE
SL-a UR-1 SL-b UR-2 UR-3 Y-a CSE
SL-a UR-1 SL-b UR-2 UR-3 Y-a UR-4 Y-b CSE
SL-a UR-1 SL-b UR-2 UR-3 Y-a UR-4 Y-b CSE
SL-a UR-1 SL-b UR-2 SL-b UR-3 UR-4 Y-a UR-5 Y-b CSE
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
100 200 300 400 500 600 700 800 900
"lunp.csv"

41. CHIKV 3’UTR Overview
SL-b UR SL-b UR UR SL-Y CSE
SL-a UR SL-b UR UR SL-Y CSE
SL-a UR SL-b UR UR SL-Y CSE
SL-a UR SL-b UR SL-b UR UR SL-Y UR SL-Y CSE
SL-a UR SL-b UR UR SL-Y UR SL-Y CSE
SL-a UR SL-b UR UR SL-Y UR SL-Y CSE
WA
ECSA-IOL
ECSA-MASA
AUL-Cbn
AUL-Am
AUL-As
500 9007000 250
CHIKV 3'UTRs
AUL-Cbn specific duplication
!41
U G
A
C
A
A
C
U
A
G
G
U
A
U
G
A
A
G
G
C
A
U
A
C G
C G
U
C
C C
U A
A
A
G
AG
A
C
AC
A
C
C
G
C
A
U
A
U
A
G
C
U
A
G
G
A
A
U
C
GC
UA
CG
A G G
U
A
U
A
U
G
U
G
U
CC
CC
U
A A
G A
G
A
C
A
C A
C
C
A
U
A
U
A
U A G C U
AU
C G
G
A
A
G
A
G
A
U
G
U
A G
G
U
A
C
U
U
A
A
G
C
U
U
C
U
U A
A
A
A
G
C A
G
C
C
G
A
A C
U
C
A
C U
U
U
G
A
G
A
U
G
UAG
G
C
A
U
A
G
C
AUA
C
C
G
A
A
C
U
C
U
U
C
C ACG
A
U
C G
G
A
A
G
A
G
A
U
G
U
A G
G
U
A
C
U
U
A
A
G
C
U
U
C
U
U A
A
A
A
G
C A
G
C
C
G
A
A C
U
C
A
C U
U
U
G
A
G
A
U
G
UAG
G
C
A
U
A
G
C
AUA
C
C
G
A
A
C
U
C
U
U
C
C ACG
A
U
G
A
G
A
U
G
U
U
A
U
U
U
U G U
U
U
U
U
A
A
U
A
U
U
U
C
UG
UA
AU
UG
5′
1
A
C
U
2
U
A
3
U
4
A
5
G
6
A
7
U
8
C
9
U
A
10
A
11
A
12
G
13
G
14
G
15
C
16
C
U
17
A
G
18
U
19
U
C
A
20
U
G
A
21
U
C
22
A
23
A
24
C
25
C
26
C
27
C
28
U
29
G
30
A
31
A
32
U
33
A
34
A
G
35
U
36
A
37
A
38
C
39
A
40
A
41
A
42
A
43
C
U
44
A
45
C
U
46
G
A
47
A
48
A
49
A
50
A
51
C
U
52
C
53
A
54
C
A
55
C
U
56
A
57
A
58
A
59
A
60
A
61
C
U
62
U
C
63
A
64
C
U
65
A
66
A
67
A
68
A
69
A
U
70
C
71
G
A
72
A
G
73
A
74
A
75
A
76
A
3′ 5′
1
A
C
U
2
U
A
3
U
4
A
5
G
6
A
7
U
8
C
9
U
A
10
A
11
A
12
G
13
G
14
G
15
C
16
C
U
17
A
G
18
U
19
U
C
A
20
U
G
A
21
U
C
22
A
23
A
24
C
25
C
26
C
27
C
28
U
29
G
30
A
31
A
32
U
33
A
34
A
G
35
U
36
A
37
A
38
C
39
A
40
A
41
A
42
A
43
C
U
44
A
45
C
U
46
G
A
47
A
48
A
49
A
50
A
51
C
U
52
C
53
A
54
C
A
55
C
U
56
A
57
A
58
A
59
A
60
A
61
C
U
62
U
C
63
A
64
C
U
65
A
66
A
67
A
68
A
69
A
U
70
C
71
G
A
72
A
G
73
A
74
A
75
A
76
A
3′ 5′
1
A
C
U
2
U
A
3
U
4
A
5
G
6
A
7
U
8
C
9
U
A
10
A
11
A
12
G
13
G
14
G
15
C
16
C
U
17
A
G
18
U
19
U
C
A
20
U
G
A
21
U
C
22
A
23
A
24
C
25
C
26
C
27
C
28
U
29
G
30
A
31
A
32
U
33
A
34
A
G
35
U
36
A
37
A
38
C
39
A
40
A
41
A
42
A
43
C
U
44
A
45
C
U
46
G
A
47
A
48
A
49
A
50
A
51
C
U
52
C
53
A
54
C
A
55
C
U
56
A
57
A
58
A
59
A
60
A
61
C
U
62
U
C
63
A
64
C
U
65
A
66
A
67
A
68
A69
A
U70
C71
G
A72
A
G73
A74
A75
A
76
A
3′ 5′
1
A
C
U
2
U
A
3
U
4
A
5
G
6
A
7
U
8
C
9
U
A
10
A
11
A
12
G
13
G
14
G
15
C
16
C
U
17
A
G
18
U
19
U
C
A
20
U
G
A
21
U
C
22
A
23
A
24
C
25
C
26
C
27
C
28
U
29
G
30
A
31
A
32
U
33
A
34
A
G
35
U
36
A
37
A
38
C
39
A
40
A
41
A
42
A
43
C
U
44
A
45
C
U
46
G
A
47
A
48
A
49
A
50
A
51
C
U
52
C
53
A
54
C
A
55
C
U
56
A
57
A
58
A
59
A
60
A
61
C
U
62
U
C
63
A
64
C
U
65
A
66
A
67
A
68
A
69
A
U
70
C
71
G
A
72
A
G
73
A
74
A
75
A
76
A
3′

42. Summary
SL-b UR SL-b UR UR SL-Y CSE
SL-a UR SL-b UR UR SL-Y CSE
SL-a UR SL-b UR UR SL-Y CSE
SL-a UR SL-b UR SL-b UR UR SL-Y UR SL-Y CSE
SL-a UR SL-b UR UR SL-Y UR SL-Y CSE
SL-a UR SL-b UR UR SL-Y UR SL-Y CSE
WA
ECSA-IOL
ECSA-MASA
AUL-Cbn
AUL-Am
AUL-As
500 9007000 250
CHIKV 3'UTRs
AUL-Cbn specific duplication
• Lineage-specific non-coding RNA architecture
• Conclusive mapping of RSEs / DRs to structured and unstructured regions
• Individual lineages show varied replication potential in mosquito / vertebrate cells
• CHIKV 3’UTR stem-loops do not stall exoribonucleases
• A-rich unstructured repeats are likely sponge RNA-binding proteins like HuR
• Whether or not miRNAs are encoded needs to be established
KY703952.1
KY703924.1
KY703926.1
KY703974.1
KY703891.1
KY703986.1
KY703982.1
KY703971.1
KY703907.1
KY703914.1
KY703892.1
KY703889.1
KY703995.1
KY703943.1
KY703944.1
KY703890.1
KY703965.1KY703905.1KY703988.1KY703950.1KY703997.1KY703966.1KY703933.1KY703951.1KY703923.1KY703983.1KY703987.1KY703925.1KY703990.1KY703992.1KY703975.1KY703960.1KY703999.1KY703921.1
KY703968.1
KY703972.1
KY704002.1
KY703978.1
KY680375.1
KY703955.1
KY703948.1
KY703919.1
KY703959.1
KY703996.1
KY703935.1
KY703920.1
MF773560.1
KY680379.1
KY435459.1
KY435480.1
KY435456.1
KY435470.1
KY680399.1
KR559488.1
KR559475.1
KY703956.1
KY703898.1
KY680352.1
KY680361.1
KY680398.1
KY680384.1
KY703922.1
KY703908.1
KY680389.1
KR559476.1
KY703928.1
KY703909.1
KY703918.1
KY703901.1
KY703984.1
KY703947.1
KY680359.1
KT192707.1
KY680363.1
KY680355.1
KY703970.1
KY703994.1
KY703934.1
KY703899.1
KR559484.1
KR046232.1
KY703903.1
KY703902.1
KY703897.1
KY703927.1
KR559472.1
KY415985.1
KY415982.1
KY415978.1
KY415981.1
KY415979.1
KY415980.1
KY415983.1
KX702402.1
KY415984.1
KR559478.1
KX702401.1
KY680402.1
KY680356.1
KY680372.1
KY704001.1
KY435471.1
KY680382.1
KY435461.1
KY680386.1
KY680413.1
MG967666.1
KY680405.1
KY435455.1
KY703993.1
KY703940.1
KY703991.1
KY703894.1
KY703893.1
KY703976.1
KY703958.1
KY703937.1
KY703900.1
KY703973.1
KY703946.1
KY703954.1
KY703915.1
KY703938.1
KY703989.1
KY703979.1
KY703957.1
KY703942.1
KY703916.1
KY703985.1
KY703931.1
KY703888.1
KY680367.1
KR046230.1
KY703930.1
KY703895.1
KY703911.1
KY703980.1
KY703998.1
KY703967.1
KR559471.1
KR046229.1
KY680393.1
KY703917.1
KY703910.1
KY703962.1
KY703896.1
KR559481.1
KY703961.1
KY703936.1
KY703977.1
KY703913.1
MG921596.1
KY703945.1
KP851709.1
KY703906.1
KY680366.1
KY435476.1
KY680347.1
KY703904.1
KR046231.1
KY680364.1
KX262994.1
KY680373.1
KY680388.1
KY435472.1KY435469.1KY435475.1KY435454.1KY704000.1KY703932.1KY703963.1KY703969.1KY703949.1KY680370.1KX262992.1KP164572.1KY435458.1KR046228.1KY435474.1KY575573.1KR559492.1
KY435481.1
KR559473.1
KY435483.1
KY435484.1
KR559494.1
KR559495.1
KR559485.1
KY435482.1
KY435462.1
KR559497.1
KR559491.1
KX496989.1
KY435465.1
KY435464.1
KY435477.1
KY680396.1
KY435473.1
KY435460.1
KY680376.1
KX262991.1
KY680407.1
KR559489.1
KP164567.1
KY435479.1
KY435457.1
KY435468.1
KR046227.1
KP851710.1
KY435467.1
KY435485.1
KY435478.1
KR559490.1
KR046234.1
KR046233.1
KR559496.1
KY435463.1
KY435486.1
KJ451624.1
KT327165.1
KT327166.1
KT327163.1
KT327164.1
KT327167.1
M
G
208125.1
KY680412.1
KY680368.1
KY272969.1
KY680397.1
KY272962.1
KY680414.1
KY680411.1
KY680362.1
KY680377.1
MF001512.1
KP164571.1
KY680378.1
KY680395.1
KY272963.1
KY680351.1
KU355832.1
KY680349.1
KY272970.1
KY272968.1
MF001519.1
MF001509.1
KY272965.1
KY435466.1
KY680403.1
KY680409.1
KR559482.1
KR559480.1
MF001506.1
KY680350.1
KY680348.1
MF001508.1
KY680380.1
MF001516.1
MF001518.1
KR264950.1
KR264949.1
KY575572.1
KY680404.1
KY680381.1
MF001517.1
KY680394.1
MF001514.1
KY680410.1
KR559486.1
KR559479.1
KY680390.1
KY680354.1
KY272967.1
MF001515.1
MF001505.1
KY680400.1
KY575566.1
MF001507.1
KY680387.1
KY680374.1
MF001510.1
KY680357.1
KY680358.1
KY680360.1
KY575569.1
KY680369.1
MF001513.1
KY272966.1
KY272961.1
KY575565.1
MF001511.1
KY680391.1
KY680353.1
KY680408.1
KY680385.1KY680401.1
KR559470.1
KY680406.1
KR559477.1
KR559498.1
KY680383.1
KY680371.1
KY272964.1
KY680392.1
KR264951.1
KR559474.1
KR559487.1
KR559483.1
MF773564.1
MF773563.1
KJ689453.1
KJ689452.1
KJ451623.1
KJ451622.1
MF773559.1
KR559493.1
MF773562.1
KT308162.1
KT308159.1
KT308160.1
KC488650.1
KF318729.1
KT308163.1
KT308161.1
MF773561.1
FJ807897.1
MF773565.1
KM923920.1
KM923917.1
KM923919.1
KM923918.1
KX168429.1
EU703761.1
EU703759.1
EU703760.1
KX097988.1
KX097982.1
KY883764.1
KM673291.1
MG664851.1
HM045790.1
HM045800.1
HM045791.1
HM045797.1
HM045802.1
HM045796.1
HM045787.1
KX262987.1
KX262988.1
HM045789.1
HM045808.1
HM045814.1
EF452493.1
HM045810.1
HM045803.1
EF027140.1
HM
045813.1
HM
045788.1
EF027141.1
M
F076575.1
M
F076573.1
M
F076574.1
M
F076572.1
JQ
861257.1
JQ
861255.1
JQ
861258.1
JQ
861253.1
JQ
861256.1
JQ
861254.1
M
F076570.1
M
F076569.1
M
F076568.1
M
F076577.1
M
F076576.1
M
F076571.1
JQ861260.1
JQ861259.1
KJ579186.1
KJ579184.1
KJ579185.1
KJ579187.1
GU301779.1
KT324224.1
FJ445430.1
KT324228.1
FJ445431.1
KX009167.1
KX009171.1
MF773567.1
KX262997.1
GU301781.1
MF773569.1
KC862329.1
FJ445463.1
FJ445433.1
FJ445432.1
FJ445445.1
KT324225.1
FJ445502.1
FJ445443.1
FJ445484.1
HQ846359.1
HQ846358.1
KF151175.1
KF151174.1
KF590565.1
KF590567.1
GU908223.1KF590566.1KF590564.1KT324226.1KX009168.1HQ846357.1HQ846356.1JX088705.1KX009170.1GU199353.1GU199352.1KT324227.1KP164869.1GU301780.1MF773568.1KX009169.1FJ807899.1GQ428213.1KT336782.1KT336781.1
GQ428214.1GQ428212.1
FJ000069.1
KX262989.1
KP003811.1
EU244823.1
KX262993.1
KP003810.1
FJ513657.1
GU013530.1
FJ513629.1
FJ513628.1
GU013528.1
FJ513679.1
FJ513637.1
GU013529.1
FJ513635.1
HM045801.1
GU199350.1
GU189061.1
FJ445428.1
HM045799.1
GQ428211.1
MF774616.1
MF774615.1
MF774617.1
MF774619.1
MF774618.1
MF740874.1
MF774613.1
MF773566.1
MG049915.1
KY057363.1
KY751908.1
KX619426.1
MG925665.1
MF499120.1
MG137428.1
KT336780.1
KT336779.1
KT336778.1
JQ065890.1
MG664850.1
JQ065891.1
KJ679578.1
JQ065892.1
KX619423.1
KX619422.1
KX619425.1
KX619424.1
KX881784.1
JN558836.1
JN558835.1
JN558834.1
GQ428210.1
FJ513654.1
FJ513645.1
FJ513673.1
FJ445426.1
FJ513632.1
KY575570.1
GU199351.1
FJ513675.1
FJ000067.1
KT336777.1
FJ807898.1
EU372006.1
GQ428215.1
EF027134.1
KJ941050.1
HM045794.1
KY575568.1
KY575567.1
FJ000068.1
FJ000062.1
FJ000064.1
FJ000063.1
FJ445427.1
KC614648.1
JF274082.1
EU564335.1
FJ807896.1
FJ445511.1
FJ445510.1
FJ000065.1
FJ000066.1
EF027137.1
EF210157.1
EF027138.1
EF027136.1
EF027135.1
KY575571.1
KP003808.1
KP003807.1
KP003809.1
EU037962.1
EU564334.1
EF012359.1
KT449801.1
DQ443544.1
KP702297.1
KF283986.1
MG649985.1
MG649973.1
MG649971.1
MG649974.1
MG649972.1
MG649984.1
MG649983.1
MG649981.1
KY124329.1
MG649982.1
MG649978.1
MG649977.1
MG649976.1
MG649970.1
MG649975.1
KY124328.1
MG649980.1
MG649979.1
KY055011.1
KY704952.1
KY704942.1
KU940225.1
KP164569.1
KP164568.1
KP164570.1
MG000876.1
KY704955.1
MG000875.1KY704954.1KX228391.1HM045823.1KY575574.1HM045784.1HM045812.1EF027139.1KY038947.1HM045793.1KY038946.1HM045822.1KP003813.1KP003812.1KX262996.1KJ679577.1HM045821.1HM045806.1HM045811.1HM045805.1
HM045792.1
HM045809.1
HM045819.1
HM045818.1
HM045820.1
HM045817.1
KX262995.1
AY726732.1
KX262986.1
HM045786.1
HM045804.1
HM045816.1
HM045785.1
HM045815.1

43. Acknowledgements
• Roman Ochsenreiter (TBI)
• Ivo L. Hofacker (TBI)
• Adriano de Bernardi Schneider (UCSD)
Updated phylogeny of Chikungunya virus
reveals lineage-specific non-coding RNAs
Adriano de Bernardi Schneider1,†
, Roman Ochsenreiter2,†
, Reilly Hostager1
,
Ivo L. Hofacker2,3
, Daniel Janies4
, and Michael T. Wolfinger2,3,
1
AntiViral Research Center, Department of Medicine, University of California San Diego, San Diego, 92103, United States of America
2
Department of Theoretical Chemistry, University of Vienna, Währingerstraße 17, 1090 Vienna, Austria
3
Research Group BCB, Faculty of Computer Science, University of Vienna, Währingerstraße 29, 1090 Vienna, Austria
4
Department of Bioinformatics and Genomics, University of North Carolina at Charlotte, Charlotte, 28223, United States of America
†
These authors have contributed equally to this work.
Chikungunya virus (CHIKV), a mosquito-borne alphavirus of paralysis, Guillain Barré Syndrome (GBS) and renal com-