evolutionary trajectory analysis-atlanta-2010.graffle

0.02

M63758-A/swine/Jamesburg/1942

AF455704-A/Swine/Indiana/P12439/00

CY022112-A/swine/Tennessee/19/1977

CY026470-A/swine/Iowa/100/1977

AB434393-A/swine/Hokkaido/2/1981

AF222768-A/Swine/Wisconsin/125/97

CY036802-A/swine/Wisconsin/30954/1976

U49091-A/swine/Beij ing/94/1991

CY022360-A/swine/Minnesota/27/1976


AB434401-A/swine/Niigata/1/1977

M63759-A/swine/Iowa/1946



CY022368-A/swine/Minnesota/5892_7/1979


M63763-A/swine/Wisconsin/1/1961

AF455702-A/Swine/Minnesota/55551/00

CY028783-A/swine/California/T9001707/1991



M76609-A/turkey/North Carolina/1790/1988

EU743154-A/turkey/IA/10271_3/1990



AF455705-A/Swine/Il l inois/100085A/01

AY744935-A/Brevig Mission/1/1918

AF342819-A/Wisconsin/10/98


CY022344-A/swine/I l l inois/1/1975

M63755-A/Wisconsin/3523/1988

AF397199-A/swine/Quebec/5393/91





M30747-A/swine/Iowa/15/1930

CY022376-A/swine/Nebraska/123/1977









AF397198-A/swine/Quebec/192/81

FJ966083-A/California/04/2009

CY025013-A/swine/Kansas/3024/1987


EU743162-A/turkey/IA/21089_3/1992


CY022973-A/swine/Iowa/31483/1988

M76606-A/New Jersey/8/1976



M22578-A/swine/Iowa/1976/1931



L24394-A/MD/12/1991


M76602-A/Ohio/3523/1988

M63767-A/swine/Ontaria/2/1981

AF251423-A/Swine/Iowa/569/99


AF455706-A/Swine/Il l inois/100084/01




L46850-A/swine/WI/1915/1988

M63757-A/swine/29/1937




M22570-A/swine/Hong Kong/127/1982



M63761-A/swine/May/1954


AF455701-A/Swine/North Carolina/93523/01

EU735789-A/turkey/NC/19762/1988

CY027158-A/swine/Iowa/24297/1991

FJ357107-A/turkey/NC/17026/1988



CY026430-A/swine/Jamesburg/1942

AF251407-A/Swine/Nebraska/209/98

EU742639-A/turkey/KS/4880/1980



CY025005-A/swine/Arizona/148/1977

EU743178-A/chicken/NY/21665_73/1998

M63768-A/swine/Iowa/17672/1988


CY009631-A/swine/1931



AF251431-A/Swine/Minnesota/593/99

CY024928-A/Ohio/3559/1988


CY022336-A/swine/Iowa/17672/1988


AF250127-A/Swine/Indiana/9K035/99

CY022384-A/swine/Ontario/4/1981


CY022480-A/swine/Maryland/23239/1991




CY024936-A/swine/Minnesota/24/1975



M76610-A/Wisconsin/3623/1988


CY026142-A/Wisconsin/301/1976



CY022472-A/swine/Kansas/3228/1987

M63766-A/swine/Italy/141/1981



CY035073-A/swine/Memphis/1/1990




M30748-A/swine/Tennessee/24/1977





AF455699-A/Swine/Ohio/891/01


M76607-A/swine/Wisconsin/1/1967CY022440-A/swine/Wisconsin/2/1970


AB434385-A/swine/Kyoto/3/1979




CY027294-A/swine/Ohio/23/1935


L46849-A/swine/IN/1726/1988


CY022352-A/swine/Kentucky/1/1976




EU735797-A/chicken/PA/35154/1991


CY014763-A/turkey/Minnesota/12537/1989

M63765-A/swine/I taly/2/1979





U49092-A/swine/Hong Kong/273/1994

M63756-A/swine/Ohio/23/1935


AB434409-A/swine/Ehime/1/1980

M63760-A/swine/41/1949


AY233394-A/Duck/NC/91347/01AF455700-A/Swine/North Carolina/98225/01

L11164-A/swine/Nebraska/1/1992

M63754-A/New Jersey/8/1976


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EF551046-A/turkey/Il l inois/2004

AB434361-A/swine/Nakhon pathom/NIAH586_1/2005

DQ469959-A/Ontario/RV1273/2005

DQ666928-A/Swine/Korea/S5/2005

FJ789828-A/swine/Shanghai/2/2005

AB441173-A/swine/Miyazaki/1/2006

EU798852-A/swine/Korea/CAS09/2006

DQ469991-A/swine/Ontario/33853/2005

EU697205-A/turkey/Minnesota/366767/2005

AY363542-A/Swine/Hong Kong/9745/01

EU798840-A/swine/Korea/Hongsong2/2004

DQ889686-A/Iowa/CEID23/2005

EU735821-A/turkey/OH/313053/2004

FJ966083-A/California/04/2009

EU850624-A/swine/Hainan/1/2005

EU258936-A/swine/Missouri/2124514/2006


EU502887-A/swine/Shanghai/1/2005

DQ923513-A/swine/Korea/CN22/2006

DQ469967-A/swine/Alberta/14722/2005

EU798843-A/swine/Korea/JL04/2005

EU798847-A/swine/Korea/PZ14/2006

FJ638307-A/swine/NC/00573/2005

AB434377-A/swine/Ratchaburi/NIAH874/2005

DQ150434-A/swine/IN/PU542/04

EF551054-A/swine/North Carolina/2003

DQ280201-A/swine/Alberta/56626/03

AY623834-A/swine/Pingtung/199_2/2002


DQ145541-A/swine/Minnesota/00395/2004


AY129159-A/Swine/Korea/CY02/02


DQ150426-A/swine/MI/PU243/04

EU015990-A/swine/Guangxi/13/2006

EU301304-A/swine/Korea/JNS06/2004




EU798857-A/swine/Korea/CY10/2007

AY623836-A/swine/Pingtung/92_2/2003


EU850621-A/swine/Guangxi/17/2005


FJ638299-A/swine/IL/00685/2005




DQ335774-A/turkey/Ohio/313053/04


DQ469983-A/swine/Manitoba/12707/2005

EU399754-A/Ontario/1252/2007



EU604694-A/swine/OH/511445/2007

AB434417-A/swine/Saitama/1996


DQ469975-A/swine/British Columbia/28103/2005

EU409950-A/swine/Ohio/24366/07

AB434345-A/swine/Chachoengsao/2003


FJ638315-A/swine/IL/07003243/2007

DQ923512-A/swine/Korea/PZ72_1/2006


EU798838-A/swine/Korea/CAN01/2004

EU798844-A/swine/Korea/Asan04/2006


DQ139324-A/Swine/Zhejiang/1/2004


DQ469999-A/turkey/Ontario/31232/2005



AY623835-A/swine/Taichung/200_8/2002

EU258946-A/swine/Missouri/4296424/2006

EU798850-A/swine/Korea/CAN04/2005

EU743213-A/turkey/MN/366767/2005


AY623833-A/swine/Chiai/77_10/2001


FJ461599-A/swine/Korea/C13/2008

EU697210-A/turkey/North Carolina/353568/2005

Introduction

In late April and early May 2009 the first pandemic strain of the 21st Century arrived in the form of the Pandemic H1N1 2009 influenza virus. Initially named "Swine Flu" the origin of the Pandemic H1N1 2009 sequence presented many initial questions about each of the segments of the virus.

We began by examining the relationship between nucleotide differences of the A/California/04/2009 and a number of top BLAST hits versus the isolation year differences. Although we expected that a correlation would exist between the nucleotides difference and isolation year difference would exist, we were surprised by the triangular pattern, shown in Figure 1

Plot Results Three clusters of sequences were noted:

• Cluster 1, diagonal triangle side labeled "1" in Figure 1• Cluster 2, vertical triangle side labeled "2"• Cluster 3, top triangle side labeled "3"

R. Burke Squires1, Elizabeth McClellan3, Jyothi Noronha1, Victoria Hunt1, Richard H. Scheuermann1,2

1Department of Pathology, 2Division of Biomedical Informatics, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 2 Department of Statistics, Southern Methodist University, Dallas, Texas.

Evolutionary Trajectory Analysis of Pandemic H1N1 2009

Quantitate Alignments

To quantify the difference we were observing in the alignments, we developed a metric to count the number of amino acid changes at each position

• Any change in an amino acid, within a single column, is counted.• If an amino acid persists then counts would be low, while amino acids that

changed often would show a higher count.

The fact that this metric was consistently lower for cluster 1 segments (Table 1) is consistent with the relative persistence of the amino acid substitutions over time.

Table 3. Slope of the evolutionary trajectory clusters and the corresponding mutation rates for influenza A virus segments.

Segment ET Slope

SUR Slope

Exp. Determined Mutations Rate.

PB2 6.9 24.9 4.3 (1)PB1 7.7 26.9PA 6.0 23.2HA 5.7 28.8 5.7 (2)NP 3.2 18.2 3.6 (3)NA 3.9 23.1 3.2 (4)M 1.4 5.6 1.5 (5)NS 2.1 12.5 1.5 - 2.6 (6, 7)

Lastly, we compared the slopes of the trend lines of the ET and SUR clusters to experimentally determined mutations rates for each of the 8 segments and found that the slopes of the ET cluster, as shown in Table 3, match published mutation rates closely.

Conclusion In conclusion, we have shown that sequence similar to the Pandemic H1N1 strain as identified by BLAST analysis fall into different categories based on other measures of sequence comparison. We propose that:

• Cluster 1 represents the true "Evolutionary Trajectory" (ET) of each segment of the A/California/04/2009 pandemic H1N1 2009 strain.

• Cluster 2 contains sequences that are "Similar, but Unrelated" (SUR).

• Cluster 3 contains sequences that are only moderately similar and also unrelated.

It has become apparent through this analysis that sequence similarity, in the absence of a time component, is not a stringent enough criterion to determine relationships among sequences.

Figure 4. Nucleotide difference versus isolation year difference between A/California/04/2009 with the three clusters of sequences highlighted with different symbols

Figure 2. Alignment of Evolutionary Trajectory strains, sorted by isolation year, which shows the persistence of amino acids changes (white background).

Figure 3. Alignment of Similar but unrelated (SUR) strains, sorted by isolation year, which shows a lack of persistence of amino acids changes (white background).

Table 1. Average number of amino acids changes per strain in

alignments of cluster 1 and 2.

IRD fludb.orgInfluenza Research Database

10

Figure 1. Nucleotide difference versus isolation year difference between A/California/04/2009 and the top 1000 BLAST hits (performed June, 2009).

1

2

3

Alignments

To further examine the sequence relationships in the different strain clusters we assembled working set of sequences for each cluster in the Influenza Research Database (IRD) (www.fludb.org). We aligned representative sequences from strains in cluster 1:

• Sorted sequences by ascending isolation years, (Figure 2).• We observed a persistence of amino acid changes in the sequences over time.

We aligned representative sequences from strains in cluster 2 (Figure 3):

• We observed no persistence. Amino acid changes seemed to appear randomly

Cluster 27.897.4

8.5511.21

11.92

7.0138.75

2.03

HA

M

Cluster 1

PB1

NA

PA

NS

5.092.22

3.2812.18

5.19

5.53

NP9.73

PB2

1.3

Segment

Phylogenic Trees

Phylogenetic trees for cluster 1 and 2 sequences were generated

• Trees are rooted at the earliest strain with ordered subtrees.• Observed a gradual progression of strains with short branch lengths

in the cluster 1 tree

In contrast, cluster 2 trees lacked the gradual progression of strains and appeared to possess longer branch lengths.

Tree Quantitation We compared the average branch lengths of each tree and performed a T-test with the results shown in Table 2.

Results confirm that cluster 1 show a gradual accumulation of sequence alterations as evidenced by relatively short branch lengths. In addition, the gradual transition in branches seemed to reflect the normal progression of time based on the year of isolation.

2.21E-05NS 6.422.879.13E-01M 4.36 4.144.44E-01NA 5.39 5.55

NP 1.50E-052.87 6.693.55HA 4.42E-089.55

2.855.57PA 2.93E-068.57 9.13E-116.92PB1

3.47PB2 4.23 1.84E-02P-valueCluster 2 Avg.Cluster 1 Avg.Segment

Table 2. Analysis of branch length of the phylogenetic trees of the cluster 1 and 2. T-test results show a low probability that the two

groups are related (Average values are 10^-3).

(A)

Overall Method

Figure 5 (A) (Above). Phylogenetic Tree of cluster 2 rooted but the earliest 2001 strain, with A/California/04/2009 indicated by the red arrow.

(B) (Left). Phylogenetic tree of cluster 1 of the A/California/04/2009 (red arrow), rooted at A/Brevig Mission/1918 in the lower left, with ordered subtrees.

(B)

• BLAST each segment returning top 1000 hits. BLAST analysis was performed with all influenza segments on June, 2009.

• Normalize for short sequence lengths, assuming a constant rate of evolution.• Plot nucleotide differences versus isolation year differences.• Determine one-phase association equation to separate cluster 1, 2.• Align cluster 1, 2. • Quantitate alignment, counting amino acids changes per column • Determine phylogenetic trees for cluster 1, 2.• Quantitate trees averaging branch lengths and T-test.

1. Gorman et al. Evolution of influenza A virus PB2 genes: implications for evolution of the ribonucleoprotein complex and origin of human influenza A virus. The Journal of Virology (1990) vol. 64 (10).

2. Fitch et al. Long term trends in the evolution of H(3) HA1 human influenza type A. Proceedings of the National Academy of Sciences of the United States of America (1997) vol. 94 (15).

3. Shu et al. Analysis of the evolution and variation of the human influenza A virus nucleoprotein gene from 1933 to 1990. The Journal of Virology (1993) vol. 67 (5).

4. Xu et al. Genetic Variation in Neuraminidase Genes of Influenza A (H3N2) Viruses. Virology (1996) vol. 224 (1).

5. Saitou and Nei. Polymorphism and evolution of influenza A virus genes. Molecular Biology and Evolution (1986) vol. 3 (1).

6. Nobusawa and Sato. Comparison of the Mutation Rates of Human Influenza A and B Viruses. The Journal of Virology (2006) vol. 80 (7).

7. Parvin, J. D., A. Moscona, W. T. Pan, J. M. Leider, and P. Palese. 1986. Measurement of the mutation rates of animal viruses: influenza A virus and poliovirus type 1. J. Virol. 59:377-383.

<= A/California/04/2009

A/California/04/2009 =>

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