Relationship of SARS-CoV to other pathogenic RNA viruses explored by tetranucleotide usage profiling

1471-2105-4-43 1471-2105 Research article Relationship of SARS-CoV to other pathogenic RNA viruses explored by tetranucleotide usage profiling Yap Leng Yee daniely@hkusua.hku.hk Zhang Wu Xue xwzhang@hkucc.hku.hk Danchin Antoine adanchin@pasteur.fr

HKU-Pasteur Research Centre, Dexter H.C. Man Building, 8 Sassoon Road Pokfulam, Hong Kong

Institute Pasteur, Unité de Génétique des Génomes Bactériens, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France

BMC Bioinformatics 1471-2105 2003 4 1 43 http://www.biomedcentral.com/1471-2105/4/43 14499005 10.1186/1471-2105-4-43 08 7 2003 20 9 2003 20 9 2003 2003 Yap et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. RNA virus convergent evolution horizontal gene transfer factor analysis SARS

Abstract

Background

The exact origin of the cause of the Severe Acute Respiratory Syndrome (SARS) is still an open question. The genomic sequence relationship of SARS-CoV with 30 different single-stranded RNA (ssRNA) viruses of various families was studied using two non-standard approaches. Both approaches began with the vectorial profiling of the tetra-nucleotide usage pattern V for each virus. In approach one, a distance measure of a vector V, based on correlation coefficient was devised to construct a relationship tree by the neighbor-joining algorithm. In approach two, a multivariate factor analysis was performed to derive the embedded tetra-nucleotide usage patterns. These patterns were subsequently used to classify the selected viruses.

Results

Both approaches yielded relationship outcomes that are consistent with the known virus classification. They also indicated that the genome of RNA viruses from the same family conform to a specific pattern of word usage. Based on the correlation of the overall tetra-nucleotide usage patterns, the Transmissible Gastroenteritis Virus (TGV) and the Feline CoronaVirus (FCoV) are closest to SARS-CoV. Surprisingly also, the RNA viruses that do not go through a DNA stage displayed a remarkable discrimination against the CpG and UpA di-nucleotide (z = -77.31, -52.48 respectively) and selection for UpG and CpA (z = 65.79,49.99 respectively). Potential factors influencing these biases are discussed.

Conclusion

The study of genomic word usage is a powerful method to classify RNA viruses. The congruence of the relationship outcomes with the known classification indicates that there exist phylogenetic signals in the tetra-nucleotide usage patterns, that is most prominent in the replicase open reading frames.

Background

Severe Acute Respiratory Syndrome (SARS), a newly identified infectious disease, has imperilled the health of human population in more than 30 nations. It has claimed over 812 lives and infected more than 8442 (9.61% death rate) by July 2, 2003 1 since its outbreak in November 2002 in the province of GuangDong, People's Republic of China. By May 15, 2003, the primary etiological agent for SARS was found to fulfil Koch's postulate through experimental infection of cynomolgus macaques (Macaca fascicularis) 2. Chronicles for the discovery of SARS CoronaVirus (SARS-CoV) can be found in articles [e.g. 34] and websites [e.g. 5].

A common question is often asked when investigating viral evolution: what hallmark, in term of genome sequence or RNA word usage, could be used to trace back the emergence of a new pathogen in humans/animals? In particular, CoronaViruses are prone to recombination 67 and like all other viruses they mutate at a high frequency 8. This makes extremely hazardous to try to trace the origin of the virus. Nevertheless, this prompted us to investigate their relationships using the RNA word usage hoping to identify some RNA viruses that display similar word usage pattern. Such RNA viruses might hint about the origin of SARS-CoV. This study will contribute to our understanding of the RNA word usage of SARS-CoV and some other pathogenic RNA viruses. In the present study, we explored the relationships of 31 RNA viruses, which are known to cause diseases to their corresponding hosts with either similar symptoms or infectiousness, including SARS-CoV, based on their global tetra-nucleotide usage pattern.

Preliminary analysis of the sequence data indicated that there are 11–14 open reading frames in the SARS-CoV genome 91011. The overall gene order for this novel pathogen supported its placement in the family of Coronaviridae which includes the animal/human CoronaViruses. It should be emphasized that the sequence similarity shown is attributed mainly to the large RNA-dependent RNA polymerase (replication enzyme or RdRp) residing in the first two open reading frames (ORFs). These two ORFs constitute more than 65% (>20 kb) of the total genome size and these regions are more conserved in their nucleotide sequences due to their specialized role for viral RNA replication. Therefore, the possible relationship based on the sequence of the replication enzyme alone was also investigated.

Results and Discussion

Mono-nucleotide bias

Table 1 presents the breakdown of the RNA sequence into mononucleotide frequencies for the 31 viral genomes in our dataset. Except for the Rabbit Hemorrhagic disease Virus (RHV) that shows a fair usage of the four nucleotides in approximately equal number, the other RNA viruses have a biased genome composition. Bovine CoronaVirus (BCoV) and Human CoronaVirus 229E (HCoV) favor the U nucleotide (35.5% and 34.6%) at the expense of the C nucleotide (15.3% and 16.7%). Relatively strong nucleotide biases are visible in the other genomes and we will mention a few of the extremes. The highest base count is 28.4% G in the Yellow Fever Virus (YFV), 38.9% A in the Respiratory Syncytial Virus (RSV), 35.5% U in the Bovine CoronaVirus (BCoV) and 28.5% C count in the Foot-and-Mouth disease Virus (FMV). The lowest base counts are 15.8% G in the Human Respiratory syncytial Virus (HRV), 21.2% A in the Equine arteritis Virus (EV1), 20.9% U in the Igbo Ora Virus (IOV) and 13.6% C in the Bovine ephemeral Fever Virus (BFV). The A nucleotide is the most popular base among RNA viruses (ranging from 21.2% to 38.9%), and C is the most variable nucleotide (ranging from 13.6% to 33.1%).

Table 1

RNA virus in current study.

Virus Name

Type

Acession Number

DNA Stage

Segment

Acronym

Size (nt)

A+U%

ssRNA positive-strand viruses

Avian infectious bronchitis virus

ss-RNA

NC_001451

ABV

27608

21.7

28.9

33.2

16.2

62.1

Bovine coronavirus

ss-RNA

NC_003045

BCoV

31028

21.8

27.4

35.5

15.3

62.9

Equine arteritis virus

ss-RNA

NC_002532

EV1

12704

26.0

21.2

27.1

25.6

48.3

Human coronavirus 229E

ss-RNA

NC_002645

HCoV

27317

21.6

27.2

34.6

16.7

61.7

Lactate dehydrogenase-elevating virus

ss-RNA

NC_002534

LDV

14225

25.9

23.1

28.2

22.6

51.3

Murine hepatitis virus

ss-RNA

NC_001846

MHV

31357

23.9

26.0

32.3

17.9

58.2

Porcine epidemic diarrhea virus

ss-RNA

NC_003436

PDV

28033

22.8

24.7

33.2

19.2

58.0

Porcine reproductive and respiratory syndrome virus

ss-RNA

NC_001961

PRV

15428

26.2

21.7

25.3

26.7

47.0

SARS coronavirus

ss-RNA

NC_004718

SAR

29751

20.8

28.5

30.7

20.0

59.2

Feline coronavirus

ss-RNA

AY204704

FCoV

9979

22.6

27.9

29.2

20.3

57.2

Simian hemorrhagic fever virus

ss-RNA

NC_003092

SFV

15717

22.6

22.5

27.4

27.5

49.9

Transmissible gastroenteritis virus

ss-RNA

NC_002306

TGV

28586

20.6

29.5

32.9

17.0

62.4

Avian encephalomyelitis virus

ss-RNA

NC_003990

AEV

7055

25.7

27.0

28.3

19.0

55.3

Bovine viral diarrhea virus genotype 2

ss-RNA

NC_002032

BDV

12255

25.2

32.7

22.3

19.8

54.9

Foot-and-mouth disease virus C

ss-RNA

NC_002554

FMV

8115

25.6

24.8

21.2

28.5

45.9

Igbo Ora virus

ss-RNA

NC_001924

IOV

11821

24.1

31.1

20.9

24.0

51.9

Poliovirus

ss-RNA

NC_002058

PV1

7440

23.0

29.7

24.0

23.3

53.7

Rabbit hemorrhagic disease virus

ss-RNA

NC_001543

RHV

7437

25.5

25.9

23.9

24.7

49.8

Tamana bat virus

ss-RNA

NC_003996

TBV

10053

21.5

33.2

28.3

16.9

61.6

Yellow fever virus

ss-RNA

NC_002031

YFV

10862

0.28

0.27

0.23

0.21

0.50

ssRNA negative-strand viruses

Avian paramyxovirus 6

ss-RNA

NC_003043

APV

16236

0.23

0.29

0.25

0.23

0.54

Bovine ephemeral fever virus

ss-RNA

NC_002526

BFV

14900

0.20

0.38

0.28

0.14

0.66

Bovine respiratory syncytial virus

ss-RNA

NC_001989

BRV

15140

0.17

0.38

0.29

0.17

0.66

Canine distemper virus

ss-RNA

NC_001921

CDV

15690

0.22

0.31

0.26

0.21

0.57

Human respiratory syncytial virus

ss-RNA

NC_001781

HRV

15225

0.16

0.39

0.28

0.18

0.67

Hantaan virus

ss-RNA

AF345636

Yes

HV1

11772

0.21

0.33

0.29

0.17

0.62

Influenza B virus

ss-RNA

NC_002208

Yes

IBV

14452

0.22

0.36

0.24

0.18

0.60

Measles virus

ss-RNA

NC_001498

MV1

15894

0.24

0.29

0.23

0.24

0.53

Respiratory syncytial virus

ss-RNA

NC_001803

RSV

15191

0.16

0.39

0.28

0.18

0.67

Reston Ebola virus

ss-RNA

NC_004161

REV

18891

0.20

0.31

0.28

0.21

0.59

Tioman virus

ss-RNA

NC_004074

TV2

15522

0.21

0.30

0.26

0.22

0.57

The information about 31 RNA viruses being investigated in this study. Their accession number, abbreviation, genome size, number of segments and whether they undergo DNA stage are tabulated. The breakdown of the RNA nucleic acids and A+U contents are also shown.

From the standpoint of the overall genomic composition analysis, the G+C content is an interesting property for a genome, in that the overall content often correlates with the organism pathogenicity 12. Most of the pathogens genomes have a low G+C content, while some such as Mycobacterium tuberculosis has a relatively high G+C content. Therefore, as expected in Table 1, we noted that most of the pathogenic viruses are A+U-rich (>50%), except for Porcine reproductive and Respiratory syndrome Virus (PRV), Equine arteritis virus (EV1), Rabbit hemorrhagic disease virus (RHV), Simian hemorrhagic Fever Virus (SFV) and Foot-and-Mouth disease Virus C (FMV).

Di-nucleotide bias

The frequencies of occurrence for di-nucleotides were compared to the random RNA counterparts having the same base proportion in order to compute the z value that reflected their di-nucleotide bias (Table 2). Among the 31 virus sequences examined, the frequencies of occurrence for di-nucleotide were not randomly distributed, with only a few exceptional di-nucleotides starting with a purine residue present at the expected frequencies (ApC, ApG, GpC, |z| < 3). A remarkable deviation from the expected frequencies occurs for the di-nucleotide pairs CpG and UpA (suppression or under-representation, z < -50) as well as di-nucleotides pairs CpA and UpG (enhancement or over-representation, z > 40). These di-nucleotide biases, together with mono-nucleotide bias 13, have a direct impact on the codon usage of viruses. For example, in the codon usage for the 24 protein coding sequences in human CoronaVirus 229E (Table 3), only 2.85% of codons contain the under-represented subword CpG di-nucleotide whereas 11.26% of the codons contain the over-represented CpA di-nucleotide (the aggregate codon usage containing each di-nucleotide subword without mono- and di-nucleotide bias is close to 6.25%).

Table 2

Di-nucleotide bias for six RNA viruses.

BCoV

MHV

SARS

ABV

HCoV

PDV

Average z value

across 31 viruses

Di-nucleotide

N(w)

E(w)

N(w)

E(w)

N(w)

E(w)

N(w)

E(w)

N(w)

E(w)

N(w)

E(w)

497

1034

-103.81

798

1342

-104.39

566

1235

-121-14

486

976

-109.69

487

979

-95.94

684

1226

-102.03

-77.31

1344

1037

62.19

1694

1341

62.33

1432

1236

36.12

1147

970

35.96

1164

976

37.86

1416

1228

35.05

5.74

2845

3007

-26.44

2499

2614

-19.25

2234

2594

-58.91

2200

2642

-76.99

2092

2556

-81.97

1976

2296

-55.43

-15.54

2818

3000

-30.10

2404

2616

-35.12

2080

2594

-87.64

2409

2641

-42.42

2033

2554

-84.51

1965

2299

-53.83

-52.48

1824

1848

-4.25

1968

1941

4.77

1749

1760

-2.00

1844

1728

21.13

1416

1601

-34.78

1537

1579

-7.12

3.80

1629

1849

-39.08

1745

1941

-32.74

1677

1764

-16.43

1505

1730

-39.47

1397

1598

-36.09

1358

1581

-38.05

-1.33

1371

1303

12.93

1384

1458

-13.50

1978

1695

50.18

1474

1292

35.28

1558

1236

58.96

1594

1332

50.25

5.42

1594

1297

56.03

1705

1453

46.19

2203

1695

87.29

1603

1290

59.90

1638

1234

74.68

1783

1327

83.96

49.99

1801

1674

22.52

1874

1806

12.28

2190

1814

67.50

1661

1487

31.50

1724

1568

28.13

1953

1784

29.95

16.50

1179

1674

-88.35

1296

1802

-94.30

1552

1815

-46.36

1127

1482

-65.41

1130

1568

-79.37

1410

1781

-67.80

-17.49

2449

2394

9.10

2473

2402

11.92

1868

1898

-5.35

2154

1982

29.46

2240

2044

34.60

2262

2119

23.86

-7.13

3101

2392

120.25

3146

2408

128.13

2663

1897

137.30

2476

1983

87.74

2898

2040

152.24

2814

2117

126.99

65.79

The di-nucleotide bias in six RNA viruses. The z value quantifies the di-nucleotide bias as defined in equation 1. N (w) and E (w) are actual and expected frequency of occurrence for word w. The last column is the average z value across 31 RNA viruses.

Table 3

Codon usage for Human CoronaVirus 229E (HCoV).

Amino Acid

Codon

Usage/%

Amino Acid

Codon

Usage/%

Arg

CGU

1.04

Ile

AUU

3.34

CGC

0.41

AUC

0.74

CGA

0.17

AUA

1.35

CGG

0.13

Gly

GGU

4.12

AGA

1.23

GGC

1.43

AGG

0.36

GGA

0.67

Leu

UUA

1.49

GGG

0.22

UUG

2.96

Val

GUU

6.00

CUU

2.48

GUC

1.23

CUC

0.46

GUA

1.09

CUA

0.65

GUG

1.90

CUG

0.63

Lys

AAA

3.15

Ser

UCU

2.70

AAG

2.31

UCC

0.66

Asn

AAU

4.15

UCA

1.37

AAC

1.82

UCG

0.20

Gln

CAA

2.04

AGU

1.86

CAG

1.17

AGC

0.71

his

CAU

1.14

Thr

ACU

3.23

CAC

0.46

ACC

0.76

Glu

GAA

2.81

ACA

2.21

GAG

1.21

ACG

0.29

Asp

GAU

3.09

Pro

CCU

1.6S

GAC

1.96

CCC

0.35

Tyr

UAU

3.00

CCA

1.07

UAC

1.46

CCG

0.19

Cys

UGU

2.26

Ala

GCU

3.58

UGC

0.95

GCC

0.83

Phe

UUU

4.59

GCA

1.80

UUC

1.10

GCG

0.42

The relative usage of synonymous codons in the 24 known CDSs of Human Corona Virus 229E (HCoV).

In double stranded DNA genomes the deficiency in di-nucleotide CpG is often supposed to be due to the fact that they are the targets for methyltransferase activity that leads to cytosine deamination 1415. It is however unlikely that the mechanism of deamination that alters the genetic contents at the DNA level would affect the viral RNA content of most RNA viruses without a DNA stage. There might exist specific cytosine RNA methylases that could be responsible for this effect 16. However it is more consistent to propose that, unlike the mechanism of cytosine deamination in the DNA realm, the dominating process is cytosine deamination in RNA viruses, converting cytosine to uracil (C ♦ U) instead of thymine (T). As a consequence of this mechanism, di-nucleotide CpG changes to either di-nucleotide UpG or CpA in the direct/complementary strands of RNA viruses and causes the over-representation in di-nucleotide UpG and CpA (z > 19). Interestingly, there is experimental evidence in vitro that the rate of cytosine deamination is faster (>100 times) in the single stranded than in double-stranded state 17. Apart from the under-representation in di-nucleotide CpG and over-representation in di-nucleotide CpA and UpG, the reason for the observed di-nucleotide UpA scarcity in RNA may be explained by its chemical lability 18. The UpA dinucleotide is chemically the most unstable among the 16 dinucleotides. Furthermore, UpA appears to be a preferential target for ribonucleases 19. This lability would create a selection pressure against di-nucleotide UpA in RNA viruses.

If we choose a critical value for z (|z| = 3.29) that only allows a chance of 1 in 1000 error for classifying a word as biased (over/under-represented), all di-nucleotides show some kind of bias in their usage pattern across 31 different viruses (Table 4, derived from the complete form of Table 2 provided as the additional file 1). The causes for these biases await further investigation.

Table 4

Overall statistics for biased di-nucleotides and tetra-nucleotides.

Percentage of di-nucleotide that can be used to discriminate between vi ruses(|z| > 3.29)

Percentage of tetra-nucleotide that can be used to discriminate between vi ruses(|z| > 3.29)

Virus

Percentage of biased di-nucleotide (|z| > 3.29)/%

Percentage of biased tetra-nucleotide (|z| > 3.29)/%

100%

96.09%

BCoV

93.8

29.7

MHV

93.8

28.1

SARS

81.3

34.4

ABV

81.3

27.3

HCoV

93.8

31.3

PDV

81.3

28.5

TGV

87.5

31.6

LDV

93.8

19.5

PRV

93.8

15.6

SFV

93.8

16.0

FCoV

75.0

11.7

EV1

87.5

14.5

TBV

75.0

21.9

AEV

93.8

11.7

PV1

87.5

11.7

YFV

93.8

29.3

BDV

87.5

17.6

RHV

93.8

9.4

FMV

87.5

12.1

IOV

75.0

9.8

HV1

62.5

12.5

RSV

87.5

18.8

HRV

87.5

19.1

BRV

93.8

19.9

TV2

81.3

15.2

REV

87.5

18.4

MV1

81.3

15.2

CDV

75.0

16.0

APV

93.8

11.7

BFV

81.3

15.2

IBV

87.5

23.4

The percentage of biased di-nucleotides and tetra-nucleotides that shows strong biases (lzl > 3.29) in 31 RNA viruses (right). For di-nucleotides, all 16 (100%) of them show strong biases in part of or all 31 RNA viruses. For tetra-nucleotides, 246 (96%) of the tetra-nucleotides show strong biases in part of or all 31 RNA viruses.

Additional File 1

The RNA word biases of different sizes in RNA viruses. These tables show the di-nucleotide, tetra-nucleotide and penta-nucleotide biases for 31 RNA viruses.

Click here for file

Tetra-nucleotide bias

Inspection of the tetra-nucleotide usage pattern for RNA viruses (additional file 2) reveals considerable differences. The frequencies of occurrence for tetra-nucleotides were compared to artificial chromosomes constructed as random RNA sequences having the same nucleotide succession up to order three to compute the z values that reflect their tetra-nucleotide bias in the corresponding virus (Table 5). If we choose a critical value for z (|z| = 3.29) that only allows a chance of 1 in 1000 error for classifying a word as over/under-represented, 96% of the tetra-nucleotides show a strong bias in their usage pattern across 31 viruses (shown in Table 4, derived from the complete form of Table 5 provided as the additional file 1). This indicated strongly that tetra-nucleotides are being used in a different manner between different viruses, providing us with a tool to study the relationships between viruses based on the tetra-nucleotide bias exhibited in their genomes.

Additional File 2

Vectorial profiling of tetra-nucleotide usage pattern in seven RNA viruses. The tetra-nucleotide frequencies of occurrence in seven viral genomes. Each column represents a tetra-nucleotide usage pattern V_ifor a single virus. We derived correlation coefficient (r) by comparing any two columns simultaneously. This parameter r indicates the likeness of word usage patterns in any two viruses.

Click here for file

Table 5

Tetra-nucleotide bias for three RNA viruses. The tetra-nucleotide bias in three viruses. z value quantifies the tetra-nucleotide bias, as defined in equation (1). N (w) and E (w) are actual and expected frequency of occurrence for word w.

BCoV

MHV

SARS

BCoV

MHV

SARS

Tetra-nucleotide

N(w)

E(w)

N(w)

E(w)

N(w)

E(w)

Tetra-nucleotide

N(w)

E(w)

N(w)

E(w)

N(w)

E(w)

AAAA

148

206.2

-7.4

147

145.8

0.2

222

216.5

0.7

UAAA

264

226.2

4.6

187

170.9

2.2

170

183

-1.7

AAAC

110

103.7

1.1

91.2

1.3

154

148.1

0.9

UAAC

105.1

-4.8

122.4

-6.1

123

128

-0.8

AAAG

184

169.7

2.0

173

133.6

6.2

165

158.8

0.9

UAAG

205

171.7

4.6

193

165.3

3.9

107

134.4

-4.3

AAAU

217

220

-0.4

179

164.9

2.0

213

200.6

1.6

UAAU

322

309.9

1.3

245

259.8

-1.7

166

193.9

-3.6

AACA

133

114.1

3.2

113

112.1

0.2

215

175.7

5.4

UACA

178

163.7

2.0

122

123.4

-0.2

230

200.3

3.8

AACC

61.3

3.4

107

75.2

6.7

102

92.5

1.8

UACC

82.9

2.8

106

98.5

1.4

118

97.1

3.8

AACG

40.7

-3.3

61.8

-6.2

66.3

-5.0

UACG

54.4

-1.1

72.6

-3.1

63.3

-3.9

AACU

121.5

-5.0

106.5

-4.0

171

168.5

0.4

UACU

196

205.2

-1.2

153

168.2

-2.1

195

192.2

0.4

AAGA

172

157.9

2.0

176

136.4

6.2

184

161.8

3.2

UAGA

128

123.4

0.8

119

124.7

-0.9

102

119.6

-2.9

AAGC

137

103.8

5.9

140

103

6.6

112.8

-2.9

UAGC

98.7

-3.6

118.7

-6.1

84.8

-2.7

AAGG

133

121.3

1.9

159

122.4

6.0

140

117.3

3.8

UAGG

78.8

-1.2

121.3

-9.0

75.6

-0.3

AAGU

191

180.6

1.4

179

163.1

2.3

136

139.4

-0.5

UAGU

171

213

-5.2

161

190.7

-3.9

101

126.8

-4.2

AAUA

189

215.2

-3.2

148

182.1

-4.6

113

154.1

-6.0

UAUA

251

237

1.7

192

189

0.4

136.5

-5.8

AAUC

100

104.5

-0.8

93.2

-3.4

121.9

-4.8

UAUC

112.3

-4.9

99.9

-2.5

116.1

-5.4

AAUG

246

229.3

2.0

234

232.1

0.2

230

201.5

3.7

UAUG

310

271.5

4.3

278

238.1

4.7

189

190.3

-0.2

AAUU

265

265.5

-0.1

212

207.8

0.5

211

212

-0.1

UAUU

314

345

-3.0

253

248.5

0.5

190

211.8

-2.7

ACAA

144

137.1

1.1

115

114.1

0.2

269

204.1

8.3

UCAA

131

130

0.2

136

117.7

3.1

202

174.1

3.8

ACAC

66.4

3.9

75.4

2.6

168

142.2

3.9

UCAC

60.4

-1.7

67.1

-2.2

130

109.6

3.5

ACAG

118

105.7

2.2

108

104.9

0.5

151

145.1

0.9

UCAG

107

122.1

-2.5

105

106.7

-0.3

110

121.4

-1.9

ACAU

128

123.5

0.7

106

122.7

-2.7

186

172.9

1.8

UCAU

124.6

-6.6

117.8

-5.0

153

146.7

0.9

ACCA

105

76.9

5.8

116

6.1

161

117.7

7.3

UCCA

73.4

-1.1

80.6

-1.3

95.4

-3.6

ACCC

37.3

5.6

57.6

6.3

60.7

-1.6

UCCC

37.4

-1.9

-2.7

44.6

-3.7

ACCG

35.5

-3.5

57.4

-1.3

48.8

-4.6

UCCG

-3.9

55.1

-4.4

29.7

-3.5

ACCU

77.7

1.1

97.7

-0.1

139

111.2

4.8

UCCU

101.4

-4.9

103

102.4

0.1

107.1

-4.8

ACGA

44.5

-3.4

56.5

-6.2

64.4

-5.5

UCGA

41.4

-6.6

50.4

-2.2

67.5

-5.4

ACGC

34.1

-1.6

52.5

-0.9

54.8

-5.8

UCGC

34.1

-1.3

-2.2

56.2

-4.4

ACGG

31.3

-1.7

48.5

-0.7

-4.2

UCGG

29.1

-3.4

49.8

-4.3

39.9

-6.9

ACGU

60.5

-3.1

72.1

-4.1

72.6

-4.2

UCGU

74.1

-4.9

74.8

-3.3

84.4

-2.3

ACUA

141

127.9

2.1

119

121.3

-0.4

166

167.3

-0.2

UCUA

116

130.2

-2.3

115

124.5

-1.5

130

140.9

-1.7

ACUC

68.2

-4.2

68.9

-1.7

119

115

0.7

UCUC

67.1

-3.3

69.6

-0.1

108.4

-4.6

ACUG

144

131.5

2.0

126

141.4

-2.4

159

163.3

-0.6

UCUG

119

135.5

-2.6

117

135.5

-2.9

133

141.4

-1.3

ACUU

142

160.9

-2.7

116

132.1

-2.5

207

184.9

3.0

UCUU

195

191.8

0.4

153

142.8

1.6

219

182.6

4.9

AGAA

147

141.6

0.8

162

126.6

5.7

144

158.7

-2.1

UGAA

174

195.3

-2.8

154

176.8

-3.1

164

180.2

-2.2

AGAC

71.9

-1.1

80.6

1.3

114

117.2

-0.5

UGAC

101.8

-2.8

118

127.6

-1.5

153

151.9

0.2

AGAG

107

88.9

3.5

115

103.4

2.1

146

112.3

5.8

UGAG

127.7

-5.1

144

167

-3.2

117

136.2

-3.0

AGAU

177

170.4

0.9

158

145.2

1.9

128

141.1

-2.0

UGAU

314

311.7

0.2

243

261

-2.0

215

196.5

2.4

AGCA

113

105.8

1.3

102

112.1

-1.7

105

118.8

-2.3

UGCA

181

166.3

2.1

187

161.5

3.6

166

182

-2.2

AGCC

54.1

5.7

80.7

2.1

55.4

3.1

UGCC

102

81.6

4.1

144

122.4

3.6

114

81.6

6.5

AGCG

44.9

0.8

71.2

-2.0

46.4

-3.8

UGCG

65.5

-3.0

97.6

-2.1

58.5

-0.1

AGCU

126

122.6

0.6

132

0.0

140

146.9

-1.0

UGCU

270

218.4

6.4

254

226.8

3.3

315

224.5

11.0

AGGA

116

96.4

3.6

114

117.8

-0.6

138

99.5

7.0

UGGA

171

154.4

2.4

187

147.4

5.9

152

126

4.2

AGGC

61.9

0.7

114

104.6

1.7

80.3

2.4

UGGC

144

103.8

7.2

184

144.4

6.0

141

103.4

6.7

AGGG

59.1

-1.0

79.5

1.7

60.7

-1.8

UGGG

105

-4.3

118.9

-4.8

74.4

-3.2

AGGU

137

143.8

-1.0

128

150

-3.3

129

119.5

1.6

UGGU

307

302.3

0.5

260

236.3

2.8

200

173.5

3.7

AGUA

137

159.2

-3.2

124

155.6

-4.6

115

116.9

-0.3

UGUA

228

233.8

-0.7

202

215.5

-1.7

161

165.6

-0.6

AGUC

77.6

-3.2

93.9

-3.5

87.3

-2.2

UGUC

116

116.5

-0.1

159

143.6

2.3

141

129.8

1.8

AGUG

152

156.2

-0.6

187

173.8

1.8

127

120.7

1.0

UGUG

266

246.9

2.2

300

255.8

5.0

214

170.9

6.0

AGUU

222

239.6

-2.1

214

206

1.0

126

161.7

-5.1

UGUU

498

407.8

8.1

415

346.1

6.7

274

252

2.5

AUAA

228

220.7

0.9

189

188.5

0.1

129

152.4

-3.4

UUAA

322

269.8

5.8

258

235.5

2.7

195

202.8

-1.0

AUAC

124

129.2

-0.8

100

112.8

-2.2

100

132.3

-5.1

UUAC

185

173.5

1.6

158

155.6

0.3

186

179.8

0.8

AUAG

120

141.9

-3.3

120

135

-2.3

91.7

-5.1

UUAG

141

177.5

-5.0

131

183

-7.0

112

119.5

-1.2

AUAU

205

237.9

-3.9

151

185.9

-4.7

144.4

-6.9

UUAU

397

385.6

1.1

309

269.1

4.4

191

226.5

-4.3

AUCA

105

122

-2.8

99.2

-4.1

139

136.8

0.3

UUCA

127

155

-4.1

132

126.1

1.0

206

180

3.5

AUCC

62.9

-0.9

65.5

-0.6

65.9

-2.7

UUCC

80.1

-2.9

90.9

-3.0

87.9

-3.3

AUCG

46.9

-4.2

57.2

-3.7

59.6

-6.7

UUCG

55.3

-5.5

69.3

-1.2

71.8

-3.4

AUCU

108

129.1

-3.4

109.2

-3.9

108

137.1

-4.5

UUCU

193

201.6

-1.1

133

151.2

-2.7

226

189.5

4.8

AUGA

204

212.3

-1.0

203

202.8

0.0

189

198.6

-1.2

UUGA

237

239.3

-0.3

197

213.4

-2.0

189

186.8

0.3

AUGC

186

151.1

5.2

194

164.2

4.2

179

154.3

3.6

UUGC

197

174.1

3.2

188

184.4

0.5

185

162.5

3.2

AUGG

211

180.8

4.1

197

179.4

2.4

185

143.1

6.4

UUGG

213

230.6

-2.1

208

185.6

3.0

153

143.2

1.5

AUGU

296

273.3

2.5

275

269.9

0.6

218

197.4

2.7

UUGU

415

363.3

4.9

368

298

7.4

245

204.3

5.2

AUUA

239

253.9

-1.7

191

216

-3.1

190

192

-0.3

UUUA

407

345.3

6.0

303

257.1

5.2

204

204.1

0.0

AUUC

106

126.1

-3.3

100

110.2

-1.8

127

136.8

-1.5

UUUC

141

161.3

-2.9

109

146.1

-5.6

187

162.4

3.5

AUUG

245

253.5

-1.0

206

211.6

-0.7

208

176

4.4

UUUG

367

357.7

0.9

318

271.3

5.2

207

194.8

1.6

AUUU

361

337.8

2.3

287

251.6

4.1

197

205.6

-1.1

UUUU

454

495.8

-3.4

296

325

-2.9

215

245.2

-3.5

GAAA

118

124

-1.0

104

111.4

-1.3

142

140.8

0.2

CAAA

128

133.8

-0.9

160

108.5

9.0

221

182.3

5.2

GAAC

64.6

-1.5

75.1

-2.5

96.9

-1.5

CAAC

2.6

74.9

3.8

166

128.5

6.0

GAAG

136

125.8

1.7

153

123.2

4.9

126

117.7

1.4

CAAG

108

111

-0.5

135

111.5

4.0

158

132.5

4.0

GAAU

118

140.8

-3.5

119

142.4

-3.6

125.7

-5.8

CAAU

144

147.5

-0.5

126

139.1

-2.0

178

170.2

1.1

GACA

-0.2

1.5

162

128.5

5.4

CACA

78.3

0.8

80.6

0.7

168

150.9

2.5

GACC

39.2

-0.6

69.1

-1.8

68.5

-0.5

CACC

4.1

59.3

3.9

80.1

3.6

GACG

-1.9

-1.3

52.9

-5.0

CACG

31.5

-1.1

-1.6

50.4

-6.0

GACU

86.6

-0.9

94.7

-1.3

101

115.3

-2.4

CACU

108

75.3

6.8

93.1

0.7

184

151.4

4.8

GAGA

77.8

-1.0

104

100.3

0.7

105

111.8

-1.2

CAGA

125

106.4

3.3

123

98.1

4.6

141

128.8

2.0

GAGC

60.1

-1.9

90.9

-4.8

74.9

1.7

CAGC

71.9

5.2

82.8

3.2

96.6

-0.3

GAGG

68.6

1.0

112

108.6

0.6

89.1

1.1

CAGG

84.9

1.8

106

93.4

2.4

102

93.4

1.6

GAGU

103

100.9

0.4

128

134

-0.9

108

98.6

1.7

CAGU

108

128.7

-3.3

132

127.4

0.7

127.2

-4.7

GAUA

149

172.1

-3.2

127

145.4

-2.8

111

-5.2

CAUA

110.8

-3.9

112.5

-3.5

118.2

-3.2

GAUC

86.1

-3.2

-2.1

75.7

-4.3

CAUC

56.5

-1.8

67.1

-4.9

100

91.9

1.5

GAUG

231

209.7

2.7

237

199.5

4.8

198

159

5.6

CAUG

110

117.8

-1.3

119

143.9

-3.8

153

138.3

2.3

GAUU

205

201.7

0.4

159

176.8

-2.4

125

128.6

-0.6

CAUU

166

149

2.5

160

159

0.1

196

173

3.2

GCAA

104

114.7

-1.8

137

123.3

2.2

133

131

0.3

CCAA

81.4

0.5

126

90.8

6.7

119

107.2

2.1

GCAC

65.3

1.1

74.2

0.6

1.3

CCAC

41.7

8.2

55.9

4.4

77.7

0.7

GCAG

131

102.4

5.1

157

113.3

7.5

100.5

-3.7

CCAG

64.5

0.6

80.7

1.9

77.5

3.6

GCAU

120

109.4

1.8

128

140.9

-2.0

112

104.2

1.4

CCAU

71.9

1.9

97.4

-0.6

1.1

GCCA

57.8

6.2

111

97.4

2.5

76.5

4.7

CCCA

41.6

1.2

62.7

4.7

60.1

-1.0

GCCC

34.5

-0.2

63.8

2.5

36.6

-0.5

CCCC

22.2

2.2

47.5

-1.2

28.8

-3.6

GCCG

29.7

-0.2

60.5

-2.2

31.7

-3.4

CCCG

20.4

-1.4

39.4

1.6

20.1

-1.6

GCCU

66.8

3.8

122

106.2

2.8

71.9

0.7

CCCU

43.9

3.8

-0.4

60.7

-3.0

GCGA

38.9

-2.6

57.3

-3.7

43.5

-2.1

CCGA

27.9

-1.0

47.1

-0.6

36.2

-6.0

GCGC

31.7

-0.2

57.4

1.8

-0.8

CCGC

21.8

-0.7

0.8

32.1

-3.6

GCGG

31.7

-3.4

56.8

-3.3

29.6

-2.2

CCGG

20.9

-3.9

44.9

-2.4

21.2

-3.2

GCGU

55.9

1.7

82.1

1.0

52.9

-1.5

CCGU

38.2

-2.7

68.1

-3.1

41.8

-1.3

GCUA

165

131.3

5.4

162

144.3

2.7

153

140.7

1.9

CCUA

1.7

96.5

-2.5

104

88.6

3.0

GCUC

58.8

-0.2

80.5

-1.1

98.1

-1.7

CCUC

40.1

-0.6

58.6

4.8

65.2

-0.5

GCUG

136

131.5

0.7

187

173.4

1.9

196

145.3

7.6

CCUG

80.4

1.7

118

108.1

1.7

-3.7

GCUU

167

147.3

3.0

158

162.5

-0.6

180

149.5

4.5

CCUU

97.4

-2.1

119

113.3

1.0

105

104.6

0.1

GGAA

82.1

0.8

103.4

-3.7

103

3.3

CGAA

42.7

-5.5

58.5

-4.4

55.5

-4.5

GGAC

48.4

0.7

67.7

-2.4

72.3

-0.9

CGAC

22.5

0.6

34.3

-0.7

46.9

-5.3

GGAG

66.6

3.2

109

95.9

2.4

70.1

4.8

CGAG

29.5

-4.2

53.5

-2.9

49.8

-3.8

GGAU

122

127

-0.8

139

124.7

2.3

83.7

-0.7

CGAU

63.1

-5.0

63.3

-4.0

-4.9

GGCA

5.0

142

99.4

7.7

108

83.7

4.8

CGCA

40.7

-0.8

63.3

0.8

58.2

-2.9

GGCC

33.7

0.1

74.8

-0.2

39.1

-1.8

CGCC

17.1

0.8

45.6

1.2

27.1

-4.2

GGCG

32.2

-1.3

62.9

-1.4

40.5

-2.1

CGCG

14.9

1.0

39.2

-2.1

23.4

-0.9

GGCU

88.7

1.2

135

117.9

2.9

115

94.9

3.8

CGCU

44.4

-2.3

73.5

-2.7

74.1

-5.9

GGGA

-3.7

65.7

-3.1

48.5

-3.3

CGGA

26.4

-4.0

-4.1

33.8

-4.9

GGGC

37.4

-5.1

68.8

-1.1

38.3

-0.7

CGGC

0.8

47.2

-0.6

29.3

-3.1

GGGG

41.9

-4.5

53.8

-7.6

31.4

-3.7

CGGG

19.9

-4.0

39.1

-3.5

17.5

-2.4

GGGU

-1.3

100.4

-2.2

63.8

-2.7

CGGU

50.2

-4.9

67.2

-3.4

55.5

-6.7

GGUA

147

153.8

-1.0

113

130.8

-2.8

106

102.8

0.6

CGUA

53.6

0.3

71.6

-4.2

60.2

-1.9

GGUC

70.4

-4.2

76.8

-3.3

71.3

-6.7

CGUC

24.9

-3.2

41.9

-3.6

41.9

-1.6

GGUG

160

161.8

-0.3

179

171.9

1.0

135

119.9

2.5

CGUG

64.9

-1.1

90.6

-1.3

71.3

-0.5

GGUU

205

201.3

0.5

175

181.2

-0.8

127

123.2

0.6

CGUU

83.2

-4.8

104.4

-2.9

81.6

-5.8

GUAA

165

174.4

-1.3

135

160.2

-3.6

101

130.5

-4.7

CUAA

154

145.5

1.3

128

140

-1.8

141

153.8

-1.9

GUAC

109.2

-1.8

110.2

-4.2

143

109

5.9

CUAC

112

88.1

4.6

87.5

1.5

160

140.2

3.0

GUAG

112

118.4

-1.1

104

136.9

-5.1

88.6

1.4

CUAG

86.6

-1.7

103.6

-5.3

99.2

-4.4

GUAU

191

195.4

-0.6

166

172

-0.8

118.6

-4.1

CUAU

163

148.3

2.2

182

150.6

4.7

177

162

2.1

GUCA

95.2

-1.9

105

98.5

1.2

114

113.3

0.1

CUCA

-3.4

82.5

-1.9

137

130.2

1.1

GUCC

52.2

-5.6

73.4

-3.1

52.7

-4.4

CUCC

-2.3

58.7

0.8

62.3

-3.7

GUCG

39.6

-1.9

-5.7

51.3

-2.9

CUCG

-3.5

-1.9

59.9

-4.0

GUCU

109.1

-2.1

125

120.1

0.8

104

112.9

-1.5

CUCU

0.4

110

94.8

2.8

126

136.9

-1.7

GUGA

122

162.3

-5.8

152

163.3

-1.6

131

127

0.6

CUGA

107

124.3

-2.8

108

139.4

-4.8

141

150.6

-1.4

GUGC

113

115.2

-0.4

149

148.5

0.1

130

110.9

3.3

CUGC

109

91.9

3.2

141

110.9

5.2

159

128.2

4.9

GUGG

158

146.3

1.8

180

158.8

3.1

109

101

1.4

CUGG

121

98.1

4.2

136

123.2

2.1

106

104.8

0.2

GUGU

245

218.3

3.3

269

223.8

5.5

174

129.3

7.2

CUGU

151

157.5

-0.9

164

179.7

-2.1

152

159.3

-1.1

GUUA

255

244.6

1.2

237

225.1

1.4

126

168.7

-6.0

CUUA

143

152.3

-1.4

125

150.3

-3.8

164

163.1

0.1

GUUC

104

123

-3.1

119

116.6

0.4

114.8

-3.0

CUUC

80.6

-2.5

81.3

-1.1

148

124.3

3.9

GUUG

280

254.7

2.9

283

248

4.0

165

169.1

-0.6

CUUG

168

147.9

3.0

154

152.9

0.2

190

154.7

5.2

GUUU

344

316.4

2.8

253

239.5

1.6

192

171

2.9

CUUU

211

212.4

-0.2

191

177.1

1.9

209

183.8

3.4

Approach one – Sequence Relationship of Viruses based on The Correlation of Tetra-nucleotide Bias

Two relationship trees were derived, one from the entire genome and the other from the replication enzyme (Figure 1). The result based on the replication enzyme sequence was included because these regions in RNA viruses are submitted to a strong selective pressure to ensure successful replication of their own RNA in the host cell. The two distance trees can be clustered distinctly into two major groups of viruses. Interestingly, this clustering validates our approach, since these clusters are consistent with biological properties of the viruses: Group #1 corresponds to all positive strand ssRNA viruses while Group #2 corresponds to negative strand ssRNA viruses. Each group must undergo different evolutionary paths which lead to their distinct pattern in tetra-nucleotide usage. The classification for the two main groups of viruses (positive/negative strand ssRNA viruses) demonstrate a level of congruence with the taxonomy of the viruses 20 and indicated that there exists a relationship signal in tetra-nucleotide usage patterns.

Figure 1

Two Relationship trees based on the correlation coefficients of tetra-nucleotide usage bias

Two Relationship trees based on the correlation coefficients of tetra-nucleotide usage bias The distance tree for 31 RNA viruses based on tetra-nucleotide usage pattern for the entire genome (right) and the replication enzyme (left). The correlation distances are shown on top of each branch.

Inside both relationship trees, Avian Encephalomyelitis Virus (AEV), Lactate Dehydrogenase-elevating Virus (LDV), Porcine Reproductive and respiratory syndrome Virus (PRV), Equine arteritis Virus (EV1), Rabbit Hemorrhagic disease Virus (RHV), Yellow Fever Virus (YFV), are the outermost group of viruses, exhibiting differences in their tetra-nucleotide usage pattern. From the family of positive strand ssRNA viruses, CoronaViruses form the largest cluster. The SARS-CoV is found to be at the basal position of other CoronaVirus types and remains closest to the Transmissible Gastroenteritis Virus (TGV) and Feline CoronaVirus (FCoV). This placement is consistent with the findings from two seminal papers 910 where the SARS-CoV was classified in a separate group from the rest of the known CoronaViruses. In addition, both distance trees suggested that the Bovine CoronaVirus (BCoV) and the Mouse Hepatitis Virus (MHV) should be grouped together whereas the Human CoronaVirus 229E (HCoV) is the closest to the Porcine epidemic Diarrhea Virus (PDV). For the family of negative strand ssRNA viruses, there are two obvious classes that have evolved through different branches of word usage pattern. The first class covers Hantaan Virus (HV1), Reston Ebola Virus (REV), Bovine Ephemeral Fever Virus (BFV), Bovine Respiratory syncytial Virus (BRV), Respiratory Syncytial Virus (RSV) and Human Respiratory syncytial Virus (HRV). The second class covers the remaining negative strand ssRNA viruses.

Approach two – Sequence Relationship of Viruses based on The Factors of the Tetra-nucleotide Usage Pattern 212223

The overall tetra-nucleotide usage pattern (additional file 2) was decomposed into several eigen-vectors using a factor analysis algorithm. They are the uncorrelated components of the original usage pattern embedded within the overall tetra-nucleotide usage pattern. Three eigen-vectors, which carry 83.3% of the variance for the viral tetra-nucleotide usage patterns, were retained (Figure 2). From the three dimensional figures (Figure 3, Figure 4, Figure 5 and Figure 6) plotted against these retained eigen-vectors, the negative strand ssRNA viruses stemmed clearly out from the positive strand ssRNA viruses. This is most obvious when the axes of projection were the 1^stand 3^rdeigen-vectors. This indicated that both types of viruses have a complex component of tetra-nucleotide usage patterns and that these patterns changes with different family of viruses.

Figure 2

Relationship between the number of eigen-vectors retained and the percentage of the variance they represent in the entire usage patterns for 31 viruses

Relationship between the number of eigen-vectors retained and the percentage of the variance they represent in the entire usage patterns for 31 viruses. As each consecutive factor is defined to identify a usage pattern that is not captured by the preceding eigen-vectors, each consecutive factors are therefore independent of each other. In addition, the order for the consecutive eigen-vectors is extracted with diminishing importance.

Figure 3

3-D plot for the vectorial profiling of each virus onto the three eigen-vectors

3-D plot for the vectorial profiling of each virus onto the three eigen-vectors. The tetra-nucleotide usage patterns V for the replicase open reading frame in each virus have been redisplayed on the 1^st, 2^ndand 3^rdeigen-vector axes ('o' represents positive strand ssRNA virus; x represents negative strand ssRNA virus). The two families of viruses clustered into two different regions of the plot.

Figure 4

2-D plots for Figure 3 with different viewpoint specifications

2-D plots for Figure 3 with different viewpoint specifications. The tetra-nucleotide usage patterns for the replicase open reading frame in each virus have been redisplayed on the (1^stvs 2^nd), (1^stvs 3^rd) and (2^ndvs 3^rd) eigen-vector axes ('o' represents positive strand ssRNA virus; 'x' represents negative strand ssRNA virus). For the top figure, the order for 'o' is [15,17,12,16,8,14,9,11,13,4,7,3,10,6,2,5,1]* (left to right), whereas 'x' is [24,27,25,28,22,26,18,23,20,19,21]* (left to right). For the middle figure, the order for 'o' is [15,17,12,16,8,14,9,11,13,4,7,3,10,6,2,5,1]* (left to right), whereas 'x' is [24,27,25,28,22,26,18,23,20,19,21]* (left to right). For the bottom figure, the order for 'o' is [15,17,12,16,8,14,9,11,13,4,7,3,10,6,2,5,1]* (left to right), whereas 'x' is [24,27,25,28,22,26,18,23,20,19,21]* (left to right). *The corresponded virus for each number follows Figure 3.

Figure 5

3-D plot for the vectorial profiling of each virus onto the three eigen-vectors

3-D plot for the vectorial profiling of each virus onto the three eigen-vectors. The tetra-nucleotide usage patterns table in the additional file 2 (entire genome) for each virus have been redisplayed on the 1^st, 2^ndand 3^rdeigen-vector axes ('o' represents positive strand ssRNA virus; 'x' represents negative strand ssRNA virus). The two families of viruses clustered into three different regions of the plot.

Figure 6

2-D plots for Figure 5 with different viewpoint specifications

2-D plots for Figure 5 with different viewpoint specifications. The tetra-nucleotide usage patterns table in the additional file 2 (entire genome) for each virus have been redisplayed on the (1^stvs 2^nd), (1^stvs 3^rd) and (2^ndvs 3^rd) eigen-vector axes ('o' represents positive strand ssRNA virus, 'x' represents negative strand ssRNA virus). For the top figure, the order for 'o' is [3,7,1,4,2,5,6,17,13,20,10,16,9,8,11,15,12,14,18,19]* (left to right), whereas 'x' is [26,22,30,23,24,28,31,27,25,29,21]* (left to right). For the middle figure, the order for 'o' is [3,7,1,4,2,5,6,17,13,20,10,16,9,8,11,15,12,14,18,19]* (left to right), whereas 'x' is [26,22,30,23,24,28,31,27,25,29,21]* (left to right). For the bottom figure, the order for 'o' is [3,7,1,4,2,5,6,17,13,20,10,16,9,8,11,15,12,14,18,19]* (left to right), whereas 'x' is [26,22,30,23,24,28,31,27,25,29,21]* (left to right). *The corresponded virus for each number follows Figure 5.

In the result based on replication enzyme sequence (Figure 3 and Figure 4), we observed a clear splitting between two main families of RNA viruses (positive/negative strand ssRNA virus). All viruses that belong to a specific family were clustered together closely. This pointed to an interesting hypothesis that the replication enzyme sequence between closely related RNA viruses adopt a common word usage pattern that are closely linked. In addition, it is clear that the viruses from different family groups adopt different strategy of word usage.

However in Figure 5 and Figure 6, when we project the tetra-nucleotide usage patterns (entire genome) for each virus on the 1^st, 2^ndand 3^rdeigen-vector axes, the separation between viruses showed a different outcome when V was derived from the entire genome. The two main families of viruses were grouped into three clusters, two being allocated to the positive strand ssRNA viruses. It is particularly interesting that all viruses in the upper left corner corresponded to the viruses originating from the CoronaVirus family. Unexpectedly, the Hantaan Virus (HV1) is the only negative strand ssRNA virus to have a high loading on the eigen-vector that corresponded to the tetra-nucleotide usage pattern for the positive strand ssRNA viruses.

It is important to realize what factor analysis will provide and how this analysis is different from the previous method of relationship tree generation using correlation coefficient. For the previous method that is based on correlation coefficient of word usage patterns, it treats the vectorial profiling V for each virus as a whole entity, However, the factor analysis considered the vectorial profiling V as a superposition of many patterns which can be separated into mutually uncorrelated patterns of word usage. Each eigen-vector represents the embedded component of RNA word usage patterns communalised by a group of viruses presumably under the same selection pressures. By projecting the overall usage patterns on these eigen-vectors, it is possible to determine a group of viruses that adopt a common strategy of word usage.

Conclusion

Using the two approaches to study the tetra-nucleotide usage pattern in RNA viruses, we reached the following conclusions:

1. Based on the correlation of the overall tetra-nucleotide usage patterns, the Transmissible Gastroenteritis Virus (TGV) and the Feline CoronaVirus (FCoV) are closest to SARS-CoV.

2. Based on the three most significant eigen-vectors, the genomes of the viruses from the same family conform to a similar tetra-nucleotide usage pattern, irrespective of their genome size.

3. The study of word usage is a powerful method to classify RNA viruses. The congruence of the relationship trees with the known classification indicates that there exist phylogenetic signals in tetra-nucleotide usage patterns, and this signal is most prominent in the replicase open reading frames.

Methods

Dataset

We focused our study on the genomic sequences (their translated strand) of ssRNA viruses (Table 1), which incorporated 20 species from the family of positive strand ssRNA viruses and 11 species from the family of negative strand ssRNA viruses. We are aware of the fact that these viruses constitute completely different species, most probably unrelated to one another. They are included in a common study in order to try to have means to identify relevant features from purely statistical background properties. The coverage included the viruses that are known to cause diseases to their corresponding hosts. The acronym for each virus is shown in the table and is referred to throughout this study. All sequences corresponding to their translated strand were retrieved from GenBank, and the accession numbers and genomic size (in nucleotides) for individual virus were provided for reference. For the present study, two sets of data were generated from the complete sequence for each virus. Dataset 1 covered the entire genome and dataset 2 covered only their replicase open reading frame. The flowchart for studying the tetra-nucleotide usage pattern in 31 viruses is shown in Figure 7.

Figure 7

Flowchart for studying the tetra-nucleotide usage pattern

Flowchart for studying the tetra-nucleotide usage pattern. The FA and NJ algorithms stand for factor analysis 212223 and neighbor joining 29 algorithm.

Computer hardware and software

Sun Fire 6800 Server with 24 CPUs (each running with a clock speed of 900 MHz) was employed throughout this study. The computation of correlation coefficient and factor analysis algorithm were implemented using Matlab Technical Programming language.

Method for counting the frequency of occurrence for RNA words

It is necessary to address the question of how we counted the number of time each tetra-nucleotide (for example 'GAGA' or any other tetra-nucleotide), appeared in a given genome. For this study, we adopted the convention of not counting overlapping words 24. Take a sequence "UAUGAGAGAUCCGAGA' as example. With second or higher overlapping words not counted, the tetra-nucleotide 'GAGA' is counted as occurring only twice, namely in position 4–7 and 13–16. Positions 6–9 are omitted because they overlap with 'GAGA' at position 4–7.

However, when we counted tetra-nucleotide 'UGAG', position 3–6 would also be registered as position 4–6 already recorded when counting tetra-nucleotide 'GAGA'. In short, all frequency counting of tetra-nucleotide were started anew when we changed from counting the frequency of one tetra-nucleotide to another; this was to preserve the correlation of tetra-nucleotides which have overlapping subword (e.g: 'UAGA' and 'GACA'). A table showing the frequencies of tetra-nucleotides is shown in the additional file 2.

Vectorial profiling (V) of the viral RNA genome word usage pattern

The nucleotide composition has being suggested to be a specific characteristic in different virus phylogeny 25. Because most viral genomes are short, and because we lack a prior information on the tempo and modes of evolution of RNA viruses, we proceeded as follows. We created a vector, V = [C₁,C₂, ... C_i, ... C_k], with each element representing the frequency for a specific RNA word of length n. The number of components (k) in V increases exponentially with word size (n) - k = 4ⁿ. In order to use V for discrimination between viruses, two criteria must be met. First, V must contain sufficient components (di-nucleotide k = 16; tri-nucleotide k = 64; tetra-nucleotide k = 256); second, the frequencies for tetra-nucleotides must show a prominent bias (over/under-representation) that is unique for a family of viruses.

For the first criteria, there are pros and cons for choosing either longer or shorter words. When the shorter words are used, they inherit the problem of inadequate representation of the viral genome because the long motifs will be neglected. But the shorter words have an advantage of saving computational time. On the other hand, when the longer words are used, they cause a problem of computer tractability due to a larger word set to explore (k = 4ⁿ). However, the larger words have an advantage of accounting for the correlation of their sub-words. In contrast the number of their occurrences falls down rapidly, preventing accurate statistical analysis. We chose tetra-nucleotides for our study because they provide 256 vector components (additional file 2) and account for correlation of sub-words up to the order three.

For the second criteria, the bias in RNA word usage was examined. The bias in word usage (of size n) is influenced by the bias of word with sizes less than n 26. Therefore, in order to evaluate the true bias of word size m, it is required to compare the frequencies of word usage in the original sequence to that of model chromosomes that take into account the biases of word size m - 1, m - 2 ... 1. These model chromosomes were generated by obeying the Markov model of the order (m - 1)^th. This can be achieved by shuffling m - 1 viral nucleotides as one whole unit so that the nucleotide successions up to order (m - 1)^thwere being preserved. Several statistical approaches have been proposed for quantifying word biases 2728. In this study, we employed the z statistics (Equation 1) for di-nucleotide and tetra-nucleotide biases 2728. The z value is a measure of the bias of a word, with values close to zero meaning no bias, negative values meaning under-representation and positive values meaning over-representation of the word w in the RNA text.

where w is a word of size m; N(w) is observed count in actual viral RNA; E(w) and Var(w) are expected count and variance for w derived from the 100 artificial chromosomes that preserved the nucleotide succession up to order m - 1.

Approach one – sequence relationship of viruses based on the correlation of tetra-nucleotide bias

A scale-invariant parameter, the correlation coefficient r, was employed to compare between word usage patterns of viruses. The correlation coefficient r measures the degree of linear relationship between two vectors. Here, the two vectors are the tetra-nucleotide word usage pattern V corresponding to each viral genome. The magnitude of r would indicate how much of the change of pattern in the tetra-nucleotide word usage in one virus is explained by the change in another. The magnitude of r is always between -1 and +1 and the relationship between the two variables will approach perfect linearity as the magnitude of correlation coefficient approaches to extreme values (+/-1). However, perfect positive correlation (r = 1) does not mean identity of the paired V_i, but, rather, identity up to positive linearity, that is, identity between the paired standardized values. This is a crucial property of r (scale-invariant) that enables the comparison of viral genome despite their differences in genomic sizes. Positive magnitude of r indicates positive association whereas negative magnitude of r indicates negative association between two usage patterns. For this study, correlation coefficient, r, for let say virus 1 and virus 2, is defined as follow:

where V₁, V₂are vector representing the tetra-nucleotide usage pattern; S_V1and S_V2standard deviation of V₁, V₂; are the mean of V₁, V₂.

Then, the distance between the tetra-nucleotide usage patterns of two viruses is defined as follows:

Distance D_ij= 1 - r_ij; (3)

where D_ijis the distance between the tetra-nucleotide usage patterns of virus i and virus j; r_ijis the correlation coefficient between the tetra-nucleotide usage patterns of virus i and virus j

Prior to the construction of a relationship tree, the pair-wise distance matrix M of size 31 by 31 was constructed (see additional file 3). Pair-wise distance between two viral genomes is measured by the value of (1 - r). Each row/column corresponds to a specific virus and an entry at the intersection of row X and column Y corresponds to the distance between virus X and virus Y. Such matrix has a diagonal entry of value 0. For the purpose of constructing a relationship tree, only the lower/upper triangular matrix of M is required. After obtaining lower/upper triangular matrix of M, the neighbor-joining method (NJ) algorithm was used to construct the relationship tree (Figure 1). The neighbor-joining method is based on minimum-distance principle. Details of the NJ algorithm are available in 29.

Additional File 3

The distance matrices. Each entry in matrix M is computed using Equation 3. The correlation coefficient (r) in equation 3 is obtained by comparing any two columns in the tetra-nucleotide usage patterns table in the additional file 2 simultaneously.

Click here for file

Approach two – sequence relationship of viruses based on the factors of the tetra-nucleotide usage pattern

The factor analysis is a statistical method that reveals simpler patterns within a complex set of tetra-nucleotide usage patterns V (additional file 2). It seeks to discover if the observed usage patterns can be explained in terms of a much smaller number of un-correlated pattern sets called factors (eigen-vectors). Suppose we take a simple case where there are 31 viruses each represented by two components (x,y) in vector V (x,y represent the frequencies of occurrence for two specific tetra-nucleotides). Then, in a scatter-plot we can think of the regression line as the original X-axis, rotated so that it approximates the regression line. This type of rotation maximize the variance of the variables (x,y) on the eigen-vector. The remaining variability around this the first eigen-vector was captured in the subsequent eigen-vectors. In this manner, consecutive eigen-vectors are extracted but with a diminishing importance. What each eigen-vector represents is the embedded RNA word usage patterns communalised by a group of viruses presumably under the same selection pressures.

We implemented the factor analysis algorithm 212223 in Matlab Technical Programming Language and computed a set of eigen-vectors. Then, the original usage pattern V was re-mapped for each virus onto the new coordinate system based on these derived eigen-vectors. The difference between approach two and approach one is discussed in the results and discussion section.

Authors' contributions

YLY participated in the design and performed the statistical analysis.

AD participated in the design and overall coordination of this study.

XWZ participated in the design of the study.

All authors read and approved the final manuscript.

Acknowledgements

Indispensable support was provided by the doctoral fellowship from The University of Hong Kong (HKU). We wish to thank the Hong Kong Innovation and Technology Fund for supporting work upstream of the present study, that made it possible at a time when the unexpected SARS outbreak reached Hong Kong. Finally, we wish to thank Dr Ralf Altmeyer for his critical interest for this work as he came at the head of the HKU-Pasteur Research Centre.

Cumulative Number of Reported Probable Cases of SARS http://www.who.int/csr/sarscountry/en/ Aetiology: Koch's postulates fulfilled for SARS virus Fouchier RA Kuiken T Schutten M Van Amerongen G Van Doornum GJ Van Den Hoogen BG Peiris M Lim W Stohr K Osterhaus AD Nature 2003 423 6937 240 12748632 10.1038/423240a SARS update Hoey J Maskalyk J CMAJ 2003 168 10 1294 5 154192 12743077 SARS Web information James JS AIDS Treat News 2003 390 6 Situation Updates – SARS http://www.who.int/csr/sars/archive/en/ Identification of a Novel CoronaVirus in Patients with Severe Acute Respiratory Syndrome Drosten C Gunther S Preiser W Van Der Werf S Brodt HR Becker S Rabenau H Panning M Kolesnikova L Fouchier RA Berger A Burguiere AM Cinatl J Eickmann M Escriou N Grywna K Kramme S Manuguerra JC Muller S Rickerts V Sturmer M Vieth S Klenk HD Osterhaus AD Schmitz H Doerr HW N Engl J Med 2003 348 20 1967 76 12690091 10.1056/NEJMoa030747 High frequency RNA recombination in porcine reproductive and respiratory syndrome virus occurs preferentially between parental sequences with high similarity Van Vugt JJ Storgaard T Oleksiewicz MB Botner A J Gen Virol 2001 82 Pt 11 2615 20 11602772 Increased mutation frequency of feline immunodeficiency virus lacking functional deoxyuridine-triphosphatase Lerner DL Wagaman PC Phillips TR Prospero-Garcia O Henriksen SJ Fox HS Bloom FE Elder JH Proc Natl Acad Sci USA 92 16 7480 4 1995 Aug 1 41363 7638216 The Genome Sequence of the SARS-Associated CoronaVirus Marra MA Jones SJ Astell CR Holt RA Brooks-Wilson A Butterfield YS Khattra J Asano JK Barber SA Chan SY Cloutier A Coughlin SM Freeman D Girn N Griffith OL Leach SR Mayo M McDonald H Montgomery SB Pandoh PK Petrescu AS Robertson AG Schein JE Siddiqui A Smailus DE Stott JM Yang GS Plummer F Andonov A Artsob H Bastien N BeRNArd K Booth TF Bowness D Drebot M FeRNAndo L Flick R Garbutt M Gray M Grolla A Jones S Feldmann H Meyers A Kabani A Li Y Normand S Stroher U Tipples GA Tyler S Vogrig R Ward D Watson B Brunham RC Krajden M Petric M Skowronski DM Upton C Roper RL Science 2003 300 5624 1399 404 12730501 10.1126/science.1085953 Characterization of a Novel CoronaVirus Associated with Severe Acute Respiratory Syndrome Rota PA Oberste MS Monroe SS Nix WA Campagnoli R Icenogle JP Penaranda S Bankamp B Maher K Chen MH Tong S Tamin A Lowe L Frace M DeRisi JL Chen Q Wang D Erdman DD Peret TC Burns C Ksiazek TG Rollin PE Sanchez A Liffick S Holloway B Limor J McCaustland K Olsen-Rassmussen M Fouchier R Gunther S Osterhaus AD Drosten C Pallansch MA Anderson LJ Bellini WJ Science 2003 300 5624 1394 9 12730500 10.1126/science.1085952 A Novel CoronaVirus Associated with Severe Acute Respiratory Syndrome Ksiazek TG Erdman D Goldsmith CS Zaki SR Peret T Emery S Tong S Urbani C Comer JA Lim W Rollin PE Dowell SF Ling AE Humphrey CD Shieh WJ Guarner J Paddock CD Rota P Fields B DeRisi J Yang JY Cox N Hughes JM LeDuc JW Bellini WJ Anderson LJ N Engl J Med 2003 348 20 1953 66 12690092 10.1056/NEJMoa030781 Ecological fitness, genomic islands and bacterial pathogenicity. A Darwinian view of the evolution of microbes Hacker J Carniel E EMBO Rep 2001 2 5 376 81 11375927 The tendency of lentiviral open reading frames to become A-rich: constraints imposed by viral genome organization and cellular tRNA availability van Hemert FJ Berkhout B J Mol Evol 1995 41 2 132 40 7666442 Biological function of DNA methylation Hubacek J Folia Microbiol (Praha) 1992 37 5 323 9 1337330 Why is CpG suppressed in the genomes of virtually all small eukaryotic viruses but not in those of large eukaryotic viruses? Karlin S Doerfler W Cardon LR J Virol 1994 68 5 2889 97 8151759 Specific RNA methylase associated with avian myeloblastosis virus Gantt RR Stromberg KJ Montes de Oca F Nature 1971 234 35 37 4331547 A sensitive genetic assay for the detection of cytosine deamination: determination of rate constants and the activation energy Frederico LA Kunkel TA Shaw BR Biochemistry 2001 29 10 2532 7 The nonenzymatic hydrolysis of oligoribonucleotides. VII. Structural elements affecting hydrolysis Bibillo A Figlerowicz M Ziomek K Kierzek R Nucleosides Nucleotides Nucleic Acids 2000 19 5–6 977 94 10893716 Evolution of the genome and the genetic code: selection at the dinucleotide level by methylation and polyribonucleotide cleavage Beutler E Gelbart T Han JH Koziol JA Beutler B Proc Natl Acad Sci USA 1989 86 1 192 6 2463621 Regenmortel MHV van Fauquet CM Bishop DHL Carstens EB Estes MK Lemon SM Maniloff J Mayo MA McGeoch DJ Pringle CR Wickner RB Virus Taxonomy: Classification and Nomenclature of Viruses. Seventh Report of the International Committee on Taxonomy of Viruses Academic Press, San Diego 2000 Factor Analysis for Categorical Data, Journal of the Royal Statistical Society. Series B (Methodological) Bartholomew DJ 1980 42 3 293 321 Kim J Mueller Charles W Introduction to factor analysis: What it is and how to do it Newbury Park, CA: Sage Publications 1978 Factor Analysis for Categorical Data, Journal of the Royal Statistical Society. Series B (Methodological) Bartholomew DJ 1980 42 3 293 321 Ewens WJ Grant GR Statistical Methods in Bioinformatics Springer-Verlag New York, Inc., New York 2001 Nucleotide composition as a driving force in the evolution of retroviruses Bronson EC Anderson JN J Mol Evol 1994 38 5 506 32 8028030 Oligo-nucleotide bias in Bacillus subtilis: general trends and taxonomic comparisons Rocha EP Viari A Danchin A Nucleic Acids Res 1998 26 12 2971 80 9611243 10.1093/nar/26.12.2971 147636 Over- and underrepresentation of short DNA words in herpesvirus genomes Leung MY Marsh GM Speed TP J Comput Biol 1996 3 3 345 60 8891954 Exceptional motifs in different Markov chain models for a statistical analysis of DNA sequences Schbath S Prum B de Turckheim E J Comput Biol 1995 2 3 417 37 8521272 The neighbor-joining method: A new method for reconstructing trees Saitou N Nei M Mol Biol and Evol 1987 4 406 425