**5.2 C-prM mutations**

The phylogenetic analysis of 433 base pair region (nucleotides 180—612) of the DEN-3 *CprM*  gene junction showed that sequences of Delhi isolates (2006 outbreak) were closely related to sequences from Guatemala (1998) and presented a nucleotide identity of 95.9—98.2% (mean 97.05%). On comparison of Delhi 2006 sequences with other Indian sequences from years 2003, 2004, and 2005, mean sequence divergence of 2.85%, 2.15%, and 1.6%, respectively, were observed (Kukreti, H. et al.,2008. Common amino acid mutations observed in 2006 DENV-3 sequences are given in table 3. Similar study performed on DEN-3 isolates of 2003-04 outbreaks in New Delhi, found them to be closely related and belonged to subtype III from Sri Lanka (Dash, P.K.et al.,2006). Moreover, Phylogenetic analysis of C/PrM/M region of

Genetic Diversity of Dengue Virus and

Associated Clinical Severity During Periodic Epidemics in South East Asia 103

with specific serotype, as viruses isolated from DHF patients fell at different locations on the

Using maximum likelihood and Bayesian approaches, phylogenetic analysis of Taiwan's indigenous DENV-3 isolated from 1994 and 1998 dengue/DHF epidemics were found to be of three different genotypes –I, II and III each associated with DEN-3 circulating in Indonesia, Thailand and Sri Lanka, respectively(King, C.C. et al.,2008). The authors of this study analyzed complete nucleotide sequence of DEN-3 for its mutation and its relation with regional evolution. The highest level of nucleotide sequence diversity, and the positive selection site was detected at position 178 of the NS1 gene.Although the authors have identified the NS 1 gene as the positive selection site and the envelope protein site for purifying selection pressure, however direct association of these changes with disease severity was not determined. Study from Bangkok Thailand performed sequence analysis on E/NS-1 region of Thai isolates to determine if viral strains from less severe DENV infections had distinct evolutionary nucleotide pattern then those with more severe form (Rico-Hesse R. et al.,1998). This study found that two distinct genotypes were identifiable from both DF and DHF cases, suggesting its evolution from common progenitor that

**year aa change (positions) Relation to** 

11 nucleotides (4.6% divergence) between Strain PUO-218-280. 22 nt or 9.2%

divergence PUO-218-

3' UTR trinucleotide change 297± 299 (two transversions and one

D80141

2311–2550)

transition)

substitutions

Table 3. Genetic Characteristics and relation to disease severity in patients with DEN-2

Variable secondary structures were detected

Srilanka 2003-06 239-nt (from positions

Thailand 1996-97 5' NCR homologus

Thailand 2006 approx 10-3

2003

**Disease severity**

No specific association with disease severity

Could not be ascertained

Trinucleotide change may alter the functional characteristic of Secondary structure

no apparent association

No clear association **Ref** 

C)

(Klungthong

(Kanakaratne et al)

(Mangada MNM)

(Zhang C)

(Zhou Y)

phylogenetic tree (Kukreti, H. et al.,2008;Dash, P.K.et al.,2006).

perhaps shares the potential to cause severe disease.

Thailand 1998

from 1980

**Geographical Origin** 

3'-UTR Thailand 1973 to

infections reports from South East Asian Region

**DENV-Protein** 

E/NS1 (77 DEN-2 virus strains studied)

E/NS1 junction Den 2

3' and 5' UTR Den 2

Den 2 E /C/NS2A


DEN-3 isolates from Pakistan (2004-05 outbreak isolates) also showed sequence homology with 2003-04 New Delhi outbreak strains suggesting that circulation of common isolates of DEN-3 subtype III in the region . There was no clear statistical association of disease severity

Table 2. Genetic Characteristics and relation to disease severity in patients with DEN-3 infections reports from South East Asian Region

DEN-3 isolates from Pakistan (2004-05 outbreak isolates) also showed sequence homology with 2003-04 New Delhi outbreak strains suggesting that circulation of common isolates of DEN-3 subtype III in the region . There was no clear statistical association of disease severity

(positions)

analysis= multiple genetic

variants (mutational positions not mentioned)

E140 (I-T) E127 (I-V)

analysis= multiple genetic

variants (mutational positions not mentioned)

CprM 121(A-A) CprM127 (I-P) CprM122 (G-G) CprM55 (A-L) CprM 128(V-G)

C-prM112(T-A )

PrM 57 (T-A)

Table 2. Genetic Characteristics and relation to disease severity in patients with DEN-3

(H-T) E172 (I-V) E479 (V-A)

Taiwan 2008 E301 (L to T) (King, C.C.

Relation to Disease severity

Could not be ascertained

Could not be ascertained

Distinct clade causing epidemic

2 distinct clades linked to mild (IIIA) and severe (IIIB) disease epidemics

No association of any particular variant with serious dengue

may be attributed to increased incidence of DHF & DSS in India

Delhi strain 2004

No association with disease severity could be determined

disease

C-prM Similar to New

outbreak

Ref

(Rico-Hesse R *et al*)

(Jarman RG)

et al.,2008)

(Islam, M.A.et al.,2006)

(Kanakaratne et al)

(Kukreti H)

(Dash PK)

Jamil B, et

(King CC)

al

year aa change

Thailand 2002 E124 (P-S) E132

Thailand 2008 Phylogenetic

Bangladesh 2005/6 E81 (I-T)

Srilanka 2003-06 Phylogenetic

India 2005/6 CprM88 (I-V)

2006

Taiwan 2008 CprM 55 (L-H)

C-prM India 2006 C-prM108(M-I)

Pakistan 2005-

infections reports from South East Asian Region

DENV-Protein

Envelope (E) Den 3

Envelope (E) Den 3

DEN 3 E region

Den 3 C-preM/E

CprM Den 3

CprM Den 3

DEN 3 prM region

Envelope (E) Den 3

Geographical Origin

with specific serotype, as viruses isolated from DHF patients fell at different locations on the phylogenetic tree (Kukreti, H. et al.,2008;Dash, P.K.et al.,2006).

Using maximum likelihood and Bayesian approaches, phylogenetic analysis of Taiwan's indigenous DENV-3 isolated from 1994 and 1998 dengue/DHF epidemics were found to be of three different genotypes –I, II and III each associated with DEN-3 circulating in Indonesia, Thailand and Sri Lanka, respectively(King, C.C. et al.,2008). The authors of this study analyzed complete nucleotide sequence of DEN-3 for its mutation and its relation with regional evolution. The highest level of nucleotide sequence diversity, and the positive selection site was detected at position 178 of the NS1 gene.Although the authors have identified the NS 1 gene as the positive selection site and the envelope protein site for purifying selection pressure, however direct association of these changes with disease severity was not determined. Study from Bangkok Thailand performed sequence analysis on E/NS-1 region of Thai isolates to determine if viral strains from less severe DENV infections had distinct evolutionary nucleotide pattern then those with more severe form (Rico-Hesse R. et al.,1998). This study found that two distinct genotypes were identifiable from both DF and DHF cases, suggesting its evolution from common progenitor that perhaps shares the potential to cause severe disease.


Table 3. Genetic Characteristics and relation to disease severity in patients with DEN-2 infections reports from South East Asian Region

Genetic Diversity of Dengue Virus and

Associated Clinical Severity During Periodic Epidemics in South East Asia 105

deeply entrenched in many cities. The distribution of DHF outbreaks in SEA correlates with emergence of mosquito *A. egypti* in South East Asian countries due to uncontrolled

Phylogenetic analysis suggests that there are foci of virus extinction and selection in South East Asian region, one such region is Thailand where the indigenous DEN-3 virus circulating up to 1992 has disappeared and replaced by two new lineages perhaps from a common ancestor. These studies point towards potential of regular extinctions of strains of dengue virus particularly DEN-3 virus and replacement by new variants in the region. Natural selection and / or genetic bottle neck are plausible causes for this variation. Since the extinction of pre 1992 strains and appearance of new epidemic strain in Thailand occurred during inter-epidemic period we therefore hypothesize that the genetic bottleneck is perhaps major cause of regional replacement. This is further supported by studies from India reporting shifting and dominance of the dengue virus serotype-3 (subtype III) replacing the earlier circulating serotype-2 (subtype IV) with emergence of increased incidence of DHF and DSS in subsequent outbreaks. Strains from the 2005 outbreak in Karachi (Pakistan) were found to be similar to those from Indian strains of dengue serotype

Despite the growing genomic data base in the gene bank there are fundamental gaps in our understanding of epidemiological and evolutionary dynamics and its relation with disease severity. There are two possibilities that explain the association between clade replacement and increased viral virulence. The first is the possibility of these viruses to be better fit and therefore produce high viremia in infected humans, consequently with better transmission of virus by the vector. The other hypothesis to explain the possible virulence of emerging clades in the region is its improved ability to avoid neutralization by serotypes cross reactive antibodies (Kochel et al., 2005). Thus there is relative abundance of different serotypes and viral linage is continually changing in South East Asia. In face changing threshold of host immunity, periodic epidemics of DHF and DSS is due to local extinction and emergence of new clades. Over the period 1989 and 2000, a new genotype of DENV-1 and new clades of DENV-3 genotype III viruses have replaced older genotype and clades in this region and

Thus South East Asia displays greatest degree of genetic diversity, suggesting that it is the hub for the evolution of new epidemic strain. However, selection of specific clade and association of specific sequence variation with disease severity at various genomic levels reported in the literature reviewed in this study lacks strength of association i.e. reporting Relative Risk (RR)/ Odds Ratio (OR) limits our interpretation regarding causality or pin pointing specific clade with virus virulence, and therefore further studies are recommended.

This work was supported by University Research Council of the Aga Khan University, special thanks are extended to Mr. Faisal Malik for expert help in formatting of figures and tables

Aiken S R, Leigh C H, (1978),Dengue haemorrhagic fever in South East Asia.Transactions of the institute of British geaographers,New series ,Vol. 3, No. 4, (1978), pp.476-497

urbanization leading to displacement of indigenous *A. albpictus* from the region.

3, and were responsible for deadly outbreak in 2005-06.

emergence of new clades coincided with severe epidemics.

**7. Acknowledgment** 

**8. References** 
