**3. Distribution of dengue virus serotypes in SEA**

Dengue like other RNA viruses is prone to genetic mutations as it replicates using RNA-Polymerase; enzyme that lacks proof reading mechanism. The mutation rates in the order of 10-3has been reported for dengue (ElodieDes et al 2009) in different host settings. Such mutations often result in variants that become targets of selection; an outcome of underlying genotype and its environment. Despite these facts dengue virus do not evolve as fast as other RNA viruses. The only **macro evolutionary** divergence is perhaps the radiations in its four serotypes in its primate host (sylvatic strains) around one thousand years ago (ElodieDes

East Asia where mean age of cases under fifteen, and the modal age of five or slightly higher was reported from countries such as Thailand, Philippines and Malaysia, however, recent reports are now documenting increasing number of DHF and DSS in adult population as well (Khan E et al 2007). The precise cause of DHF/DSS remains elusive despite enormous research in this area. Evidences suggest interplay of multiple factors such as host genetic make-up with unique immune response and viral virulence may play a role in determining

There are two form of Severe disease, namely dengue shock syndrome (DSS) and DHF without shock. It is proposed that devastating coagulation derangements due to host immune response leads to heamorrhage and shock in severe cases. The concept of original antibody sin leading to immune enhancement is considered to be the main reason whereby infection with one type of dengue virus sensitizes an individual and that subsequent infection with different virus type elicits a hypersensitivity reaction (secondary infection). Various studies have been conducted to show the association of elevated cytokines in patients presenting with DHF and DSS. Elevated serum levels of cytokine and chemokines such as IL-2, IL-8, IL-6, IL-10, IL-13, TNF and INF-γ have been found to be significantly associated with patients presenting with DHF and DSS in clinical setting (Azeredo et al., 2001; Hung, et al., 2004, Clyde. K. et al., 2006). It has been proposed that the pro-inflammatory cytokines released by the cross reactive memory T-cellls, induce plasma leakage by its effects on the endothelial cells (Eva.H. et al 2004; Aviruntanan et al., 1998). In fact in-vitro studies have rendered endothelial cell monolayers permeable by the application of chemokine such as IL-1β (Cardier et al.,2005). In vitro-and in-vivo models of studies also suggest role of decreased nitric oxide levels and its relation with IL-10 and raised viral load (Simmons et al., 2007). There is evidence that suggests relation of increased expression of certain cytokines such as IL-1β,

With the advances in genomic and bioinformatics tools the scope of genetic studies has greatly expanded particularly in depth data on genomic changes and its association with disease epidemiology, seasonality and severity has been made available. Growing availability of comparative genome sequence data has provided important insights into the molecular evolution of dengue virus. Evidence strongly suggests appearance of new strains correlating with DHF/DSS epidemics. Despite the wealth of genomic data now available the exact cause and effect of viral virulence and clade changes is yet to be proven, however it is quite evident that different serotypes and viral linage is continually changing with local extinction and emergence of new clade and that the introduction of new clade in the region

Dengue like other RNA viruses is prone to genetic mutations as it replicates using RNA-Polymerase; enzyme that lacks proof reading mechanism. The mutation rates in the order of 10-3has been reported for dengue (ElodieDes et al 2009) in different host settings. Such mutations often result in variants that become targets of selection; an outcome of underlying genotype and its environment. Despite these facts dengue virus do not evolve as fast as other RNA viruses. The only **macro evolutionary** divergence is perhaps the radiations in its four serotypes in its primate host (sylvatic strains) around one thousand years ago (ElodieDes

the severity of the disease.

**2.4 Pathogenesis of severe dengue disease** 

TNF-γ, and IL-6 with elevated NO production (Guzik et al., 2003).

translates in form of outbreaks of DHF and DSS.

**3. Distribution of dengue virus serotypes in SEA** 

et al 2009). There after genetic mutation in the envelope protein and receptor binding domains resulted in its emergence as infectious pathogen in human population. The divergent forms of these sylvatic strains are often found to be circulating in human habitat, suggesting that enzootic cycles with some spill over in the surrounding human population. This has been shown in Malaysian populations settled near forest and marshy habitats (Wang, E. et al., 2000). The phylogenetic studies conducted based on envelope gene sequences of basal portion of sylvatic linage, DENV 1,-2,-4 of Malaysian descent suggest that endemic /epidemic strains of these viruses diverged from sylvatic ancestors more than 1000 years ago (Wang, E. et al., 2000). Thereafter, only micro evolutionary change within dengue serotypes have taken place, these changes have nevertheless resulted in substantial genetic diversity with emergence of endemic and epidemic strains in different parts of the region.

Fig. 1. The effects of climatic and social change on vector evolution and disease severity

Genetic Diversity of Dengue Virus and

with clinical severity of the disease.

**Author's name Why excluded?** 

east Asia)

U.S.A.

virus gene mutations

virus gene mutations

virus gene mutations

Does not give sequence analysis in detail

**4.1 Methods** 

**4.1.1 Literature search** 

Araujo J.M.G. *et al,* 

Lanciotti RS.*et al*,

Soundravally R, *et al* 

Soundravally R., *et* 

Soundravally R., *et* 

Gibbons RV. *et al,* 

Table 1. Features of the excluded studies

(2009)

(1994)

(2007)

*al* (2008 a)

*al,* (2008b)

(2007)

Associated Clinical Severity During Periodic Epidemics in South East Asia 97

available the exact cause and effect of viral virulence and clade changes is yet to be proven, however, viral linage is continually changing with local extinction and emergence of new clade. The introduction of new clade in the region translates in form of outbreaks of DHF and DSS. In order to analyze if there is a selection of specific clade in South East Asia that is circulating in the region and causing DHF outbreaks we conducted a meta-analysis. Studies conducted from 1950 to 2009 in South East Asian region that have investigated association of disease severity with specific sequence mutations in the dengue virus genome were retrieved. The objective was to analyze association of disease severity with the specific genomic mutation in the clade circulating and causing periodic epidemics in South East Asia. Since DENV-2 and DENV-3 are more common in this region our study was focused on these two genotypes only. Objectives of the metaanalysis were to identify association of specific genetic mutation in DENV-2 and DENV-3 with clinical severity seen during periodic epidemics in South East Asia. The specific review question was:Is clinical severity of dengue in the South East Asian region associated with emergence of specific mutations in genomes of DENV-2 and DENV-3 genotypes? We hypothesized that there is changing pattern of dengue virus genotypes in South East Asia and these mutations are associated

The literature search was performed from February 2010 to June 2010. Data sources include Medline via Pubmed (1950-February 2010), Cochrane data base of systematic reviews, Google scholar and experts in the field. Secondary references and review articles were scanned for thematic review. Hand search of the journal was also carried out. However, unpublished and ongoing studies could not be explored. Terminologies i.e. dengue type 1-4,

> Conducted in Brazil, (also include dengue strains from regions other than South east Asia) but country of origin of these isolates

> Conducted in USA, origin of isolates not clear (also include dengue strains from other geographical regions other than South

> The DEN-3 viruses used in this study were obtained from the collection at the Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colo.,

> Focused on host factors as cause of disease severity rather than on

Focused on host factors as cause of disease severity rather than on

Focused on host factors as cause of disease severity rather than on

was not clear, Sequences were selected from gene bank.

DEN-3 viruses have undergone independent evolution which has resulted in emergence of four genetic subtypes of which subtype I-III circulate in the South East Asian Region. Subtype I comprises of viruses from Indonesia, Malaysia and the Philippines; subtype II of viruses from Thailand and subtype III includes viruses from Sri Lanka India and Pakistan. The genetic evolution in these subtypes is primarily reported mutations in the prM/M and E structural protein genes. In spite of these mutations, the genomic region has retained greater than 95% amino acid sequence similarity (Lanciotti, R.S et al.,1994), suggesting that these are highly conserved regions responsible for protein architecture and / or biological function.

Phylogenetic studies suggest that there are regional foci of virus extinction and selection, one such region is Thailand where the indigenous DEN-3 virus circulating up to 1992 disappeared and was replaced by two new lineages perhaps from a common ancestor (Wittke, V. et al. 2002). The sequence of all Thai DEN-3 isolates recovered after 1992 had T at position 2370 in contrast to the C at this site in the pre-1992 samples(Wittke, V. et al. 2002), and nucleotides difference was observed in at least 45 sites of total 96 sites studied. It appears that the post-1992 strains have replaced the pre-1992 strains). These studies point towards potential of regular extinctions of strains of DEN-3 virus and replacement by new variants in the region (Wittke, V. et al. 2002). Natural selection and / or genetic bottle neck could be the 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 it is therefore hypothesized that the genetic bottleneck is perhaps the cause of regional replacement. This is further supported by studies from India reporting shift 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 (Dash, P.K.et al. 2006). Strains from the 2005 outbreak in Karachi (Pakistan) were found to be similar to those from Indian strains of dengue serotype 3, and were responsible for deadly outbreak in 2005-06 (Jamil. B. et al. 2007). Thus over the period 1989 and 2000, a new clades of DENV-3 genotype III viruses have replaced older genotype and clades in this region and emergence of new clades coincided with severe epidemics. The epidemiologic data suggests that the DEN-3 virus responsible for recent epidemic outbreaks in Mozambiques, Gutamealla, Pakistan and SriLanka may have been introduced from India, and changing age structure of dengue patients from 1996–2005 may also be indicative of the selected virus moving into new areas(Kanakaratne, N. et al.2009 ).
