title: Evolution and heterogeneity of multiple serotypes of Dengue virus in Pakistan, 2006-2011.
abstract: BACKGROUND
  Even though dengue has been recognized as one of the major public health threats in Pakistan, the understanding of its molecular epidemiology is still limited. The genotypic diversity of Dengue virus (DENV) serotypes involved in dengue outbreaks since 2005 in Pakistan is not well studied. Here, we investigated the origin, diversity, genetic relationships and geographic distribution of DENV to understand virus evolution during the recent expansion of dengue in Pakistan.
  
  
  METHODS
  The study included 200 sera obtained from dengue-suspected patients from 2006 to 2011. DENV infection was confirmed in 94 (47%) sera by a polymerase chain reaction assay. These included 36 (38.3%) DENV-2, 57 DENV-3 (60.6%) and 1 DENV-4 (1.1%) cases. Sequences of 13 whole genomes (6 DENV-2, 6 DENV-3 and 1 DENV-4) and 49 envelope genes (26 DENV-2, 22 DENV-3 and 1 DENV-4) were analysed to determine the origin, phylogeny, diversity and selection pressure during virus evolution.
  
  
  RESULTS
  DENV-2, DENV-3 and DENV-4 in Pakistan from 2006 to 2011 shared 98.5-99.6% nucleotide and 99.3-99.9% amino acid similarity with those circulated in the Indian subcontinent during the last decade. Nevertheless, Pakistan DENV-2 and DENV-3 strains formed distinct clades characterized by amino acid signatures of NS2A-I116T + NS5-K861R and NS3-K590R + NS5-S895L respectively. Each clade consisted of a heterogenous virus population that circulated in Southern (2006-2009) and Northern Pakistan (2011).
  
  
  CONCLUSIONS
  DENV-2, DENV-3 and DENV-4 that circulated during 2006-2011 are likely to have first introduced via the southern route of Pakistan. Both DENV-2 and DENV-3 have undergone in-situ evolution to generate heterogenous populations, possibly driven by sustained local DENV transmission during 2006-2011 periods. While both DENV-2 and DENV-3 continued to circulate in Southern Pakistan until 2009, DENV-2 has spread in a Northern direction to establish in Punjab Province, which experienced a massive dengue outbreak in 2011.
authors.affiliation: Environmental Health Institute, National Environment Agency, [as part of its work as a] WHO Collaborating Centre for Reference and Research of Arbovirus and their Associated Vectors, 11, Biopolis Way, #06-05-08, 138667 Singapore, Singapore. david_lee@nea.gov.sg.
authors.forename: Carmen
authors.initials: C
authors.lastname: Koo
authors.forename: Amna
authors.initials: A
authors.lastname: Nasir
authors.forename: Hapuarachchige Chanditha
authors.initials: HC
authors.lastname: Hapuarachchi
authors.forename: Kim-Sung
authors.initials: KS
authors.lastname: Lee
authors.forename: Zahra
authors.initials: Z
authors.lastname: Hasan
authors.forename: Lee-Ching
authors.initials: LC
authors.lastname: Ng
authors.forename: Erum
authors.initials: E
authors.lastname: Khan
mesh_terms: D016000:Cluster Analysis; D015331:Cohort Studies; D003715:Dengue / Q000453:epidemiology / Q000821:virology*; D003716:Dengue Virus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D014644:Genetic Variation; D006801:Humans; D017720:Molecular Epidemiology; D008969:Molecular Sequence Data; D010154:Pakistan / Q000453:epidemiology; D058974:Phylogeography; D012367:RNA, Viral / Q000235:genetics; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
chemical_list: D012367:RNA, Viral