MOLECULAR EPIDEMIOLOGY OF DENGUE VIRUS INFECTION AND FULL LENGTH CHARACTERIZATION OF PREVALENT GENOTYPES IN PAKISTAN

Abstract

Dengue fever is one of the important and most common mosquitoe borne diseases in the tropical and subtropical areas of the world. Since 1994, the dengue fever is appearing as a fatal health hazard in Pakistan. Two severe outbreaks of dengue were recorded during the years-2011 in Punjab Province and 2013 in Swat District, wherein hundreds of people died and 1000s more remained with the potential risk of the fetal fever. This thesis encompasses molecular biology of the major dengue outbreaks of Pakistan in general and the first hand information on the dengue outbreak of the year 2013 in Swat District. In order to characterize the dengue virus (DENV), blood samples from 1795 dengue patients were obtained and analyzed through RT-PCR, for its epidemiology, evolutionary history and prevalent genotypes/serotypes of the outbreaks. Phylogenetic analyses were carried out through MEGA and PhyML software while selection pressure was determined through SLAC, REL and FEL methods implemented in Datamonkey software. Whereas Recombination Event Detection was analysed through GARD method and nucleotide and amino acid identities were determined throgu Clustal Omega. Results of the study were based upon the characterization of 4 complete sequences of DENV-2, 7 NS3 genome sequences of DENV-2 and 10 each of the C, E and NS3 gene sequences, of DENV-3. The results revealed that the active dengue infection in Swat District was 30.5% with the prevalence of DENV-2 and DENV-3 as 36.6 and 62.8 percent, respectively which shows that the major serotypes circulating in Swat were DENV-2 and DENV-3. Furthermore DENV-1 and DENV-4 were not detected in any of the sample collected from Swat. In Punjab Province during the 2011 outbreak the active infection was 58.5%, of which the prevalence of DENV-2 and DENV-3 was 41.8 and 38.0 percent, respectively, which figured-out that these two serotypes were mainly involved in the dengue outbreak of the year-2011. A limited incidence of DENV-4 and DENV-1 in 2011 outbreak in Punjab at the rate of 9.5 and 4.5 percent respectively was also recorded. Phylogenetic analysis based on both complete genomes and the NS3 gene sequences of DENV-2 grouped the Pakistani isolates as cosmopolitan genotypes. Wheras no recombination was detected in the entire genome of DENV-2 of Pakistani isolates. The phylogenetic analyses, percent nucleotides and amino acids identities of the complete genomes of DENV-2 indicated that the 2013 dengue outbreak in Swat was a continuation of the 2011 dengue infections in Punjab. Furthermore the Pakistani isolates were found to be genetically more close to the Indian isolates based upon the nucleotide (97.1%) and amino acid (98.5%) identities. The selection pressure analyses revealed no positively selected sites in any of the genes of the DENV-2 among Pakistani isolates. However codon 65 of C, 155 of PrM, 203 of E and 254 of NS1 genes were under negative selection pressure. The highest substitution rate observed among amino acids residues of the ORF of all DENV-2 Pakistani isolates, was between arginine and lysine residues, followed by valine and isoleucine. Phylogenetic analysis of DENV-3 on the basis of the complete E gene and NS3 gene sequences grouped the Pakistani isolates into the genotype III; recognized that the year’s 2013 outbreak in Swat was the continuation of the previous outbreaks in Pakistan and the Pakistani DENV-3 isolates are more closely related to Indian and Chinese isolates. Analyses of the C gene of DENV-3 of the year-2011 were mostly conserved for lysine, arginine and asparagine. The viruses of the 2013 outbreak of Swat District showed the amino acids change for the nonpolar P (proline) and polar K (lysine) instead of R and N in the C gene. All the viruses isolated from the year-2011 dengue outbreak of Punjab showed a conserved pattern of nucleotides/amino acids as compared to the DENV-3 isolates of the dengue outbreak in Swat. This pattern may be due to one of the reasons that viruses established a severe infection in the relatively colder area of Swat and its adaptation to the new environment. Selection pressure analyses of NS3 genes of DENV-3 revealed no negatively or positively selected sites using all the three analytical tools. However codon 94 and 477 of the E genes turned out to be under negative selection pressure. From the selection pressure analysis it is assumed that the emergence and spread of dengue in Pakistan is more due to permissive ecological conditions than the drastic genetic changes in the viral gemome. Hence it is recommended that concerted efforts in terms of public awareness, vector control and household protection measures against dengue should be adopted to check its spread to other areas of Pakistan.