Isolation and molecular characterization of dengue virus clinical isolates from pediatric patients in New Delhi

Abstract

Objectives To characterize the in vitro replication fitness, viral diversity and phylogeny of dengue viruses (DENV) isolated from Indian patients. Methods DENV was isolated from whole blood collected from patients by passaging in cell culture. Passage 3 viruses were used for growth kinetics in C6/36 mosquito cells. Parallel efforts also focused on isolation of DENV RNA from plasma samples of the same patients and processed for next generation sequencing. Results We were able to isolate 64 clinical isolates, mostly DENV-2, of which 25 were further used for growth curve analysis in vitro which showed a wide range of replication kinetics. Highest viral titers were of isolates from dengue with warning signs and severe dengue cases. We obtained full genome sequences of 21 DENV isolates. Genome analysis mapped the circulating DENV-2 strains to the Cosmopolitan genotype. Conclusions The replication kinetics of isolates from patients with mild or severe infection was not significantly different but the viral titers between the isolates varied by two orders of magnitude suggesting differences in replication fitness among the circulating DENV-2 isolates.

Detection of dengue virus type 2 of Indian origin in acute febrile patients in rural Kenya

“…Consistently with the complete coding sequence analysis, all the Kenyan sequences formed a monophyletic group most closely related to sequences from New Delhi, India, in the year 2014 [43] (tMRCA: 2012 [95% HPD [2011][2012]). In addition to the strains mentioned above, the sister group included sequences originating from India and Singapore.…”

Molecular and evolutionary analysis of dengue virus serotype 2 isolates from Korean travelers in 2015

“…The DENV isolates described in this report possessed variable motifs and showed evidence of recombination in the NS5 region, which encodes the RNA-dependent RNA polymerase and therefore could affect replication capacity (Table 3, Fig. 3) [36][37][38][39]. Three motifs in the E protein were differentiated among the Korean isolates (Table 3).…”

“…The 43,248 strain proved to be similar to strains endemic in Thailand [ 44 ]. Only the KBPV-VR29 strain belonged to the American genotype and exhibited the most similarity to the JHA-1 strain, which is highly neurovirulent in mice [ 38 ].…”

A importância da utilização de técnicas moleculares no diagnóstico laboratorial de Dengue virus: uma revisão

“…Novas técnicas, como o sequenciamento de nova geração (Next Generation Sequencing -NGS) foi utilizado em 5 estudos na identificação de mutações a nível de nucleotídeos e aminoácidos (Perry;Tai, 2018;Jerome et al, 2019;Kar et al, 2019;Cai et al, 2020;Queiroz et al, 2020). A diferença desta técnica para as demais baseia-se detecção de todas as alterações existentes no DNA sequenciado, por meio de softwares de computadores, comparando com os DNAs de amostras controle.…”

“…No estudo realizado por Kar (2019) foi possível identificar 64 isolados clínicos de DENV, principalmente DENV-2, identificado como o mais virulento, o que corrobora com as manifestações clínicas graves e atípicas apresentadas pelos pacientes positivos para o sorotipo detectado, onde os maiores títulos virais foram associados a isolados de pacientes com dengue com sinais de alerta e casos graves de dengue. Análises filogenéticas com base no sequenciamento do genoma completo caracterizaram as cepas circulantes de DENV-2 para o genótipo Cosmopolita.…”