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Cell-specific adaptation of two flaviviruses following serial passage in mosquito cell culture.

ABSTRACT: West Nile Virus (WNV) is a mosquito-borne flavivirus that was introduced into the U.S. in the New York City area in 1999. Despite its successful establishment and rapid spread in a naive environment, WNV has undergone limited evolution since its introduction. This evolutionary stability has been attributed to compromises made to permit alternating cycles of viral replication in vertebrate hosts and arthropod vectors. Outbreaks of a close relative of WNV, St. Louis encephalitis virus (SLEV), occur in the U.S. periodically and are also characterized by limited genetic change overtime. We measured both phenotypic and genotypic changes in WNV and SLEV serially passaged in mosquito cell culture in order to clarify the role of an individual host cell type in flavivirus adaptation and evolution. Genetic changes in passaged WNV and SLEV were minimal but led to increased relative fitness and replicative ability of the virus in the homologous cell line C6/36 mosquito cells. Similar increases were not measured in the heterologous cell line DF-1 avian cells. These phenotypic changes are consistent with the concept of cell-specific adaptation in flaviviruses.

SUBMITTER: Ciota AT

PROVIDER: S-EPMC3249649 | biostudies-literature | 2007 Jan

REPOSITORIES: biostudies-literature

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Cell-specific adaptation of two flaviviruses following serial passage in mosquito cell culture.

Ciota Alexander T AT Lovelace Amy O AO Ngo Kiet A KA Le An N AN Maffei Joseph G JG Franke Mary A MA Payne Anne F AF Jones Susan A SA Kauffman Elizabeth B EB Kramer Laura D LD

Virology 20060911 2

West Nile Virus (WNV) is a mosquito-borne flavivirus that was introduced into the U.S. in the New York City area in 1999. Despite its successful establishment and rapid spread in a naive environment, WNV has undergone limited evolution since its introduction. This evolutionary stability has been attributed to compromises made to permit alternating cycles of viral replication in vertebrate hosts and arthropod vectors. Outbreaks of a close relative of WNV, St. Louis encephalitis virus (SLEV), occu ...[more]

PMID: 16963095

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Project description:BackgroundZika virus (ZIKV) is an arthropod-borne virus (arbovirus) with an urban transmission cycle that primarily involves humans and Aedes aegypti. Evidence suggests that the evolution of some arboviruses is constrained by their dependency on alternating between disparate (vertebrate and invertebrate) hosts. The goals of this study are to compare the genetic changes that occur in ZIKV after serial passaging in mosquito or vertebrate cell lines or alternate passaging in both cell types and to compare the replication, dissemination, and transmission efficiencies of the cell culture-derived viruses in Ae. aegypti.MethodsAn isolate of ZIKV originally acquired from a febrile patient in Yucatan, Mexico, was serially passaged six times in African green monkey kidney (Vero) cells or Aedes albopictus (C6/36) cells or both cell types by alternating passage. A colony of Ae. aegypti from Yucatan was established, and mosquitoes were challenged with the cell-adapted viruses. Midguts, Malpighian tubules, ovaries, salivary glands, wings/legs and saliva were collected at various times after challenge and tested for evidence of virus infection.ResultsGenome sequencing revealed the presence of two non-synonymous substitutions in the premembrane and NS1 regions of the mosquito cell-adapted virus and two non-synonymous substitutions in the capsid and NS2A regions of both the vertebrate cell-adapted and alternate-passaged viruses. Additional genetic changes were identified by intrahost variant frequency analysis. Virus maintained by continuous C6/36 cell passage was significantly more infectious in Ae. aegypti than viruses maintained by alternating passage and consecutive Vero cell passage.ConclusionsMosquito cell-adapted ZIKV displayed greater in vivo fitness in Ae. aegypti compared to the other viruses, indicating that obligate cycling between disparate hosts carries a fitness cost. These data increase our understanding of the factors that drive ZIKV adaptation and evolution and underscore the important need to consider the in vivo passage histories of flaviviruses to be evaluated in vector competence studies.

Project description:Passage in mice of opportunistic pathogens such as Cryptococcus neoformans is known to increase virulence, but little is known about the molecular mechanisms involved in virulence adaptation. Serial mouse passage of nine environmental strains of serotype A C. neoformans identified two highly adapted virulent strains that showed a 4-fold reduction in time to death after four passages. Transcriptome sequencing expression studies demonstrated increased expression of a FRE3-encoded iron reductase in the two strains but not in a control strain that did not demonstrate increased virulence during mouse passage. FRE3 was shown to express an iron reductase activity and to play a role in iron-dependent growth of C. neoformans. Overexpression of FRE3 in the two original environmental strains increased growth in the macrophage cell line J774.16 and increased virulence. These data demonstrate a role for FRE3 in the virulence of C. neoformans and demonstrate how the increased expression of such a "virulence acquisition gene" during the environment-to-mammal transition, can optimize the virulence of environmental strains in mammalian hosts. IMPORTANCE Cryptococcus neoformans is a significant global fungal pathogen that also resides in the environment. Recent studies have suggested that the organism may undergo microevolution in the host. However, little is known about the permitted genetic changes facilitating the adaptation of environmental strains to mammalian hosts. The present studies subjected environmental strains isolated from several metropolitan areas of the United States to serial passages in mice. Transcriptome sequencing expression studies identified the increased expression of an iron reductase gene, FRE3, in two strains that adapted in mice to become highly virulent, and overexpression of FRE3 recapitulated the increased virulence after mouse passage. Iron reductase in yeast is important to iron uptake in a large number of microbial pathogens. These studies demonstrate the capacity of C. neoformans to show reproducible changes in the expression levels of small numbers of genes termed "virulence adaptation genes" to effectively increase pathogenicity during the environment-to-mammal transition.

Dataset Information

Cell-specific adaptation of two flaviviruses following serial passage in mosquito cell culture.

Publications

Cell-specific adaptation of two flaviviruses following serial passage in mosquito cell culture.

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