Abstract
BackgroundMost known flaviviruses, including West Nile virus (WNV), are maintained in natural transmission cycles between hematophagous arthropods and vertebrate hosts. Other flaviviruses such as Modoc virus (MODV) and Culex flavivirus (CxFV) have host ranges restricted to vertebrates and insects, respectively. The genetic elements that modulate the differential host ranges and transmission cycles of these viruses have not been identified.MethodsFusion polymerase chain reaction (PCR) was used to replace the capsid (C), premembrane (prM) and envelope (E) genes and the prM-E genes of a full-length MODV infectious cDNA clone with the corresponding regions of WNV and CxFV. Fusion products were directly transfected into baby hamster kidney-derived cells that stably express T7 RNA polymerase. At 4 days post-transfection, aliquots of each supernatant were inoculated onto vertebrate (BHK-21 and Vero) and mosquito (C6/36) cells which were then assayed for evidence of viral infection by reverse transcription-PCR, Western blot and plaque assay.ResultsChimeric virus was recovered in cells transfected with the fusion product containing the prM-E genes of WNV. The virus could infect vertebrate but not mosquito cells. The in vitro replication kinetics and yields of the chimeric virus were similar to MODV but the chimeric virus produced larger plaques. Chimeric virus was not recovered in cells transfected with any of the other fusion products.ConclusionsOur data indicate that genetic elements outside of the prM-E gene region of MODV condition its vertebrate-specific phenotype.
Highlights
All viruses in the genus Flavivirus possess a single-stranded, positive-sense RNA genome of approximately 11 kb [1]
The overall goal of this study is to characterize the in vitro host ranges of chimeric viruses constructed using representative viruses from the vertebrate-specific, insect-specific and arthropod/vertebrate flavivirus groups (MODV, Culex flavivirus (CxFV) and West Nile virus (WNV), respectively) in order to increase our knowledge of the genetic elements that condition the vastly different host ranges and transmissibilities of these viruses
CxFV was originally isolated from Culex pipiens in Iowa in 2007 [28]. cDNAs were generated from WNV and CxFV RNA and used as template for fusion polymerase chain reaction (PCR) reactions as described below
Summary
All viruses in the genus Flavivirus (family Flaviviridae) possess a single-stranded, positive-sense RNA genome of approximately 11 kb [1]. The genome contains a single open reading frame (ORF) flanked by 5’ and 3’ untranslated regions (UTRs) of ~100 and 400–700 nt, respectively [2]. The flavivirus genome is packaged in an icosahedral nucleocapsid with multiple copies of the C protein [1]. The nucleocapsid is surrounded by a lipid envelope, acquired from the host cell, in which the prM(M) and E proteins are embedded. Most known flaviviruses, including West Nile virus (WNV), are maintained in natural transmission cycles between hematophagous arthropods and vertebrate hosts. Other flaviviruses such as Modoc virus (MODV) and Culex flavivirus (CxFV) have host ranges restricted to vertebrates and insects, respectively. The genetic elements that modulate the differential host ranges and transmission cycles of these viruses have not been identified
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