Characterization of an infectious clone of the wild-type yellow fever virus Asibi strain that is able to infect and disseminate in mosquitoes

Abstract

Infectious clone technology provides an opportunity to study the molecular basis of arthropod-virus interactions in detail. This study describes the development of an infectious clone of the prototype yellow fever virus Asibi strain (YFV-As) with the purpose of identifying sequences or domains that influence infection dynamics in the mosquito vector. The full-length cDNA of YFV-As virus was produced from RT-PCR products of parental viral RNA. These were cloned into a low-copy-number plasmid previously used to develop the YFV-17D infectious clone (pACNR/FLYF-17D). Virus recovered from the infectious clone exhibited biological characteristics similar to those of the parental YFV-As, including replication kinetics, reactivity to flavivirus cross-reactive and YFV-specific antibodies and infection and dissemination rates in Aedes aegypti, the principal mosquito vector of YFV. These data provide the basis for future studies with chimeric Asibi/17D viruses to identify the determinants of vaccine attenuation in the vector.Yellow fever virus (YFV) was the first mosquito-borne virus to be identified (Stokes et al., 1928). It causes a severe and often fatal haemorrhagic disease in humans, for which there is no specific treatment. Despite the existence of two safe and efficacious vaccines, YFV 17D-204 and 17DD, the incidence of YFV infections is increasing, and the disease has re-emerged in many parts of Africa and South America where incidence had previously been reduced largely through mosquito control programmes (Barrett & Monath, 2003;Mutebi & Barrett, 2002;Robertson et al., 1996). The remoteness of many regions in which YFV occurs hinders accurate data collection, but it is estimated that there are 200 000 infections and 30 000 deaths annually.YFV is the prototype member of the genus Flavivirus, family Flaviviridae, which includes other important human pathogens, for example, dengue viruses (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV) and tick-borne encephalitis virus (TBEV). The prototype YFV strain, Asibi (Ghana27), was isolated from Mr Asibi in 1927 (Stokes et al., 1928). Flaviviruses are single-stranded positive-sense RNA viruses with genomes of approximately 11 kb that encode three structural proteins, capsid, membrane and envelope, and seven non-structural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5, between 59 and 39 non-coding regions (NCRs). Virus is transcribed and translated as one long polyprotein precursor that is processed co-and posttranslationally by host and viral proteases .Infectious cDNA clones provide a useful platform on which to investigate the genetic determinants of flavivirus virulence. Initially, flavivirus infectious clones, including one for YFV (Rice et al., 1989), depended on a two-plasmid system with in vitro ligation. However, a full-length YFV infectious clone, pACNR/FLYF-17D, based on YFV 17D-204, was recently described (Bredenbeek et al., 2003). Infectious clones based on virulent virus strains and the capacity to generate chimeric viruses with components o...

Vector Competence of Australian Mosquitoes for Yellow Fever Virus

“…Infectious clone-derived YFV Asibi was recovered from recombinant genome pYFV-As-RBZ IC, which contained the viral coding sequence derived from the parental YFV Asibi i.c. (pYFV-As IC) 12 and was modified to incorporate a Hepatitis Delta Rybozyme sequence. Briefly, pYFV-As-RBZ IC was linearized, purified, and transcribed in vitro using the mMessage mMachine SP6 kit (Ambion, Austin, TX) in accordance with manufacturer protocols.…”

“…Viral antigen in mosquito samples and the blood/virus mixture titrations was detected using a modification of the indirect immunofluorescence assay described previously. 9,12 The YFV NS1-reactive monoclonal antibody 863 was used as the primary antibody, 16 and Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen, Eugene, OR) was used as the secondary antibody. Samples were scored using an Olympus IX70 inverted epifluorescence microscope (Olympus, Tokyo, Japan) fitted with a fluorescein filter.…”

“…Three strains of YFV that had been characterized previously in mosquitoes were used for the experiments. [12][13][14] These viruses were obtained from the National Institutes of Health-supported World Reference Center for Emerging Viruses at UTMB. An African strain of YFV, BA-55, was isolated from a fatal yellow fever case during an epidemic in Nigeria in 1987 and had been passaged three times in suckling mouse brains.…”

Construction of an infectious cDNA clone for Omsk hemorrhagic fever virus, and characterization of mutations in NS2A and NS5

“…This vector has been used to construct stable infectious clones of several flaviviruses and pestiviruses (Bredenbeek et al, 2003;McElroy et al, 2005;Mendez et al, 1998;Ruggli et al, 1996). The OHF-IC plasmids were stable during passaging in bacteria, indicating that this infectious clone can be useful for genetic manipulations.…”

“…The overall strategy to construct the full-length infectious clone of OHFV strain Guriev is assembled into the low-copy plasmid pACNR, which has been used successfully to construct stable infectious clones in several flaviviruses (Bredenbeek et al, 2003;McElroy et al, 2005;Rossi et al, 2005). The complete genome sequence of this plasmid (designated OHF-IC-ori) was determined and compared with the parent virus.…”