The flavivirus NS2A protein is involved in the assembly of infectious particles. To further understand its role in this process, a charged-to-alanine scanning analysis was performed on NS2A encoded by an infectious cDNA clone of yellow fever virus (YFV). Fifteen mutants containing single, double, or triple charged-to-alanine changes were tested. Five of them did not produce infectious particles, whereas efficient RNA replication was detectable for two of the five NS2A mutants (R22A-K23A-R24A and R99A-E100A-R101A mutants). Prolonged cultivation of transfected cells resulted in the recovery of pseudorevertants. Besides suppressor mutants in NS2A, a compensating second-site mutation in NS3 (D343G) arose for the NS2A R22A-K23A-R24A mutant. We found this NS3 mutation previously to be suppressive for the NS2A␣ cleavage site Q189S mutant, also deficient in virion assembly. In this study, the subsequently suggested interaction between NS2A and NS3 was proven by coimmunoprecipitation analyses. Using selectively permeabilized cells, we could demonstrate that the regions encompassing R22A-K23A-R24A and Q189S in NS2A are localized to the cytoplasm, where NS3 is also known to reside. However, the defect in particle production observed for the NS2A R22A-K23A-R24A and Q189S mutants was not due to a defect in physical interaction between NS2A and NS3, as the NS2A mutations did not interrupt NS3 interaction. In fact, a region just upstream of R22-K23-R24 was mapped to be critical for NS2A-NS3 interaction. Taken together, these data support a complex interplay between YFV NS2A and NS3 in virion assembly and identify a basic cluster in the NS2A N terminus to be critical in this process. IMPORTANCEDespite an available vaccine, yellow fever remains endemic in tropical areas of South America and Africa. To control the disease, antiviral drugs are required, and an understanding of the determinants of virion assembly is central to their development. In this study, we identified a basic cluster of amino acids in the N terminus of YFV NS2A which inhibited virion assembly upon mutation. The defect was rescued by a spontaneously occurring mutation in NS3. Our study proves an interaction between NS2A and NS3, which, remarkably, was maintained for the NS2A mutant in the presence and absence of the NS3 mutation. This suggests a role for other viral and/or cellular proteins in virion assembly. Residues important for YFV virion production reported here only partially coincided with those reported for other flaviviruses, suggesting that the determinants for particle production are virus specific. Reconstruction of a YFV encoding tagged NS2A paves the way to identify further NS2A interaction partners. Y ellow fever virus (YFV) belongs to the genus flavivirus within the family Flaviviridae. Clinical disease associated with this prototype flavivirus can result in acute viral hemorrhagic fever (1). Other important human flaviviruses include dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus, and tick-borne encephalitis v...

Most viruses of the genus Flavivirus, e.g., YFV and dengue virus, are mosquito borne and transmitted to vertebrates during blood feeding of mosquitoes. Within the last decade, an increasing number of viruses with a host range exclusively restricted to insects in close relationship to the vertebrate-pathogenic flaviviruses were discovered in mosquitoes. To identify barriers that could block the arboviral vertebrate tropism, we set out to identify the steps at which the ISF replication cycle fails in vertebrates. Our studies revealed blocks at several levels, suggesting that flavivirus host range expansion from insects to vertebrates was a complex process that involved overcoming multiple barriers.

Chikungunya virus (CHIKV) has caused massive epidemics in the Indian Ocean region since 2005. It belongs to the genus Alphavirus and possesses a positive-stranded RNA genome of nearly 12 kb in size. To produce genetically modified viruses for the study of various aspects of the CHIKV life cycle, a reverse genetic system is needed. We report the generation of a T7 RNA polymerase-driven infectious cDNA clone of CHIKV. Electroporation of in vitro-transcribed RNA resulted in the recovery of a recombinant virus with growth characteristics comparable to the parental strain. Using the established cDNA clone, the red fluorescent marker gene mCherry was introduced into two different sites within the CHIKV nsP3 gene. Both constructs allowed the rescue of stable fluorescent reporter viruses with growth characteristics similar to the wild-type virus. The latter reporter viruses represent valuable tools for easy follow-up of replicating CHIKV useful in several applications of CHIKV research.

Due to high genome plasticity, the evolutionary fate and geographical history of picornaviruses is hard to follow. Here, we determined the complete coding sequences of eight human parechoviruses (HPeV) of types 1, 5 and 6 directly from clinical samples from Brazil. The capsid genes of these strains were not remarkably different from European, North American and Japanese HPeV. Full genome analysis revealed frequent intertypic recombination in the nonstructural genome region. In addition, evidence of recombination between viruses of the same type in the capsid-encoding genome region among HPeV1 and HPeV4 was obtained. Bayesian phylogenetic analysis indicated that strains without evidence of recombination with each other in any genome region were separated by no more than 35 years of circulation. Interestingly, in the 3C gene, all Brazilian parechoviruses grouped together regardless of serotype. The most recent common ancestor of these strains dated back 108 years, suggesting long-term endemicity of this particular P3 genome lineage in South America. Our results support the idea that picornavirus replicative genes acquire capsid proteins introduced by new strains. Under certain epidemiological conditions, replicative genes may be maintained in circumscript geographical regions. INTRODUCTIONHuman parechoviruses (HPeV) are small non-enveloped viruses that define a genus of the family Picornaviridae. Their positive-sense RNA genome encodes a single polyprotein that is cleaved into three structural proteins, VP0, VP3 and VP1 (defining the P1 genome region), and seven non-structural proteins, 2A-2C (P2) and 3A-3D (P3). The first two HPeV serotypes were isolated in the 1950s and designated echovirus 22 and 23. In the 1990s, parechoviruses were reclassified into a new genus based on their distinct genome organization (Hyypiä et al., 1992). High prevalence of HPeV was not truly appreciated until the last few years. Recently, HPeVs were shown to be highly diverse, with up to 14 provisionally assigned types (AlSunaidi et al., 2007;Benschop et al., 2008a; Drexler et al., 2009;Ito et al., 2004; Kim Pham et al., 2010;Li et al., 2009;Watanabe et al., 2007). HPeV also proved highly prevalent in the human population, with seropositivity rates reaching 88 % at the age of 2 years (Joki-Korpela & Hyypiä, 1998;Takao et al., 2001), and an important cause of meningitis and sepsis-like disease of newborns (Baumgarte et al., 2008; 3These authors contributed equally to this work.The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are HQ696570-HQ696577. Wolthers et al., 2008). In general, recent reports showed that epidemiological and clinical features of HPeV are similar to the most common picornavirus genus, Enterovirus. HPeV genetic and evolutionary properties also resemble those of enteroviruses. They feature both high mutation rate (Faria et al., 2009) and frequent natural recombination (Benschop et al., 2008bCalvert et al., 2010;Williams et al., 2009;Zoll et al., 2009). Unfortunately, analyses of re...

Flavivirus outbreaks require fast and reliable diagnostics that can be easily adapted to newly emerging and re-emerging flaviviruses. Due to the serological cross-reactivity among flavivirus antibodies, neutralization tests (NT) are considered the gold standard for sero-diagnostics. Here, we first established wild-type single-round infectious virus replicon particles (VRPs) by packaging a yellow fever virus (YFV) replicon expressing Gaussia luciferase (Gluc) with YFV structural proteins in trans using a double subgenomic Sindbis virus (SINV) replicon. The latter expressed the YFV envelope proteins prME via the first SINV subgenomic promoter and the capsid protein via a second subgenomic SINV promoter. VRPs were produced upon co-electroporation of replicon and packaging RNA. Introduction of single restriction enzyme sites in the packaging construct flanking the prME sequence easily allowed to exchange the prME moiety resulting in chimeric VRPs that have the surface proteins of other flaviviruses including dengue virus 1-4, Zika virus, West Nile virus, and tick-borne encephalitis virus. Besides comparing the YF-VRP based NT assay to a YF reporter virus NT assay, we analyzed the neutralization efficiencies of different human anti-flavivirus sera or a monoclonal antibody against all established VRPs. The assays were performed in a 96-well high-throughput format setting with Gluc as readout in comparison to classical plaque reduction NTs indicating that the VRP-based NT assays are suitable for high-throughput analyses of neutralizing flavivirus antibodies.