Aura Virus Is a New World Representative of Sindbis-like Viruses

Aura virus is an alphavirus present in Brazil and Argentina that is serologically related to Sindbis virus (present throughout the Old World) and to Western equine encephalitis (WEE) virus (present in the Americas). We have previously shown that WEE is a recombinant virus whose glycoproteins and part of whose 3' nontranslated region (NTR) are derived from a Sindbis-like virus, but the remainder of whose genome is derived from Eastern equine encephalitis (EEE) virus. We show here that Aura virus is a Sindbis-like virus that shares considerable organizational and sequence identity with Sindbis virus. Certain nucleotide sequence elements present in Aura RNA that are believed to function as promoters are almost identical to their Sindbis counterparts, repeated elements in the 3' nontranslated region are shared with Sindbis virus, and important antigenic epitopes are conserved between the two viruses. Despite their close relationship, the two viruses have diverged significantly, sharing 73% amino acid sequence identity in the nonstructural proteins and 62% identity in the structural proteins. This is about the same as the identities between EEE and Venezuelan equine encephalitis virus, whose promoter elements, 3' NTRs, and antigenic epitopes have diverged more radically, such that these two viruses are considered to belong to different subgroups. Importantly, the glycoproteins of WEE are more closely related to those of Sindbis than to those of Aura virus. From this we propose that an ancestral Sindbis-like virus present in the Americas (probably South America) diverged 1000-2000 years ago into a lineage that gave rise to Aura virus and a lineage that gave rise to Sindbis virus and to the Sindbis-like parent of WEE. At some time after this divergence, a Slndbis-like virus belonging to the latter lineage was transferred to the Old World where it gave rise to Sindbis viruses distributed throughout the Old World, and in a separate event a Sindbis-like virus belonging to the same lineage underwent recombination with EEE to give rise to WEE

Regulation of Semliki Forest virus RNA replication: a model for the control of alphavirus pathogenesis in invertebrate hosts

Alphavirus nonstructural proteins are translated as a polyprotein that is ultimately cleaved into four mature proteins called nsP1, nsP2, nsP3, and nsP4 from their order in the polyprotein. The role of this nonstructural polyprotein, of cleavage intermediates, and of mature proteins in synthesis of Semliki Forest virus (SFV) RNA has been studied using mutants unable to cleave one or more of the sites in the nonstructural polyprotein or that had the arginine sense codon between nsP3 and nsP4 changed to an opal termination codon. The results were compared with those obtained for Sindbis virus (SINV), which has a naturally occurring opal codon between nsP2 and nsP3. We found that (1) an active nonstructural protease in nsP2 is required for RNA synthesis. This protease is responsible for all three cleavages in the nonstructural polyprotein. (2) Cleavage between nsP3 and nsP4 (the viral RNA polymerase) is required for RNA synthesis by SFV. (3) SFV mutants that are able to produce only polyprotein P123 and nsP4 synthesize minus-strand RNA early after infection as efficiently as SF wild type but are defective in the synthesis of plus-strand RNA. The presence of sense or opal following nsP3 did not affect this result. At 30 °C, they give rise to low yields of virus after a delay, but at 39°C, they are nonviable. (4) SFV mutants that produce nsP1, P23, nsP4, as well as the precursor P123 are viable but produce an order of magnitude less virus than wild type at 30 °C and two orders of magnitude less virus at 39°C. The ratio of subgenomic mRNA to genomic RNA is much reduced in these mutants relative to the parental viruses. (5) At 30°C, the variants containing an opal codon grow as well as or slightly better than the corresponding virus with a sense codon. At 39 °C, however, the opal variants produce significantly more virus. These results support the conclusion that SFV and SINV, and by extension all alphaviruses, regulate their RNA synthesis in the same fashion after infection. P123 and nsP4 form a minus-strand replicase that synthesizes plus-strand RNA only inefficiently, especially at the higher temperatures found in mammals and birds. A replicase containing nsP1, P23, and nsP4 can make both plus and minus strands, but prefers the promoter for genomic plus sense RNA to that for subgenomic mRNA. The fully cleaved replicase can make only plus-strand RNA, and prefers the promoter for subgenomic mRNA to that for genomic RNA. Alphaviruses alternate between infection of hematophagous arthropods and higher vertebrates. Although the infection of higher vertebrates is acute and often accompanied by disease, continuing transmission of the virus in nature requires that infection of arthropods be persistent and relatively asymptomatic. We propose that this mechanism for control of RNA synthesis evolved to moderate the pathogenicity of the viruses in their arthropod hosts

Temperature and blood leukocyte counts of pigs infected with 5×10⁶ TCID₅₀ Vp447 or Vp447_ΔcN₂₁₇₇Y.

<p>After infection, rectal temperature (A) was recorded daily and EDTA blood was collected on days 2, 4, 7, 10, 14, 21 and 28 and the number of leukocytes (B) was determined in Giga/l. wt = Vp447 infected; Δc = Vp447_ΔcN₂₁₇₇Y infected; wtS = sentinel animal in Vp447 infected group; ΔcS = sentinel in Vp447_ΔcN₂₁₇₇Y infected group.</p

Distribution of infectivity and genomes of Vp447 and Vp447_ΔcN₂₁₇₇Y upon size exclusion chromatography.

<p>10⁸ ffu/ml of Vp447 and Vp447_ΔcN₂₁₇₇Y each were simultaneously applied to a size exclusion chromatography column and virus titer was determined for all fractions. Genomes of both viruses were quantified in the different fractions by virus specific real-time RT-PCRs. The column was calibrated employing IgM as a size marker. wt = Vp447; Δc = Vp447_ΔcN₂₁₇₇Y.</p