Biological transmission of arthropod-borne viruses (arboviruses) to vertebrate hosts by hematophagous insects poses a global threat because such arboviruses can result in a range of serious public health infectious diseases. Sindbis virus (SINV), the prototype Alphavirus, was used to track infections in the posterior midgut (PMG) of Aedes aegypti adult mosquitoes. Females were fed viremic blood containing a virus reporter, SINV [Thosea asigna virus-green fluorescent protein (TaV-GFP)], that leaves a fluorescent signal in infected cells. We assessed whole-mount PMGs to identify primary foci, secondary target tissues, distribution, and virus persistence. Following a viremic blood meal, PMGs were dissected and analyzed at various days of post blood-feeding. We report that virus foci indicated by GFP in midgut epithelial cells resulted in a 9.8% PMG infection and a 10.8% dissemination from these infected guts. The number of virus foci ranged from 1 to 3 per individual PMG and was more prevalent in the PMG-middle > PMG-frontal > PMG-caudal regions. SINV TaV-GFP was first observed in the PMG (primary target tissue) at 3 days post blood-feeding, was sequestered in circumscribed foci, replicated in PMG peristaltic muscles (secondary target tissue) following dissemination, and GFP was observed to persist in PMGs for 30 days postinfection.

Prolonged fatigue is increasingly reported among chikungunya virus (CHIKV)-infected populations. We investigated the relationships between CHIKV exposure, long-lasting rheumatic musculoskeletal pain (LRMSP) and chronic fatigue. 1094 participants (512 CHIKV seropositive and 582 seronegative) of the TELECHIK population-based cohort were analysed considering the duration of the manifestations throughout an average 2-year follow-up. Weighted prevalence rates and prevalence ratios for LRMSP, idiopathic chronic fatigue (ICF), and chronic fatigue syndrome (CFS)-like illness, both latter syndromes adapted from Centers for Disease Control (CDC)-1994/Fukuda criteria, were compared. Population attributable fractions (PAF) were estimated to assess the contribution of CHIKV infection to each of the three phenotypes. Among 362 adult subjects who had reported either rheumatic pain or fatigue at the onset of the infection, weighted prevalence rates of LRMSP, ICF and CFS-like illness were respectively of 32.9%, 38.7% and 23.9%, and of 8.7%, 8.5% and 7.4% among initially asymptomatic peers (P < 0.01, respectively). Each of the three outcomes was highly attributable to chikungunya (PAF of 43.2%, 36.2% and 41.0%, respectively). In the sub-cohort of CHIKV-infected subjects, LRMSP, ICF and CFS-like illness, which overlapped in 70%, accounted for 53% of the chronic manifestations. In addition to rheumatic disease, chronic fatigue could be considered in caring for patients with chronic chikungunya disease.

In Australia, Ross River virus (RRV) is predominantly identified and managed through passive health surveillance. Here, the proactive use of environmental datasets to improve community-scale public health interventions in southeastern Tasmania is explored. Known environmental drivers (temperature, rainfall, tide) of the RRV vector Aedes camptorhynchus are analysed against cumulative case records for five adjacent local government areas (LGAs) from 1993 to 2009. Allowing for a 0- to 3-month lag period, temperature was the most significant driver of RRV cases at 1-month lag, contributing to a 23·2% increase in cases above the long-term case average. The potential for RRV to become an emerging public health issue in Tasmania due to projected climate changes is discussed. Moreover, practical outputs from this research are proposed including the development of an early warning system for local councils to implement preventative measures, such as public outreach and mosquito spray programmes.

Alphavirus genome replication is a multistep asymmetric process. Several lines of evidence suggest that the template preference of the RNA replicase is regulated by proteolytic cleavage of the viral nonstructural polyprotein. Cis-acting RNA elements in the viral genome also play crucial roles in regulating genome replication and subgenomic RNA transcription. In this report, a series of RNA templates were analyzed in vitro and in vivo to define functional elements in the 5′ end of the genome. The 5′ UTR was shown to contain distinct core promoter elements for both minus- and plus-strand synthesis. In addition, two conserved stem-loop structures within the nsP1 coding sequence enhanced RNA replication but were not required. Studies with chimeric templates and trans-competition experiments suggest that the 5′ determinant for minus-strand initiation can differ among alphaviruses and binds to one or more limiting replicase components. The results provide compelling evidence that the 5′ and 3′ ends of alphavirus genome RNAs must interact to initiate replication and we propose one model for how this interaction might occur. In addition to providing new insight into the initiation of alphavirus genome replication, these results have implications for the development of improved alphavirus vector systems with reduced recombination potential.

The brome mosaic virus (BMV) RNA-dependent RNA polymerase (RdRp) directs template-specific synthesis of (−)-strand genomic and (+)-strand subgenomic RNAs in vitro. Although the requirements for (−)-strand RNA synthesis have been characterized previously, the mechanism of subgenomic RNA synthesis has not. Mutational analysis of the subgenomic promoter revealed that the +1 cytidylate and the +2 adenylate are important for RNA synthesis. Unlike (−)-strand RNA synthesis, which required only a high GTP concentration, subgenomic RNA synthesis required high concentrations of both GTP and UTP. Phylogenetic analysis of the sequences surrounding the initiation sites for subgenomic and genomic (+)-strand RNA synthesis in representative members of the alphavirus-like superfamily revealed that the +1 and +2 positions are highly conserved as a pyrimidine–adenylate. GDP and dinucleotide primers were able to more efficiently stimulate (−)-strand synthesis than subgenomic synthesis under conditions of limiting GTP. Oligonucleotide products of 6-, 7-, and 9-nt were synthesized and released by RdRp in 3–20-fold molar excess to full-length subgenomic RNA. Termination of RNA synthesis by RdRp was not induced by template sequence alone. Our characterization of the stepwise mechanism of subgenomic and (−)-strand RNA synthesis by RdRp permits comparisons to the mechanism of DNA-dependent RNA synthesis.