The mosquito-borne alphavirus, chikungunya virus (CHIKV), has recently reemerged, producing the largest epidemic ever recorded for this virus, with up to 6.5 million cases of acute and chronic rheumatic disease. There are currently no licensed vaccines for CHIKV and current anti-inflammatory drug treatment is often inadequate. Here we describe the isolation and characterization of two human monoclonal antibodies, C9 and E8, from CHIKV infected and recovered individuals. C9 was determined to be a potent virus neutralizing antibody and a biosensor antibody binding study demonstrated it recognized residues on intact CHIKV VLPs. Shotgun mutagenesis alanine scanning of 98 percent of the residues in the E1 and E2 glycoproteins of CHIKV envelope showed that the epitope bound by C9 included amino-acid 162 in the acid-sensitive region (ASR) of the CHIKV E2 glycoprotein. The ASR is critical for the rearrangement of CHIKV E2 during fusion and viral entry into host cells, and we predict that C9 prevents these events from occurring. When used prophylactically in a CHIKV mouse model, C9 completely protected against CHIKV viremia and arthritis. We also observed that when administered therapeutically at 8 or 18 hours post-CHIKV challenge, C9 gave 100% protection in a pathogenic mouse model. Given that targeting this novel neutralizing epitope in E2 can potently protect both in vitro and in vivo, it is likely to be an important region both for future antibody and vaccine-based interventions against CHIKV.

In Arabidopsis thaliana Columbia (Col-0) plants, the restriction of Tobacco etch virus (TEV) long-distance movement involves at least three dominant RTM (restricted TEV movement) genes named RTM1, RTM2, and RTM3. Previous work has established that, while the RTM-mediated resistance is also effective against other potyviruses, such as Plum pox virus (PPV) and Lettuce mosaic virus (LMV), some isolates of these viruses are able to overcome the RTM mechanism. In order to identify the viral determinant of this RTM-resistance breaking, the biological properties of recombinants between PPV-R, which systemically infects Col-0, and PPV-PSes, restricted by the RTM resistance, were evaluated. Recombinants that contain the PPV-R coat protein (CP) sequence in an RTM-restricted background are able to systemically infect Col-0. The use of recombinants carrying chimeric CP genes indicated that one or more PPV resistance-breaking determinants map to the 5' half of the CP gene. In the case of LMV, sequencing of independent RTM-breaking variants recovered after serial passages of the LMV AF199 isolate on Col-0 plants revealed, in each case, amino acid changes in the CP N-terminal region, close to the DAG motif. Taken together, these findings demonstrate that the potyvirus CP N-terminal region determines the outcome of the interaction with the RTM-mediated resistance.

Chikungunya virus (CHIKV) is an alphavirus responsible for a number of large outbreaks. Here we describe the efficient incorporation of CHIKV envelope glycoproteins into lentiviral and rhabdoviral particles. Vectors pseudotyped with CHIKV envelope proteins efficiently transduced many cell types from different species. However, hematopoietic cell types were either partially or completely refractory. A mutation in E1 (A226V) has been linked with expansion of tropism for mosquito species, although differences in in vitro infection of mosquito cell lines have not been noted. However, pseudovirion infectivity assays detected subtle differences in infection of mosquito cells, suggesting an explanation for the changes in mosquito tropism. The presence of C-type lectins increased CHIKV pseudotyped vector infectivity, but not infection of refractory cells, suggesting that they act as attachment factors rather than primary receptors. CHIKV pseudotypes will serve as an important tool for the study of neutralizing antibodies and the analysis of envelope glycoprotein functions.

Plum pox virus (PPV) is a member of the genus Potyvirus that is able to infect a large variety of plant species, including trees of the genus Prunus, its natural host. When some PPV isolates are propagated for an extended time in herbaceous plants, their ability to infect trees is reduced. The molecular basis of this change in host infectivity is poorly understood. We report the construction of hybrid viruses from cDNA clones of two D-strain isolates of PPV, PPV-D and PPV-R, which differ in their host range. PPV-D can infect GF305 peach seedlings efficiently, however, it is unable to infect Nicotiana clevelandii plants. Conversely, PPV-R infects N. clevelandii, but not GF305 peach seedlings. The analyses of the hybrid viruses showed that, although determinants of PPV pathogenicity are extensively spread throughout the PPV genome, the 3' terminal region of the PPV-R genome, including the 3' noncoding region and the coding regions for the coat protein (CP), NIb, and part of NIa protein, is sufficient to confer infectivity of N. clevelandii in a PPV-D background. Our data demonstrate a high concentration of amino acid substitutions in the CP and a host-specific effect of a deletion at the N terminus of this protein in PPV pathogenicity in peach and N. clevelandii infectivity experiments. These results suggest that relevant host specificity determinants are located in the N-terminal region of the CP. The analyses of the PPV-R and PPV-D chimeras also showed that key host-specific pathogenicity determinants lie in the 5' terminal third of the PPV genome, a region that spans proteins P1, HCPro, and P3. The selection of mutations in only a few specific residues in proteins P1, P3, and 6K1 after partial adaptation of a chimeric virus (BD-GFP) to N. clevelandii further suggests a relevant role for these proteins in host adaptation.