Comparison of dengue virus type 2-specific small RNAs from RNA interference-competent and -incompetent mosquito cells.

The exogenous RNA interference (RNAi) pathway is an important antiviral defense against arboviruses in mosquitoes, and virus-specific small interfering (si)RNAs are key components of this pathway. Understanding the biogenesis of siRNAs in mosquitoes could have important ramifications in using RNAi to control arbovirus transmission. Using deep sequencing technology, we characterized dengue virus type 2 (DENV2)-specific small RNAs produced during infection of Aedes aegypti mosquitoes and A. aegypti Aag2 cell cultures and compared them to those produced in the C6/36 Aedes albopictus cell line. We show that the size and mixed polarity of virus-specific small RNAs from DENV-infected A. aegypti cells indicate that they are products of Dicer-2 (Dcr2) cleavage of long dsRNA, whereas C6/36 cells generate DENV2-specific small RNAs that are longer and predominantly positive polarity, suggesting that they originate from a different small RNA pathway. Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus (CFAV) corroborated these findings. An in vitro assay also showed that Aag2 A. aegypti cells are capable of siRNA production, while C6/36 A. albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA. Defective expression or function of Dcr2, the key initiator of the RNAi pathway, might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line, which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts.

Phage display selection of cyclic peptides that inhibit Andes virus infection.

Specific therapy is not available for hantavirus cardiopulmonary syndrome caused by Andes virus (ANDV). Peptides capable of blocking ANDV infection in vitro were identified using antibodies against ANDV surface glycoproteins Gn and Gc to competitively elute a cyclic nonapeptide-bearing phage display library from purified ANDV particles. Phage was examined for ANDV infection inhibition in vitro, and nonapeptides were synthesized based on the most-potent phage sequences. Three peptides showed levels of viral inhibition which were significantly increased by combination treatment with anti-Gn- and anti-Gc-targeting peptides. These peptides will be valuable tools for further development of both peptide and nonpeptide therapeutic agents.

Multivalent presentation of antihantavirus peptides on nanoparticles enhances infection blockade.

Viral entry into susceptible host cells typically results from multivalent interactions between viral surface proteins and host entry receptors. In the case of Sin Nombre virus (SNV), a New World hantavirus that causes hantavirus cardiopulmonary syndrome, infection involves the interaction between viral membrane surface glycoproteins and the human integrin alpha(v)beta(3). Currently, there are no therapeutic agents available which specifically target SNV. To address this problem, we used phage display selection of cyclic nonapeptides to identify peptides that bound SNV and specifically prevented SNV infection in vitro. We synthesized cyclic nonapeptides based on peptide sequences of phage demonstrating the strongest inhibition of infection, and in all cases, the isolated peptides were less effective at blocking infection (9.0% to 27.6% inhibition) than were the same peptides presented by phage (74.0% to 82.6% inhibition). Since peptides presented by the phage were pentavalent, we determined whether the identified peptides would show greater inhibition if presented in a multivalent format. We used carboxyl linkages to conjugate selected cyclic peptides to multivalent nanoparticles and tested infection inhibition. Two of the peptides, CLVRNLAWC and CQATTARNC, showed inhibition that was improved over that of the free format when presented on nanoparticles at a 4:1 nanoparticle-to-virus ratio (9.0% to 32.5% and 27.6% to 37.6%, respectively), with CQATTARNC inhibition surpassing 50% when nanoparticles were used at a 20:1 ratio versus virus. These data illustrate that multivalent inhibitors may disrupt polyvalent protein-protein interactions, such as those utilized for viral infection of host cells, and may represent a useful therapeutic approach.

Early innate immune responses to Sin Nombre hantavirus occur independently of IFN regulatory factor 3, characterized pattern recognition receptors, and viral entry.

Sin Nombre virus (SNV) is a highly pathogenic New World virus and etiologic agent of hantavirus cardiopulmonary syndrome. We have previously shown that replication-defective virus particles are able to induce a strong IFN-stimulated gene (ISG) response in human primary cells. RNA viruses often stimulate the innate immune response by interactions between viral nucleic acids, acting as a pathogen-associated molecular pattern, and cellular pattern-recognition receptors (PRRs). Ligand binding to PRRs activates transcription factors which regulate the expression of antiviral genes, and in all systems examined thus far, IFN regulatory factor 3 (IRF3) has been described as an essential intermediate for induction of ISG expression. However, we now describe a model in which IRF3 is dispensable for the induction of ISG transcription in response to viral particles. IRF3-independent ISG transcription in human hepatoma cell lines is initiated early after exposure to SNV virus particles in an entry- and replication-independent fashion. Furthermore, using gene knockdown, we discovered that this activation is independent of the best-characterized RNA- and protein-sensing PRRs including the cytoplasmic caspase recruitment domain-containing RNA helicases and the TLRs. SNV particles engage a heretofore unrecognized PRR, likely located at the cell surface, and engage a novel IRF3-independent pathway that activates the innate immune response.

Selection and characterization of scFv antibodies against the Sin Nombre hantavirus nucleocapsid protein.

Rodent-borne hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) in the old world and hantavirus cardio-pulmonary syndrome (HCPS) in the new. Most cases of HCPS in North America are caused by Sin Nombre Virus (SNV). Current viral detection technologies depend upon the identification of anti-viral antibodies in patient serum. Detection of viral antigen may facilitate earlier detection of the pathogen. We describe here the characterization of two single-chain Fv antibodies (scFvs), selected from a large naïve phage antibody library, which are capable of identifying the Sin Nombre Virus nucleocapsid protein (SNV-N), with no cross reactivity with the nucleocapsid protein from other hantaviruses. The utility of such selected scFvs was increased by the creation of an scFv-alkaline phosphatase fusion protein which was able to directly detect virally produced material without the need for additional reagents.