Structural and functional basis of VLDLR usage by Eastern equine encephalitis virus.

The very-low-density lipoprotein receptor (VLDLR) comprises eight LDLR type A (LA) domains and supports entry of distantly related alphaviruses, including Eastern equine encephalitis virus (EEEV) and Semliki Forest virus (SFV). Here, by resolving multiple cryo-electron microscopy structures of EEEV-VLDLR complexes and performing mutagenesis and functional studies, we show that EEEV uses multiple sites (E1/E2 cleft and E2 A domain) to engage more than one LA domain simultaneously. However, no single LA domain is necessary or sufficient to support efficient EEEV infection. Whereas all EEEV strains show conservation of two VLDLR-binding sites, the EEEV PE-6 strain and a few other EEE complex members feature a single amino acid substitution that enables binding of LA domains to an additional site on the E2 B domain. These structural and functional analyses informed the design of a minimal VLDLR decoy receptor that neutralizes EEEV infection and protects mice from lethal challenge.

Comprehensive Assessment of Inactivation Methods for Madariaga Virus.

The Eastern Equine Encephalitis Virus (EEEV) is an emerging public health threat, with the number of reported cases in the US increasing in recent years. EEEV is a BSL3 pathogen, and the North American strain is a US Federal Select Agent (SA). These restrictions make experiments with EEEV difficult to perform, as high-tech equipment is often unavailable in BSL3 spaces and due to concerns about generating aerosols during manipulations. Therefore, a range of inactivation methods suitable for different downstream analysis methods are essential for advancing research on EEEV. We used heat, chemical, and ultraviolet (UV)-based methods for the inactivation of infected cells and supernatants infected with the non-select agent Madariaga virus (MADV). Although the MADV and EEEV strains are genetically distinct, differing by 8-11% at the amino acid level, they are expected to be similarly susceptible to various inactivation methods. We determined the following to be effective methods of inactivation: heat, TRIzol LS, 4% PFA, 10% formalin, and UV radiation for infected supernatants; TRIzol, 2.5% SDS with BME, 0.2% NP40, 4% PFA, and 10% formalin for infected cells. Our results have the potential to expand the types and complexity of experiments and analyses performed by EEEV researchers.

Host associations of Culex panocossa (Diptera: Culicidae) in southern Florida and its implications for arbovirus transmission.

Culex panocossa, Dyar and Knab, an important enzootic vector of Venezuelan equine encephalitis virus subtype ID in Central and South America, was found to have invaded and become established in southern Florida in 2016. No information is currently available regarding the ecology of this invasive mosquito in the United States. Here, we use PCR-based blood meal analysis to investigate vertebrate host associations of Cx. panocossa from Florida to provide information necessary for determining the potential importance of this mosquito for arbovirus transmission in the United States. Culex panocossa fed mainly upon birds (49.5%) but took a substantial fraction of blood meals from mammals (33.3%) and reptiles (17.1%). By feeding upon amplifying hosts of Everglades virus (hispid cotton rat) and eastern equine encephalitis virus (wading birds) and humans, Cx. panocossa could act as a bridge vector for these pathogenic Alphaviruses in Florida, potentially resulting in increased human disease.