In Vitro and In Vivo Phenotypes of Venezuelan, Eastern and Western Equine Encephalitis Viruses Derived from cDNA Clones of Human Isolates

Abstract

The Department of Defense recently began an effort to improve and standardize virus challenge materials and efficacy determination strategies for testing therapeutics and vaccines. This includes stabilization of virus genome sequences in cDNA form where appropriate, use of human-derived virus isolates, and noninvasive strategies for determination of challenge virus replication. Eventually, it is desired that these approaches will satisfy the FDA “Animal Rule” for licensure, which substitutes animal efficacy data when human data are unlikely to be available. To this end, we created and examined the virulence phenotype of cDNA clones of prototypic human infection-derived strains of the alphaviruses, Venezuelan (VEEV INH9813), eastern (EEEV V105) and western (WEEV Fleming) equine encephalitis viruses, and created fluorescent and luminescent reporter expression vectors for evaluation of replication characteristics in vitro and in vivo. Sequences of minimally passaged isolates of each virus were used to synthesize full-length cDNA clones along with a T7 transcription promoter-based bacterial propagation vector. Viruses generated from the cDNA clones were compared with other “wild type” strains derived from cDNA clones and GenBank sequences to identify and eliminate putative tissue culture artifacts accumulated in the cell passaged biological stocks. This was followed by examination of aerosol and subcutaneous infection and disease in mouse models. A mutation that increased heparan sulfate binding was identified in the VEEV INH9813 biological isolate sequence and eliminated from the cDNA clone. Viruses derived from the new human isolate cDNA clones showed similar mouse virulence to existing clone-derived viruses after aerosol or subcutaneous inoculation.

Phagocyte-expressed glycosaminoglycans promote capture of alphaviruses from the blood circulation in a host species-specific manner

“…pretreatments of mice with heparinase. In contrast with in vitro evidence that our panel of enhanced GAG-binding viruses bind to HS ( 3 , 17 , 21 , 24 ), we found heparinase treatment did not impede the vascular clearance of all the enhanced GAG-binding alphavirus particles evaluated (Fig. 4 ).…”

“…Previous in vitro studies with our alphavirus panel identified specific E2 glycoprotein residues that promote virion interactions with HS (Table 1 ) ( 3 , 17 , 18 , 21 , 22 , 24 ). Therefore, to more definitively define the role of host HS in vivo, we assessed the effect of transient vascular HS depletion on the vascular clearance of enhanced GAG-binding alphaviruses.…”

“…1 C, D, and S1 ). These enhanced GAG-binding VEEV variants are characterized by the E2 glycoprotein mutations T120R in TC-83 (a derivative of TrD) ( 3 ) and E3K in INH9813 ( 24 ). WT EEEV FL93 is intrinsically capable of enhanced GAG-binding in vitro ( 17 , 18 ), and this enhanced GAG-binding phenotype can be abrogated by alanine substitution of E2 lysines 71, 74, and 77 (K71-74-77A) ( 17 , 18 ).…”

“…SINV-VEEV-IAB was previously described ( 68 ) and used to generate SINV-VEEV-IAB E2-T120R, also referred to as TrD (T120R), by site-directed mutagenesis with the following primers: 5′-GAGCAGGAGTGTCTGACGGAATCTTTCTTAAATTCC-3′ and 5′-GGAATTTAAGAAAGATTCCGTCAGACACTCCTGCTC-3′. The enhanced GAG-binding SINV-VEEV-IC mutant was generated based on the previously described full-length VEEV-IC recombinant virus ( 24 ) and constructed similarly as described for SINV-VEEV-IAB ( 68 ). The wildtype SINV-VEEV-IC sequence was based on the previously described full-length SINV-VEEV-IC ( 24 ) and generated by site-directed mutagenesis of the enhanced GAG-binding variant using primers 5′-GAAGAAAAAGGAGATCTACCGAGGAGCTGTTTAAGGAGTAT-3′ and 5′-ATACTCCTTAAACAGCTCCTCGGTAGATCTCCTTTTTCTTC-3′.…”

Sequelae and Animal Modeling of Encephalitic Alphavirus Infections

“…Use of these models will significantly aid in the development of detailed mechanistic studies as well as preclinical testing of vaccines and therapeutics. Finally, the virus strain specificity observed in the past with encephalitis alphavirus model infections can be overcome with the use of epidemiologically relevant alphavirus strains derived from cDNA clones to eliminate the effect of cell culture passages on virulence [ 91 , 92 ].…”