Antibodies generated against West Nile virus (WNV) during infection are essential for controlling dissemination. Recent studies have demonstrated that epitopes in all three domains of the flavivirus envelope protein (E) are targets for neutralizing antibodies, with determinants in domain III (DIII) eliciting antibodies with strong inhibitory properties. In order to increase the magnitude and quality of the antibody response against the WNV E protein, DNA vaccines with derivatives of the WNV E gene (full length E, truncated E, or DIII region, some in the context of the pre-membrane [prM] gene) were conjugated to the molecular adjuvant P28. The P28 region of the complement protein C3d is the minimum CR2-binding domain necessary for the adjuvant activity of C3d. Delivery of DNA-based vaccines by gene gun and intramuscular routes stimulated production of IgG antibodies against the WNV DIII region of the E protein. With the exception of the vaccine expressing prM/E given intramuscularly, only mice that received DNA vaccines by gene gun produced protective neutralizing antibody titers (FRNT₈₀ titer >1/40). Correspondingly, mice vaccinated by the gene gun route were protected to a greater level from lethal WNV challenge. In general, mice vaccinated with P28-adjuvated vaccines produced higher IgG titers than mice vaccinated with non-adjuvanted vaccines.

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.

SARS-CoV-2, the causative agent of COVID-19, emerged at the end of 2019 and by mid-June 2020, the virus has spread to at least 215 countries, caused more than 8,000,000 confirmed infections and over 450,000 deaths, and overwhelmed healthcare systems worldwide. Like SARS-CoV, which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low passage (P) strains of SARS-CoV-2 (Wash1: P4 and Munich: P1) were cultured twice in Vero-E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1: P6 and minor variants in the Munich: P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero-E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies we investigated the development of neutralizing antibody titers in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

Eastern equine encephalitis virus (EEEV), a mosquito-borne RNA virus, is one of the most acutely virulent viruses endemic to the Americas, causing between 30% and 70% mortality in symptomatic human cases. A major factor in the virulence of EEEV is the presence of four binding sites for the hematopoietic cell-specific microRNA, miR-142-3p, in the 3’ untranslated region (3’ UTR) of the virus. Three of the sites are “canonical” with all 8 seed sequence residues complimentary to miR-142-3p while one is “non-canonical” and has a seed sequence mismatch. Interaction of the EEEV genome with miR-142-3p limits virus replication in myeloid cells and suppresses the systemic innate immune response, greatly exacerbating EEEV neurovirulence. The presence of the miRNA binding sequences is also required for efficient EEEV replication in mosquitoes and, therefore, essential for transmission of the virus. In the current studies, we have examined the role of each binding site by point mutagenesis of seed sequences in all combinations of sites followed by infection of mammalian myeloid cells, mosquito cells and mice. The resulting data indicate that both canonical and non-canonical sites contribute to cell infection and animal virulence, however, surprisingly, all sites are rapidly deleted from EEEV genomes shortly after infection of myeloid cells or mice. Finally, we show that the virulence of a related encephalitis virus, western equine encephalitis virus, is also dependent upon miR-142-3p binding sites. Author Summary: Eastern equine encephalitis virus (EEEV) is one of the most acutely virulent mosquito-borne viruses in the Americas. A major determinant of EEEV virulence is a mammalian microRNA (miRNA) that is primarily expressed in myeloid cells, miR-142-3p. Like miRNA suppression of host mRNA, miR-142-3p binds to the 3’ untranslated region (UTR) of the EEEV genome only in myeloid cells suppressing virus replication and the induction of the innate immune response. In this study, we used point mutations in all four miR-142-3p binding sites in the EEEV 3’ UTR to understand the mechanism behind this miRNA suppression. We observed that decreasing the number of miR-142-3p binding sites leads to virus escape and ultimately attenuation in vivo . Furthermore, another virus, western equine encephalitis virus, also encodes miR-142-3p binding sites that contribute to virulence in vivo . These results provide insight into the mechanism of how cell-specific miRNAs can mediate suppression of virus replication.