Faculty Bibliography

Soluble forms of angiotensin-converting enzyme 2 (ACE2) have recently been shown to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We report on an improved soluble ACE2, termed a "microbody," in which the ACE2 ectodomain is fused to Fc domain 3 of the immunoglobulin (Ig) heavy chain. The protein is smaller than previously described ACE2-Ig Fc fusion proteins and contains an H345A mutation in the ACE2 catalytic active site that inactivates the enzyme without reducing its affinity for the SARS-CoV-2 spike. The disulfide-bonded ACE2 microbody protein inhibits entry of SARS-CoV-2 spike protein pseudotyped virus and replication of live SARS-CoV-2 in vitro and in a mouse model. Its potency is 10-fold higher than soluble ACE2, and it can act after virus bound to the cell. The microbody inhibits the entry of β coronaviruses and virus with the variant D614G spike. The ACE2 microbody may be a valuable therapeutic for coronavirus disease 2019 (COVID-19) that is active against viral variants and future coronaviruses.

The COVID-19 pandemic remains a global threat, and host immunity remains the main mechanism of protection against the disease. The spike protein on the surface of SARS-CoV-2 is a major antigen and its engagement with human ACE2 receptor plays an essential role in viral entry into host cells. Consequently, antibodies targeting the ACE2-interacting surface (ACE2IS) located in the receptor-binding domain (RBD) of the spike protein can neutralize the virus. However, the understanding of immune responses to SARS-CoV-2 is still limited, and it is unclear how the virus protects this surface from recognition by antibodies. Here, we designed an RBD mutant that disrupts the ACE2IS and used it to characterize the prevalence of antibodies directed to the ACE2IS from convalescent sera of 94 COVID19-positive patients. We found that only a small fraction of RBD-binding antibodies targeted the ACE2IS. To assess the immunogenicity of different parts of the spike protein, we performed in vitro antibody selection for the spike and the RBD proteins using both unbiased and biased selection strategies. Intriguingly, unbiased selection yielded antibodies that predominantly targeted regions outside the ACE2IS, whereas ACE2IS-binding antibodies were readily identified from biased selection designed to enrich such antibodies. Furthermore, antibodies from an unbiased selection using the RBD preferentially bound to the surfaces that are inaccessible in the context of whole spike protein. These results suggest that the ACE2IS has evolved less immunogenic than the other regions of the spike protein, which has important implications in the development of vaccines against SARS-CoV-2.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus known to cause epidemics resulting in predominantly symptomatic infections, which in rare cases cause long term debilitating arthritis and arthralgia. Significant progress has been made in understanding the roles of canonical RNA sensing pathways in the host recognition of CHIKV; however, less is known regarding antagonism of CHIKV by cytosolic DNA sensing pathways like that of cyclic GMP-AMP synthase (cGAS) and Stimulator of Interferon Genes (STING). With the use of cGAS or STING null cells we demonstrate that the pathway restricts CHIKV replication in fibroblasts and immune cells. We show that DNA accumulates in the cytoplasm of infected cells and that CHIKV blocks DNA dependent IFN-β transcription. This antagonism of DNA sensing is via an early autophagy-mediated degradation of cGAS and expression of the CHIKV capsid protein is sufficient to induce cGAS degradation. Furthermore, we identify an interaction of CHIKV nsP1 with STING and map the interaction to 23 residues in the cytosolic loop of the adaptor protein. This interaction stabilizes the viral protein and increases the level of palmitoylated nsP1 in cells. Together, this work supports previous publications highlighting the relevance of the cGAS-STING pathway in the early detection of (+)ssRNA viruses and provides direct evidence that CHIKV interacts with and antagonizes cGAS-STING signaling.

The fundamental basis of how arboviruses evolve in nature and what regulates the adaptive process remain unclear. To address this problem, we established a Zika virus (ZIKV) vector-borne transmission system in immunocompromised mice to study the evolutionary characteristics of ZIKV infection. Using this system, we defined factors that influence the evolutionary landscape of ZIKV infection and show that transmission route and specific organ microenvironments impact viral diversity and defective viral genome production. In addition, we identified in mice the emergence of ZIKV mutants previously seen in natural infections, including variants present in currently circulating Asian and American strains, as well as mutations unique to the mouse infections. With these studies, we have established an insect-to-mouse transmission model to study ZIKV evolution in vivo. We also defined how organ microenvironments and infection route impact the ZIKV evolutionary landscape, providing a deeper understanding of the factors that regulate arbovirus evolution and emergence.

In an increasingly interconnected world, the exposure and subsequent spread of emergent viruses has become inevitable. This is particularly true for Aedes (Ae.) mosquito-vectored viruses, whose range has increased over the past decade from tropical to temperate regions. However, it is unclear if all populations of Ae. mosquitoes in temperate New York City are able to successfully replicate and transmit arboviruses. To answer this question, we reared Ae. albopictus mosquitoes living in a temperate climate from three locations in New York City. We first sequenced the salivary antiviral protein D7 from individual mosquitoes in each population and found single nucleotide variants that are both shared and unique for each Ae. albopictus population. We then fed each population chikungunya virus (CHIKV) via an artificial blood meal. All three mosquito populations could be infected with CHIKV, yet viral titers differed between populations at 7 days post infection. Moreover, we found that these mosquitoes could transmit CHIKV to mice, and that virus RNA reached the saliva as early as two days post infection. Upon sequencing of the saliva CHIKV genomic RNA, we found mutations at sites correlated with increased transmission and virulence. These studies show that NYC Ae. albopictus populations can be infected with and transmit CHIKV, CHIKV is able to evolve in these mosquitoes, and that host salivary factors display population-specific diversity. Taken together, these studies highlight the need to study how distinct mosquito populations control viral infections, both at the virus and host level.

At the close of this Special Issue of Viruses on the Transmission Dynamics of Insect Viruses, we would like to thank all of the authors for their submissions and the great work expanding our knowledge of insect virus biology and transmission [...].

Chikungunya virus (CHIKV) is a reemerged arbovirus, a member of the Togaviridae family. It circulates through mosquito vectors mainly of the Aedes family and a mammalian host. CHIKV causes chikungunya fever, a mild to severe disease characterized by arthralgia, with some fatal outcomes described. In the past years, several outbreaks mainly caused by enhanced adaptation of the virus to the vector and ineffective control of the contacts between infected mosquito populations and the human host have been reported. Vaccines represent the best solution for the control of insect-borne viruses, including CHIKV, but are often unavailable. We designed live attenuated CHIKVs by applying a rational genomic design based on multiple replacements of synonymous codons. In doing so, the virus mutational robustness (capacity to maintain phenotype despite introduction of mutations to genotype) is decreased, driving the viral population toward deleterious evolutionary trajectories. When the candidate viruses were tested in the insect and mammalian hosts, we observed overall strong attenuation in both and greatly diminished signs of disease. Moreover, we found that the vaccine candidates elicited protective immunity related to the production of neutralizing antibodies after a single dose. During an experimental transmission cycle between mosquitoes and naive mice, vaccine candidates could be transmitted by mosquito bite, leading to asymptomatic infection in mice with compromised dissemination. Using deep-sequencing technology, we observed an increase in detrimental (stop) codons, which confirmed the effectiveness of this genomic design. Because the approach involves hundreds of synonymous modifications to the genome, the reversion risk is significantly reduced, rendering the viruses promising vaccine candidates.IMPORTANCE Chikungunya fever is a debilitating disease that causes severe pain to the joints, which can compromise the patient's lifestyle for several months and even in some grave cases lead to death. The etiological agent is chikungunya virus, an alphavirus transmitted by mosquito bite. Currently, there are no approved vaccines or treatments against the disease. In our research, we developed novel live attenuated vaccine candidates against chikungunya virus by applying an innovative genomic design. When tested in the insect and mammalian host, the vaccine candidates did not cause disease, elicited strong protection against further infection, and had low risk of reversion to pathogenic phenotypes.

Understanding the fundamental mechanisms of arbovirus transmission and pathogenesis is essential to develop strategies for treatment and prevention. We previously took an in vivo evolution-based approach and identified the chikungunya virus E1 glycoprotein residue 80 to play a critical role in viral transmission and pathogenesis. In this study, we address the genetic conservation and function of position 80 and demonstrate that this residue is a key determinant in alphavirus infectivity and dissemination through modulation of viral fusion and cholesterol dependence. In addition, in studying the evolution of position 80, we identified a network of glycoprotein residues, including epidemic determinants, that regulate virus dissemination and infectivity. These studies underscore the importance of taking evolution-based approaches to not only identify key viral determinants driving arbovirus transmission and pathogenesis but also to uncover fundamental aspects of arbovirus biology.