Outbreaks from zoonotic sources represent a threat to both human disease as well as the global economy. Despite a wealth of metagenomics studies, methods to leverage these datasets to identify future threats are underdeveloped. In this study, we describe an approach that combines existing metagenomics data with reverse genetics to engineer reagents to evaluate emergence and pathogenic potential of circulating zoonotic viruses. Focusing on the severe acute respiratory syndrome (SARS)-like viruses, the results indicate that the WIV1-coronavirus (CoV) cluster has the ability to directly infect and may undergo limited transmission in human populations. However, in vivo attenuation suggests additional adaptation is required for epidemic disease. Importantly, available SARS monoclonal antibodies offered success in limiting viral infection absent from available vaccine approaches. Together, the data highlight the utility of a platform to identify and prioritize prepandemic strains harbored in animal reservoirs and document the threat posed by WIV1-CoV for emergence in human populations.A lthough previously associated with upper respiratory infections, the emergence of severe acute respiratory coronavirus (SARS-CoV) in [2002][2003], and more recently, Middle East respiratory syndrome (MERS)-CoV underscores the threat of cross-species transmission leading to virulent pandemic viral infections (1, 2). Whereas prevailing research suggests that SARSCoV emerged from viruses in the Chinese horseshoe bat, identifying a progenitor strain that used human angiotensin converting enzyme 2 (ACE2) had proven elusive (3, 4). However, recent metagenomics studies isolated several SARS-like virus sequences that share ≥90% genome-wide homology and represented the closest sequences to the epidemic strains (5, 6). Importantly, researchers also isolated replication competent virus; WIV1-CoV, part of the Rs3306 cluster, could use ACE2 orthologs and mediated low-level replication in human cells (5). Overall, the evidence indicates that SARS-CoV likely emerged from Chinese horseshoe bats and that similar viruses are still harbored in these populations.The identification of WIV1-CoV and its capacity to use ACE2 orthologs offers a warning for possible reemergence and provides an opportunity to prepare for a future CoV outbreak. To achieve this goal, a new platform is required to translate metagenomics findings; the approach must generate critical diagnostic reagents, define emergence potential of novel strains, and determine efficacy of current therapeutics. Building on this premise, we developed a framework to examine circulating CoVs using reverse genetic systems to construct full-length and chimeric viruses. The results indicate that viruses using WIV1-CoV spike are poised to emerge in human populations due to efficient replication in primary human airway epithelial cell cultures. However, additional adaptation, potentially independent of the spike protein receptor-binding domain, is required for pathogenesis and epidemic disease. Importantly,...

Significance Dengue virus is the most important arthropod-borne viral disease of humans worldwide, with an estimated 390 million acute infections annually. The best means to control this global health threat is a vaccine, but dengue vaccine development has progressed slowly, partly because the antigenic targets required to stimulate long-term immunity are not well-defined. Here, we show a specific region on the viral surface (the envelope domain I/II hinge) that is the target of protective antibodies after primary human infections. These results are critically important for dengue vaccine design, because we hypothesize that a successful dengue vaccine will stimulate antibodies that target this region. More broadly, this study establishes a template for similar approaches for improving vaccines for influenza, HIV, hepatitis C virus, and other clinically important viral pathogens.

The four dengue virus (DENV) serotypes, DENV1 through 4, are endemic throughout tropical and subtropical regions of the world. While first infection confers long-term protective immunity against viruses of the infecting serotype, a second infection with virus of a different serotype carries a greater risk of severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. Recent studies demonstrate that humans exposed to DENV infections develop neutralizing antibodies that bind to quaternary epitopes formed by the viral envelope (E) protein dimers or higher-order assemblies required for the formation of the icosahedral viral envelope. Here we show that the quaternary epitope target of the human DENV3-specific neutralizing monoclonal antibody (MAb) 5J7 can be partially transplanted into a DENV1 strain by changing the core residues of the epitope contained within a single monomeric E molecule. MAb 5J7 neutralized the recombinant DENV1/3 strain in cell culture and was protective in a mouse model of infection with the DENV1/3 strain. However, the 5J7 epitope was only partially recreated by transplantation of the core residues because MAb 5J7 bound and neutralized wild-type (WT) DENV3 better than the DENV1/3 recombinant. Our studies demonstrate that it is possible to transplant a large number of discontinuous residues between DENV serotypes and partially recreate a complex antibody epitope, while retaining virus viability. Further refinement of this approach may lead to new tools for measuring epitopespecific antibody responses and new vaccine platforms. IMPORTANCEDengue virus is the most important mosquito-borne pathogen of humans worldwide, with approximately one-half the world's population living in regions where dengue is endemic. Dengue immunity following infection is robust and thought to be conferred by antibodies raised against the infecting virus. However, the specific viral components that these antibodies recognize and how they neutralize the virus have been incompletely described. Here we map a region on dengue virus serotype 3 recognized by the human neutralizing antibody 5J7 and then test the functional significance of this region by transplanting it into a serotype 1 virus. Our studies demonstrate a region on dengue virus necessary for 5J7 binding and neutralization. Our work also demonstrates the technical feasibility of engineering dengue viruses to display targets of protective antibodies. This technology can be used to develop new dengue vaccines and diagnostic assays.T he four dengue virus serotypes (DENV1 to DENV4), transmitted by Aedes species mosquitoes, are endemic throughout tropical and subtropical regions of the world (1). Primary infection with virus of one serotype confers long-term immunity against viruses of that serotype, but subsequent infection with virus of a different serotype results in an increased risk of potentially fatal severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome (2-4). This risk has been attributed, at least...

Dengue vaccine trials have revealed deficits in our understanding of the mechanisms of protective immunity, demonstrating a need to measure epitope-specific antibody responses against each DENV serotype. HmAb 5J7 binds to a complex, 3-monomer spanning quaternary epitope in the DENV3 envelope (E) protein, but it is unclear whether all interactions are needed for neutralization. Structure guided design and reverse genetics were used to sequentially transplant larger portions of the DENV3-specific 5J7 mAb epitope into dengue virus serotype 4 (DENV4). We observed complete binding and neutralization only when the entire 3 monomer spanning epitope was transplanted into DENV4, providing empirical proof that cooperative monomer-hmAb 5J7 interactions maximize activity. The rDENV4/3 virus containing the most expanded 5J7 epitope was also significantly more sensitive than WT DENV4 to neutralization by DENV3 primary immune sera. We conclude that the hinge-spanning region of the 5J7 quaternary epitope is a target for serotype-specific neutralizing antibodies after DENV3 infection.

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