Structure and immunogenicity of the murine astrovirus capsid spike.

Human astroviruses (HAstVs) are small, non-enveloped icosahedral RNA viruses that are a significant cause of diarrhoea in young children. Despite their worldwide prevalence, HAstV pathogenesis studies and vaccine development remain challenging due to the lack of an animal model for HAstV infection. The recent development of a murine astrovirus (MuAstV) infection model in mice provides the opportunity to test proof-of-concept vaccines based on MuAstV antigens. To help establish a system in which an astrovirus capsid spike-based vaccine could be tested in vivo, we designed and produced a recombinant MuAstV capsid spike protein based on predicted secondary structure homology to HAstV spike proteins. The recombinant MuAstV spike can be expressed with high efficiency in Escherichia coli and retains antigenicity to polyclonal antibodies elicited by MuAstV infection. We determined the crystal structure of the MuAstV spike to 1.75 Å and assessed its structural conservation with HAstV capsid spike. Despite low sequence identity between the MuAstV and HAstV spikes and differences in their overall shapes, they share related structural folds. Additionally, we found that vaccination with MuAstV spike induced anti-MuAstV-spike antibodies, highlighting that the recombinant spike is immunogenic. These studies lay a foundation for future in vivo MuAstV challenge studies to test whether MuAstV spike can be the basis of an effective vaccine.

Structures of Two Human Astrovirus Capsid/Neutralizing Antibody Complexes Reveal Distinct Epitopes and Inhibition of Virus Attachment to Cells.

Human astrovirus is an important cause of viral gastroenteritis worldwide. Young children, the elderly, and the immunocompromised are especially at risk for contracting severe disease. However, no vaccines exist to combat human astrovirus infection. Evidence points to the importance of antibodies in protecting healthy adults from reinfection. To develop an effective subunit vaccine that broadly protects against diverse astrovirus serotypes, we must understand how neutralizing antibodies target the capsid surface at the molecular level. Here, we report the structures of the human astrovirus capsid spike domain bound to two neutralizing monoclonal antibodies. These antibodies bind two distinct conformational epitopes on the spike surface. We add to existing evidence that the human astrovirus capsid spike contains a receptor-binding domain and demonstrate that both antibodies neutralize human astrovirus by blocking virus attachment to host cells. We identify patches of conserved amino acids which overlap or border the antibody epitopes and may constitute a receptor-binding site. Our findings provide a basis for developing therapies to prevent and treat human astrovirus gastroenteritis. IMPORTANCE Human astroviruses infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. Despite the prevalence of this virus, little is known about how antibodies block astrovirus infection. Here, we determined the crystal structures of the astrovirus capsid protein in complex with two virus-neutralizing antibodies. We show that the antibodies bind to two distinct sites on the capsid spike domain, however, both antibodies block virus attachment to human cells. Importantly, our findings support the use of the human astrovirus capsid spike as an antigen in a subunit-based vaccine to prevent astrovirus disease.

Structure and Antigenicity of the Porcine Astrovirus 4 Capsid Spike.

Porcine astrovirus 4 (PoAstV4) has been recently associated with respiratory disease in pigs. In order to understand the scope of PoAstV4 infections and to support the development of a vaccine to combat PoAstV4 disease in pigs, we designed and produced a recombinant PoAstV4 capsid spike protein for use as an antigen in serological assays and for potential future use as a vaccine antigen. Structural prediction of the full-length PoAstV4 capsid protein guided the design of the recombinant PoAstV4 capsid spike domain expression plasmid. The recombinant PoAstV4 capsid spike was expressed in Escherichia coli, purified by affinity and size-exclusion chromatography, and its crystal structure was determined at 1.85 Å resolution, enabling structural comparisons to other animal and human astrovirus capsid spike structures. The recombinant PoAstV4 capsid spike protein was also used as an antigen for the successful development of a serological assay to detect PoAstV4 antibodies, demonstrating that the recombinant PoAstV4 capsid spike retains antigenic epitopes found on the native PoAstV4 capsid. These studies lay a foundation for seroprevalence studies and the development of a PoAstV4 vaccine for swine.

Discovery of three novel neutralizing antibody epitopes on the human astrovirus capsid spike and mechanistic insights into virus neutralization.

Human astroviruses (HAstVs) are a leading cause of viral childhood diarrhea that infect nearly every individual during their lifetime. Although human astroviruses are highly prevalent, no approved vaccine currently exists. Antibody responses appear to play an important role in protection from HAstV infection, however knowledge about the neutralizing epitope landscape is lacking, as only 3 neutralizing antibody epitopes have previously been determined. Here, we structurally define the epitopes of 3 uncharacterized HAstV-neutralizing monoclonal antibodies: antibody 4B6 with X-ray crystallography to 2.67 Å, and antibodies 3H4 and 3B4 simultaneously with single-particle cryogenic-electron microscopy to 3.33 Å. We assess the epitope locations relative to conserved regions on the capsid spike and find that while antibodies 4B6 and 3B4 target the upper variable loop regions of the HAstV spike protein, antibody 3H4 targets a novel region near the base of the spike that is more conserved. Additionally, we found that all 3 antibodies bind with high affinity, and they compete with receptor FcRn binding to the capsid spike. These studies inform which regions of the HAstV capsid can be targeted by monoclonal antibody therapies and could aid in rational vaccine design.

Indoleamine 2,3-dioxygenase 1 regulates cell permissivity to astrovirus infection.

Astroviruses cause a spectrum of diseases spanning asymptomatic infections to severe diarrhea, but little is understood about their pathogenesis. We previously determined that small intestinal goblet cells were the main cell type infected by murine astrovirus-1. Here, we focused on the host immune response to infection and inadvertently discovered a role for indoleamine 2,3-dioxygenase 1 (Ido1), a host tryptophan catabolizing enzyme, in the cellular tropism of murine and human astroviruses. We identified that Ido1 expression was highly enriched among infected goblet cells, and spatially corresponded to the zonation of infection. Because Ido1 can act as a negative regulator of inflammation, we hypothesized it could dampen host antiviral responses. Despite robust interferon signaling in goblet cells, as well as tuft cell and enterocyte bystanders, we observed delayed cytokine induction and suppressed levels of fecal lipocalin-2. Although we found Ido-/- animals were more resistant to infection, this was not associated with fewer goblet cells nor could it be rescued by knocking out interferon responses, suggesting that IDO1 instead regulates cell permissivity. We characterized IDO1-/- Caco-2 cells and observed significantly reduced human astrovirus-1 infection. Together this study highlights a role for Ido1 in astrovirus infection and epithelial cell maturation.

Impacts of preharvest factors during kernel development on rice quality and functionality.

Rice quality and functionality are characterized in many ways, depending largely on the industry segment using the rice. These characteristics include appearance, milling, and cooking parameters. Recently, variable quality of rice grown in the United States has been reported, but the cause was not well documented. Agronomic impacts include planting time, irrigation and fertility, cultivar selection, and harvest conditions. However, recent research suggests that ambient air temperature, specifically elevated nighttime air temperature (NTAT) during grain filling, dramatically affects the variability of rice milling quality, in terms of milled- and head-rice yields; appearance, in terms of chalkiness; and functional characteristics, including viscosity profiles, gelatinization temperatures, and proximate concentrations. Future research is needed to develop cultivars that are resistant to stress resulting from elevated NTAT during the critical period of grain filling, and, for the near term, to develop altered production management practices that mitigate elevated-temperature stress.