Porcine Sapovirus Protease Controls the Innate Immune Response and Targets TBK1.

Human sapoviruses (HuSaVs) and noroviruses are considered the leading cause of acute gastroenteritis worldwide. While extensive research has focused on noroviruses, our understanding of sapoviruses (SaVs) and their interactions with the host's immune response remains limited. HuSaVs have been challenging to propagate in vitro, making the porcine sapovirus (PSaV) Cowden strain a valuable model for studying SaV pathogenesis. In this study we show, for the first time, that PSaV Cowden strain has mechanisms to evade the host's innate immune response. The virus 3C-like protease (NS6) inhibits type I IFN production by targeting TBK1. Catalytically active NS6, both during ectopic expression and during PSaV infection, targets TBK1 which is then led for rapid degradation by the proteasome. Moreover, deletion of TBK1 from porcine cells led to an increase in PSaV titres, emphasizing its role in regulating PSaV infection. Additionally, we successfully established PSaV infection in IPEC-J2 cells, an enterocytic cell line originating from the jejunum of a neonatal piglet. Overall, this study provides novel insights into PSaV evasion strategies, opening the way for future investigations into SaV-host interactions, and enabling the use of a new cell line model for PSaV research.

An optimized protocol for the extraction and quantification of cytosolic DNA in mammalian cells.

Leakage of mitochondrial or nuclear DNA into the cytosol can occur following viral infections, radiation damage, and some cancers. Here, we present an optimized protocol for isolating and quantifying cytosolic DNA from mammalian cells. We describe steps for collecting cytosolic fractions from cells, extracting DNA using columns, and quantifying extracted DNA using qPCR. This straightforward protocol can be completed in as little as 5 hours, and allows for the identification of the source of DNA. For complete details on the use and execution of this protocol, please refer to Jahun et al.1.

Leaked genomic and mitochondrial DNA contribute to the host response to noroviruses in a STING-dependent manner.

The cGAS-STING pathway is central to the interferon response against DNA viruses. However, recent studies are increasingly demonstrating its role in the restriction of some RNA viruses. Here, we show that the cGAS-STING pathway also contributes to the interferon response against noroviruses, currently the commonest causes of infectious gastroenteritis worldwide. We show a significant reduction in interferon-ß induction and a corresponding increase in viral replication in norovirus-infected cells after deletion of STING, cGAS, or IFI16. Further, we find that immunostimulatory host genome-derived DNA and mitochondrial DNA accumulate in the cytosol of norovirus-infected cells. Lastly, overexpression of the viral NS4 protein is sufficient to drive the accumulation of cytosolic DNA. Together, our data find a role for cGAS, IFI16, and STING in the restriction of noroviruses and show the utility of host genomic DNA as a damage-associated molecular pattern in cells infected with an RNA virus.

Microfluidic antibody profiling after repeated SARS-CoV-2 vaccination links antibody affinity and concentration to impaired immunity and variant escape in patients on anti-CD20 therapy.

Background: Patients with autoimmune/inflammatory conditions on anti-CD20 therapies, such as rituximab, have suboptimal humoral responses to vaccination and are vulnerable to poorer clinical outcomes following SARS-CoV-2 infection. We aimed to examine how the fundamental parameters of antibody responses, namely, affinity and concentration, shape the quality of humoral immunity after vaccination in these patients. Methods: We performed in-depth antibody characterisation in sera collected 4 to 6 weeks after each of three vaccine doses to wild-type (WT) SARS-CoV-2 in rituximab-treated primary vasculitis patients (n = 14) using Luminex and pseudovirus neutralisation assays, whereas we used a novel microfluidic-based immunoassay to quantify polyclonal antibody affinity and concentration against both WT and Omicron (B.1.1.529) variants. We performed comparative antibody profiling at equivalent timepoints in healthy individuals after three antigenic exposures to WT SARS-CoV-2 (one infection and two vaccinations; n = 15) and in convalescent patients after WT SARS-CoV-2 infection (n = 30). Results: Rituximab-treated patients had lower antibody levels and neutralisation titres against both WT and Omicron SARS-CoV-2 variants compared to healthy individuals. Neutralisation capacity was weaker against Omicron versus WT both in rituximab-treated patients and in healthy individuals. In the rituximab cohort, this was driven by lower antibody affinity against Omicron versus WT [median (range) KD: 21.6 (9.7-38.8) nM vs. 4.6 (2.3-44.8) nM, p = 0.0004]. By contrast, healthy individuals with hybrid immunity produced a broader antibody response, a subset of which recognised Omicron with higher affinity than antibodies in rituximab-treated patients [median (range) KD: 1.05 (0.45-1.84) nM vs. 20.25 (13.2-38.8) nM, p = 0.0002], underpinning the stronger serum neutralisation capacity against Omicron in the former group. Rituximab-treated patients had similar anti-WT antibody levels and neutralisation titres to unvaccinated convalescent individuals, despite two more exposures to SARS-CoV-2 antigen. Temporal profiling of the antibody response showed evidence of affinity maturation in healthy convalescent patients after a single SARS-CoV-2 infection, which was not observed in rituximab-treated patients, despite repeated vaccination. Discussion: Our results enrich previous observations of impaired humoral immune responses to SARS-CoV-2 in rituximab-treated patients and highlight the significance of quantitative assessment of serum antibody affinity and concentration in monitoring anti-viral immunity, viral escape, and the evolution of the humoral response.

Improving the efficiency and effectiveness of an industrial SARS-CoV-2 diagnostic facility.

On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories.

Genomic epidemiology of SARS-CoV-2 in a UK university identifies dynamics of transmission.

Understanding SARS-CoV-2 transmission in higher education settings is important to limit spread between students, and into at-risk populations. In this study, we sequenced 482 SARS-CoV-2 isolates from the University of Cambridge from 5 October to 6 December 2020. We perform a detailed phylogenetic comparison with 972 isolates from the surrounding community, complemented with epidemiological and contact tracing data, to determine transmission dynamics. We observe limited viral introductions into the university; the majority of student cases were linked to a single genetic cluster, likely following social gatherings at a venue outside the university. We identify considerable onward transmission associated with student accommodation and courses; this was effectively contained using local infection control measures and following a national lockdown. Transmission clusters were largely segregated within the university or the community. Our study highlights key determinants of SARS-CoV-2 transmission and effective interventions in a higher education setting that will inform public health policy during pandemics.

B cell receptor repertoire kinetics after SARS-CoV-2 infection and vaccination.

B cells are important in immunity to both severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and vaccination, but B cell receptor (BCR) repertoire development in these contexts has not been compared. We analyze serial samples from 171 SARS-CoV-2-infected individuals and 63 vaccine recipients and find the global BCR repertoire differs between them. Following infection, immunoglobulin (Ig)G1/3 and IgA1 BCRs increase, somatic hypermutation (SHM) decreases, and, in severe disease, IgM and IgA clones are expanded. In contrast, after vaccination, the proportion of IgD/M BCRs increase, SHM is unchanged, and expansion of IgG clones is prominent. VH1-24, which targets the N-terminal domain (NTD) and contributes to neutralization, is expanded post infection except in the most severe disease. Infection generates a broad distribution of SARS-CoV-2-specific clones predicted to target the spike protein, while a more focused response after vaccination mainly targets the spike's receptor-binding domain. Thus, the nature of SARS-CoV-2 exposure differentially affects BCR repertoire development, potentially informing vaccine strategies.

Evolution of enhanced innate immune evasion by SARS-CoV-2.

The emergence of SARS-CoV-2 variants of concern suggests viral adaptation to enhance human-to-human transmission1,2. Although much effort has focused on the characterization of changes in the spike protein in variants of concern, mutations outside of spike are likely to contribute to adaptation. Here, using unbiased abundance proteomics, phosphoproteomics, RNA sequencing and viral replication assays, we show that isolates of the Alpha (B.1.1.7) variant3 suppress innate immune responses in airway epithelial cells more effectively than first-wave isolates. We found that the Alpha variant has markedly increased subgenomic RNA and protein levels of the nucleocapsid protein (N), Orf9b and Orf6-all known innate immune antagonists. Expression of Orf9b alone suppressed the innate immune response through interaction with TOM70, a mitochondrial protein that is required for activation of the RNA-sensing adaptor MAVS. Moreover, the activity of Orf9b and its association with TOM70 was regulated by phosphorylation. We propose that more effective innate immune suppression, through enhanced expression of specific viral antagonist proteins, increases the likelihood of successful transmission of the Alpha variant, and may increase in vivo replication and duration of infection4. The importance of mutations outside the spike coding region in the adaptation of SARS-CoV-2 to humans is underscored by the observation that similar mutations exist in the N and Orf9b regulatory regions of the Delta and Omicron variants.

Interferon responses to norovirus infections: current and future perspectives.

Human noroviruses (HuNoVs) are increasingly becoming the main cause of transmissible gastroenteritis worldwide, with hundreds of thousands of deaths recorded annually. Yet, decades after their discovery, there is still no effective treatment or vaccine. Efforts aimed at developing vaccines or treatment will benefit from a greater understanding of norovirus-host interactions, including the host response to infection. In this review, we provide a concise overview of the evidence establishing the significance of type I and type III interferon (IFN) responses in the restriction of noroviruses. We also critically examine our current understanding of the molecular mechanisms of IFN induction in norovirus-infected cells, and outline the diverse strategies deployed by noroviruses to supress and/or avoid host IFN responses. It is our hope that this review will facilitate further discussion and increase interest in this area.

Murine norovirus virulence factor 1 (VF1) protein contributes to viral fitness during persistent infection.

Murine norovirus (MNV) is widely used as a model for studying norovirus biology. While MNV isolates vary in their pathogenesis, infection of immunocompetent mice mostly results in persistent infection. The ability of a virus to establish a persistent infection is dependent on its ability to subvert or avoid the host immune response. Previously, we described the identification and characterization of virulence factor 1 (VF1) in MNV, and demonstrated its role as an innate immune antagonist. Here, we explore the role of VF1 during persistent MNV infection in an immunocompetent host. Using reverse genetics, we generated MNV-3 viruses carrying a single or a triple termination codon inserted in the VF1 ORF. VF1-deleted MNV-3 replicated to comparable levels to the wildtype virus in tissue culture. Comparative studies between MNV-3 and an acute MNV-1 strain show that MNV-3 VF1 exerts the same functions as MNV-1 VF1, but with reduced potency. C57BL/6 mice infected with VF1-deleted MNV-3 showed significantly reduced replication kinetics during the acute phase of the infection, but viral loads rapidly reached the levels seen in mice infected with wildtype virus after phenotypic restoration of VF1 expression. Infection with an MNV-3 mutant that had three termination codons inserted into VF1, in which reversion was suppressed, resulted in consistently lower replication throughout a 3 month persistent infection in mice, suggesting a role for VF1 in viral fitness in vivo. Our results indicate that VF1 expressed by a persistent strain of MNV also functions to antagonize the innate response to infection. We found that VF1 is not essential for viral persistence, but instead contributes to viral fitness in mice. These data fit with the hypothesis that noroviruses utilize multiple mechanisms to avoid and/or control the host response to infection and that VF1 is just one component of this.

Murine Norovirus Infection Results in Anti-inflammatory Response Downstream of Amino Acid Depletion in Macrophages.

Murine norovirus (MNV) infection results in a late translation shutoff that is proposed to contribute to the attenuated and delayed innate immune response observed both in vitro and in vivo. Recently, we further demonstrated the activation of the α subunit of eukaryotic initiation factor 2 (eIF2α) kinase GCN2 during MNV infection, which has been previously linked to immunomodulation and resistance to inflammatory signaling during metabolic stress. While viral infection is usually associated with activation of double-stranded RNA (dsRNA) binding pattern recognition receptor PKR, we hypothesized that the establishment of a metabolic stress in infected cells is a proviral event, exploited by MNV to promote replication through weakening the activation of the innate immune response. In this study, we used multi-omics approaches to characterize cellular responses during MNV replication. We demonstrate the activation of pathways related to the integrated stress response, a known driver of anti-inflammatory phenotypes in macrophages. In particular, MNV infection causes an amino acid imbalance that is associated with GCN2 and ATF2 signaling. Importantly, this reprogramming lacks the features of a typical innate immune response, with the ATF/CHOP target GDF15 contributing to the lack of antiviral responses. We propose that MNV-induced metabolic stress supports the establishment of host tolerance to viral replication and propagation. IMPORTANCE During viral infection, host defenses are typically characterized by the secretion of proinflammatory autocrine and paracrine cytokines, potentiation of the interferon (IFN) response, and induction of the antiviral response via activation of JAK and Stat signaling. To avoid these and propagate, viruses have evolved strategies to evade or counteract host sensing. In this study, we demonstrate that murine norovirus controls the antiviral response by activating a metabolic stress response that activates the amino acid response and impairs inflammatory signaling. This highlights novel tools in the viral countermeasures arsenal and demonstrates the importance of the currently poorly understood metabolic reprogramming occurring during viral infections.

Patterns of within-host genetic diversity in SARS-CoV-2.

Monitoring the spread of SARS-CoV-2 and reconstructing transmission chains has become a major public health focus for many governments around the world. The modest mutation rate and rapid transmission of SARS-CoV-2 prevents the reconstruction of transmission chains from consensus genome sequences, but within-host genetic diversity could theoretically help identify close contacts. Here we describe the patterns of within-host diversity in 1181 SARS-CoV-2 samples sequenced to high depth in duplicate. 95.1% of samples show within-host mutations at detectable allele frequencies. Analyses of the mutational spectra revealed strong strand asymmetries suggestive of damage or RNA editing of the plus strand, rather than replication errors, dominating the accumulation of mutations during the SARS-CoV-2 pandemic. Within- and between-host diversity show strong purifying selection, particularly against nonsense mutations. Recurrent within-host mutations, many of which coincide with known phylogenetic homoplasies, display a spectrum and patterns of purifying selection more suggestive of mutational hotspots than recombination or convergent evolution. While allele frequencies suggest that most samples result from infection by a single lineage, we identify multiple putative examples of co-infection. Integrating these results into an epidemiological inference framework, we find that while sharing of within-host variants between samples could help the reconstruction of transmission chains, mutational hotspots and rare cases of superinfection can confound these analyses.

The COVID-19 pandemic has had major health impacts across the globe. The scientific community has focused much attention on finding ways to monitor how the virus responsible for the pandemic, SARS-CoV-2, spreads. One option is to perform genetic tests, known as sequencing, on SARS-CoV-2 samples to determine the genetic code of the virus and to find any differences or mutations in the genes between the viral samples. Viruses mutate within their hosts and can develop into variants that are able to more easily transmit between hosts. Genetic sequencing can reveal how genetically similar two SARS-CoV-2 samples are. But tracking how SARS-CoV-2 moves from one person to the next through sequencing can be tricky. Even a sample of SARS-CoV-2 viruses from the same individual can display differences in their genetic material or within-host variants. Could genetic testing of within-host variants shed light on factors driving SARS-CoV-2 to evolve in humans? To get to the bottom of this, Tonkin-Hill, Martincorena et al. probed the genetics of SARS-CoV-2 within-host variants using 1,181 samples. The analyses revealed that 95.1% of samples contained within-host variants. A number of variants occurred frequently in many samples, which were consistent with mutational hotspots in the SARS-CoV-2 genome. In addition, within-host variants displayed mutation patterns that were similar to patterns found between infected individuals. The shared within-host variants between samples can help to reconstruct transmission chains. However, the observed mutational hotspots and the detection of multiple strains within an individual can make this challenging. These findings could be used to help predict how SARS-CoV-2 evolves in response to interventions such as vaccines. They also suggest that caution is needed when using information on within-host variants to determine transmission between individuals.

Superspreaders drive the largest outbreaks of hospital onset COVID-19 infections.

SARS-CoV-2 is notable both for its rapid spread, and for the heterogeneity of its patterns of transmission, with multiple published incidences of superspreading behaviour. Here, we applied a novel network reconstruction algorithm to infer patterns of viral transmission occurring between patients and health care workers (HCWs) in the largest clusters of COVID-19 infection identified during the first wave of the epidemic at Cambridge University Hospitals NHS Foundation Trust, UK. Based upon dates of individuals reporting symptoms, recorded individual locations, and viral genome sequence data, we show an uneven pattern of transmission between individuals, with patients being much more likely to be infected by other patients than by HCWs. Further, the data were consistent with a pattern of superspreading, whereby 21% of individuals caused 80% of transmission events. Our study provides a detailed retrospective analysis of nosocomial SARS-CoV-2 transmission, and sheds light on the need for intensive and pervasive infection control procedures.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, presents a global public health challenge. Hospitals have been at the forefront of this battle, treating large numbers of sick patients over several waves of infection. Finding ways to manage the spread of the virus in hospitals is key to protecting vulnerable patients and workers, while keeping hospitals running, but to generate effective infection control, researchers must understand how SARS-CoV-2 spreads. A range of factors make studying the transmission of SARS-CoV-2 in hospitals tricky. For instance, some people do not present any symptoms, and, amongst those who do, it can be difficult to determine whether they caught the virus in the hospital or somewhere else. However, comparing the genetic information of the SARS-CoV-2 virus from different people in a hospital could allow scientists to understand how it spreads. Samples of the genetic material of SARS-CoV-2 can be obtained by swabbing infected individuals. If the genetic sequences of two samples are very different, it is unlikely that the individuals who provided the samples transmitted the virus to one another. Illingworth, Hamilton et al. used this information, along with other data about how SARS-CoV-2 is transmitted, to develop an algorithm that can determine how the virus spreads from person to person in different hospital wards. To build their algorithm, Illingworth, Hamilton et al. collected SARS-CoV-2 genetic data from patients and staff in a hospital, and combined it with information about how SARS-CoV-2 spreads and how these people moved in the hospital . The algorithm showed that, for the most part, patients were infected by other patients (20 out of 22 cases), while staff were infected equally by patients and staff. By further probing these data, Illingworth, Hamilton et al. revealed that 80% of hospital-acquired infections were caused by a group of just 21% of individuals in the study, identifying a 'superspreader' pattern. These findings may help to inform SARS-CoV-2 infection control measures to reduce spread within hospitals, and could potentially be used to improve infection control in other contexts.

Evolution of enhanced innate immune evasion by the SARS-CoV-2 B.1.1.7 UK variant.

Emergence of SARS-CoV-2 variants, including the globally successful B.1.1.7 lineage, suggests viral adaptations to host selective pressures resulting in more efficient transmission. Although much effort has focused on Spike adaptation for viral entry and adaptive immune escape, B.1.1.7 mutations outside Spike likely contribute to enhance transmission. Here we used unbiased abundance proteomics, phosphoproteomics, mRNA sequencing and viral replication assays to show that B.1.1.7 isolates more effectively suppress host innate immune responses in airway epithelial cells. We found that B.1.1.7 isolates have dramatically increased subgenomic RNA and protein levels of Orf9b and Orf6, both known innate immune antagonists. Expression of Orf9b alone suppressed the innate immune response through interaction with TOM70, a mitochondrial protein required for RNA sensing adaptor MAVS activation, and Orf9b binding and activity was regulated via phosphorylation. We conclude that B.1.1.7 has evolved beyond the Spike coding region to more effectively antagonise host innate immune responses through upregulation of specific subgenomic RNA synthesis and increased protein expression of key innate immune antagonists. We propose that more effective innate immune antagonism increases the likelihood of successful B.1.1.7 transmission, and may increase in vivo replication and duration of infection.

Longitudinal analysis reveals that delayed bystander CD8+ T cell activation and early immune pathology distinguish severe COVID-19 from mild disease.

The kinetics of the immune changes in COVID-19 across severity groups have not been rigorously assessed. Using immunophenotyping, RNA sequencing, and serum cytokine analysis, we analyzed serial samples from 207 SARS-CoV2-infected individuals with a range of disease severities over 12 weeks from symptom onset. An early robust bystander CD8+ T cell immune response, without systemic inflammation, characterized asymptomatic or mild disease. Hospitalized individuals had delayed bystander responses and systemic inflammation that was already evident near symptom onset, indicating that immunopathology may be inevitable in some individuals. Viral load did not correlate with this early pathological response but did correlate with subsequent disease severity. Immune recovery is complex, with profound persistent cellular abnormalities in severe disease correlating with altered inflammatory responses, with signatures associated with increased oxidative phosphorylation replacing those driven by cytokines tumor necrosis factor (TNF) and interleukin (IL)-6. These late immunometabolic and immune defects may have clinical implications.

A luciferase-based approach for measuring HBGA blockade antibody titers against human norovirus.

BACKGROUND: Noroviruses are the most common cause of viral gastroenteritis worldwide, yet there is a deficit in the understanding of protective immunity. Surrogate neutralization assays have been widely used that measure the ability of antibodies to block virus-like particle (VLP) binding to histo-blood group antigens (HBGAs). However, screening large sample sets against multiple antigens using the traditional HBGA blocking assay requires significant investment in terms of time, equipment, and technical expertise, largely associated with the generation of purified VLPs. METHODS: To address these issues, a luciferase immunoprecipitation system (LIPS) assay was modified to measure the norovirus-specific HBGA blockade activity of antibodies. The assay (designated LIPS-Blockade) was validated using a panel of well-characterized homotypic and heterotypic hyperimmune sera as well as strain-specific HBGA blocking monoclonal antibodies. RESULTS: The LIPS-Blockade assay was comparable in specificity to a standard HBGA blocking protocol performed with VLPs. Using time-ordered patient sera, the luciferase-based approach was also able to detect changes in HBGA blocking titers following viral challenge and natural infection with norovirus. CONCLUSION: In this study we developed a rapid, robust, and scalable surrogate neutralization assay for noroviruses that circumvented the need for purified VLPs. This LIPS-Blockade assay should streamline the process of large-scale immunological studies, ultimately aiding in the characterization of protective immunity to human noroviruses.

Single-dose BNT162b2 vaccine protects against asymptomatic SARS-CoV-2 infection.

The BNT162b2 mRNA COVID-19 vaccine (Pfizer-BioNTech) is being utilised internationally for mass COVID-19 vaccination. Evidence of single-dose protection against symptomatic disease has encouraged some countries to opt for delayed booster doses of BNT162b2, but the effect of this strategy on rates of asymptomatic SARS-CoV-2 infection remains unknown. We previously demonstrated frequent pauci- and asymptomatic SARS-CoV-2 infection amongst healthcare workers (HCWs) during the UK's first wave of the COVID-19 pandemic, using a comprehensive PCR-based HCW screening programme (Rivett et al., 2020; Jones et al., 2020). Here, we evaluate the effect of first-dose BNT162b2 vaccination on test positivity rates and find a fourfold reduction in asymptomatic infection amongst HCWs ≥12 days post-vaccination. These data provide real-world evidence of short-term protection against asymptomatic SARS-CoV-2 infection following a single dose of BNT162b2 vaccine, suggesting that mass first-dose vaccination will reduce SARS-CoV-2 transmission, as well as the burden of COVID-19 disease.

The Cryo-EM Structure of Vesivirus 2117 Highlights Functional Variations in Entry Pathways for Viruses in Different Clades of the Vesivirus Genus.

Vesivirus 2117 is an adventitious agent that has been responsible for lost productivity in biopharmaceutical production following contamination of Chinese hamster ovary cell cultures in commercial bioreactors. A member of the Caliciviridae, 2117 is classified within the Vesivirus genus in a clade that includes canine and mink caliciviruses but is distinct from the vesicular exanthema of swine virus (VESV) clade, which includes the extensively studied feline calicivirus (FCV). We have used cryogenic electron microscopy (cryo-EM) to determine the structure of the capsid of this small, icosahedral, positive-sense-RNA-containing virus. We show that the outer face of the dimeric capsomeres, which contains the receptor binding site and major immunodominant epitopes in all caliciviruses studied thus far, is quite different from that of FCV. This is a consequence of a 22-amino-acid insertion in the sequence of the FCV major capsid protein that forms a "cantilevered arm" that both plays an important role in receptor engagement and undergoes structural rearrangements thought to be important for genome delivery to the cytosol. Our data highlight a potentially important difference in the attachment and entry pathways employed by the different clades of the Vesivirus genus. IMPORTANCE Vesivirus 2117 has caused significant losses in manufacturing of biopharmaceutical products following contamination of cell cultures used in their production. We report the structure of the vesivirus 2117 capsid, the shell that encloses the virus's genome. Comparison of this structure with that of a related vesivirus, feline calicivirus (FCV), highlighted potentially important differences related to virus attachment and entry. Our findings suggest that these two viruses may bind differently to receptors at the host cell surface. We also show that a region of the capsid protein of FCV that rearranges following receptor engagement is not present in vesivirus 2117. These structural changes in the FCV capsid have been shown to allow the assembly of a portal-like structure that is hypothesized to deliver the viral genome to the cell's interior. Our data suggest that the 2117 portal assembly may employ a different means of anchoring to the outer face of the capsid.