Using Species a Rotavirus Reverse Genetics to Engineer Chimeric Viruses Expressing SARS-CoV-2 Spike Epitopes.

Species A rotavirus (RVA) vaccines based on live attenuated viruses are used worldwide in humans. The recent establishment of a reverse genetics system for rotoviruses (RVs) has opened the possibility of engineering chimeric viruses expressing heterologous peptides from other viral or microbial species in order to develop polyvalent vaccines. We tested the feasibility of this concept by two approaches. First, we inserted short SARS-CoV-2 spike peptides into the hypervariable region of the simian RV SA11 strain viral protein (VP) 4. Second, we fused the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, or the shorter receptor binding motif (RBM) nested within the RBD, to the C terminus of nonstructural protein (NSP) 3 of the bovine RV RF strain, with or without an intervening Thosea asigna virus 2A (T2A) peptide. Mutating the hypervariable region of SA11 VP4 impeded viral replication, and for these mutants, no cross-reactivity with spike antibodies was detected. To rescue NSP3 mutants, we established a plasmid-based reverse genetics system for the bovine RV RF strain. Except for the RBD mutant that demonstrated a rescue defect, all NSP3 mutants delivered endpoint infectivity titers and exhibited replication kinetics comparable to that of the wild-type virus. In ELISAs, cell lysates of an NSP3 mutant expressing the RBD peptide showed cross-reactivity with a SARS-CoV-2 RBD antibody. 3D bovine gut enteroids were susceptible to infection by all NSP3 mutants, but cross-reactivity with SARS-CoV-2 RBD antibody was only detected for the RBM mutant. The tolerance of large SARS-CoV-2 peptide insertions at the C terminus of NSP3 in the presence of T2A element highlights the potential of this approach for the development of vaccine vectors targeting multiple enteric pathogens simultaneously. IMPORTANCE We explored the use of rotaviruses (RVs) to express heterologous peptides, using SARS-CoV-2 as an example. Small SARS-CoV-2 peptide insertions (<34 amino acids) into the hypervariable region of the viral protein 4 (VP4) of RV SA11 strain resulted in reduced viral titer and replication, demonstrating a limited tolerance for peptide insertions at this site. To test the RV RF strain for its tolerance for peptide insertions, we constructed a reverse genetics system. NSP3 was C-terminally tagged with SARS-CoV-2 spike peptides of up to 193 amino acids in length. With a T2A-separated 193 amino acid tag on NSP3, there was no significant effect on the viral rescue efficiency, endpoint titer, and replication kinetics. Tagged NSP3 elicited cross-reactivity with SARS-CoV-2 spike antibodies in ELISA. We highlight the potential for development of RV vaccine vectors targeting multiple enteric pathogens simultaneously.

A2B-COVID: A Tool for Rapidly Evaluating Potential SARS-CoV-2 Transmission Events.

Identifying linked cases of infection is a critical component of the public health response to viral infectious diseases. In a clinical context, there is a need to make rapid assessments of whether cases of infection have arrived independently onto a ward, or are potentially linked via direct transmission. Viral genome sequence data are of great value in making these assessments, but are often not the only form of data available. Here, we describe A2B-COVID, a method for the rapid identification of potentially linked cases of COVID-19 infection designed for clinical settings. Our method combines knowledge about infection dynamics, data describing the movements of individuals, and evolutionary analysis of genome sequences to assess whether data collected from cases of infection are consistent or inconsistent with linkage via direct transmission. A retrospective analysis of data from two wards at Cambridge University Hospitals NHS Foundation Trust during the first wave of the pandemic showed qualitatively different patterns of linkage between cases on designated COVID-19 and non-COVID-19 wards. The subsequent real-time application of our method to data from the second epidemic wave highlights its value for monitoring cases of infection in a clinical context.

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.

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.

A functional assay for serum detection of antibodies against SARS-CoV-2 nucleoprotein.

The humoral immune response to SARS-CoV-2 results in antibodies against spike (S) and nucleoprotein (N). However, whilst there are widely available neutralization assays for S antibodies, there is no assay for N-antibody activity. Here, we present a simple in vitro method called EDNA (electroporated-antibody-dependent neutralization assay) that provides a quantitative measure of N-antibody activity in unpurified serum from SARS-CoV-2 convalescents. We show that N antibodies neutralize SARS-CoV-2 intracellularly and cell-autonomously but require the cytosolic Fc receptor TRIM21. Using EDNA, we show that low N-antibody titres can be neutralizing, whilst some convalescents possess serum with high titres but weak activity. N-antibody and N-specific T-cell activity correlates within individuals, suggesting N antibodies may protect against SARS-CoV-2 by promoting antigen presentation. This work highlights the potential benefits of N-based vaccines and provides an in vitro assay to allow the antibodies they induce to be tested.

Genomic epidemiology of COVID-19 in care homes in the east of England.

COVID-19 poses a major challenge to care homes, as SARS-CoV-2 is readily transmitted and causes disproportionately severe disease in older people. Here, 1167 residents from 337 care homes were identified from a dataset of 6600 COVID-19 cases from the East of England. Older age and being a care home resident were associated with increased mortality. SARS-CoV-2 genomes were available for 700 residents from 292 care homes. By integrating genomic and temporal data, 409 viral clusters within the 292 homes were identified, indicating two different patterns - outbreaks among care home residents and independent introductions with limited onward transmission. Approximately 70% of residents in the genomic analysis were admitted to hospital during the study, providing extensive opportunities for transmission between care homes and hospitals. Limiting viral transmission within care homes should be a key target for infection control to reduce COVID-19 mortality in this population.

Viral nucleoprotein antibodies activate TRIM21 and induce T cell immunity.

Nucleoprotein (N) is an immunodominant antigen in many enveloped virus infections. While the diagnostic value of anti-N antibodies is clear, their role in immunity is not. This is because while they are non-neutralising, they somehow clear infection by coronavirus, influenza and LCMV in vivo. Here, we show that anti-N immune protection is mediated by the cytosolic Fc receptor and E3 ubiquitin ligase TRIM21. Exploiting LCMV as a model system, we demonstrate that TRIM21 uses anti-N antibodies to target N for cytosolic degradation and generate cytotoxic T cells (CTLs) against N peptide. These CTLs rapidly eliminate N-peptide-displaying cells and drive efficient viral clearance. These results reveal a new mechanism of immune synergy between antibodies and T cells and highlights N as an important vaccine target.

Rapid implementation of SARS-CoV-2 sequencing to investigate cases of health-care associated COVID-19: a prospective genomic surveillance study.

BACKGROUND: The burden and influence of health-care associated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is unknown. We aimed to examine the use of rapid SARS-CoV-2 sequencing combined with detailed epidemiological analysis to investigate health-care associated SARS-CoV-2 infections and inform infection control measures. METHODS: In this prospective surveillance study, we set up rapid SARS-CoV-2 nanopore sequencing from PCR-positive diagnostic samples collected from our hospital (Cambridge, UK) and a random selection from hospitals in the East of England, enabling sample-to-sequence in less than 24 h. We established a weekly review and reporting system with integration of genomic and epidemiological data to investigate suspected health-care associated COVID-19 cases. FINDINGS: Between March 13 and April 24, 2020, we collected clinical data and samples from 5613 patients with COVID-19 from across the East of England. We sequenced 1000 samples producing 747 high-quality genomes. We combined epidemiological and genomic analysis of the 299 patients from our hospital and identified 35 clusters of identical viruses involving 159 patients. 92 (58%) of 159 patients had strong epidemiological links and 32 (20%) patients had plausible epidemiological links. These results were fed back to clinical, infection control, and hospital management teams, leading to infection-control interventions and informing patient safety reporting. INTERPRETATION: We established real-time genomic surveillance of SARS-CoV-2 in a UK hospital and showed the benefit of combined genomic and epidemiological analysis for the investigation of health-care associated COVID-19. This approach enabled us to detect cryptic transmission events and identify opportunities to target infection-control interventions to further reduce health-care associated infections. Our findings have important implications for national public health policy as they enable rapid tracking and investigation of infections in hospital and community settings. FUNDING: COVID-19 Genomics UK funded by the Department of Health and Social Care, UK Research and Innovation, and the Wellcome Sanger Institute.