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Development of a prophylactic vaccine for hepatitis C virus (HCV) remains a global health challenge. Cumulative evidence supports the importance of antibodies targeting the HCV E2 envelope glycoprotein to facilitate viral clearance. However, a significant challenge for a B cell-based vaccine is focusing the immune response on conserved E2 epitopes capable of eliciting neutralizing antibodies not associated with viral escape. We hypothesized that glycosylation might influence the antigenicity and immunogenicity of E2. Accordingly, we performed head-to-head molecular, antigenic and immunogenic comparisons of soluble E2 (sE2) produced in (i) mammalian (HEK293) cells, which confer mostly complex and high mannose type glycans; and (ii) insect (Sf9) cells, which impart mainly paucimannose type glycans. Mass spectrometry demonstrated that all 11 predicted N-glycosylation sites were utilized in both HEK293- and Sf9-derived sE2, but that N-glycans in insect sE2 were on average smaller and less complex. Both proteins bound CD81 and were recognized by conformation-dependent antibodies. Mouse immunogenicity studies revealed that similar polyclonal antibody responses were generated against antigenic domains A-E of E2. Although neutralizing antibody titers showed that Sf9-derived sE2 induced moderately stronger responses than HEK293-derived sE2 against the homologous HCV H77c isolate, the two proteins elicited comparable neutralization titers against heterologous isolates. Given that global alteration of HCV E2 glycosylation by expression in different hosts did not appreciably affect antigenicity or overall immunogenicity, a more productive approach to increasing the antibody response to neutralizing epitopes may be complete deletion, rather than just modification, of specific N-glycans proximal to these epitopes. [ABSTRACT FROM AUTHOR]

Trichodysplasia spinulosa polyomavirus causes trichodysplasia spinulosa, a skin infection, in immunocompromised persons, but the virus is rarely detected in respiratory samples. Using PCR, we detected persistent virus in respiratory and skin samples from an immunocompromised boy with respiratory signs but no characteristic skin spicules. This virus may play a role in respiratory illness. [ABSTRACT FROM AUTHOR]

The broadly neutralizing antibodies HIV 2F5 and 4E10, which bind to overlapping epitopes in the membrane-proximal external region of the fusion protein gp41, have been proposed to use a two-step mechanism for neutralization; first, they bind and preconcentrate at the viral membrane through their long, hydrophobic CDRH3 loops, and second, they form a high affinity complex with the protein epitope. Accordingly, mutagenesis of the CDRH3 can abolish their neutralizing activity, with no change in the affinity for the peptide epitope. We show here that we can mimic this mechanism by conjugating a cholesterol group outside of the paratope of an antibody. Cholesterol-conjugated antibodies bind to lipid raft domains on the membrane, and because of this enrichment, they show increased antiviral potency. In particular, we find that cholesterol conjugation (i) rescues the antiviral activity of CDRH3-mutated 2F5, (ii) increases the antiviral activity of WT 2F5, (iii) potentiates the non-membrane-binding HIV antibody D5 10-100-fold (depending on the virus strain), and (iv) increases synergy between 2F5 and D5. Conjugation can be made at several positions, including variable and constant domains. Cholesterol conjugation therefore appears to be a general strategy to boost the potency of antiviral antibodies, and, because membrane affinity is engineered outside of the antibody paratope, it can complement affinity maturation strategies. [ABSTRACT FROM AUTHOR]

A new generation of drugs targeting the non-structural (NS) proteins of the Hepatitis C virus (HCV) will substantially increase treatment success rates, reducing global infections. Amongst the NS proteins, the NS3 protease represents an important drug target, responsible for liberation of mature NS proteins from the nascent HCV polyprotein and suppression of host innate immunity. Despite this, the evolutionary stability of the genomic locus encoding the NS3 protease is poorly characterized in chronic HCV infection. To address this shortfall, we developed a high-throughput amplicon pyrosequencing protocol and utilised it to monitor NS3 protease coding-sequence evolution for over a decade in two patients. Although patient-specific evolutionary trends were apparent, the protease amino acid population consensus remained stable with a massive excess of synonymous mutations observed, confirming this locus is under strong purifying selection during chronic infection within individual patients. No evidence for continuous immune escape was detected. Additionally, both patients failed protease inhibitor (PI) therapy and protease sequence diversity pre- and post-therapy were also assessed. No baseline resistance associated variants (RAVs) contributed to treatment failure. Significant reductions in viral diversity were observed post-PI therapy, indicating a population bottleneck occurred. The genetic vestiges of this bottleneck were still detectable 18 months after therapy discontinuation. Although significant enrichment of the Q80L mutation was observed in one patient, genetic and phenotypic data reveal no detectable RAV persistence post-therapy failure. Together this investigation provides a sensitive and reproducible high-throughput framework to interrogate viral sequence diversity at high-resolution, with potential applications for routine monitoring of treatment regimens. This study also reveals novel insights into the evolutionary processes that shape NS3 sequence divergence in both chronic HCV infection and post PI-therapy failure. [ABSTRACT FROM AUTHOR]

An abstract of the article "Definition of a Conserved Immunodominant Domain on Hepatitis C Virus E2 Glycoprotein by Neutralizing Human Monoclonal Antibodies," by Zhen-Yong Keck, Ta-Kai Li, Jinming Xia, Meital Gal-Tanamy, Oakley Olson, Sophia H. Li, Arvind H. Patel, and colleagues is presented.

One way to dissect the antibody response to an invading microorganism is to clone the antibody repertoire from immune donors and subsequently characterize the specific antibodies. Recently, methodological advances have allowed investigations of neutralizing antibodies against hepatitis C virus (HCV) in vitro. We have investigated three human mAbs, previously isolated from an individual infected with HCV of genotype 2b, that are known to cross-react in a binding assay to the envelope E2 protein of genotypes 1a and 1b. We now report that two of them have a neutralizing activity with a breadth not previously observed. Indeed. mAbs 1:7 and A8 recognized E2 from all of the six major genotypes, and they neutralized retroviral pseudoparticles [HCV pseudoparticles (HCVpp)] carrying genetically equally diverse HCV envelope glycoproteins. Importantly, these antibodies were also able to neutralize the cell culture infectious HCV clone JFH-1 in vitro, with IC50 values of 60 ng/ml and 560 ng/ml, respectively. The conformational epitopes of these two broadly reactive antibodies were overlapping yet distinct and involved amino acid residues in the 523–535 region of E2, known to be important for the E2-CD81 interaction. The third antibody clone, representing a dominant population in the initial screen for these antibodies, was less broadly reactive and was unable to neutralize the genotype 2a infectious clone JFH-1. Our results confirm at the clonal level that broadly neutralizing human anti-HCV antibodies can be elicited and that the region amino acids 523–535 of the HCV envelope glycoprotein E2 carries neutralizing epitopes conserved across all genotypes. [ABSTRACT FROM AUTHOR]

Abstract: Affinity selection of phage display peptide libraries is routinely used for isolating peptides capable of binding a range of molecules, including antibodies and receptors. This process is most successful when the selecting molecule is relatively pure, for example, a monoclonal antibody. However, isolation of peptides able to bind to target molecules present in a complex mixture is more difficult because the affinity selection process isolates peptides capable of binding to all molecules present in the mixture. Here we describe the development of a tagged polymerase chain reaction (PCR) subtractive hybridization method that is universally applicable for the targeted isolation of peptides able to bind to unique molecules within a complex mixture. We also describe a discriminatory limiting dilution PCR method that can be used to optimize hybridization conditions. [Copyright &y& Elsevier]

Reference 1 Napolitano F, Merone R, Abbate A, Ammendola V, Horncastle E, Lanzaro F, et al. (2020) A next generation vaccine against human rabies based on a single dose of a chimpanzee adenovirus vector serotype C. PLoS Negl Trop Dis14(7): e0008459. https://doi.org/10.1371/journal.pntd.0008459, 32667913 The twelfth author's name is spelled incorrectly. [Extracted from the article]

A major problem in hepatitis C virus (HCV) immunotherapy or vaccine design is the extreme variability of the virus. We identified human monoclonal antibodies (mAbs) that neutralize genetically diverse HCV isolates and protect against heterologous HCV quasispecies challenge in a human liver–chimeric mouse model. The results provide evidence that broadly neutralizing antibodies to HCV protect against heterologous viral infection and suggest that a prophylactic vaccine against HCV may be achievable. [ABSTRACT FROM AUTHOR]

Despite available treatments, a prophylactic HCV vaccine is needed to achieve elimination targets. HCV vaccine development has faltered largely because the extreme diversity of the virus limits the protective breadth of vaccine elicited antibodies. It is believed that the principle neutralizing epitope in natural infection, HVR1, which is the most variable epitope in HCV, mediates humoral immune escape. So far, efforts to circumvent HVR1 interference in the induction and function of conserved targeting Ab have failed. Efforts to understand factors contributing to cross-neutralization of HVR1 variants have also been limited. Here, following mouse immunizations with two patient-derived HVR1 peptides, we observe cross-genotype neutralization of variants differing at 15/21 positions. Surprisingly, sequence similarity was not associated with cross-neutralization. It appeared neutralization sensitivity was an intrinsic feature of each variant, rather than emergent from the immunogen specific Ab response. These findings provide novel insight into HVR1-mediated immune evasion, with important implications for HCV vaccine design. [ABSTRACT FROM AUTHOR]

Orthohantaviruses are globally distributed viruses, associated with rodents and other small mammals. However, data on the circulation of orthohantaviruses within the UK, particularly the UK-endemic Tatenale virus, is sparse. In this study, 531 animals from five rodent species were collected from two locations in northern and central England and screened using a degenerate, pan- orthohantavirus RT-PCR assay. Tatenale virus was detected in a single field vole (Microtus agrestis) from central England and twelve field voles from northern England. Unbiased high-throughput sequencing of the central English strain resulted in the recovery of the complete coding sequence of a novel strain of Tatenale virus, whilst PCR-primer walking of the northern English strain recovered almost complete coding sequence of a previously identified strain. These findings represented the detection of a third lineage of Tatenale virus in the United Kingdom and extended the known geographic distribution of these viruses from northern to central England. Furthermore, the recovery of the complete coding sequence revealed that Tatenale virus was sufficiently related to the recently identified Traemersee virus, to meet the accepted criteria for classification as a single species of orthohantavirus. [ABSTRACT FROM AUTHOR]

Summary The 2013–2016 outbreak of Ebola virus (EBOV) in West Africa was the largest recorded. It began following the cross-species transmission of EBOV from an animal reservoir, most likely bats, into humans, with phylogenetic analysis revealing the co-circulation of several viral lineages. We hypothesized that this prolonged human circulation led to genomic changes that increased viral transmissibility in humans. We generated a synthetic glycoprotein (GP) construct based on the earliest reported isolate and introduced amino acid substitutions that defined viral lineages. Mutant GPs were used to generate a panel of pseudoviruses, which were used to infect different human and bat cell lines. These data revealed that specific amino acid substitutions in the EBOV GP have increased tropism for human cells, while reducing tropism for bat cells. Such increased infectivity may have enhanced the ability of EBOV to transmit among humans and contributed to the wide geographic distribution of some viral lineages. [ABSTRACT FROM AUTHOR]

Background & Aims: An optimal HCV vaccine requires the induction of antibodies that neutralise the infectivity of many heterogenous viral isolates. In this study, we have focused on determining the optimal recombinant envelope glycoprotein component to elicit cross-neutralising antibodies against global HCV genotypes. We compared the immunoreactivity and antigenicity of the HCV genotype 1a strain H77C-derived envelope glycoprotein heterodimer gpE1/gpE2 with that of recombinant gpE2 alone., Methods: Characterisation of the envelope glycoproteins was accomplished by determining their ability to bind to a panel of broadly cross-neutralising monoclonal antibodies. Immunogenicity was determined by testing the ability of vaccine antisera to neutralise the infectivity in vitro of a panel of pseudotyped HCV particles in which gpE1/gpE2 derived from representative isolates of the major global HCV genotypes were displayed., Results: gpE1/gpE2 binds to more diverse broadly cross-neutralising antibodies than gpE2 alone and elicits a broader profile of cross-neutralising antibodies in animals, especially against more heterologous, non-1a genotypes. While not all heterologous HCV strains can be potently inhibited in vitro by gpE1/gpE2 antisera derived from a single HCV strain, the breadth of heterologous cross-neutralisation is shown to be substantial., Conclusions: Our work supports the inclusion of gpE1/gpE2 in an HCV vaccine in order to maximise the cross-neutralisation of heterogenous HCV isolates. Our data also offers future directions in formulating a cocktail of gpE1/gpE2 antigens from a small selection of HCV genotypes to further enhance cross-neutralisation of global HCV strains and hopefully advance the development of a globally effective HCV vaccine., Impact and Implications: An HCV vaccine is urgently required to prevent the high global incidence of HCV infection and disease. Since HCV is a highly heterogeneous virus, it is desirable for a vaccine to elicit antibodies that neutralise the infectivity of most global strains. To this end, we have compared the immunoreactivity and antigenicity of recombinant H77C E1E2 heterodimer with that of H77C E2 alone and show that the former exhibits more cross-neutralising epitopes and demonstrates a broader cross-neutralisation profile in vitro. In addition, our data suggests a way to further broaden cross-neutralisation using a combination of E1E2 antigens derived from a few different HCV clades. Our work is relevant for the development of an effective global HCV vaccine., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

We report the retrospective identification and subsequent recovery of a near-complete West Nile Virus lineage 2 genomes from a hospitalized patient with acute febrile illness in Uganda, using a combination of degenerate primer polymerase chain reaction screening and a novel 1200bp nanopore-based whole-genome amplicon sequencing scheme. This represents the first West Nile virus genome to be recovered from a human in Uganda since its discovery in 1937. Basic molecular rather than serological surveillance methods could be more widely deployed in the region to better diagnose febrile infections., Competing Interests: The authors have no competing interests to declare., (© 2024 The Author(s).)

A hepatitis C virus (HCV) vaccine is urgently needed. Vaccine development has been hindered by HCV's genetic diversity, particularly within the immunodominant hypervariable region 1 (HVR1). Here, we developed a strategy to elicit broadly neutralizing antibodies to HVR1, which had previously been considered infeasible. We first applied a unique information theory-based measure of genetic distance to evaluate phenotypic relatedness between HVR1 variants. These distances were used to model the structure of HVR1's sequence space, which was found to have five major clusters. Variants from each cluster were used to immunize mice individually, and as a pentavalent mixture. Sera obtained following immunization neutralized every variant in a diverse HCVpp panel (n = 10), including those resistant to monovalent immunization, and at higher mean titers (1/ID 50 = 435) than a glycoprotein E2 (1/ID 50 = 205) vaccine. This synergistic immune response offers a unique approach to overcoming antigenic variability and may be applicable to other highly mutable viruses.

Background: Arboviruses are endemic in Uganda; however, little is known about their epidemiology, seasonality and spatiotemporal distribution. Our study sought to provide information on arbovirus outbreaks from acute clinical presentations., Methods: Immunoglobulin M (IgM) and confirmatory Plaque Reduction Neutralisation Test (PRNT) results for arbovirus diagnosis of samples collected from patients attending sentinel sites from 2016-19 were analysed retrospectively. Demographic data were analysed with SaTScan and SPSS software to determine the epidemiology and spatiotemporal distribution of arboviruses., Results: Arbovirus activity peaked consistently during March-May rainy seasons. Overall, arbovirus seroprevalence was 9.5%. Of 137 IgM positives, 52.6% were confirmed by PRNT, of which 73.6% cases were observed in central Uganda with Yellow Fever Virus had the highest prevalence (27.8%). The 5-14 age group were four times more likely to be infected with an arbovirus p=0.003, 4.1 (95% CI 1.3-12.3). Significant arboviral activity was observed among outdoor workers(p=0.05) . Spatiotemporal analysis indicated arboviral activity in 23 of the 85 districts analysed.., Interpretation: Our study shows that arbovirus activity peaks during the March-May rainy season and highlights the need for YFV mass vaccination to reduce the clinical burden of arboviruses transmitted within the region., Competing Interests: All authors declare no conflict of interest., (© 2023 The Authors.)

BACKGROUND AND AIMS: The newly developed direct-acting antivirals have revolutionized the treatment of chronic hepatitis C virus (HCV), with cure rates as high as 98% in some cohorts. Although genome sequencing has demonstrated that some subtypes of HCV naturally harbor drug resistance associated substitutions (RAS), these are often overlooked as "rarities." Furthermore, commercial subtyping assays and associated epidemiological findings are skewed towards Western cohorts and whole-genome sequencing can be problematic to deploy without significant infrastructure and training support. We thus aimed to develop a simple, robust and accurate HCV subtyping pipeline, to optimize and streamline molecular detection and sequence-based typing of diverse RAS-containing subtypes., Methods: HCV serum derived from 146 individuals, whose likely source of infection was from sub-Saharan Africa (SSA) was investigated with a novel panel of single round polymerase chain reaction (PCR) assays targeting NS5B and NS5A genomic regions. Virus subtype assignments were determined by pairwise-distance analysis and compared to both diagnostic laboratory assignments and free-to-use online typing tools., Results: Partial NS5A and NS5B sequences were respectively obtained from 131 to 135 HCV-positive patients born in 19 different countries from SSA but attending clinics in the UK. We determined that routine clinical diagnostic methods incorrectly subtyped 59.0% of samples, with a further 6.8% incorrectly genotyped. Of five commonly used online tools, Geno2Pheno performed most effectively in determining a subtype in agreement with pairwise distance analysis., Conclusion: This study provides a simple low-cost pathway to accurately subtype in SSA, guide regional therapeutic choice and assist global surveillance and elimination initiatives., (© 2022 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Analysis of host genetic polymorphisms is an increasingly important tool for understanding and predicting pathogenesis and treatment response of viral diseases. The gene locus of scavenger receptor class B type I (SR-BI), encoding a cell entry factor and receptor for hepatitis C virus (HCV), contains several genetic polymorphisms. We applied a probe extension assay to determine the frequency of six single nucleotide polymorphisms (SNPs) within the SR-BI gene locus in 374 individuals with history of HCV infection. In addition, SR-BI messenger RNA (mRNA) levels were analyzed in liver biopsy specimens of chronically infected HCV subjects. The rs5888 variant allele T was present at a higher frequency in subjects with advanced fibrosis (χ 2 , p = 0.016) and after adjusting for age, duration of infection and alcohol intake as confounding factors. Haplotype analysis of SNP frequencies showed that a haplotype consisting of rs61932577 variant allele C and rs5888 variant allele T was associated with an increased risk of advanced liver fibrosis (defined by an Ishak score 4-6) (adjusted odds ratio 2.81; 95% confidence interval 1.06-7.46. p = 0.038). Carriers of the rs5888 variant allele T displayed reduced SR-BI mRNA expression in liver biopsy specimens. In conclusion the rs5888 polymorphism variant is associated with decreased SR-BI expression and an increased risk of development of advanced fibrosis in chronic HCV infection. These findings provide further evidence for a role of SR-BI in HCV pathogenesis and provides a genetic marker for prediction of those infected individuals at greater risk of developing severe disease., (© 2022 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Background: Human Parainfluenza viruses (HPIV) comprise of four members of the genetically distinct genera of Respirovirus (HPIV1&3) and Orthorubulavirus (HPIV2&4), causing significant upper and lower respiratory tract infections worldwide, particularly in children. However, despite frequent molecular diagnosis, they are frequently considered collectively or with HPIV4 overlooked entirely. We therefore investigated clinical and viral epidemiological distinctions of the relatively less prevalent Orthorubulaviruses HPIV2&4 at a regional UK hospital across four autumn/winter epidemic seasons., Methods: A retrospective audit of clinical features of all HPIV2 or HPIV4 RT-PCR-positive patients, diagnosed between 1st September 2013 and 12th April 2017 was undertaken, alongside sequencing of viral genome fragments in a representative subset of samples., Results: Infection was observed across all age groups, but predominantly in children under nine and adults over 40, with almost twice as many HPIV4 as HPIV2 cases. Fever, abnormal haematology, elevated C-reactive protein and hospital admission were more frequently seen in HPIV2 than HPIV4 infection. Each of the four seasonal peaks of either HPIV2, HPIV4 or both, closely matched that of RSV, occurring in November and December and preceding that of Influenza A. A subset of viruses were partially sequenced, indicating co-circulation of multiple subtypes of both HPIV2&4, but with little variation between each epidemic season or from limited global reference sequences., Conclusions: Despite being closest known genetic relatives, our data indicates a potential difference in associated disease between HPIV2 and HPIV4, with more hospitalisation seen in HPIV2 mono-infected individuals, but a greater overall number of HPIV4 cases., (© 2022 The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

A better understanding of the antibody response during natural infection and the effect on disease progression and reinfection is necessary for the development of a protective hepatitis C virus (HCV) vaccine. The HCV pseudoparticle (HCVpp) system enables the study of viral entry and inhibition by antibody neutralization. A robust and comparable neutralization assay is crucial for the development and evaluation of experimental vaccines.With the aim of optimizing the HCVpp-murine leukaemia virus (MLV) system, we tested the neutralization of HCVpp-harbouring E1E2 from 21 HCV isolates representing 6 different genotypes by several monoclonal antibodies (mAbs). HCVpps are generated by expressing functional envelope glycoproteins (E1E2) onto pseudoparticles derived from env-deleted MLV. Adjustments of E1E2, gag-pol and luciferase plasmid ratios resulted in increased yields for most HCVpps and recovery of one non-infectious HCVpp. We simplified and improved the protocol to achieve higher signal/noise ratios and minimized the amount of HCVpps and mAbs needed for the detection of neutralization. Using our optimized protocol, we demonstrated comparable results to previously reported data with both diluted and freeze-thawed HCVpps.In conclusion, we successfully established a simplified and reproducible HCVpp neutralization protocol for studying a wide range of HCV variants. This simplified protocol provides highly consistent results and could be easily adopted by others to evaluate precious biological material. This will contribute to a better understanding of the antibody response during natural infection and help evaluate experimental HCV vaccines.

Asymptomatic testing for SARS-CoV-2 RNA has been used to prevent and manage COVID-19 outbreaks in university settings, but few studies have explored their implementation. The aim of the study was to evaluate how an accredited asymptomatic SARS-CoV-2 testing service (ATS) was implemented at the University of Nottingham, a multi-campus university in England, to identify barriers and enablers of implementation and to draw out lessons for implementing pandemic response initiatives in higher education settings. A qualitative interview study was conducted with 25 ATS personnel between May and July 2022. Interviews were conducted online, audio-recorded, and transcribed. Participants were asked about their experience of the ATS, barriers and enablers of implementation. Transcripts were thematically analysed. There were four overarching themes: (1) social responsibility and innovation, (2) when, how and why people accessed testing, (3) impact of the ATS on the spread of COVID-19, and (4) lessons learned for the future. In establishing the service, the institution was seen to be valuing its community and socially responsible. The service was viewed to be broadly successful as a COVID-19 mitigation approach. Challenges to service implementation were the rapidly changing pandemic situation and government advice, delays in service accreditation and rollout to staff, ambivalence towards testing and isolating in the target population, and an inability to provide follow-up support for positive cases within the service. Facilitators included service visibility, reduction in organisational bureaucracy and red tape, inclusive leadership, collaborative working with regular feedback on service status, flexibility in service delivery approaches and simplicity of saliva testing. The ATS instilled a perception of early 'return to normality' and impacted positively on staff feelings of safety and wellbeing, with wider benefits for healthcare services and local communities. In conclusion, we identified common themes that have facilitated or hindered the implementation of a SARS-CoV-2 testing service at a university in England. Lessons learned from ATS implementation will inform future pandemic response interventions in higher education settings., Competing Interests: CD is Academic lead and co-founder of the ATS. JKB is co-founder of, and virology advisor to the ATS. HB is behavioural advisor to the ATS. No other conflicts of interest are declared.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the causative agent of coronavirus disease 2019 (COVID-19)-has caused a global public health emergency. Personal protective equipment (PPE) is the primary defence against viral exposure in healthcare and community settings. However, the surfaces of PPE materials may trap virus for contact transmission or through laden aerosols generated during removal of PPE, through cleaning or during movement. In this study, the relative efficacy of current PPE materials in terms of virion adsorption to materials and their antiviral potency, has been evaluated on a wide range of PPE for the first time, including four polymer glove types, two types of scrubs, apron material, a mask, visor and a selection of other commercial polymers and products. Although differences in virion adsorption to the test materials were observed, none of the existing polymer-based PPE resulted in more than tenfold reduction in the SARS-CoV-2 titre within either 10 min or 30 min contact period. The wettability and surface chemistry of the test materials were analysed to investigate any correlations with their surface physicochemical properties. While no correlation was found between wettability and viral retention under air flow challenge, one secondary ion of m/z 101.03 (+) and three secondary ions of m/z 31.98 (-), 196.93 (-) and 394.33 (+) in ToF-SIMS data of the test materials showed positive and negative correlations with the viral retention, respectively, which was identified by PLS regression model, suggesting that the surface chemistry plays a role in determining the extent of virion adsorption. Our findings outline the material aspects that influence the efficacy of current PPE against SARS-CoV-2 transmission and give suggestions on the development of novel simple polymer-based PPE for better infection protection., (© 2022. The Author(s).)

The aim of the study was to explore workforce experiences of the rapid implementation of a SARS-CoV-2 asymptomatic testing service (ATS) in a higher education setting during the COVID-19 pandemic. The setting was a multi-campus university in the UK, which hosted a testing service for employees and students over two years. Qualitative semi-structured videoconference interviews were conducted. We contacted 58 participants and 25 were interviewed (43% response rate). Data were analysed thematically. The analysis produced four overarching themes: (1) feelings relating to their involvement in the service, (2) perceptions of teamwork, (3) perceptions of ATS leadership, (4) valuing the opportunity for career development. Agile and inclusive leadership style created psychological safety and team cohesion, which facilitated participants in the implementation of a rapid mitigation service, at pace and scale. Specific features of the ATS (shared vision, collaboration, networking, skills acquisition) instilled self-confidence, value and belonging, meaningfully impacting on professional development and career opportunities. This is the first qualitative study to explore the experiences of university employees engaged in the rapid deployment of a service as part of a pandemic outbreak and mitigation strategy within a higher education setting. Despite pressures and challenges of the task, professional growth and advancement were universal. This has implications for workforce engagement and creating workplaces across the sector that are well-prepared to respond to future pandemics and other disruptive events.

Background: Chronic hepatitis C virus (HCV) infection affects 71 million individuals, mostly residing in low- and middle-income countries (LMICs). Direct-acting antivirals (DAAs) give high rates of sustained virological response (SVR) in high-income countries where a restricted range of HCV genotypes/subtypes circulate., Methods: We studied United Kingdom-resident patients born in Africa to examine DAA effectiveness in LMICs where there is far greater breadth of HCV genotypes/subtypes. Viral genome sequences were determined from 233 patients., Results: Full-length viral genomic sequences for 26 known subtypes and 5 previously unidentified isolates covering 5 HCV genotypes were determined. From 149 patients who received DAA treatment/retreatment, the overall SVR was 93%. Treatment failure was associated primarily with 2 subtypes, gt1l and gt4r, using sofosbuvir/ledipasvir. These subtypes contain natural resistance-associated variants that likely contribute to poor efficacy with this drug combination. Treatment failure was also significantly associated with hepatocellular carcinoma., Conclusions: DAA combinations give high SVR rates despite the high HCV diversity across the African continent except for subtypes gt1l and gt4r, which respond poorly to sofosbuvir/ledipasvir. These subtypes are widely distributed across Western, Central, and Eastern Africa. Thus, in circumstances where accurate genotyping is absent, ledipasvir and its generic compounds should not be considered as a recommended treatment option., Competing Interests: Potential conflicts of interest. W. L. I. has received speaker and consultancy fees from Roche Products, Janssen-Cilag, and Novartis; educational grants from Boehringer Ingelheim, MSD, and Gilead Sciences; and research grant support from GlaxoSmithKline, Pfizer, Gilead Sciences, and Janssen-Cilag. K. Ag. has received speaker, scientific advisory board, and consultancy fees from Aligos Therapeutics, Assemblybio, Arbutus Biopharma, Biotest, Gilead Sciences, Immunocore, GLG Pharma, Shionogi, Sobi, and Vir Biotechnology; and grants from Abbott and MSD. M. E. C. has received speaker and consultancy fees from AbbVie, BMS, Gilead Sciences, Janssen, and MSD. J. M. has received speaker and consultancy fees from AbbVie, Gilead Sciences, and BMS. All other authors report no potential conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America.)

Emerging SARS-CoV-2 variants are creating major challenges in the ongoing COVID-19 pandemic. Being able to predict mutations that could arise in SARS-CoV-2 leading to increased transmissibility or immune evasion would be extremely valuable in development of broad-acting therapeutics and vaccines, and prioritising viral monitoring and containment. Here we use in vitro evolution to seek mutations in SARS-CoV-2 receptor binding domain (RBD) that would substantially increase binding to ACE2. We find a double mutation, S477N and Q498H, that increases affinity of RBD for ACE2 by 6.5-fold. This affinity gain is largely driven by the Q498H mutation. We determine the structure of the mutant-RBD:ACE2 complex by cryo-electron microscopy to reveal the mechanism for increased affinity. Addition of Q498H to SARS-CoV-2 RBD variants is found to boost binding affinity of the variants for human ACE2 and confer a new ability to bind rat ACE2 with high affinity. Surprisingly however, in the presence of the common N501Y mutation, Q498H inhibits binding, due to a clash between H498 and Y501 side chains. To achieve an intermolecular bonding network, affinity gain and cross-species binding similar to Q498H alone, RBD variants with the N501Y mutation must acquire instead the related Q498R mutation. Thus, SARS-CoV-2 RBD can access large affinity gains and cross-species binding via two alternative mutational routes involving Q498, with route selection determined by whether a variant already has the N501Y mutation. These mutations are now appearing in emerging SARS-CoV-2 variants where they have the potential to influence human-to-human and cross-species transmission., Competing Interests: The authors have declared that no competing interests exist.

The global COVID-19 pandemic has impacted on the mental well-being of university students, but little attention has been given to international students, who may have a unique experience and perspective. The aim of this study was to explore the views of international students and university staff towards COVID-19 restrictions, self-isolation, their well-being, and support needs, through eight online focus groups with international students ( n = 29) and semi-structured interviews with university staff ( n = 17) at a higher education institution in England. Data were analysed using an inductive thematic approach, revealing three key themes and six subthemes: (1) practical, academic, and psychological challenges faced during self-isolation and the COVID-19 pandemic; (2) coping strategies to self-isolation and life during the pandemic; and (3) views on further support needed for international students. International students faced practical, academic, and psychological challenges during the COVID-19 pandemic, particularly relating to the rapid transition to online learning and the impact of social restrictions on integration with peers and well-being. Online social connections with peers, family, or new acquaintances reduced feelings of isolation and encouraged involvement in university life. Despite raising mental health concerns, most international students did not access mental health support services. Staff related this to perceived stigma around mental health in certain cultural groups. In conclusion, international students experienced specific practical and emotional challenges during the pandemic, and are at risk of mental ill-health, but may not actively seek out support from university services. Proactive and personalised approaches to student support will be important for positive student experiences and the retention of students who are studying abroad in the UK higher education system.

Background: Psychological factors can influence susceptibility to viral infections. We examined whether such influences are evident in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection., Methods: Participants (n = 102) completed measures of anxiety, depression, positive mood, and loneliness and provided a blood sample for the measurement of antibodies to the SARS-CoV-2 spike and nucleocapsid proteins., Results: SARS-CoV-2 was significantly negatively associated with anxiety and depression. The model remained significant after adjustment for age and gender, although anxiety and depression were no longer significant independent predictors., Conclusions: These findings offer early support for the hypothesis that psychological factors may influence susceptibility to SARS-CoV-2 infection., (© The Author(s) 2022. Published by Oxford University Press for the Infectious Diseases Society of America.)

Enterovirus D68 (EV-D68) has recently been identified in biennial epidemics coinciding with diagnoses of non-polio acute flaccid paralysis/myelitis (AFP/AFM). We investigated the prevalence, genetic relatedness and associated clinical features of EV-D68 in 193 EV-positive samples from 193 patients in late 2018, UK. EV-D68 was detected in 83 (58 %) of 143 confirmed EV-positive samples. Sequencing and phylogenetic analysis revealed extensive genetic diversity, split between subclades B3 ( n =50) and D1 ( n =33), suggesting epidemiologically unrelated infections. B3 predominated in children and younger adults, and D1 in older adults and the elderly ( P =0.0009). Clinical presentation indicated causation or exacerbation of respiratory distress in 91.4 % of EV-D68-positive individuals, principally cough (75.3 %), shortness of breath (56.8 %), coryza (48.1 %), wheeze (46.9 %), supplemental oxygen required (46.9 %) and fever (38.9 %). Two cases of AFM were observed, one with EV-D68 detectable in the cerebrospinal fluid, but otherwise neurological symptoms were rarely reported ( n =4). Both AFM cases and all additional instances of intensive care unit (ICU) admission ( n =5) were seen in patients infected with EV-D68 subclade B3. However, due to the infrequency of severe infection in our cohort, statistical significance could not be assessed.

Following acute HCV infection, the virus establishes a chronic disease in the majority of patients whilst few individuals clear the infection spontaneously. The precise mechanisms that determine chronic HCV infection or spontaneous clearance are not completely understood but are proposed to be driven by host and viral genetic factors as well as HCV encoded immunomodulatory proteins. Using the HIV-1 LTR as a tool to measure NF-κB activity, we identified that the HCV E1E2 glycoproteins and more so the E2 protein down-modulates HIV-1 LTR activation in 293T, TZM-bl and the more physiologically relevant Huh7 liver derived cell line. We demonstrate this effect is specifically mediated through inhibiting NF-κB binding to the LTR and show that this effect was conserved for all HCV genotypes tested. Transcriptomic analysis of 293T cells expressing the HCV glycoproteins identified E1E2 mediated stimulation of the endoplasmic reticulum (ER) stress response pathway and upregulation of stress response genes such as ATF3. Through shRNA mediated inhibition of ATF3, one of the components, we observed that E1E2 mediated inhibitory effects on HIV-1 LTR activity was alleviated. Our in vitro studies demonstrate that HCV Env glycoprotein activates host ER Stress Pathways known to inhibit NF-κB activity. This has potential implications for understanding HCV induced immune activation as well as oncogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 McKay, Thomas, Albalawi, Fattaccioli, Dieu, Ruggiero, McKeating, Ball, Tarr, Renard, Pollakis and Paxton.)

Hepatitis C virus (HCV) is responsible for more than 180 million infections worldwide, and about 80 % of infections are reported in Low and Middle-income countries (LMICs). Therapy is based on the administration of interferon (INF), ribavirin (RBV) or more recently Direct-Acting Antivirals (DAAs). However, amino acid substitutions associated with resistance (RAS) have been extensively described and can contribute to treatment failure, and diagnosis of RAS requires considerable infrastructure, not always locally available. Dried serum spots (DSS) sampling is an alternative specimen collection method, which embeds drops of serum onto filter paper to be transported by posting to a centralized laboratory. Here, we assessed feasibility of genotypic analysis of HCV from DSS in a cohort of 80 patients from São Paulo state Brazil. HCV RNA was detected on DSS specimens in 83 % of samples of HCV infected patients. HCV genotypes 1a, 1b, 2a, 2c and 3a were determined using the sequence of the palm domain of NS5B region, and RAS C316N/Y, Q309R and V321I were identified in HCV 1b samples. Concerning therapy outcome, 75 % of the patients who used INF +RBV as a previous protocol of treatment did not respond to DAAs, and 25 % were end-of-treatment responders. It suggests that therapy with INF plus RBV may contribute for non-response to a second therapeutic protocol with DAAs. One patient that presented RAS (V321I) was classified as non-responder, and combination of RAS C316N and Q309R does not necessarily imply in resistance to treatment in this cohort of patients. Data presented herein highlights the relevance of studying circulating variants for a better understanding of HCV variability and resistance to the therapy. Furthermore, the feasibility of carrying out genotyping and RAS phenotyping analysis by using DSS card for the potential of informing future treatment interventions could be relevant to overcome the limitations of processing samples in several location worldwide, especially in LMICs., Competing Interests: The authors declare that they have no conflicts of interest., (© 2022 The Authors.)

Background & Aims: Development of a prophylactic hepatitis C virus (HCV) vaccine will require accurate and reproducible measurement of neutralizing breadth of vaccine-induced antibodies. Currently available HCV panels may not adequately represent the genetic and antigenic diversity of circulating HCV strains, and the lack of standardization of these panels makes it difficult to compare neutralization results obtained in different studies. Here, we describe the selection and validation of a genetically and antigenically diverse reference panel of 15 HCV pseudoparticles (HCVpps) for neutralization assays., Methods: We chose 75 envelope (E1E2) clones to maximize representation of natural polymorphisms observed in circulating HCV isolates, and 65 of these clones generated functional HCVpps. Neutralization sensitivity of these HCVpps varied widely. HCVpps clustered into 15 distinct groups based on patterns of relative sensitivity to 7 broadly neutralizing monoclonal antibodies. We used these data to select a final panel of 15 antigenically representative HCVpps., Results: Both the 65 and 15 HCVpp panels span 4 tiers of neutralization sensitivity, and neutralizing breadth measurements for 7 broadly neutralizing monoclonal antibodies were nearly equivalent using either panel. Differences in neutralization sensitivity between HCVpps were independent of genetic distances between E1E2 clones., Conclusions: Neutralizing breadth of HCV antibodies should be defined using viruses spanning multiple tiers of neutralization sensitivity rather than panels selected solely for genetic diversity. We propose that this multitier reference panel could be adopted as a standard for the measurement of neutralizing antibody potency and breadth, facilitating meaningful comparisons of neutralization results from vaccine studies in different laboratories., (Copyright © 2022 AGA Institute. All rights reserved.)

Nosocomial severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have severely affected bed capacity and patient flow. We utilized whole-genome sequencing (WGS) to identify outbreaks and focus infection control resources and intervention during the United Kingdom's second pandemic wave in late 2020. Phylogenetic analysis of WGS and epidemiological data pinpointed an initial transmission event to an admission ward, with immediate prior community infection linkage documented. High incidence of asymptomatic staff infection with genetically identical viral sequences was also observed, which may have contributed to the propagation of the outbreak. WGS allowed timely nosocomial transmission intervention measures, including admissions ward point-of-care testing and introduction of portable HEPA14 filters. Conversely, WGS excluded nosocomial transmission in 2 instances with temporospatial linkage, conserving time and resources. In summary, WGS significantly enhanced understanding of SARS-CoV-2 clusters in a hospital setting, both identifying high-risk areas and conversely validating existing control measures in other units, maintaining clinical service overall., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America.)

Understanding the impact of prior infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the response to vaccination is a priority for responding to the coronavirus disease 2019 (COVID-19) pandemic. In particular, it is necessary to understand how prior infection plus vaccination can modulate immune responses against variants of concern. To address this, we sampled 20 individuals with and 25 individuals without confirmed previous SARS-CoV-2 infection from a large cohort of health care workers followed serologically since April 2020. All 45 individuals had received two doses of the Pfizer-BioNTech BNT162b2 vaccine with a delayed booster at 10 weeks. Absolute and neutralizing antibody titers against wild-type SARS-CoV-2 and variants were measured using enzyme immunoassays and pseudotype neutralization assays. We observed antibody reactivity against lineage A, B.1.351, and P.1 variants with increasing antigenic exposure, through either vaccination or natural infection. This improvement was further confirmed in neutralization assays using fixed dilutions of serum samples. The impact of antigenic exposure was more evident in enzyme immunoassays measuring SARS-CoV-2 spike protein–specific IgG antibody concentrations. Our data show that multiple exposures to SARS-CoV-2 spike protein in the context of a delayed booster expand the neutralizing breadth of the antibody response to neutralization-resistant SARS-CoV-2 variants. This suggests that additional vaccine boosts may be beneficial in improving immune responses against future SARS-CoV-2 variants of concern.

Introduction. Zika virus (ZIKV) emerged as a public health concern on the American continent during late 2015. As the number of infected grew so did the concerns about its capability to cause long-term damage especially with the appearance of the congenital Zika syndrome (CZS). Proteins from the TAM family of receptor tyrosine kinases (RTKs) were proposed as the cellular receptors, however, due to the ability of the virus to infect a variety of cell lines different strategies to elucidate the tropism of the virus should be investigated. Hypothesis. Pseudotyping is a powerful tool to interrogate the ability of the glycoprotein (GP) to permit entry of viruses. Aim. We aimed to establish a highly tractable pseudotype model using lenti- and retro-viral backbones to investigate the entry pathway of ZIKV. Methodology. We used different glycoprotein constructs and different lenti- or retro-viral backbones, in a matrix of ratios to investigate production of proteins and functional pseudotypes. Results. Varying the ratio of backbone and glycoprotein plasmids did not yield infectious pseudotypes. Moreover, the supplementation of the ZIKV protease or the substitution of the backbone had no positive impact on the infectivity. We showed production of the proteins in producer cells implying the lack of infectious pseudotypes is due to a lack of successful glycoprotein incorporation, rather than lack of protein production. Conclusion. In line with other reports, we were unable to successfully produce infectious pseudotypes using the variety of methods described. Other strategies may be more suitable in the development of an efficient pseudotype model for ZIKV and other flaviviruses.

The coronavirus disease 2019 (COVID-19) pandemic caused by the emergent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health, and there is an urgent need to develop safe and effective vaccines. Here, we report the generation and the preclinical evaluation of a novel replication-defective gorilla adenovirus-vectored vaccine encoding the pre-fusion stabilized Spike (S) protein of SARS-CoV-2. We show that our vaccine candidate, GRAd-COV2, is highly immunogenic both in mice and macaques, eliciting both functional antibodies that neutralize SARS-CoV-2 infection and block Spike protein binding to the ACE2 receptor, and a robust, T helper (Th)1-dominated cellular response. We show here that the pre-fusion stabilized Spike antigen is superior to the wild type in inducing ACE2-interfering, SARS-CoV-2-neutralizing antibodies. To face the unprecedented need for vaccine manufacturing at a massive scale, different GRAd genome deletions were compared to select the vector backbone showing the highest productivity in stirred tank bioreactors. This preliminary dataset identified GRAd-COV2 as a potential COVID-19 vaccine candidate, supporting the translation of the GRAd-COV2 vaccine in a currently ongoing phase I clinical trial (ClinicalTrials.gov: NCT04528641)., Competing Interests: Declaration of interests S. Capone, A.R., M.G., S.B., A.S., A.M.C., R.S., F.B., A. Leuzzi, E.L., G. Miselli, A.N., M.F., F.T., M.S., A.F., S. Colloca, and A.V. are employees of ReiThera Srl. A.F. and S. Colloca are also shareholders of Keires AG. A. Lahm is a consultant for ReiThera Srl. S. Colloca, A. Lahm, A.R., and A.V. are inventors of the patent application number 20183515.4, titled “Gorilla Adenovirus Nucleic Acid- and Amino Acid-Sequences, Vectors Containing Same, and Uses Thereof.” The other authors declare no competing interests., (Copyright © 2021 The American Society of Gene and Cell Therapy. All rights reserved.)

In the early phases of the SARS coronavirus type 2 (SARS-CoV-2) pandemic, testing focused on individuals fitting a strict case definition involving a limited set of symptoms together with an identified epidemiological risk, such as contact with an infected individual or travel to a high-risk area. To assess whether this impaired our ability to detect and control early introductions of the virus into the UK, we PCR-tested archival specimens collected on admission to a large UK teaching hospital who retrospectively were identified as having a clinical presentation compatible with COVID-19. In addition, we screened available archival specimens submitted for respiratory virus diagnosis, and dating back to early January 2020, for the presence of SARS-CoV-2 RNA. Our data provides evidence for widespread community circulation of SARS-CoV-2 in early February 2020 and into March that was undetected at the time due to restrictive case definitions informing testing policy. Genome sequence data showed that many of these early cases were infected with a distinct lineage of the virus. Sequences obtained from the first officially recorded case in Nottinghamshire - a traveller returning from Daegu, South Korea - also clustered with these early UK sequences suggesting acquisition of the virus occurred in the UK and not Daegu. Analysis of a larger sample of sequences obtained in the Nottinghamshire area revealed multiple viral introductions, mainly in late February and through March. These data highlight the importance of timely and extensive community testing to prevent future widespread transmission of the virus.