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At present, diagnosis of Ebola virus disease requires transport of venepuncture blood to field biocontainment laboratories for testing by real-time RT-PCR, resulting in delays that complicate patient care and infection control efforts. Therefore, an urgent need exists for a point-of-care rapid diagnostic test for this disease. In this Article, we report the results of a field validation of the Corgenix ReEBOV Antigen Rapid Test kit. We performed the rapid diagnostic test on fingerstick blood samples from 106 individuals with suspected Ebola virus disease presenting at two clinical centres in Sierra Leone. Adults and children who were able to provide verbal consent or assent were included; we excluded patients with haemodynamic instability and those who were unable to cooperate with fingerstick or venous blood draw. Two independent readers scored each rapid diagnostic test, with any disagreements resolved by a third. We compared point-of-care rapid diagnostic test results with clinical real-time RT-PCR results (RealStar Filovirus Screen RT-PCR kit 1·0; altona Diagnostics GmbH, Hamburg, Germany) for venepuncture plasma samples tested in a Public Health England field reference laboratory (Port Loko, Sierra Leone). Separately, we performed the rapid diagnostic test (on whole blood) and real-time RT-PCR (on plasma) on 284 specimens in the reference laboratory, which were submitted to the laboratory for testing from many clinical sites in Sierra Leone, including our two clinical centres. In point-of-care testing, all 28 patients who tested positive for Ebola virus disease by RT-PCR were also positive by fingerstick rapid diagnostic test (sensitivity 100% [95% CI 87·7–100]), and 71 of 77 patients who tested negative by RT-PCR were also negative by the rapid diagnostic test (specificity 92·2% [95% CI 83·8–97·1]). In laboratory testing, all 45 specimens that tested positive by RT-PCR were also positive by the rapid diagnostic test (sensitivity 100% [95% CI 92·1–100]), and 214 of 232 specimens that tested negative by RT-PCR were also negative by the rapid diagnostic test (specificity 92·2% [88·0–95·3]). The two independent readers agreed about 95·2% of point-of-care and 98·6% of reference laboratory rapid diagnostic test results. Cycle threshold values ranged from 15·9 to 26·3 (mean 22·6 [SD 2·6]) for the PCR-positive point-of-care cohort and from 17·5 to 26·3 (mean 21·5 [2·7]) for the reference laboratory cohort. Six of 16 banked plasma samples from rapid diagnostic test-positive and altona-negative patients were positive by an alternative real-time RT-PCR assay (the Trombley assay); three (17%) of 18 samples from individuals who were negative by both the rapid diagnostic test and altona test were also positive by Trombley. The ReEBOV rapid diagnostic test had 100% sensitivity and 92% specificity in both point-of-care and reference laboratory testing in this population (maximum cycle threshold 26·3). With two independent readers, the test detected all patients who were positive for Ebola virus by altona real-time RT-PCR; however, this benchmark itself had imperfect sensitivity. Abundance Foundation.

The virions of enteroviruses such as poliovirus undergo a global conformational change after binding to the cellular receptor, characterized by a 4% expansion, and by the opening of holes at the two and quasi-three-fold symmetry axes of the capsid. The resultant particle is called a 135S particle or A-particle and is thought to be on the pathway to a productive infection. Previously published studies have concluded that the membrane-interactive peptides, namely VP4 and the N-terminus of VP1, are irreversibly externalized in the 135S particle. However, using established protocols to produce the 135S particle, and single particle cryo-electron microscopy methods, we have identified at least two unique states that we call the early and late 135S particle. Surprisingly, only in the \"late\" 135S particles have detectable levels of the VP1 N-terminus been trapped outside the capsid. Moreover, we observe a distinct density inside the capsid that can be accounted for by VP4 that remains associated with the genome. Taken together our results conclusively demonstrate that the 135S particle is not a unique conformation, but rather a family of conformations that could exist simultaneously.

The envelope glycoprotein (Env) of the Human Immunodeficiency Virus Type-1 (HIV-1) is a critical determinant of viral infectivity, tropism and is the main target for humoral immunity; however, little is known about the cellular machinery that directs Env trafficking and its incorporation into nascent virions. Here we identify the mammalian retromer complex as a novel and important cellular factor regulating Env trafficking. Retromer mediates endosomal sorting and is most closely associated with endosome-to-Golgi transport. Consistent with this function, inactivating retromer using RNAi targeting the cargo selective trimer complex inhibited retrograde trafficking of endocytosed Env to the Golgi. Notably, in HIV-1 infected cells, inactivating retromer modulated plasma membrane expression of Env, along with Env incorporation into virions and particle infectivity. Mutagenesis studies coupled with coimmunoprecipitations revealed that retromer-mediated trafficking requires the Env cytoplasmic tail that we show binds directly to retromer components Vps35 and Vps26. Taken together these results provide novel insight into regulation of HIV-1 Env trafficking and infectious HIV-1 morphogenesis and show for the first time a role for retromer in the late-steps of viral replication and assembly of a virus.

Accurate and rapid point-of-care (PoC) diagnostics are critical to the control of the COVID-19 pandemic. The current standard for accurate diagnosis of SARS-CoV-2 is laboratory-based reverse transcription polymerase chain reaction (RT-PCR) assays. Here, a preliminary prospective performance evaluation of the QuantuMDx Q-POC SARS-CoV-2 RT-PCR assay is reported. Between November 2020 and March 2021, 49 longitudinal combined nose/throat (NT) swabs from 29 individuals hospitalised with RT-PCR confirmed COVID-19 were obtained at St George’s Hospital, London. In addition, 101 mid-nasal (MN) swabs were obtained from healthy volunteers in June 2021. These samples were used to evaluate the Q-POC SARS-CoV-2 RT-PCR assay. The primary analysis was to compare the sensitivity and specificity of the Q-POC test against a reference laboratory-based RT-PCR assay. The overall sensitivity of the Q-POC test compared with the reference test was 96.88% (83.78– 99.92% CI) for a cycle threshold (Ct) cut-off value for the reference test of 35 and 80.00% (64.35–90.95% CI) without altering the reference test’s Ct cut-off value of 40. The Q-POC test is a sensitive, specific and rapid PoC test for SARS-CoV-2 at a reference Ct cut-off value of 35. The Q-POC test provides an accurate option for RT-PCR at PoC without the need for sample pre-processing and laboratory handling, enabling rapid diagnosis and clinical triage in acute care and other settings.

HIV-1 can disseminate between susceptible cells by two mechanisms: cell-free infection following fluid-phase diffusion of virions and by highly-efficient direct cell-to-cell transmission at immune cell contacts. The contribution of this hybrid spreading mechanism, which is also a characteristic of some important computer worm outbreaks, to HIV-1 progression in vivo remains unknown. Here we present a new mathematical model that explicitly incorporates the ability of HIV-1 to use hybrid spreading mechanisms and evaluate the consequences for HIV-1 pathogenenesis. The model captures the major phases of the HIV-1 infection course of a cohort of treatment naive patients and also accurately predicts the results of the Short Pulse Anti-Retroviral Therapy at Seroconversion (SPARTAC) trial. Using this model we find that hybrid spreading is critical to seed and establish infection, and that cell-to-cell spread and increased CD4+ T cell activation are important for HIV-1 progression. Notably, the model predicts that cell-to-cell spread becomes increasingly effective as infection progresses and thus may present a considerable treatment barrier. Deriving predictions of various treatments' influence on HIV-1 progression highlights the importance of earlier intervention and suggests that treatments effectively targeting cell-to-cell HIV-1 spread can delay progression to AIDS. This study suggests that hybrid spreading is a fundamental feature of HIV infection, and provides the mathematical framework incorporating this feature with which to evaluate future therapeutic strategies.

Picornaviruses are non-enveloped RNA viruses that enter cells via receptor-mediated endocytosis. Because they lack an envelope, picornaviruses face the challenge of delivering their RNA genomes across the membrane of the endocytic vesicle into the cytoplasm to initiate infection. Currently, the mechanism of genome release and translocation across membranes remains poorly understood. Within the enterovirus genus, poliovirus, rhinovirus 2, and rhinovirus 16 have been proposed to release their genomes across intact endosomal membranes through virally induced pores, whereas one study has proposed that rhinovirus 14 releases its RNA following disruption of endosomal membranes. For the more distantly related aphthovirus genus (e.g. foot-and-mouth disease viruses and equine rhinitis A virus) acidification of endosomes results in the disassembly of the virion into pentamers and in the release of the viral RNA into the lumen of the endosome, but no details have been elucidated as how the RNA crosses the vesicle membrane. However, more recent studies suggest aphthovirus RNA is released from intact particles and the dissociation to pentamers may be a late event. In this study we have investigated the RNase A sensitivity of genome translocation of poliovirus using a receptor-decorated-liposome model and the sensitivity of infection of poliovirus and equine-rhinitis A virus to co-internalized RNase A. We show that poliovirus genome translocation is insensitive to RNase A and results in little or no release into the medium in the liposome model. We also show that infectivity is not reduced by co-internalized RNase A for poliovirus and equine rhinitis A virus. Additionally, we show that all poliovirus genomes that are internalized into cells, not just those resulting in infection, are protected from RNase A. These results support a finely coordinated, directional model of viral RNA delivery that involves viral proteins and cellular membranes.

Introduction: Prolyl-4-hydroxylases ( P4H ) catalyse the irreversible conversion of proline to hydroxyproline, constituting a common posttranslational modification of proteins found in humans, plants, and microbes. Hydroxyproline residues can be further modified in plants to yield glycoproteins containing characteristic O-glycans. It is currently unknown how these plant endogenous modifications impact protein functionality and they cause considerable concerns for the recombinant production of therapeutic proteins in plants. In this study, we carried out host engineering to generate a therapeutic glycoprotein largely devoid of plant-endogenous O-glycans for functional characterization. Methods: Genome editing was used to inactivate two genes coding for enzymes of the P4H10 subfamily in the widely used expression host Nicotiana benthamiana . Using glycoengineering in plants and expression in human HEK293 cells we generated four variants of a potent, SARS-CoV-2 neutralizing antibody, COVA2-15 IgA1. The variants that differed in the number of modified proline residues and O-glycan compositions of their hinge region were assessed regarding their physicochemical properties and functionality. Results: We found that plant endogenous O-glycan formation was strongly reduced on IgA1 when transiently expressed in the P4H10 double mutant N. benthamiana plant line. The IgA1 glycoforms displayed differences in proteolytic stability and minor differences in receptor binding thus highlighting the importance of O-glycosylation in the hinge region of human IgA1. Discussion: This work reports the successful protein O-glycan engineering of an important plant host for recombinant protein expression. While the complete removal of endogenous hydroxyproline residues from the hinge region of plant-produced IgA1 is yet to be achieved, our engineered line is suitable for structure-function studies of O-glycosylated recombinant glycoproteins produced in plants.

Background Older adults, particularly in long-term care facilities (LTCF), remain at considerable risk from SARS-CoV-2. Data on the protective effect and mechanisms of hybrid immunity are skewed towards young adults precluding targeted vaccination strategies. Methods A single-centre longitudinal seroprevalence vaccine response study was conducted with 280 LCTF participants (median 82 yrs, IQR 76-88 yrs; 95.4% male). Screening by SARS-CoV-2 polymerase chain reaction with weekly asymptomatic/symptomatic testing (March 2020-October 2021) and serology pre-/post-two-dose Pfizer-BioNTech BNT162b2 vaccination for (i) anti-nucleocapsid, (ii) quantified anti-receptor binding domain (RBD) antibodies at three time-intervals, (iii) pseudovirus neutralisation, and (iv) inhibition by anti-RBD competitive ELISA were conducted. Neutralisation activity: antibody titre relationship was assessed via beta linear-log regression and RBD antibody-binding inhibition: post-vaccine infection relationship by Wilcoxon rank sum test. Results Here we show neutralising antibody titres are 9.2-fold (95% CI 5.8–14.5) higher associated with hybrid immunity ( p  < 0.00001); +7.5-fold (95% CI 4.6-12.1) with asymptomatic infection; +20.3-fold, 95% (CI 9.7-42.5) with symptomatic infection. A strong association is observed between antibody titre: neutralising activity ( p  < 0.00001) and rising anti-RBD antibody titre: RBD antibody-binding inhibition ( p  < 0.001), although 18/169 (10.7%) participants with high anti-RBD titre (>100BAU/ml), show inhibition <75%. Higher RBD antibody-binding inhibition values are associated with hybrid immunity and reduced likelihood of infection ( p  = 0.003). Conclusions Hybrid immunity in older adults was associated with considerably higher antibody titres, neutralisation and inhibition capacity. Instances of high anti-RBD titre with lower inhibition suggests antibody quantity and quality as independent potential correlates of protection, highlighting added value of measuring inhibition over antibody titre alone to inform vaccine strategy. Plain language summary Older adults continue to be at risk of COVID-19, particularly in residential care home settings. We investigated the effect of infection and vaccination on antibody development and subsequent SARS-CoV-2 infection in older adults. Antibodies are proteins that the immune system produces on infection or vaccination that can help respond to subsequent infection with SARS-CoV-2. We found that older adults produce antibodies to SARS-CoV-2 after 2-doses of Pfizer BioNTech BNT162b2 vaccine. The strongest immune responses were seen among those older adults who also had prior history of infection. The results highlight the importance of both antibody quality and quantity when considering possible indicators of protection against COVID-19 and supports the need for a third, booster, vaccination in this age group.. Pallett, Heskin et al. monitor longitudinal response in older adults following vaccination with the BNT162b2 COVID-19 (Pfizer-BioNTech) vaccination. Hybrid immunity is associated with higher antibody titres, neutralisation and inhibition capacity.

Equine rhinitis A virus (ERAV) is closely related to foot-and-mouth disease virus (FMDV), belonging to the genus Aphthovirus of the Picornaviridae. How picornaviruses introduce their RNA genome into the cytoplasm of the host cell to initiate replication is unclear since they have no lipid envelope to facilitate fusion with cellular membranes. It has been thought that the dissociation of the FMDV particle into pentameric subunits at acidic pH is the mechanism for genome release during cell entry, but this raises the problem of how transfer across the endosome membrane of the genome might be facilitated. In contrast, most other picornaviruses form 'altered' particle intermediates (not reported for aphthoviruses) thought to induce membrane pores through which the genome can be transferred. Here we show that ERAV, like FMDV, dissociates into pentamers at mildly acidic pH but demonstrate that dissociation is preceded by the transient formation of empty 80S particles which have released their genome and may represent novel biologically relevant intermediates in the aphthovirus cell entry process. The crystal structures of the native ERAV virus and a low pH form have been determined via highly efficient crystallization and data collection strategies, required due to low virus yields. ERAV is closely similar to FMDV for VP2, VP3 and part of VP4 but VP1 diverges, to give a particle with a pitted surface, as seen in cardioviruses. The low pH particle has internal structure consistent with it representing a pre-dissociation cell entry intermediate. These results suggest a unified mechanism of picornavirus cell entry.

IntroductionThe present study aimed to investigate if antibodies against SARS-CoV-2 can be detected in the colorectal mucus (CM) overlaying the internal surface of the gut in COVID-19 patients. This approach could potentially be used for the assessment of the immunological status of individuals recovering from COVID-19 infection.MethodsThe presence of anti-COVID antibodies (IgA and IgG) was assessed in samples of colorectal mucus (CM) non-invasively collected (self-collected) from patients hospitalized with COVID-19 infection. Samples were collected from 12 patients with COVID-19 confirmed by positive PCR test for the virus (some of these patients collected samples repeatedly every week) and 8 healthy controls (8 samples) were analysed. The samples were tested for antibodies (either IgA or IgG) against trimeric spike protein of SARS-CoV-2 using in-house ELISA assays.ResultsPreliminary results of the analyses are presented in figure 1.Abstract O38 Figure 1Detection of IgG antibodies against trimeric spike protein of SARS-CoV-2 in CM samples collected in Viral Transport Medium (VTM) from three patients with COVID-19 (cases 4, 8 and 9: two time points for patient 4 and three time points for patient 9) and one COVID-free individual (control 40).Results for serial sample dilutions are shown for each tested sample. CM samples from cases 4 and 9 collected one week following COVID-19 infection confirmation are positive for antibody presence. Moreover, sample from case 9 collected two weeks following COVID-19 infection confirmation is weakly positive for antibody presence as well.Preliminary results presented in Figure 1 clearly indicate that IgG antibodies against trimeric spike protein of SARS-CoV-2 can be detected in CM samples collected from patients recovering from COVID-19 infection. It is important to note that patients 4 and 9 found to be positive for IgG antibody one week following COVID-19 confirmation were already asymptomatic. These encouraging early results suggest that the peak of anti-SARS-CoV-2 antibodies occurs at least one week following disease development, but further analysis is needed to confirm the existence of this interesting trend. Further work is ongoing to assess IgA antibody detection in CM.ConclusionsThe preliminary results of this study indicate that the detection of anti-SARS-CoV-2 IgG antibodies in non-invasively collected CM samples may be used for developing new diagnostic techniques for assessing immunity against COVID-19 infection, especially in asymptomatic individuals. This could also provide a tool for assessing immune status of individuals undergoing anti-COVID vaccination.