Hybrid spreading mechanisms and T cell activation shape the dynamics of HIV-1 infection

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

Equine Rhinitis A Virus and Its Low pH Empty Particle: Clues Towards an Aphthovirus Entry Mechanism?

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

Accuracy of the Mologic COVID-19 rapid antigen test: a prospective multi-centre analytical and clinical evaluation

Background: The coronavirus disease 2019 (COVID-19) pandemic has highlighted the reliance on antigen detection rapid diagnostic tests (Ag-RDTs). Their evaluation at point of use is a priority. Methods: Here, we report a multi-centre evaluation of the analytical sensitivity, specificity, and clinical accuracy of the Mologic COVID-19 Ag-RDT by comparing to reverse transcriptase polymerase chain reaction (RT-qPCR) results from individuals with and without COVID-19 symptoms. Participants had attended hospitals in Merseyside, hospital and ambulance services in Yorkshire, and drive-through testing facilities in Northumberland, UK. Results: The limit of detection of the Mologic COVID-19 Ag-RDT was 5.0 x 102 pfu/ml in swab matrix with no cross-reactivity and interference for any other pathogens tested. A total of 347 participants were enrolled from 26th of November 2020 to 15th of February 2021 with 39.2% (CI 34.0-44.6) testing RT-qPCR positive for SARS-CoV-2. The overall sensitivity and specificity of the Mologic Ag-RDT compared to the reference SARS-CoV-2 RT-qPCR were 85.0% (95% CI 78.3-90.2) and 97.8% (95.0-99.3), respectively. Sensitivity was stratified by RT-qPCR cycle threshold (Ct) and 98.4% (91.3-100) of samples with a Ct less than 20 and 93.2% (86.5-97.2) of samples with a Ct less than 25 were detected using the Ag-RDT. Clinical accuracy was stratified by sampling strategy, swab type and clinical presentation. Mologic COVID-19 Ag-RDT demonstrated highest sensitivity with nose/throat swabs compared with throat or nose swabs alone; however, the differences were not statistically significant. Conclusions: Overall, the Mologic test had high diagnostic accuracy across multiple different settings, different demographics, and on self-collected swab specimens. These findings suggest the Mologic rapid antigen test may be deployed effectively across a range of use settings

Rapid outbreak sequencing of Ebola virus in Sierra Leone identifies transmission chains linked to sporadic cases.

To end the largest known outbreak of Ebola virus disease (EVD) in West Africa and to prevent new transmissions, rapid epidemiological tracing of cases and contacts was required. The ability to quickly identify unknown sources and chains of transmission is key to ending the EVD epidemic and of even greater importance in the context of recent reports of Ebola virus (EBOV) persistence in survivors. Phylogenetic analysis of complete EBOV genomes can provide important information on the source of any new infection. A local deep sequencing facility was established at the Mateneh Ebola Treatment Centre in central Sierra Leone. The facility included all wetlab and computational resources to rapidly process EBOV diagnostic samples into full genome sequences. We produced 554 EBOV genomes from EVD cases across Sierra Leone. These genomes provided a detailed description of EBOV evolution and facilitated phylogenetic tracking of new EVD cases. Importantly, we show that linked genomic and epidemiological data can not only support contact tracing but also identify unconventional transmission chains involving body fluids, including semen. Rapid EBOV genome sequencing, when linked to epidemiological information and a comprehensive database of virus sequences across the outbreak, provided a powerful tool for public health epidemic control efforts

Functional Analyses of Novel Insect Virus IRES Elements.

Rhopalosiphum padi virus (RhPV) is a member of the Dicistroviridae. The genomes of these viruses contain two open reading frames, each preceded by distinct internal ribosome entry site (IRES) elements. The activity of the RhPV 5' IRES element has previously been demonstrated in mammalian, insect and plant translation systems. It has also been shown that the RhPV 5' IRES forms 48S initiation complexes in vitro with just the mammalian initiation factors eIF2, eIF3 and elFl. It is also possible to delete large regions of the 5' IRES without affecting initiation complex formation. We have now defined the minimal sequences required for directing internal initiation in mammalian, plant and insect translation systems. Fragments of around 130 nt from the 3' portion of the 5'untranslated region (UTR) can direct translation in each of these systems. Thus, the 3' region within the 5'UTR seems to be critical for IRES function. Interestingly, this region is mainly unstructured, making the RhPV 5'IRES unique among viral IRES elements. The RhPV IRES was also compared to the 5'UTR of the insect iflavirus Kakugo virus (KV). Iflaviruses had been initially classified as insect picoma-like viruses alongside the Dicistroviridae. The KV 5'UTR was shown to contain an IRES element similar in function to that of RhPV. Since IRES elements have been shown to require trans-acting factors (ITAFs) for their activity, in addition to the canonical initiation factors, we investigated the interactions of the RhPV IRES with cellular proteins of mammalian, plant and insect origin. In our preliminary studies we identified proteins with a potential role as ITAFs. The potential advantages of the RhPV IRES in biotechnology were assessed in conjunction with a Sindbis virus-based expression system. Like the RhPV 5'IRES, this system is able to function in mammalian and insect systems. However, our preliminary experiments in mammalian cells were not able to clearly establish the advantages of the RhPV 5'IRES in such a system

Cell Entry of the Aphthovirus Equine Rhinitis A Virus Is Dependent on Endosome Acidification▿

Equine rhinitis A virus (ERAV) is genetically closely related to foot-and-mouth disease virus (FMDV), and both are now classified within the genus Aphthovirus of the family Picornaviridae. For disease security reasons, FMDV can be handled only in high-containment facilities, but these constraints do not apply to ERAV, making it an attractive alternative for the study of aphthovirus biology. Here, we show, using immunofluorescence, pharmacological agents, and dominant negative inhibitors, that ERAV entry occurs (as for FMDV) via clathrin-mediated endocytosis and acidification of early endosomes. This validates the use of ERAV as a model system to study the mechanism of cell entry by FMDV

Retromer Regulates HIV-1 Envelope Glycoprotein Trafficking and Incorporation into Virions

<div><p>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.</p></div

The gp41 cytoplasmic tail mediates retromer-dependent Golgi retrieval.

<p><b>A)</b> Immunostaining of intracellular CD8-gp41CT (green) and Vps26 (red) in HeLa cells. Panels are single <i>xy</i> slices and are a representative example from three independent experiments. Coincident staining appears yellow. Scale bar is 10 microns. <b>B)</b> CD8-gp41CT expressing cells were treated with control or Vps26 siRNA. Cells were incubated with anti-CD8 monoclonal antibody at 4°C for 30 min, washed and incubated 37°C for 10 and 120 min. Cells were fixed, permeabilized and stained for the Golgi marker giantin (red) and fluorescently conjugated anti-mouse secondary antibody to localize the pool of internalized CD8-gp41CT (green). Panels are maximum intensity projections reconstructed from serial <i>Z</i> sections through the entire volume of the cell. Data are representative of three independent experiments. Scale bar is 20 microns. <b>C)</b> Immunostaining of total intracellular CD8-gp41CT (green) and Vps35 (red) in HeLa cells. Panels are single <i>xy</i> slices and are a representative example from three independent experiments. Coincident staining appears yellow. Scale bar is 15 microns. <b>D)</b> CD8-gp41CT expressing cells were treated with control or Vps35 siRNA and antibody-feeding and Golgi retrieval performed as described in B. Data are representative of three independent experiments. Scale bar is 20 microns. Western blot confirming efficient knockdown of Vps35 in HeLa cells following transfection with control or Vps35 siRNA as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004518#ppat-1004518-g001" target="_blank">Figure 1</a>. Band intensities were quantified using ImageJ and the percentage of remaining Vps35 relative to tubulin was calculated. One representative Western blot from three independent experiments is shown.</p