Rationale: Coronavirus disease 2019 (COVID-19) is characterized by increased incidence of microthrombosis with hyperactive platelets sporadically containing viral RNA. It is unclear if SARS-CoV-2 (severe acute respiratory syndrome, corona virus-2) directly alters platelet activation or if these changes are a reaction to infection-mediated global inflammatory alterations. Importantly, the direct effect of SARS-CoV-2 on platelets has yet to be studied. Objective: To characterize the direct SARS-CoV-2–platelet interactions using in vitro studies with purified infectious virions and samples from infected patients. Methods and Results: Platelet RNA analyzed by ARTIC v3 sequencing for SARS-CoV-2 showed presence of fragmented viral genome in all patients with COVID-19. Immunofluorescent imaging of platelets from patients with COVID-19 confirmed presence of SARS-CoV-2 proteins, whereas there was no detection of viral RNA by real-time quantitative polymerase chain reaction. Transmission electron microscopy of platelets incubated with purified SARS-CoV-2 virions demonstrated rapid internalization and digestion leading to distinct morphological changes and resulted in a release of extracellular vesicles. Interactions between SARS-CoV-2 and platelets occurred with or without ACE (angiotensin-converting enzyme) 2 presence as measured by immunofluorescence. Transmission electron microscopy showed that SARS-CoV-2 virions became internalized when they were attached to microparticles, bypassing the need for ACE2. Enrichment analysis of platelet transcriptome from patients with acute COVID-19, compared with those with clinical thrombosis, suggested upregulation of pathways related to virally mediated cell death, specifically necroptosis and apoptosis. Platelets incubated with infectious virus appeared to undergo cell death in 30 minutes postincubation as assessed by transmission electron microscopy and platelets from patients with COVID-19 showed evidence of increased markers of apoptosis and necroptosis by Western blot. Immunofluorescence confirmed colocalization of SARS-CoV-2 with phospho-MLKL (mixed lineage kinase domain-like pseudokinase) and caspase 3 on nonpermeabilized platelets in vitro and in COVID-19 platelets. Conclusions: Platelets internalize SARS-CoV-2 virions, directly or attached to microparticles, and viral internalization leads to rapid digestion, programmed cell death, and extracellular vesicle release. During COVID-19, platelets mediate a rapid response to SARS-CoV-2 and this response can contribute to dysregulated immunity and thrombosis.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge, causing surges, breakthrough infections, and devastating losses—underscoring the importance of identifying SARS-CoV-2 antivirals. A simple, accessible human cell culture model permissive to SARS-CoV-2 variants is critical for identifying and assessing antivirals in a high-throughput manner. Although human alveolar A549 cells are a valuable model for studying respiratory virus infections, they lack two essential host factors for SARS-CoV-2 infection: angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). SARS-CoV-2 uses the ACE2 receptor for viral entry and TMPRSS2 to prime the SARS-CoV-2 spike protein, both of which are negligibly expressed in A549 cells. Here, we report the generation of a suitable human cell line for SARS-CoV-2 studies by transducing human ACE2 and TMPRSS2 into A549 cells. We show that subclones highly expressing ACE2 and TMPRSS2 (“ACE2plus” and the subclone “ACE2plusC3”) are susceptible to infection with SARS-CoV-2, including the delta and omicron variants. These subclones express more ACE2 and TMPRSS2 transcripts than existing commercial A549 cells engineered to express ACE2 and TMPRSS2. Additionally, the antiviral drugs EIDD-1931, remdesivir, nirmatrelvir, and nelfinavir strongly inhibit SARS-CoV-2 variants in our infection model. Our data show that ACE2plusC3 cells are highly permissive to SARS-CoV-2 infection and can be used to identify anti-SARS-CoV-2 drugs.

Background: Understanding the properties of HIV-1 variants that are transmitted from women to their infants is crucial to improving strategies to prevent transmission. In this study, 162 full-lengthenvelope(env) clones were generated from plasma RNA obtained from 5 HIV-1 Clade B infected mother-infant pairs. Following extensive genotypic and phylogenetic analyses, 35 representative clones were selected for functional studies.Results: Infant quasispecies were highly homogeneous and generally represented minor maternal variants, consistent with transmission across a selective bottleneck. Infant clones did not differ from the maternal inenvlength, or glycosylation. All infant variants utilized the CCR5 co-receptor, but were not macrophage tropic. Relatively high levels (IC₅₀≥ 100 μg/ml) of autologous maternal plasma IgG were required to neutralize maternal and infant viruses; however, all infant viruses were neutralized by pooled sera from HIV-1 infected individuals, implying that they were not inherently neutralization-resistant. All infant viruses were sensitive to the HIV-1 entry inhibitors Enfuvirtide and soluble CD4; none were resistant to Maraviroc. Sensitivity to human monoclonal antibodies 4E10, 2F5, b12 and 2G12 varied.Conclusions: This study provides extensive characterization of the genotypic and functional properties of HIV-1envshortly after transmission. We present the first detailed comparisons of the macrophage tropism of infant and maternalenvvariants and their sensitivity to Maraviroc, the only CCR5 antagonist approved for therapeutic use. These findings may have implications for improving approaches to prevent mother-to-child HIV-1 transmission.

Rapid and cost-effective methods for HIV-1 diagnosis and viral load monitoring would greatly enhance the clinical management of HIV-1 infected adults and children in limited-resource settings. Recent recommendations to treat perinatally infected infants within the first year of life are feasible only if early diagnosis is routinely available. Dried blood spots (DBS) on filter paper are an easy and convenient way to collect and transport blood samples. A rapid and cost effective method to diagnose and quantify HIV-1 from DBS is urgently needed to facilitate early diagnosis of HIV-1 infection and monitoring of antiretroviral therapy. We have developed a real-time LightCycler (rtLC) PCR assay to detect and quantify HIV-1 from DBS. HIV-1 RNA extracted from DBS was amplified in a one-step, single-tube system using primers specific for long-terminal repeat sequences that are conserved across all HIV-1 clades. SYBR Green dye was used to quantify PCR amplicons and HIV-1 RNA copy numbers were determined from a standard curve generated using serially diluted known copies of HIV-1 RNA. This assay detected samples across clades, has a dynamic range of 5 log₁₀, and %CV <8% up to 4 log₁₀ dilution. Plasma HIV-1 RNA copy numbers obtained using this method correlated well with the Roche Ultrasensitive (r = 0.91) and branched DNA (r = 0.89) assays. The lower limit of detection (95%) was estimated to be 136 copies. The rtLC DBS assay was 2.5 fold rapid as well as 40-fold cheaper when compared to commercial assays. Adaptation of the assay into other real-time systems demonstrated similar performance. The accuracy, reliability, genotype inclusivity and affordability, along with the small volumes of blood required for the assay suggest that the rtLC DBS assay will be useful for early diagnosis and monitoring of pediatric HIV-1 infection in resource-limited settings.

Coronavirus disease 2019 (COVID-19) has had devastating effects worldwide, with particularly high morbidity and mortality in outbreaks on residential care facilities. Amantadine, originally licensed as an antiviral agent for therapy and prophylaxis against influenza A virus, has beneficial effects on patients with Parkinson’s disease and is used for treatment of Parkinson’s disease, multiple sclerosis, acquired brain injury, and various other neurological disorders. Recent observational data suggest an inverse relationship between the use of amantadine and COVID-19. Adamantanes, including amantadine and rimantadine, are reported to have in vitro activity against severe acute respiratory syndrome coronavirus (SARS-CoV) and, more recently, SARS-CoV-2. We hypothesized that adamantanes have antiviral activity against SARS-CoV-2, including variant strains. To assess the activity of adamantanes against SARS-CoV-2, we used in vitro and in vivo models of infection. We established that amantadine, rimantadine, and tromantadine inhibit the growth of SARS-CoV-2 in vitro in cultured human epithelial cells. While neither rimantadine nor amantadine reduces lung viral titers in mice infected with mouse-adapted SARS-CoV-2, rimantadine significantly reduces viral titers in the lungs in golden Syrian hamsters infected with SARS-CoV-2. In summary, rimantadine has antiviral activity against SARS-CoV-2 in human alveolar epithelial cells and in the hamster model of SARS-CoV-2 lung infection. The evaluation of amantadine or rimantadine in human randomized controlled trials can definitively address applications for the treatment or prevention of COVID-19.