Cyanovirin-N binds to select SARS-CoV-2 spike oligosaccharides outside of the receptor binding domain and blocks infection by SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped positive stranded RNA virus which has caused the recent deadly pandemic called COVID-19. The SARS-CoV-2 virion is coated with a heavily glycosylated Spike glycoprotein which is responsible for attachment and entry into target cells. One, as yet unexploited strategy for preventing SARS-CoV-2 infections, is the targeting of the glycans on Spike. Lectins are carbohydrate-binding proteins produced by plants, algae, and cyanobacteria. Some lectins can neutralize enveloped viruses displaying external glycoproteins, offering an alternative therapeutic approach for the prevention of infection with virulent ß-coronaviruses, such as SARS-CoV-2. Here we show that the cyanobacterial lectin cyanovirin-N (CV-N) can selectively target SARS-CoV-2 Spike oligosaccharides and inhibit SARS-CoV-2 infection in vitro and in vivo. CV-N neutralizes Delta and Omicron variants in vitro better than earlier circulating viral variants. CV-N binds selectively to Spike with a Kd as low as 15 nM and a stoichiometry of 2 CV-N: 1 Spike but does not bind to the receptor binding domain (RBD). Further mapping of CV-N binding sites on Spike shows that select high-mannose oligosaccharides in the S1 domain of Spike are targeted by CV-N. CV-N also reduced viral loads in the nares and lungs in vivo to protect hamsters against a lethal viral challenge. In summary, we present an anti-coronavirus agent that works by an unexploited mechanism and prevents infection by a broad range of SARS-CoV-2 strains.

Copper regulation disturbance linked to oxidative stress and cell death during Zika virus infection in human astrocytes.

The Zika virus (ZIKV) caused neurological abnormalities in more than 3500 Brazilian newborns between 2015 and 2020. Data have pointed to oxidative stress in astrocytes as well as to dysregulations in neural cell proliferation and cell cycle as important events accounting for the cell death and neurological complications observed in Congenital Zika Syndrome. Copper imbalance has been shown to induce similar alterations in other pathologies, and disturbances in copper homeostasis have already been described in viral infections. Here, we investigated copper homeostasis imbalance as a factor that could contribute to the cytotoxic effects of ZIKV infection in astrocytes. Human induced pluripotent stem cell-derived astrocytes were infected with ZIKV; changes in the gene expression of copper homeostasis proteins were analyzed. The effect of the administration of CuCl2 or a copper chelator on oxidative stress, cell viability and percentage of infection were also studied. ZIKV infection leads to a downregulation of one of the transporters mediating copper release, ATP7B protein. We also observed the activation of mechanisms that counteract high copper levels, including the synthesis of copper chaperones and the reduction of the copper importer protein CTR1. Finally, we show that chelator-mediated copper sequestration in ZIKV-infected astrocytes reduces the levels of reactive oxygen species and improves cell viability, but does not change the overall percentage of infected cells. In summary, our results show that copper homeostasis imbalance plays a role in the pathology of ZIKV in astrocytes, indicating that it may also be a factor accounting for the developmental abnormalities in the central nervous system following viral infection. Evaluating micronutrient levels and the use of copper chelators in pregnant women susceptible to ZIKV infection may be promising strategies to manage novel cases of congenital ZIKV syndrome.

Intracellular host cell membrane remodelling induced by SARS-CoV-2 infection in vitro.

BACKGROUND INFORMATION: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection induces an alteration in the endomembrane system of the mammalian cells. In this study, we used transmission electron microscopy and electron tomography to investigate the main structural alterations in the cytoplasm of Vero cells infected with a SARS-CoV-2 isolate from São Paulo state (Brazil). RESULTS: Different membranous structures derived from the zippered endoplasmic reticulum were observed along with virus assembly through membrane budding. Also, we demonstrated the occurrence of annulate lamellae in the cytoplasm of infected cells and the presence of virus particles in the perinuclear space. CONCLUSIONS AND SIGNIFICANCE: This study contributes to a better understanding of the cell biology of SARS-CoV-2 and the mechanisms of the interaction of the virus with the host cell that promote morphological changes, recruitment of organelles and cell components, in a context of a virus-induced membrane remodelling.

Identification and characterisation of SARS-CoV-2 and Human alphaherpesvirus 1 from a productive coinfection in a fatal COVID-19 case.

BACKGROUND: During routine Coronavirus disease 2019 (COVID-19) diagnosis, an unusually high viral load was detected by reverse transcription real-time polymerase chain reaction (RT-qPCR) in a nasopharyngeal swab sample collected from a patient with respiratory and neurological symptoms who rapidly succumbed to the disease. Therefore we sought to characterise the infection. OBJECTIVES: We aimed to determine and characterise the etiological agent responsible for the poor outcome. METHODS: Classical virological methods, such as plaque assay and plaque reduction neutralisation test combined with amplicon-based sequencing, as well as a viral metagenomic approach, were performed to characterise the etiological agents of the infection. FINDINGS: Plaque assay revealed two distinct plaque phenotypes, suggesting either the presence of two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains or a productive coinfection of two different species of virus. Amplicon-based sequencing did not support the presence of any SARS-CoV-2 genetic variants that would explain the high viral load and suggested the presence of a single SARS-CoV-2 strain. Nonetheless, the viral metagenomic analysis revealed that Coronaviridae and Herpesviridae were the predominant virus families within the sample. This finding was confirmed by a plaque reduction neutralisation test and PCR. MAIN CONCLUSIONS: We characterised a productive coinfection of SARS-CoV-2 and Herpes simplex virus 1 (HSV-1) in a patient with severe symptoms that succumbed to the disease. Although we cannot establish the causal relationship between the coinfection and the severity of the clinical case, this work serves as a warning for future studies focused on the interplay between SARS-CoV-2 and HSV-1 coinfection and COVID-19 severity.

Prolonged SARS-CoV-2 Positivity in Immunocompetent Patients: Virus Isolation, Genomic Integrity, and Transmission Risk.

Current guidelines for patient isolation in COVID-19 cases recommend a symptom-based approach, averting the use of control real-time reverse transcription PCR (rRT-PCR) testing. However, we hypothesized that patients with persistently positive results by RT-PCR for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could be potentially infectious for a prolonged time, even if immunocompetent and asymptomatic, which would demand a longer social isolation period than presently recommended. To test this hypothesis, 72 samples from 51 mildly symptomatic immunocompetent patients with long-lasting positive rRT-PCR results for SARS-CoV-2 were tested for their infectiousness in cell culture. The serological response of samples from those patients and virus genomic integrity were also analyzed. Infectious viruses were successfully isolated from 34.38% (22/64) of nasopharynx samples obtained 14 days or longer after symptom onset. Indeed, we observed successful virus isolation up to 128 days. Complete SARS-COV-2 genome integrity was demonstrated, suggesting the presence of replication-competent viruses. No correlation was found between the isolation of infectious viruses and rRT-PCR cycle threshold values or the humoral immune response. These findings call attention to the need to review current isolation guidelines, particularly in scenarios involving high-risk individuals. IMPORTANCE In this study, we evaluated mildly symptomatic immunocompetent patients with long-lasting positive rRT-PCR results for SARS-CoV-2. Infectious viruses were successfully isolated in cell cultures from nasopharynx samples obtained 14 days or longer after symptom onset. Indeed, we observed successful virus isolation for up to 128 days. Moreover, SARS-CoV-2 genome integrity was demonstrated by sequencing, suggesting the presence of replication-competent viruses. These data point out the risk of continuous SARS-CoV-2 transmission from patients with prolonged detection of SARS-CoV-2 in the upper respiratory tract, which has important implications for current precaution guidelines, particularly in settings where vulnerable individuals may be exposed (e.g., nursing homes and hospitals).

In Vitro and In Vivo Characterization of the Anti-Zika Virus Activity of ProTides of 2'-C-ß-Methylguanosine.

The ProTide approach has emerged as a powerful tool to improve the intracellular delivery of nucleotide analogs with antiviral and anticancer activity. Here, we characterized the anti-ZIKV (ZIKV, Zika virus) activity of two ProTides of 2'-C-ß-methylguanosine. ProTide UMN-1001 is a 2'-C-ß-methylguanosine tryptamine phosphoramidate monoester, and ProTide UMN-1002 is a 2-(methylthio)-ethyl-2'-C-ß-methylguanosine tryptamine phosphoramidate diester. UMN-1002 undergoes stepwise intracellular activation to the corresponding nucleotide monophosphate followed by P-N bond cleavage by intracellular histidine triad nucleotide binding protein 1 (Hint1). UMN-1001 is activated by Hint1 but is less cell-permeable than UMN-1002. UMN-1001 and UMN-1002 were found to be more potent than 2'-C-ß-methylguanosine against ZIKV in human-derived microvascular endothelial and neuroblastoma cells and in reducing ZIKV RNA replication. Studies with a newborn mouse model of ZIKV infection demonstrated that, while treatment with 2'-C-ß-methylguanosine and UMN-1001 was lethal, treatment with UMN-1002 was nontoxic and significantly reduced ZIKV infection. Our data suggests that anchimeric activated ProTides of 2'-C-ß-methyl nucleosides should be further investigated for their potential as anti-ZIKV therapeutics.

Identification and characterisation of SARS-CoV-2 and Human alphaherpesvirus 1 from a productive coinfection in a fatal COVID-19 case

BACKGROUND During routine Coronavirus disease 2019 (COVID-19) diagnosis, an unusually high viral load was detected by reverse transcription real-time polymerase chain reaction (RT-qPCR) in a nasopharyngeal swab sample collected from a patient with respiratory and neurological symptoms who rapidly succumbed to the disease. Therefore we sought to characterise the infection. OBJECTIVES We aimed to determine and characterise the etiological agent responsible for the poor outcome. METHODS Classical virological methods, such as plaque assay and plaque reduction neutralisation test combined with amplicon-based sequencing, as well as a viral metagenomic approach, were performed to characterise the etiological agents of the infection. FINDINGS Plaque assay revealed two distinct plaque phenotypes, suggesting either the presence of two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains or a productive coinfection of two different species of virus. Amplicon-based sequencing did not support the presence of any SARS-CoV-2 genetic variants that would explain the high viral load and suggested the presence of a single SARS-CoV-2 strain. Nonetheless, the viral metagenomic analysis revealed that Coronaviridae and Herpesviridae were the predominant virus families within the sample. This finding was confirmed by a plaque reduction neutralisation test and PCR. MAIN CONCLUSIONS We characterised a productive coinfection of SARS-CoV-2 and Herpes simplex virus 1 (HSV-1) in a patient with severe symptoms that succumbed to the disease. Although we cannot establish the causal relationship between the coinfection and the severity of the clinical case, this work serves as a warning for future studies focused on the interplay between SARS-CoV-2 and HSV-1 coinfection and COVID-19 severity.