Canine real-time detection of SARS-CoV-2 infections in the context of a mass screening event.

INTRODUCTION: Previous research demonstrated that medical scent detection dogs have the ability to distinguish SARS-CoV-2 positive from negative samples with high diagnostic accuracy. To deploy these dogs as a reliable screening method, it is mandatory to examine if canines maintain their high diagnostic accuracy in real-life screening settings. We conducted a study to evaluate the performance of medical scent detection dogs under real-life circumstances. METHODS: Eight dogs were trained to detect SARS-CoV-2 RT-qPCR-positive samples. Four concerts with a total of 2802 participants were held to evaluate canines' performance in screening individuals for SARS-CoV-2 infection. Sweat samples were taken from all participants and presented in a line-up setting. In addition, every participant had been tested with a SARS-CoV-2 specific rapid antigen test and a RT-qPCR and they provided information regarding age, sex, vaccination status and medical disease history. The participants' infection status was unknown at the time of canine testing. Safety measures such as mask wearing and distance keeping were ensured. RESULTS: The SARS-CoV-2 detection dogs achieved a diagnostic specificity of 99.93% (95% CI 99.74% to 99.99%) and a sensitivity of 81.58% (95% CI 66.58% to 90.78%), respectively. The overall rate of concordant results was 99.68%. The majority of the study population was vaccinated with varying vaccines and vaccination schemes, while several participants had chronic diseases and were under chronic medication. This did not influence dogs' decisions. CONCLUSION: Our results demonstrate that SARS-CoV-2 scent detection dogs achieved high diagnostic accuracy in a real-life scenario. The vaccination status, previous SARS-CoV-2 infection, chronic disease and medication of the participants did not influence the performance of the dogs in detecting the acute infection. This indicates that dogs provide a fast and reliable screening option for public events in which high-throughput screening is required.

An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants of concern.

The ongoing evolution of SARS-CoV-2 has resulted in the emergence of Omicron, which displays notable immune escape potential through mutations at key antigenic sites on the spike protein. Many of these mutations localize to the spike protein ACE2 receptor binding domain, annulling the neutralizing activity of therapeutic antibodies that were effective against other variants of concern (VOCs) earlier in the pandemic. Here, we identified a receptor-blocking human monoclonal antibody, 87G7, that retained potent in vitro neutralizing activity against SARS-CoV-2 variants including the Alpha, Beta, Gamma, Delta, and Omicron (BA.1/BA.2) VOCs. Using cryo-electron microscopy and site-directed mutagenesis experiments, we showed that 87G7 targets a patch of hydrophobic residues in the ACE2-binding site that are highly conserved in SARS-CoV-2 variants, explaining its broad neutralization capacity. 87G7 protected mice and hamsters prophylactically against challenge with all current SARS-CoV-2 VOCs and showed therapeutic activity against SARS-CoV-2 challenge in both animal models. Our findings demonstrate that 87G7 holds promise as a prophylactic or therapeutic agent for COVID-19 that is more resilient to SARS-CoV-2 antigenic diversity.

Calu-3 cells are largely resistant to entry driven by filovirus glycoproteins and the entry defect can be rescued by directed expression of DC-SIGN or cathepsin L.

Priming of the viral glycoprotein (GP) by the cellular proteases cathepsin B and L (CatB, CatL) is believed to be essential for cell entry of filoviruses. However, pseudotyping systems that predominantly produce non-filamentous particles have frequently been used to prove this concept. Here, we report that GP-mediated entry of retroviral-, rhabdoviral and filoviral particles depends on CatB/CatL activity and that this effect is cell line-independent. Moreover, we show that the human cell line Calu-3, which expresses low amounts of CatL, is largely resistant to entry driven by diverse filovirus GPs. Finally, we demonstrate that Calu-3 cell entry mediated by certain filovirus GPs can be rescued upon directed expression of CatL or DC-SIGN. Our results identify Calu-3 cells as largely resistant to filovirus GP-driven entry and demonstrate that entry is limited at the stage of virion attachment and GP priming.

Inhibitors of signal peptide peptidase and subtilisin/kexin-isozyme 1 inhibit Ebola virus glycoprotein-driven cell entry by interfering with activity and cellular localization of endosomal cathepsins.

Emerging viruses such as severe fever and thrombocytopenia syndrome virus (SFTSV) and Ebola virus (EBOV) are responsible for significant morbidity and mortality. Host cell proteases that process the glycoproteins of these viruses are potential targets for antiviral intervention. The aspartyl protease signal peptide peptidase (SPP) has recently been shown to be required for processing of the glycoprotein precursor, Gn/Gc, of Bunyamwera virus and for viral infectivity. Here, we investigated whether SPP is also required for infectivity of particles bearing SFTSV-Gn/Gc. Entry driven by the EBOV glycoprotein (GP) and the Lassa virus glycoprotein (LASV-GPC) depends on the cysteine proteases cathepsin B and L (CatB/CatL) and the serine protease subtilisin/kexin-isozyme 1 (SKI-1), respectively, and was examined in parallel for control purposes. We found that inhibition of SPP and SKI-1 did not interfere with SFTSV Gn + Gc-driven entry but, unexpectedly, blocked entry mediated by EBOV-GP. The inhibition occurred at the stage of proteolytic activation and the SPP inhibitor was found to block CatL/CatB activity. In contrast, the SKI-1 inhibitor did not interfere with CatB/CatL activity but disrupted CatB localization in endo/lysosomes, the site of EBOV-GP processing. These results underline the potential of protease inhibitors for antiviral therapy but also show that previously characterized compounds might exert broader specificity than initially appreciated and might block viral entry via diverse mechanisms.

A Polymorphism within the Internal Fusion Loop of the Ebola Virus Glycoprotein Modulates Host Cell Entry.

The large scale of the Ebola virus disease (EVD) outbreak in West Africa in 2013-2016 raised the question whether the host cell interactions of the responsible Ebola virus (EBOV) strain differed from those of other ebolaviruses. We previously reported that the glycoprotein (GP) of the virus circulating in West Africa in 2014 (EBOV2014) exhibited reduced ability to mediate entry into two nonhuman primate (NHP)-derived cell lines relative to the GP of EBOV1976. Here, we investigated the molecular determinants underlying the differential entry efficiency. We found that EBOV2014-GP-driven entry into diverse NHP-derived cell lines, as well as human monocyte-derived macrophages and dendritic cells, was reduced compared to EBOV1976-GP, although entry into most human- and all bat-derived cell lines tested was comparable. Moreover, EBOV2014 replication in NHP but not human cells was diminished relative to EBOV1976, suggesting that reduced cell entry translated into reduced viral spread. Mutagenic analysis of EBOV2014-GP and EBOV1976-GP revealed that an amino acid polymorphism in the receptor-binding domain, A82V, modulated entry efficiency in a cell line-independent manner and did not account for the reduced EBOV2014-GP-driven entry into NHP cells. In contrast, polymorphism T544I, located in the internal fusion loop in the GP2 subunit, was found to be responsible for the entry phenotype. These results suggest that position 544 is an important determinant of EBOV infectivity for both NHP and certain human target cells.IMPORTANCE The Ebola virus disease outbreak in West Africa in 2013 entailed more than 10,000 deaths. The scale of the outbreak and its dramatic impact on human health raised the question whether the responsible virus was particularly adept at infecting human cells. Our study shows that an amino acid exchange, A82V, that was acquired during the epidemic and that was not observed in previously circulating viruses, increases viral entry into diverse target cells. In contrast, the epidemic virus showed a reduced ability to enter cells of nonhuman primates compared to the virus circulating in 1976, and a single amino acid exchange in the internal fusion loop of the viral glycoprotein was found to account for this phenotype.

The Tetherin Antagonism of the Ebola Virus Glycoprotein Requires an Intact Receptor-Binding Domain and Can Be Blocked by GP1-Specific Antibodies.

The glycoprotein of Ebola virus (EBOV GP), a member of the family Filoviridae, facilitates viral entry into target cells. In addition, EBOV GP antagonizes the antiviral activity of the host cell protein tetherin, which may otherwise restrict EBOV release from infected cells. However, it is unclear how EBOV GP antagonizes tetherin, and it is unknown whether the GP of Lloviu virus (LLOV), a filovirus found in dead bats in Northern Spain, also counteracts tetherin. Here, we show that LLOV GP antagonizes tetherin, indicating that tetherin may not impede LLOV spread in human cells. Moreover, we demonstrate that appropriate processing of N-glycans in tetherin/GP-coexpressing cells is required for tetherin counteraction by EBOV GP. Furthermore, we show that an intact receptor-binding domain (RBD) in the GP1 subunit of EBOV GP is a prerequisite for tetherin counteraction. In contrast, blockade of Niemann-Pick disease type C1 (NPC1), a cellular binding partner of the RBD, did not interfere with tetherin antagonism. Finally, we provide evidence that an antibody directed against GP1, which protects mice from a lethal EBOV challenge, may block GP-dependent tetherin antagonism. Our data, in conjunction with previous reports, indicate that tetherin antagonism is conserved among the GPs of all known filoviruses and demonstrate that the GP1 subunit of EBOV GP plays a central role in tetherin antagonism. IMPORTANCE: Filoviruses are reemerging pathogens that constitute a public health threat. Understanding how Ebola virus (EBOV), a highly pathogenic filovirus responsible for the 2013-2016 Ebola virus disease epidemic in western Africa, counteracts antiviral effectors of the innate immune system might help to define novel targets for antiviral intervention. Similarly, determining whether Lloviu virus (LLOV), a filovirus detected in bats in northern Spain, is inhibited by innate antiviral effectors in human cells might help to determine whether the virus constitutes a threat to humans. The present study shows that LLOV, like EBOV, counteracts the antiviral effector protein tetherin via its glycoprotein (GP), suggesting that tetherin does not pose a defense against LLOV spread in humans. Moreover, our work identifies the GP1 subunit of EBOV GP, in particular an intact receptor-binding domain, as critical for tetherin counteraction and provides evidence that antibodies directed against GP1 can interfere with tetherin counteraction.

The Glycoproteins of All Filovirus Species Use the Same Host Factors for Entry into Bat and Human Cells but Entry Efficiency Is Species Dependent.

Ebola and marburgviruses, members of the family Filoviridae, can cause severe hemorrhagic fever in humans. The ongoing Ebola virus (EBOV) disease epidemic in Western Africa claimed more than 11,300 lives and was associated with secondary cases outside Africa, demonstrating that filoviruses pose a global health threat. Bats constitute an important natural reservoir of filoviruses, including viruses of the recently identified Cuevavirus genus within the Filoviridae family. However, the interactions of filoviruses with bat cells are incompletely understood. Here, we investigated whether filoviruses employ different strategies to enter human and bat cells. For this, we examined host cell entry driven by glycoproteins (GP) from all filovirus species into cell lines of human and fruit bat origin. We show that all GPs were able to mediate entry into human and most fruit bat cell lines with roughly comparable efficiency. In contrast, the efficiency of entry into the cell line EidNi/41 derived from a straw-colored fruit bat varied markedly between the GPs of different filovirus species. Furthermore, inhibition studies demonstrated that filoviruses employ the same host cell factors for entry into human, non-human primate and fruit bat cell lines, including cysteine proteases, two pore channels and NPC1 (Niemann-Pick C1 molecule). Finally, processing of GP by furin and the presence of the mucin-like domain in GP were dispensable for entry into both human and bat cell lines. Collectively, these results show that filoviruses rely on the same host cell factors for entry into human and fruit bat cells, although the efficiency of the usage of these factors might differ between filovirus species.