Functional assessment of cell entry and receptor use for merbecoviruses.

The merbecovirus subgenus of coronaviruses includes Middle East Respiratory Syndrome Coronavirus (MERS-CoV), which is a zoonotic respiratory pathogen that transmits from dromedary camels to humans and causes severe respiratory disease. Viral discovery efforts have uncovered hundreds of merbecoviruses in different species across multiple continents, but few of these viruses have been isolated or studied under laboratory conditions, leaving basic questions regarding their threat to humans unresolved. Viral entry into host cells is considered an early and critical step for transmission between hosts. In this study, a scalable approach to assessing novel merbecovirus cell entry was developed and used to measure receptor use across the entire merbecovirus subgenus. Merbecoviruses are sorted into four clades based on the receptor binding domain of the spike glycoprotein. Receptor tropism is clade-specific, with only one clade using DPP4 and multiple clades using ACE2, including the entire HKU5 cluster of bat coronaviruses.

Virology-the path forward.

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

USP2 inhibition prevents infection with ACE2-dependent coronaviruses in vitro and is protective against SARS-CoV-2 in mice.

Targeting angiotensin-converting enzyme 2 (ACE2) represents a promising and effective approach to combat not only the COVID-19 pandemic but also potential future pandemics arising from coronaviruses that depend on ACE2 for infection. Here, we report ubiquitin specific peptidase 2 (USP2) as a host-directed antiviral target; we further describe the development of MS102, an orally available USP2 inhibitor with viable antiviral activity against ACE2-dependent coronaviruses. Mechanistically, USP2 serves as a physiological deubiquitinase of ACE2, and targeted inhibition with specific small-molecule inhibitor ML364 leads to a marked and reversible reduction in ACE2 protein abundance, thereby blocking various ACE2-dependent coronaviruses tested. Using human ACE2 transgenic mouse models, we further demonstrate that ML364 efficiently controls disease caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as evidenced by reduced viral loads and ameliorated lung inflammation. Furthermore, we improved the in vivo performance of ML364 in terms of both pharmacokinetics and antiviral activity. The resulting lead compound, MS102, holds promise as an oral therapeutic option for treating infections with coronaviruses that are reliant on ACE2.

Isolation of ACE2-dependent and -independent sarbecoviruses from Chinese horseshoe bats.

While the spike proteins from severe acute respiratory syndrome coronaviruses-1 and 2 (SARS-CoV and SARS-CoV-2) bind to host angiotensin-converting enzyme 2 (ACE2) to infect cells, the majority of bat sarbecoviruses cannot use ACE2 from any species. Despite their discovery almost 20 years ago, ACE2-independent sarbecoviruses have never been isolated from field samples, leading to the assumption these viruses pose little risk to humans. We have previously shown how spike proteins from a small group of ACE2-independent bat sarbecoviruses may possess the ability to infect human cells in the presence of exogenous trypsin. Here, we adapted our earlier findings into a virus isolation protocol and recovered two new ACE2-dependent viruses, RsYN2012 and RsYN2016A, as well as an ACE2-independent virus, RsHuB2019A. Although our stocks of RsHuB2019A rapidly acquired a tissue-culture adaption that rendered the spike protein resistant to trypsin, trypsin was still required for viral entry, suggesting limitations on the exogenous entry factors that support bat sarbecoviruses. Electron microscopy revealed that ACE2-independent sarbecoviruses have a prominent spike corona and share similar morphology to other coronaviruses. Our findings demonstrate a broader zoonotic threat posed by sarbecoviruses and shed light on the intricacies of coronavirus isolation and propagation in vitro. IMPORTANCE Several coronaviruses have been transmitted from animals to people, and 20 years of virus discovery studies have uncovered thousands of new coronavirus sequences in nature. Most of the animal-derived sarbecoviruses have never been isolated in culture due to cell incompatibilities and a poor understanding of the in vitro requirements for their propagation. Here, we built on our growing body of work characterizing viral entry mechanisms of bat sarbecoviruses in human cells and have developed a virus isolation protocol that allows for the exploration of these understudied viruses. Our protocol is robust and practical, leading to successful isolation of more sarbecoviruses than previous approaches and from field samples that had been collected over a 10-year longitudinal study.

ACE2-Independent Bat Sarbecovirus Entry and Replication in Human and Bat Cells.

Hundreds of sarbecoviruses have been found in bats, but only a fraction of them have the ability to infect cells using angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV and -2. To date, only ACE2-dependent sarbecoviruses have been isolated from field samples or grown in the laboratory. ACE2-independent sarbecoviruses, comprising the majority of the subgenus, have not been propagated in any type of cell culture, as the factors and conditions needed for their replication are completely unknown. Given the significant zoonotic threat posed by sarbecoviruses, cell culture models and in vitro tools are urgently needed to study the rest of this subgenus. We previously showed that the exogenous protease trypsin could facilitate cell entry of viral-like particles pseudotyped with spike protein from some of the ACE2-independent sarbecoviruses. Here, we tested if these conditions were sufficient to support bona fide viral replication using recombinant bat sarbecoviruses. In the presence of trypsin, some of the spike proteins from clade 2 viruses were capable of supporting bat sarbecovirus infection and replication in human and bat cells. Protease experiments showed a specific viral dependence on high levels of trypsin, as TMPRSS2 and furin had no effect on clade 2 virus entry. These results shed light on how sarbecoviruses transmit and coexist in their natural hosts, provide key insights for future efforts to isolate and grow these viruses from field samples, and further underscore the need for broadly protective, universal coronavirus vaccines. IMPORTANCE Our studies demonstrate that some unexplored sarbecoviruses are capable of replicating in human and bat cells in an ACE2-independent way but need a high trypsin environment. We found that trypsin is not compensated by other known proteases involved in some coronavirus entry. This work provides important information that the trypsin-dependent entry may be a widely employed mechanism for coronaviruses and will help for further understanding the biological features of the less-studied viruses.

An ACE2-dependent Sarbecovirus in Russian bats is resistant to SARS-CoV-2 vaccines.

Spillover of sarbecoviruses from animals to humans has resulted in outbreaks of severe acute respiratory syndrome SARS-CoVs and the ongoing COVID-19 pandemic. Efforts to identify the origins of SARS-CoV-1 and -2 has resulted in the discovery of numerous animal sarbecoviruses-the majority of which are only distantly related to known human pathogens and do not infect human cells. The receptor binding domain (RBD) on sarbecoviruses engages receptor molecules on the host cell and mediates cell invasion. Here, we tested the receptor tropism and serological cross reactivity for RBDs from two sarbecoviruses found in Russian horseshoe bats. While these two viruses are in a viral lineage distinct from SARS-CoV-1 and -2, the RBD from one virus, Khosta 2, was capable of using human ACE2 to facilitate cell entry. Viral pseudotypes with a recombinant, SARS-CoV-2 spike encoding for the Khosta 2 RBD were resistant to both SARS-CoV-2 monoclonal antibodies and serum from individuals vaccinated for SARS-CoV-2. Our findings further demonstrate that sarbecoviruses circulating in wildlife outside of Asia also pose a threat to global health and ongoing vaccine campaigns against SARS-CoV-2.

The Glycan-Binding Trait of the Sarbecovirus Spike N-Terminal Domain Reveals an Evolutionary Footprint.

The spike protein on sarbecovirus virions contains two external, protruding domains: an N-terminal domain (NTD) with unclear function and a C-terminal domain (CTD) that binds the host receptor, allowing for viral entry and infection. While the CTD is well studied for therapeutic interventions, the role of the NTD is far less well understood for many coronaviruses. Here, we demonstrate that the spike NTD from SARS-CoV-2 and other sarbecoviruses binds to unidentified glycans in vitro similarly to other members of the Coronaviridae family. We also show that these spike NTD (S-NTD) proteins adhere to Calu3 cells, a human lung cell line, although the biological relevance of this is unclear. In contrast to what has been shown for Middle East respiratory syndrome coronavirus (MERS-CoV), which attaches sialic acids during cell entry, sialic acids present on Calu3 cells inhibited sarbecovirus infection. Therefore, while sarbecoviruses can interact with cell surface glycans similarly to other coronaviruses, their reliance on glycans for entry is different from that of other respiratory coronaviruses, suggesting sarbecoviruses and MERS-CoV have adapted to different cell types, tissues, or hosts during their divergent evolution. Our findings provide important clues for further exploring the biological functions of sarbecovirus glycan binding and adds to our growing understanding of the complex forces that shape coronavirus spike evolution. IMPORTANCE Spike N-terminal domains (S-NTD) of sarbecoviruses are highly diverse; however, their function remains largely understudied compared with the receptor-binding domains (RBD). Here, we show that sarbecovirus S-NTD can be phylogenetically clustered into five clades and exhibit various levels of glycan binding in vitro. We also show that, unlike some coronaviruses, including MERS-CoV, sialic acids present on the surface of Calu3, a human lung cell culture, inhibit SARS-CoV-2 and other sarbecoviruses. These results suggest that while glycan binding might be an ancestral trait conserved across different coronavirus families, the functional outcome during infection can vary, reflecting divergent viral evolution. Our results expand our knowledge on the biological functions of the S-NTD across diverse sarbecoviruses and provide insight on the evolutionary history of coronavirus spike.

Sequence determinants of human-cell entry identified in ACE2-independent bat sarbecoviruses: A combined laboratory and computational network science approach.

BACKGROUND: The sarbecovirus subgenus of betacoronaviruses is widely distributed throughout bats and other mammals globally and includes human pathogens, SARS-CoV and SARS-CoV-2. The most studied sarbecoviruses use the host protein, ACE2, to infect cells. Curiously, the majority of sarbecoviruses identified to date do not use ACE2 and cannot readily acquire ACE2 binding through point mutations. We previously screened a broad panel of sarbecovirus spikes for cell entry and observed bat-derived viruses that could infect human cells, independent of ACE2. Here we further investigate the sequence determinants of cell entry for ACE2-independent bat sarbecoviruses. METHODS: We employed a network science-based approach to visualize sequence and entry phenotype similarities across the diversity of sarbecovirus spike protein sequences. We then verified these computational results and mapped determinants of viral entry into human cells using recombinant chimeric spike proteins within an established viral pseudotype assay. FINDINGS: We show ACE2-independent viruses that can infect human and bat cells in culture have a similar putative receptor binding motif, which can impart human cell entry into other bat sarbecovirus spikes that cannot otherwise infect human cells. These sequence determinants of human cell entry map to a surface-exposed protrusion from the predicted bat sarbecovirus spike receptor binding domain structure. INTERPRETATION: Our findings provide further evidence of a group of bat-derived sarbecoviruses with zoonotic potential and demonstrate the utility in applying network science to phenotypic mapping and prediction. FUNDING: This work was supported by Washington State University and the Paul G. Allen School for Global Health.

Anti-spike antibodies and neutralising antibody activity in people living with HIV vaccinated with COVID-19 mRNA-1273 vaccine: a prospective single-centre cohort study.

BACKGROUND: Vaccines against COVID-19 are a powerful tool to control the current SARS-CoV-2 pandemic. A thorough description of their immunogenicity among people living with HIV (PLWHIV) is necessary. We aimed to assess the immunogenicity of the mRNA-1273 vaccine among PLWHIV. METHODS: In this prospective cohort, adult PLWHIV outpatients were enrolled during the Italian vaccination campaign. Enrolment was allowed irrespective of ongoing combination antiretroviral therapy (ART), plasma HIV viral load and CD4+ T cell count. A two-dose regimen of mRNA-1273, with administrations performed 28 days apart, was employed. The primary outcomes were anti-spike (anti-S) antibody titres and neutralising antibody activity, assessed 28 days after completing the vaccination schedule. A convenient sample of individuals not affected by HIV was also collected to serve as control (referred as healthy-donors, HDs). FINDINGS: We enrolled 71 PLWHIV, mostly male (84·5%), with a mean age of 47 years, a median CD4+ T cell count of 747·0 cells per µL and a median HIV viral load <50 copies/mL. COVID-19-experienced PLWHIV displayed higher anti-S antibody titres (p=0·0007) and neutralising antibody activity in sera (p=0·0007) than COVID-19-naïve PLWHIV. When stratified according to CD4+ T cell count (<350 cells/µL, 350-500 cells/µL, >500 cells/µL), anti-S antibody titres (6/71, median 2173 U/mL [IQR 987-4109]; 7/71, 5763 IU/mL [IQR 4801->12500]; 58/71, 2449 U/mL [IQR 1524-5704]) were not lower to those observed among HDs (10, median 1425 U/mL [IQR 599-6131]). In addition, neutralising antibody activity, stratified according to the CD4+ T cell count (6/71, median 1314 [IQR 606-2477]; 7/71, 3329 IU/mL [IQR 1905-10508]; 58/71, 1227 U/mL [IQR 761-3032]), was like those displayed by HDs (10, median 2112 U/mL [IQR 719-8889]). INTERPRETATION: In our cohort of PLWHIV with well-controlled ART, stable viral suppression and robust CD4+ T cell count, inoculation with mRNA-1273 vaccine given 4 weeks apart produced detectable humoral immune response, similar to individuals without HIV infection, supporting vaccination in PLWHIV. FUNDING: This study was partially supported by Italian Ministry of Health Ricerca Corrente 2021, by Intesa San Paolo COVID-19 emergency 2020 funds, and by Fondazione Cariplo Grant (INNATE-CoV).

Surface‒Aerosol Stability and Pathogenicity of Diverse Middle East Respiratory Syndrome Coronavirus Strains, 2012‒2018.

Middle East respiratory syndrome coronavirus (MERS-CoV) infects humans and dromedary camels and is responsible for an ongoing outbreak of severe respiratory illness in humans in the Middle East. Although some mutations found in camel-derived MERS-CoV strains have been characterized, most natural variation found across MERS-CoV isolates remains unstudied. We report on the environmental stability, replication kinetics, and pathogenicity of several diverse isolates of MERS-CoV, as well as isolates of severe acute respiratory syndrome coronavirus 2, to serve as a basis of comparison with other stability studies. Although most MERS-CoV isolates had similar stability and pathogenicity in our experiments, the camel-derived isolate C/KSA/13 had reduced surface stability, and another camel isolate, C/BF/15, had reduced pathogenicity in a small animal model. These results suggest that although betacoronaviruses might have similar environmental stability profiles, individual variation can influence this phenotype, underscoring the need for continual global viral surveillance.

Risk Factors for Middle East Respiratory Syndrome Coronavirus Infection among Camel Populations, Southern Jordan, 2014-2018.

After the first detection of Middle East respiratory syndrome coronavirus (MERS-CoV) in camels in Jordan in 2013, we conducted 2 consecutive surveys in 2014-2015 and 2017-2018 investigating risk factors for MERS-CoV infection among camel populations in southern Jordan. Multivariate analysis to control for confounding demonstrated that borrowing of camels, particularly males, for breeding purposes was associated with increased MERS-CoV seroprevalence among receiving herds, suggesting a potential route of viral transmission between herds. Increasing age, herd size, and use of water troughs within herds were also associated with increased seroprevalence. Closed herd management practices were found to be protective. Future vaccination strategies among camel populations in Jordan could potentially prioritize breeding males, which are likely to be shared between herds. In addition, targeted management interventions with the potential to reduce transmission between herds should be considered; voluntary closed herd schemes offer a possible route to achieving disease-free herds.

Limited Genetic Diversity Detected in Middle East Respiratory Syndrome-Related Coronavirus Variants Circulating in Dromedary Camels in Jordan.

Middle East respiratory syndrome-related coronavirus (MERS-CoV) is a persistent zoonotic pathogen with frequent spillover from dromedary camels to humans in the Arabian Peninsula, resulting in limited outbreaks of MERS with a high case-fatality rate. Full genome sequence data from camel-derived MERS-CoV variants show diverse lineages circulating in domestic camels with frequent recombination. More than 90% of the available full MERS-CoV genome sequences derived from camels are from just two countries, the Kingdom of Saudi Arabia (KSA) and United Arab Emirates (UAE). In this study, we employ a novel method to amplify and sequence the partial MERS-CoV genome with high sensitivity from nasal swabs of infected camels. We recovered more than 99% of the MERS-CoV genome from field-collected samples with greater than 500 TCID50 equivalent per nasal swab from camel herds sampled in Jordan in May 2016. Our subsequent analyses of 14 camel-derived MERS-CoV genomes show a striking lack of genetic diversity circulating in Jordan camels relative to MERS-CoV genome sequences derived from large camel markets in KSA and UAE. The low genetic diversity detected in Jordan camels during our study is consistent with a lack of endemic circulation in these camel herds and reflective of data from MERS outbreaks in humans dominated by nosocomial transmission following a single introduction as reported during the 2015 MERS outbreak in South Korea. Our data suggest transmission of MERS-CoV among two camel herds in Jordan in 2016 following a single introduction event.

ChAdOx1-vectored Lassa fever vaccine elicits a robust cellular and humoral immune response and protects guinea pigs against lethal Lassa virus challenge.

Lassa virus (LASV) infects hundreds of thousands of individuals each year, highlighting the need for the accelerated development of preventive, diagnostic, and therapeutic interventions. To date, no vaccine has been licensed for LASV. ChAdOx1-Lassa-GPC is a chimpanzee adenovirus-vectored vaccine encoding the Josiah strain LASV glycoprotein precursor (GPC) gene. In the following study, we show that ChAdOx1-Lassa-GPC is immunogenic, inducing robust T-cell and antibody responses in mice. Furthermore, a single dose of ChAdOx1-Lassa-GPC fully protects Hartley guinea pigs against morbidity and mortality following lethal challenge with a guinea pig-adapted LASV (strain Josiah). By contrast, control vaccinated animals reached euthanasia criteria 10-12 days after infection. Limited amounts of LASV RNA were detected in the tissues of vaccinated animals. Viable LASV was detected in only one animal receiving a single dose of the vaccine. A prime-boost regimen of ChAdOx1-Lassa-GPC in guinea pigs significantly increased antigen-specific antibody titers and cleared viable LASV from the tissues. These data support further development of ChAdOx1-Lassa-GPC and testing in non-human primate models of infection.

Surface-aerosol stability and pathogenicity of diverse MERS-CoV strains from 2012 - 2018.

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a coronavirus that infects both humans and dromedary camels and is responsible for an ongoing outbreak of severe respiratory illness in humans in the Middle East. While some mutations found in camel-derived MERS-CoV strains have been characterized, the majority of natural variation found across MERS-CoV isolates remains unstudied. Here we report on the environmental stability, replication kinetics and pathogenicity of several diverse isolates of MERS-CoV as well as SARS-CoV-2 to serve as a basis of comparison with other stability studies. While most of the MERS-CoV isolates exhibited similar stability and pathogenicity in our experiments, the camel derived isolate, C/KSA/13, exhibited reduced surface stability while another camel isolate, C/BF/15, had reduced pathogenicity in a small animal model. These results suggest that while betacoronaviruses may have similar environmental stability profiles, individual variation can influence this phenotype, underscoring the importance of continual, global viral surveillance.

Defining the Syrian hamster as a highly susceptible preclinical model for SARS-CoV-2 infection.

Following emergence in late 2019, SARS-CoV-2 rapidly became pandemic and is presently responsible for millions of infections and hundreds of thousands of deaths worldwide. There is currently no approved vaccine to halt the spread of SARS-CoV-2 and only very few treatment options are available to manage COVID-19 patients. For development of preclinical countermeasures, reliable and well-characterized small animal disease models will be of paramount importance. Here we show that intranasal inoculation of SARS-CoV-2 into Syrian hamsters consistently caused moderate broncho-interstitial pneumonia, with high viral lung loads and extensive virus shedding, but animals only displayed transient mild disease. We determined the infectious dose 50 to be only five infectious particles, making the Syrian hamster a highly susceptible model for SARS-CoV-2 infection. Neither hamster age nor sex had any impact on the severity of disease or course of infection. Finally, prolonged viral persistence in interleukin 2 receptor gamma chain knockout hamsters revealed susceptibility of SARS-CoV-2 to adaptive immune control. In conclusion, the Syrian hamster is highly susceptible to SARS-CoV-2 making it a very suitable infection model for COVID-19 countermeasure development.

Defining the Syrian hamster as a highly susceptible preclinical model for SARS-CoV-2 infection.

Following emergence in late 2019, SARS-CoV-2 rapidly became pandemic and is presently responsible for millions of infections and hundreds of thousands of deaths worldwide. There is currently no approved vaccine to halt the spread of SARS-CoV-2 and only very few treatment options are available to manage COVID-19 patients. For development of preclinical countermeasures, reliable and well-characterized small animal disease models will be of paramount importance. Here we show that intranasal inoculation of SARS-CoV-2 into Syrian hamsters consistently caused moderate broncho-interstitial pneumonia, with high viral lung loads and extensive virus shedding, but animals only displayed transient mild disease. We determined the infectious dose 50 to be only five infectious particles, making the Syrian hamster a highly susceptible model for SARS-CoV-2 infection. Neither hamster age nor sex had any impact on the severity of disease or course of infection. Finally, prolonged viral persistence in interleukin 2 receptor gamma chain knockout hamsters revealed susceptibility of SARS-CoV-2 to adaptive immune control. In conclusion, the Syrian hamster is highly susceptible to SARS-CoV-2 making it a very suitable infection model for COVID-19 countermeasure development.

Functional assessment of cell entry and receptor usage for lineage B ß-coronaviruses, including 2019-nCoV.

Over the past 20 years, several coronaviruses have crossed the species barrier into humans, causing outbreaks of severe, and often fatal, respiratory illness. Since SARS-CoV was first identified in animal markets, global viromics projects have discovered thousands of coronavirus sequences in diverse animals and geographic regions. Unfortunately, there are few tools available to functionally test these novel viruses for their ability to infect humans, which has severely hampered efforts to predict the next zoonotic viral outbreak. Here we developed an approach to rapidly screen lineage B betacoronaviruses, such as SARS-CoV and the recent 2019-nCoV, for receptor usage and their ability to infect cell types from different species. We show that host protease processing during viral entry is a significant barrier for several lineage B viruses and that bypassing this barrier allows several lineage B viruses to enter human cells through an unknown receptor. We also demonstrate how different lineage B viruses can recombine to gain entry into human cells and confirm that human ACE2 is the receptor for the recently emerging 2019-nCoV.

Bat-borne virus diversity, spillover and emergence.

Most viral pathogens in humans have animal origins and arose through cross-species transmission. Over the past 50 years, several viruses, including Ebola virus, Marburg virus, Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory coronavirus (MERS-CoV) and SARS-CoV-2, have been linked back to various bat species. Despite decades of research into bats and the pathogens they carry, the fields of bat virus ecology and molecular biology are still nascent, with many questions largely unexplored, thus hindering our ability to anticipate and prepare for the next viral outbreak. In this Review, we discuss the latest advancements and understanding of bat-borne viruses, reflecting on current knowledge gaps and outlining the potential routes for future research as well as for outbreak response and prevention efforts.

Why is There Low Morbidity and Mortality of COVID-19 in Africa?

Three months since the detection of the first COVID-19 case in Africa, almost all countries of the continent continued to report lower morbidity and mortality than the global trend, including Europe and North America. We reviewed the merits of various hypotheses advanced to explain this phenomenon, including low seeding rate, effective mitigation measures, population that is more youthful, favorable weather, and possible prior exposure to a cross-reactive virus. Having a youthful population and favorable weather appears compelling, particularly their combined effect; however, progression of the pandemic in the region and globally may dispel these in the coming months.