The origins of SARS-CoV-2: A critical review.

Since the first reports of a novel severe acute respiratory syndrome (SARS)-like coronavirus in December 2019 in Wuhan, China, there has been intense interest in understanding how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in the human population. Recent debate has coalesced around two competing ideas: a "laboratory escape" scenario and zoonotic emergence. Here, we critically review the current scientific evidence that may help clarify the origin of SARS-CoV-2.

Emergence of an early SARS-CoV-2 epidemic in the United States.

The emergence of the COVID-19 epidemic in the United States (U.S.) went largely undetected due to inadequate testing. New Orleans experienced one of the earliest and fastest accelerating outbreaks, coinciding with Mardi Gras. To gain insight into the emergence of SARS-CoV-2 in the U.S. and how large-scale events accelerate transmission, we sequenced SARS-CoV-2 genomes during the first wave of the COVID-19 epidemic in Louisiana. We show that SARS-CoV-2 in Louisiana had limited diversity compared to other U.S. states and that one introduction of SARS-CoV-2 led to almost all of the early transmission in Louisiana. By analyzing mobility and genomic data, we show that SARS-CoV-2 was already present in New Orleans before Mardi Gras, and the festival dramatically accelerated transmission. Our study provides an understanding of how superspreading during large-scale events played a key role during the early outbreak in the U.S. and can greatly accelerate epidemics.

Convergent Structures Illuminate Features for Germline Antibody Binding and Pan-Lassa Virus Neutralization.

Lassa virus (LASV) causes hemorrhagic fever and is endemic in West Africa. Protective antibody responses primarily target the LASV surface glycoprotein (GPC), and GPC-B competition group antibodies often show potent neutralizing activity in humans. However, which features confer potent and broadly neutralizing antibody responses is unclear. Here, we compared three crystal structures of LASV GPC complexed with GPC-B antibodies of varying neutralization potency. Each GPC-B antibody recognized an overlapping epitope involved in binding of two adjacent GPC monomers and preserved the prefusion trimeric conformation. Differences among GPC-antibody interactions highlighted specific residues that enhance neutralization. Using structure-guided amino acid substitutions, we increased the neutralization potency and breadth of these antibodies to include all major LASV lineages. The ability to define antibody residues that allow potent and broad neutralizing activity, together with findings from analyses of inferred germline precursors, is critical to develop potent therapeutics and for vaccine design and assessment.

Roots, Not Parachutes: Research Collaborations Combat Outbreaks.

Recent infectious disease epidemics illustrate how health systems failures anywhere can create disease vulnerabilities everywhere. We must therefore prioritize investments in health care infrastructure in outbreak-prone regions of the world. We describe how "rooted" research collaborations can establish capacity for pathogen surveillance and facilitate rapid outbreak responses.

A Fc engineering approach to define functional humoral correlates of immunity against Ebola virus.

Protective Ebola virus (EBOV) antibodies have neutralizing activity and induction of antibody constant domain (Fc)-mediated innate immune effector functions. Efforts to enhance Fc effector functionality often focus on maximizing antibody-dependent cellular cytotoxicity, yet distinct combinations of functions could be critical for antibody-mediated protection. As neutralizing antibodies have been cloned from EBOV disease survivors, we sought to identify survivor Fc effector profiles to help guide Fc optimization strategies. Survivors developed a range of functional antibody responses, and we therefore applied a rapid, high-throughput Fc engineering platform to define the most protective profiles. We generated a library of Fc variants with identical antigen-binding fragments (Fabs) from an EBOV neutralizing antibody. Fc variants with antibody-mediated complement deposition and moderate natural killer (NK) cell activity demonstrated complete protective activity in a stringent in vivo mouse model. Our findings highlight the importance of specific effector functions in antibody-mediated protection, and the experimental platform presents a generalizable resource for identifying correlates of immunity to guide therapeutic antibody design.

After the pandemic: perspectives on the future trajectory of COVID-19.

There is a realistic expectation that the global effort in vaccination will bring the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) under control. Nonetheless, uncertainties remain about the type of long-term association that the virus will establish with the human population and, in particular, whether coronavirus disease 2019 (COVID-19) will become an endemic disease. Although the trajectory is difficult to predict, the conditions, concepts and variables that influence this transition can be anticipated. Persistence of SARS-CoV-2 as an endemic virus, perhaps with seasonal epidemic peaks, may be fuelled by pockets of susceptible individuals and waning immunity after infection or vaccination, changes in the virus through antigenic drift that diminish protection and re-entries from zoonotic reservoirs. Here we review relevant observations from previous epidemics and discuss the potential evolution of SARS-CoV-2 as it adapts during persistent transmission in the presence of a level of population immunity. Lack of effective surveillance or adequate response could enable the emergence of new epidemic or pandemic patterns from an endemic infection of SARS-CoV-2. There are key pieces of data that are urgently needed in order to make good decisions; we outline these and propose a way forward.

A human monoclonal antibody combination rescues nonhuman primates from advanced disease caused by the major lineages of Lassa virus.

There are no approved treatments for Lassa fever (LF), which is responsible for thousands of deaths each year in West Africa. A major challenge in developing effective medical countermeasures against LF is the high diversity of circulating Lassa virus (LASV) strains with four recognized lineages and four proposed lineages. The recent resurgence of LASV in Nigeria caused by genetically distinct strains underscores this concern. Two LASV lineages (II and III) are dominant in Nigeria. Here, we show that combinations of two or three pan-lineage neutralizing human monoclonal antibodies (8.9F, 12.1F, 37.D) known as Arevirumab-2 or Arevirumab-3 can protect up to 100% of cynomolgus macaques against challenge with both lineage II and III LASV isolates when treatment is initiated at advanced stages of disease on day 8 after LASV exposure. This work demonstrates that it may be possible to develop postexposure interventions that can broadly protect against most strains of LASV.

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.

Ebola Virus-Specific Neutralizing Antibody Persists at High Levels in Survivors 2 Years After Resolution of Disease in a Sierra Leonean Cohort.

Ebola virus (EBOV) infection results in Ebola virus disease (EVD), an often severe disease with a nonspecific presentation. Since its recognition, periodic outbreaks of EVD continue to occur in sub-Saharan Africa. The 2013-2016 West African EVD outbreak was the largest recorded, resulting in a substantial cohort of EVD survivors with persistent health complaints and variable immune responses. In this study, we characterize humoral immune responses in EVD survivors and their contacts in Eastern Sierra Leone. We found high levels of EBOV IgG in EVD survivors and lower yet substantial antibody levels in household contacts, suggesting subclinical transmission. Neutralizing antibody function was prevalent but variable in EVD survivors, raising questions about the durability of immune responses from natural infection with EBOV. Additionally, we found that certain discrete symptoms-ophthalmologic and auditory-are associated with EBOV IgG seropositivity, while an array of symptoms are associated with the presence of neutralizing antibody.

50 Years of Lassa Fever Research.

Lassa fever was first described as a clinical entity fifty years ago. The causative agent Lassa virus was isolated from these first known cases. This chapter reviews the key publications on Lassa fever research that appeared in the scientific literature at that time and over the ensuing decades.

Lassa Virus Structural Biology and Replication.

Lassa virus (LASV) is the causative agent of Lassa fever, an often-fatal hemorrhagic fever that is endemic in West Africa. LASV virions are enveloped and contain two single-stranded RNA genome segments. Both segments are ambisense and encode two proteins. The nucleoprotein associates with viral RNAs forming ribonucleoprotein complexes. The glycoprotein complex mediates viral attachment and entry. The Zinc protein serves as the matrix protein. Large is a polymerase that catalyzes viral RNA transcription and replication. LASV virion entry occurs via a clathrin-independent endocytic pathway usually involving alpha-dystroglycan and lysosomal associated membrane protein 1 as surface and intracellular receptors, respectively. Advances in understanding LASV structural biology and replication have facilitated development of promising vaccine and drug candidates.

Lassa Fever Natural History and Clinical Management.

Lassa fever is caused by Lassa virus (LASV), an Old World Mammarenavirus that is carried by Mastomys natalensis and other rodents. It is endemic in Sierra Leone, Nigeria, and other countries in West Africa. The clinical presentation of LASV infection is heterogenous varying from an inapparent or mild illness to a fatal hemorrhagic fever. Exposure to LASV is usually through contact with rodent excreta. After an incubation period of 1-3 weeks, initial symptoms such as fever, headache, and fatigue develop that may progress to sore throat, retrosternal chest pain, conjunctival injection, vomiting, diarrhea, and abdominal pain. Severe illness, including hypotension, shock, and multiorgan failure, develops in a minority of patients. Patient demographics and case fatality rates are distinctly different in Sierra Leone and Nigeria. Laboratory diagnosis relies on the detection of LASV antigens or genomic RNA. LASV-specific immunoglobulin G and M assays can also contribute to clinical management. The mainstay of treatment for Lassa fever is supportive care. The nucleoside analog ribavirin is commonly used to treat acute Lassa fever but is considered useful only if treatment is begun early in the disease course. Drugs in development, including a monoclonal antibody cocktail, have the potential to impact the management of Lassa fever.

The Arenaviridae Family: Knowledge Gaps, Animal Models, Countermeasures, and Prototype Pathogens.

Lassa virus (LASV), Junin virus (JUNV), and several other members of the Arenaviridae family are capable of zoonotic transfer to humans and induction of severe viral hemorrhagic fevers. Despite the importance of arenaviruses as potential pandemic pathogens, numerous gaps exist in scientific knowledge pertaining to this diverse family, including gaps in understanding replication, immunosuppression, receptor usage, and elicitation of neutralizing antibody responses, that in turn complicates development of medical countermeasures. A further challenge to the development of medical countermeasures for arenaviruses is the requirement for use of animal models at high levels of biocontainment, where each model has distinct advantages and limitations depending on, availability of space, animals species-specific reagents, and most importantly the ability of the model to faithfully recapitulate human disease. Designation of LASV and JUNV as prototype pathogens can facilitate progress in addressing the public health challenges posed by members of this important virus family.

High crossreactivity of human T cell responses between Lassa virus lineages.

Lassa virus infects hundreds of thousands of people each year across rural West Africa, resulting in a high number of cases of Lassa fever (LF), a febrile disease associated with high morbidity and significant mortality. The lack of approved treatments or interventions underscores the need for an effective vaccine. At least four viral lineages circulate in defined regions throughout West Africa with substantial interlineage nucleotide and amino acid diversity. An effective vaccine should be designed to elicit Lassa virus specific humoral and cell mediated immunity across all lineages. Most current vaccine candidates use only lineage IV antigens encoded by Lassa viruses circulating around Sierra Leone, Liberia, and Guinea but not Nigeria where lineages I-III are found. As previous infection is known to protect against disease from subsequent exposure, we sought to determine whether LF survivors from Nigeria and Sierra Leone harbor memory T cells that respond to lineage IV antigens. Our results indicate a high degree of cross-reactivity of CD8+ T cells from Nigerian LF survivors to lineage IV antigens. In addition, we identified regions within the Lassa virus glycoprotein complex and nucleoprotein that contributed to these responses while T cell epitopes were not widely conserved across our study group. These data are important for current efforts to design effective and efficient vaccine candidates that can elicit protective immunity across all Lassa virus lineages.

Post-Ebola Syndrome Presents With Multiple Overlapping Symptom Clusters: Evidence From an Ongoing Cohort Study in Eastern Sierra Leone.

BACKGROUND: Following the 2013-2016 West African Ebola outbreak, distinct, persistent health complaints were recognized in Ebola virus disease (EVD) survivors. Here we provide an in-depth characterization of post-Ebola syndrome >2.5 years after resolution of disease. Additionally, we report subphenotypes of post-Ebola syndrome with overlapping symptom clusters in survivors from Eastern Sierra Leone. METHODS: Participants in Eastern Sierra Leone were identified by the Sierra Leone Association of Ebola survivors. Survivors and their contacts were administered a questionnaire assessing self-reported symptoms and a physical examination. Comparisons between survivors and contacts were conducted using conditional logistic regression. Symptom groupings were identified using hierarchical clustering approaches. Simplified presentation of incredibly complex evaluations (SPICE), correlation analysis, logistic regression, and principal component analysis (PCA) were performed to explore the relationships between symptom clusters. RESULTS: Three hundred seventy-five EVD survivors and 1040 contacts were enrolled into the study. At enrollment, EVD survivors reported significantly more symptoms than their contacts in all categories (P < .001). Symptom clusters representing distinct organ systems were identified. Correlation and logistic regression analysis identified relationships between symptom clusters, including stronger relationships between clusters including musculoskeletal symptoms (r = 0.63, P < .001; and P < .001 for correlation and logistic regression, respectively). SPICE and PCA further highlighted subphenotypes with or without musculoskeletal symptoms. CONCLUSIONS: This study presents an in-depth characterization of post-Ebola syndrome in Sierra Leonean survivors >2.5 years after disease. The interrelationship between symptom clusters indicates that post-Ebola syndrome is a heterogeneous disease. The distinct musculoskeletal and non-musculoskeletal phenotypes identified likely require targeted therapies to optimize long-term treatment for EVD survivors.

Genomic Analysis of Lassa Virus during an Increase in Cases in Nigeria in 2018.

During 2018, an unusual increase in Lassa fever cases occurred in Nigeria, raising concern among national and international public health agencies. We analyzed 220 Lassa virus genomes from infected patients, including 129 from the 2017-2018 transmission season, to understand the viral populations underpinning the increase. A total of 14 initial genomes from 2018 samples were generated at Redeemer's University in Nigeria, and the findings were shared with the Nigerian Center for Disease Control in real time. We found that the increase in cases was not attributable to a particular Lassa virus strain or sustained by human-to-human transmission. Instead, the data were consistent with ongoing cross-species transmission from local rodent populations. Phylogenetic analysis also revealed extensive viral diversity that was structured according to geography, with major rivers appearing to act as barriers to migration of the rodent reservoir.

Broad-Spectrum Antiviral Entry Inhibition by Interfacially Active Peptides.

Numerous peptides inhibit the entry of enveloped viruses into cells. Some of these peptides have been shown to inhibit multiple unrelated viruses. We have suggested that such broad-spectrum antiviral peptides share a property called interfacial activity; they are somewhat hydrophobic and amphipathic, with a propensity to interact with the interfacial zones of lipid bilayer membranes. In this study, we further tested the hypothesis that such interfacial activity is a correlate of broad-spectrum antiviral activity. In this study, several families of peptides, selected for the ability to partition into and disrupt membrane integrity but with no known antiviral activity, were tested for the ability to inhibit multiple diverse enveloped viruses. These include Lassa pseudovirus, influenza virus, dengue virus type 2, herpes simplex virus 1, and nonenveloped human adenovirus 5. Various families of interfacially active peptides caused potent inhibition of all enveloped viruses tested at low and submicromolar concentrations, well below the range in which they are toxic to mammalian cells. These membrane-active peptides block uptake and fusion with the host cell by rapidly and directly interacting with virions, destabilizing the viral envelope, and driving virus aggregation and/or intervirion envelope fusion. We speculate that the molecular characteristics shared by these peptides can be exploited to enable the design, optimization, or molecular evolution of novel broad-spectrum antiviral therapeutics.IMPORTANCE New classes of antiviral drugs are needed to treat the ever-changing viral disease landscape. Current antiviral drugs treat only a small number of viral diseases, leaving many patients with established or emerging infections to be treated solely with supportive care. Recent antiviral peptide research has produced numerous membrane-interacting peptides that inhibit diverse enveloped viruses in vitro and in vivo Peptide therapeutics are becoming more common, with over 60 FDA-approved peptides for clinical use. Included in this class of therapeutics is enfuvirtide, a 36-residue peptide drug that inhibits HIV entry/fusion. Due to their broad-spectrum mechanism of action and enormous potential sequence diversity, peptides that inhibit virus entry could potentially fulfill the need for new antiviral therapeutics; however, a better understanding of their mechanism is needed for the optimization or evolution of sequence design to combat the wide landscape of viral disease.

Identification of Common CD8+ T Cell Epitopes from Lassa Fever Survivors in Nigeria and Sierra Leone.

Early and robust T cell responses have been associated with survival from Lassa fever (LF), but the Lassa virus-specific memory responses have not been well characterized. Regions within the virus surface glycoprotein (GPC) and nucleoprotein (NP) are the main targets of the Lassa virus-specific T cell responses, but, to date, only a few T cell epitopes within these proteins have been identified. We identified GPC and NP regions containing T cell epitopes and HLA haplotypes from LF survivors and used predictive HLA-binding algorithms to identify putative epitopes, which were then experimentally tested using autologous survivor samples. We identified 12 CD8-positive (CD8+) T cell epitopes, including epitopes common to both Nigerian and Sierra Leonean survivors. These data should be useful for the identification of dominant Lassa virus-specific T cell responses in Lassa fever survivors and vaccinated individuals as well as for designing vaccines that elicit cell-mediated immunity.IMPORTANCE The high morbidity and mortality associated with clinical cases of Lassa fever, together with the lack of licensed vaccines and limited and partially effective interventions, make Lassa virus (LASV) an important health concern in its regions of endemicity in West Africa. Previous infection with LASV protects from disease after subsequent exposure, providing a framework for designing vaccines to elicit similar protective immunity. Multiple major lineages of LASV circulate in West Africa, and therefore, ideal vaccine candidates should elicit immunity to all lineages. We therefore sought to identify common T cell epitopes between Lassa fever survivors from Sierra Leone and Nigeria, where distinct lineages circulate. We identified three such epitopes derived from highly conserved regions within LASV proteins. In this process, we also identified nine other T cell epitopes. These data should help in the design of an effective pan-LASV vaccine.