Clinical benefit of remdesivir in rhesus macaques infected with SARS-CoV-2.

Effective therapies to treat coronavirus disease 2019 (COVID-19) are urgently needed. While many investigational, approved, and repurposed drugs have been suggested as potential treatments, preclinical data from animal models can guide the search for effective treatments by ruling out those that lack efficacy in vivo. Remdesivir (GS-5734) is a nucleotide analogue prodrug with broad antiviral activity1,2 that is currently being investigated in COVID-19 clinical trials and recently received Emergency Use Authorization from the US Food and Drug Administration3,4. In animal models, remdesivir was effective against infection with Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV)2,5,6. In vitro, remdesivir inhibited replication of SARS-CoV-27,8. Here we investigate the efficacy of remdesivir in a rhesus macaque model of SARS-CoV-2 infection9. Unlike vehicle-treated animals, macaques treated with remdesivir did not show signs of respiratory disease; they also showed reduced pulmonary infiltrates on radiographs and reduced virus titres in bronchoalveolar lavages twelve hours after the first dose. Virus shedding from the upper respiratory tract was not reduced by remdesivir treatment. At necropsy, remdesivir-treated animals had lower lung viral loads and reduced lung damage. Thus, treatment with remdesivir initiated early during infection had a clinical benefit in rhesus macaques infected with SARS-CoV-2. Although the rhesus macaque model does not represent the severe disease observed in some patients with COVID-19, our data support the early initiation of remdesivir treatment in patients with COVID-19 to prevent progression to pneumonia.

Respiratory disease in rhesus macaques inoculated with SARS-CoV-2.

An outbreak of coronavirus disease 2019 (COVID-19), which is caused by a novel coronavirus (named SARS-CoV-2) and has a case fatality rate of approximately 2%, started in Wuhan (China) in December 20191,2. Following an unprecedented global spread3, the World Health Organization declared COVID-19 a pandemic on 11 March 2020. Although data on COVID-19 in humans are emerging at a steady pace, some aspects of the pathogenesis of SARS-CoV-2 can be studied in detail only in animal models, in which repeated sampling and tissue collection is possible. Here we show that SARS-CoV-2 causes a respiratory disease in rhesus macaques that lasts between 8 and 16 days. Pulmonary infiltrates, which are a hallmark of COVID-19 in humans, were visible in lung radiographs. We detected high viral loads in swabs from the nose and throat of all of the macaques, as well as in bronchoalveolar lavages; in one macaque, we observed prolonged rectal shedding. Together, the rhesus macaque recapitulates the moderate disease that has been observed in the majority of human cases of COVID-19. The establishment of the rhesus macaque as a model of COVID-19 will increase our understanding of the pathogenesis of this disease, and aid in the development and testing of medical countermeasures.

ChAdOx1 nCoV-19 vaccine prevents SARS-CoV-2 pneumonia in rhesus macaques.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 20191,2 and is responsible for the coronavirus disease 2019 (COVID-19) pandemic3. Vaccines are an essential countermeasure and are urgently needed to control the pandemic4. Here we show that the adenovirus-vector-based vaccine ChAdOx1 nCoV-19, which encodes the spike protein of SARS-CoV-2, is immunogenic in mice and elicites a robust humoral and cell-mediated response. This response was predominantly mediated by type-1 T helper cells, as demonstrated by the profiling of the IgG subclass and the expression of cytokines. Vaccination with ChAdOx1 nCoV-19 (using either a prime-only or a prime-boost regimen) induced a balanced humoral and cellular immune response of type-1 and type-2 T helper cells in rhesus macaques. We observed a significantly reduced viral load in the bronchoalveolar lavage fluid and lower respiratory tract tissue of vaccinated rhesus macaques that were challenged with SARS-CoV-2 compared with control animals, and no pneumonia was observed in vaccinated SARS-CoV-2-infected animals. However, there was no difference in nasal shedding between vaccinated and control SARS-CoV-2-infected macaques. Notably, we found no evidence of immune-enhanced disease after viral challenge in vaccinated SARS-CoV-2-infected animals. The safety, immunogenicity and efficacy profiles of ChAdOx1 nCoV-19 against symptomatic PCR-positive COVID-19 disease will now be assessed in randomized controlled clinical trials in humans.

A monoclonal antibody targeting the Nipah virus fusion glycoprotein apex imparts protection from disease.

Nipah virus (NiV) is a highly pathogenic paramyxovirus capable of causing severe respiratory and neurologic disease in humans. Currently, there are no licensed vaccines or therapeutics against NiV, underscoring the urgent need for the development of countermeasures. The NiV surface-displayed glycoproteins, NiV-G and NiV-F, mediate host cell attachment and fusion, respectively, and are heavily targeted by host antibodies. Here, we describe a vaccination-derived neutralizing monoclonal antibody, mAb92, that targets NiV-F. Structural characterization of the Fab region bound to NiV-F (NiV-F-Fab92) by cryo-electron microscopy analysis reveals an epitope in the DIII domain at the membrane distal apex of NiV-F, an established site of vulnerability on the NiV surface. Further, prophylactic treatment of hamsters with mAb92 offered complete protection from NiV disease, demonstrating beneficial activity of mAb92 in vivo. This work provides support for targeting NiV-F in the development of vaccines and therapeutics against NiV.IMPORTANCENipah virus (NiV) is a highly lethal henipavirus (HNV) that causes severe respiratory and neurologic disease in humans. Currently, there are no licensed vaccines or therapeutics against NiV, highlighting a need to develop countermeasures. The NiV surface displays the receptor binding protein (NiV-G, or RBP) and the fusion protein (NiV-F), which allow the virus to attach and enter cells. These proteins can be targeted by vaccines and antibodies to prevent disease. This work describes a neutralizing antibody (mAb92) that targets NiV-F. Structural characterization by cryo-electron microscopy analysis reveals where the antibody binds to NiV-F to neutralize the virus. This study also shows that prophylactic treatment of hamsters with mAb92 completely protected against developing NiV disease. This work shows how targeting NiV-F can be useful to preventing NiV disease, supporting future studies in the development of vaccines and therapeutics.

Compartmentalized SARS-CoV-2 Replication in the Upper vs Lower Respiratory Tract After Intranasal Inoculation or Aerosol Exposure.

Nonhuman primate models are essential for the development of vaccines and antivirals against infectious diseases. Rhesus macaques are a widely utilized infection model for SARS-CoV-2. We compared cellular tropism and virus replication in rhesus macaques inoculated with SARS-CoV-2 via the intranasal route or via exposure to aerosols. Intranasal inoculation resulted in replication in the upper respiratory tract with limited involvement in the lower respiratory tract, whereas exposure to aerosols resulted in infection throughout the respiratory tract. In comparison with multiroute inoculation, intranasal and aerosol inoculation resulted in reduced SARS-CoV-2 replication in the respiratory tract.

A bivalent Adenovirus-Vectored Vaccine induces a robust humoral response, but does not protect cynomolgus macaques against a lethal challenge with Sudan virus.

The most recent Sudan virus (SUDV) outbreak in Uganda was first detected in September 2022 and resulted in 164 laboratory-confirmed cases and 77 deaths. There are no approved vaccines against SUDV. Here, we investigated the protective efficacy of ChAdOx1-biEBOV in cynomolgus macaques using a prime or a prime-boost regimen. ChAdOx1-biEBOV is a replication-deficient simian adenovirus vector encoding SUDV and Ebola virus (EBOV) glycoproteins (GPs). Intramuscular vaccination induced SUDV and EBOV GP-specific IgG responses and neutralizing antibodies. Upon challenge with SUDV, vaccinated animals showed signs of disease like those observed in control animals, and no difference in survival outcomes were measured among all three groups. Viral load in blood samples and in tissue samples obtained after necropsy were not significantly different between groups. Overall, this study highlights the importance of evaluating vaccines in multiple animal models and demonstrates the importance of understanding protective efficacy in both animal models and human hosts.

The B.1.427/1.429 (epsilon) SARS-CoV-2 variants are more virulent than ancestral B.1 (614G) in Syrian hamsters.

As novel SARS-CoV-2 variants continue to emerge, it is critical that their potential to cause severe disease and evade vaccine-induced immunity is rapidly assessed in humans and studied in animal models. In early January 2021, a novel SARS-CoV-2 variant designated B.1.429 comprising 2 lineages, B.1.427 and B.1.429, was originally detected in California (CA) and it was shown to have enhanced infectivity in vitro and decreased antibody neutralization by plasma from convalescent patients and vaccine recipients. Here we examine the virulence, transmissibility, and susceptibility to pre-existing immunity for B 1.427 and B 1.429 in the Syrian hamster model. We find that both variants exhibit enhanced virulence as measured by increased body weight loss compared to hamsters infected with ancestral B.1 (614G), with B.1.429 causing the most marked body weight loss among the 3 variants. Faster dissemination from airways to parenchyma and more severe lung pathology at both early and late stages were also observed with B.1.429 infections relative to B.1. (614G) and B.1.427 infections. In addition, subgenomic viral RNA (sgRNA) levels were highest in oral swabs of hamsters infected with B.1.429, however sgRNA levels in lungs were similar in all three variants. This demonstrates that B.1.429 replicates to higher levels than ancestral B.1 (614G) or B.1.427 in the oropharynx but not in the lungs. In multi-virus in-vivo competition experiments, we found that B.1. (614G), epsilon (B.1.427/B.1.429) and gamma (P.1) dramatically outcompete alpha (B.1.1.7), beta (B.1.351) and zeta (P.2) in the lungs. In the nasal cavity, B.1. (614G), gamma, and epsilon dominate, but the highly infectious alpha variant also maintains a moderate size niche. We did not observe significant differences in airborne transmission efficiency among the B.1.427, B.1.429 and ancestral B.1 (614G) and WA-1 variants in hamsters. These results demonstrate enhanced virulence and high relative oropharyngeal replication of the epsilon (B.1.427/B.1.429) variant in Syrian hamsters compared to an ancestral B.1 (614G) variant.

Stability of Monkeypox Virus in Body Fluids and Wastewater.

An outbreak of human mpox infection in nonendemic countries appears to have been driven largely by transmission through body fluids or skin-to-skin contact during sexual activity. We evaluated the stability of monkeypox virus (MPXV) in different environments and specific body fluids and tested the effectiveness of decontamination methodologies. MPXV decayed faster at higher temperatures, and rates varied considerably depending on the medium in which virus was suspended, both in solution and on surfaces. More proteinaceous fluids supported greater persistence. Chlorination was an effective decontamination technique, but only at higher concentrations. Wastewater was more difficult to decontaminate than plain deionized water; testing for infectious MPXV could be a helpful addition to PCR-based wastewater surveillance when high levels of viral DNA are detected. Our findings suggest that, because virus stability is sufficient to support environmental MPXV transmission in healthcare settings, exposure and dose-response will be limiting factors for those transmission routes.

Comparative Aerosol and Surface Stability of SARS-CoV-2 Variants of Concern.

SARS-CoV-2 transmits principally by air; contact and fomite transmission may also occur. Variants of concern are more transmissible than ancestral SARS-CoV-2. We found indications of possible increased aerosol and surface stability for early variants of concern, but not for the Delta and Omicron variants. Stability changes are unlikely to explain increased transmissibility.

K18-hACE2 mice develop respiratory disease resembling severe COVID-19.

SARS-CoV-2 emerged in late 2019 and resulted in the ongoing COVID-19 pandemic. Several animal models have been rapidly developed that recapitulate the asymptomatic to moderate disease spectrum. Now, there is a direct need for additional small animal models to study the pathogenesis of severe COVID-19 and for fast-tracked medical countermeasure development. Here, we show that transgenic mice expressing the human SARS-CoV-2 receptor (angiotensin-converting enzyme 2 [hACE2]) under a cytokeratin 18 promoter (K18) are susceptible to SARS-CoV-2 and that infection resulted in a dose-dependent lethal disease course. After inoculation with either 104 TCID50 or 105 TCID50, the SARS-CoV-2 infection resulted in rapid weight loss in both groups and uniform lethality in the 105 TCID50 group. High levels of viral RNA shedding were observed from the upper and lower respiratory tract and intermittent shedding was observed from the intestinal tract. Inoculation with SARS-CoV-2 resulted in upper and lower respiratory tract infection with high infectious virus titers in nasal turbinates, trachea and lungs. The observed interstitial pneumonia and pulmonary pathology, with SARS-CoV-2 replication evident in pneumocytes, were similar to that reported in severe cases of COVID-19. SARS-CoV-2 infection resulted in macrophage and lymphocyte infiltration in the lungs and upregulation of Th1 and proinflammatory cytokines/chemokines. Extrapulmonary replication of SARS-CoV-2 was observed in the cerebral cortex and hippocampus of several animals at 7 DPI but not at 3 DPI. The rapid inflammatory response and observed pathology bears resemblance to COVID-19. Additionally, we demonstrate that a mild disease course can be simulated by low dose infection with 102 TCID50 SARS-CoV-2, resulting in minimal clinical manifestation and near uniform survival. Taken together, these data support future application of this model to studies of pathogenesis and medical countermeasure development.

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.

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.

Rousettus aegyptiacus Bats Do Not Support Productive Nipah Virus Replication.

Nipah virus (NiV) is a bat-borne zoonotic pathogen that can cause severe respiratory distress and encephalitis upon spillover into humans. NiV is capable of infecting a broad range of hosts including humans, pigs, ferrets, dogs, cats, hamsters, and at least 2 genera of bats. Little is known about the biology of NiV in the bat reservoir. In this study, we evaluate the potential for the Egyptian fruit bat (EFB), Rousettus aegyptiacus, to serve as a model organism for studying NiV in bats. Our data suggest that NiV does not efficiently replicate in EFBs in vivo. Furthermore, we show no seroconversion against NiV glycoprotein and a lack of viral replication in primary and immortalized EFB-derived cell lines. Our data show that despite using a conserved target for viral entry, NiV replication is limited in some bat species. We conclude that EFBs are not an appropriate organism to model NiV infection or transmission in bats.

A Novel Field-Deployable Method for Sequencing and Analyses of Henipavirus Genomes From Complex Samples on the MinION Platform.

Viruses in the genus Henipavirus encompass 2 highly pathogenic emerging zoonotic pathogens, Hendra virus (HeV) and Nipah virus (NiV). Despite the impact on human health, there is currently limited full-genome sequence information available for henipaviruses. This lack of full-length genomes hampers our ability to understand the molecular drivers of henipavirus emergence. Furthermore, rapidly deployable viral genome sequencing can be an integral part of outbreak response and epidemiological investigations to study transmission chains. In this study, we describe the development of a reverse-transcription, long-range polymerase chain reaction (LRPCR) assay for efficient genome amplification of NiV, HeV, and a related non-pathogenic henipavirus, Cedar virus (CedPV). We then demonstrated the utility of our method by amplifying partial viral genomes from 6 HeV-infected tissue samples from Syrian hamsters and 4 tissue samples from a NiV-infected African green monkey with viral loads as low as 52 genome copies/mg. We subsequently sequenced the amplified genomes on the portable Oxford Nanopore MinION platform and analyzed the data using a newly developed field-deployable bioinformatic pipeline. Our LRPCR assay allows amplification and sequencing of 2 or 4 amplicons in semi-nested reactions. Coupled with an easy-to-use bioinformatics pipeline, this method is particularly useful in the field during outbreaks in resource-poor environments.

Effect of Environmental Conditions on SARS-CoV-2 Stability in Human Nasal Mucus and Sputum.

We found that environmental conditions affect the stability of severe acute respiratory syndrome coronavirus 2 in nasal mucus and sputum. The virus is more stable at low-temperature and low-humidity conditions, whereas warmer temperature and higher humidity shortened half-life. Although infectious virus was undetectable after 48 hours, viral RNA remained detectable for 7 days.

Effectiveness of N95 Respirator Decontamination and Reuse against SARS-CoV-2 Virus.

The coronavirus pandemic has created worldwide shortages of N95 respirators. We analyzed 4 decontamination methods for effectiveness in deactivating severe acute respiratory syndrome coronavirus 2 virus and effect on respirator function. Our results indicate that N95 respirators can be decontaminated and reused, but the integrity of respirator fit and seal must be maintained.

Importance of Neutralizing Monoclonal Antibodies Targeting Multiple Antigenic Sites on the Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein To Avoid Neutralization Escape.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a highly lethal pulmonary infection with ∼35% mortality. The potential for a future pandemic originating from animal reservoirs or health care-associated events is a major public health concern. There are no vaccines or therapeutic agents currently available for MERS-CoV. Using a probe-based single B cell cloning strategy, we have identified and characterized multiple neutralizing monoclonal antibodies (MAbs) specifically binding to the receptor-binding domain (RBD) or S1 (non-RBD) regions from a convalescent MERS-CoV-infected patient and from immunized rhesus macaques. RBD-specific MAbs tended to have greater neutralizing potency than non-RBD S1-specific MAbs. Six RBD-specific and five S1-specific MAbs could be sorted into four RBD and three non-RBD distinct binding patterns, based on competition assays, mapping neutralization escape variants, and structural analysis. We determined cocrystal structures for two MAbs targeting the RBD from different angles and show they can bind the RBD only in the "out" position. We then showed that selected RBD-specific, non-RBD S1-specific, and S2-specific MAbs given prophylactically prevented MERS-CoV replication in lungs and protected mice from lethal challenge. Importantly, combining RBD- and non-RBD MAbs delayed the emergence of escape mutations in a cell-based virus escape assay. These studies identify MAbs targeting different antigenic sites on S that will be useful for defining mechanisms of MERS-CoV neutralization and for developing more effective interventions to prevent or treat MERS-CoV infections.IMPORTANCE MERS-CoV causes a highly lethal respiratory infection for which no vaccines or antiviral therapeutic options are currently available. Based on continuing exposure from established reservoirs in dromedary camels and bats, transmission of MERS-CoV into humans and future outbreaks are expected. Using structurally defined probes for the MERS-CoV spike glycoprotein (S), the target for neutralizing antibodies, single B cells were sorted from a convalescent human and immunized nonhuman primates (NHPs). MAbs produced from paired immunoglobulin gene sequences were mapped to multiple epitopes within and outside the receptor-binding domain (RBD) and protected against lethal MERS infection in a murine model following passive immunization. Importantly, combining MAbs targeting distinct epitopes prevented viral neutralization escape from RBD-directed MAbs. These data suggest that antibody responses to multiple domains on CoV spike protein may improve immunity and will guide future vaccine and therapeutic development efforts.