SARS-CoV-2 infection and persistence in the human body and brain at autopsy.

Coronavirus disease 2019 (COVID-19) is known to cause multi-organ dysfunction1-3 during acute infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with some patients experiencing prolonged symptoms, termed post-acute sequelae of SARS-CoV-2 (refs. 4,5). However, the burden of infection outside the respiratory tract and time to viral clearance are not well characterized, particularly in the brain3,6-14. Here we carried out complete autopsies on 44 patients who died with COVID-19, with extensive sampling of the central nervous system in 11 of these patients, to map and quantify the distribution, replication and cell-type specificity of SARS-CoV-2 across the human body, including the brain, from acute infection to more than seven months following symptom onset. We show that SARS-CoV-2 is widely distributed, predominantly among patients who died with severe COVID-19, and that virus replication is present in multiple respiratory and non-respiratory tissues, including the brain, early in infection. Further, we detected persistent SARS-CoV-2 RNA in multiple anatomic sites, including throughout the brain, as late as 230 days following symptom onset in one case. Despite extensive distribution of SARS-CoV-2 RNA throughout the body, we observed little evidence of inflammation or direct viral cytopathology outside the respiratory tract. Our data indicate that in some patients SARS-CoV-2 can cause systemic infection and persist in the body for months.

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.

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.

The virota and its transkingdom interactions in the healthy infant gut.

SignificanceMicrobes colonizing the infant gut during the first year(s) of life play an important role in immune system development. We show that after birth the (nearly) sterile gut is rapidly colonized by bacteria and their viruses (phages), which often show a strong cooccurrence. Most viruses infecting the infant do not cause clinical signs and their numbers strongly increase after day-care entrance. The infant diet is clearly reflected by identification of plant-infecting viruses, whereas fungi and parasites are not part of a stable gut microbiota. These temporal high-resolution baseline data about the gut colonization process will be valuable for further investigations of pathogenic viruses, dynamics between phages and their bacterial host, as well as studies investigating infants with a disturbed microbiota.

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.

Honey-bee-associated prokaryotic viral communities reveal wide viral diversity and a profound metabolic coding potential.

Honey bees (Apis mellifera) produce an enormous economic value through their pollination activities and play a central role in the biodiversity of entire ecosystems. Recent efforts have revealed the substantial influence that the gut microbiota exert on bee development, food digestion, and homeostasis in general. In this study, deep sequencing was used to characterize prokaryotic viral communities associated with honey bees, which was a blind spot in research up until now. The vast majority of the prokaryotic viral populations are novel at the genus level, and most of the encoded proteins comprise unknown functions. Nevertheless, genomes of bacteriophages were predicted to infect nearly every major bee-gut bacterium, and functional annotation and auxiliary metabolic gene discovery imply the potential to influence microbial metabolism. Furthermore, undiscovered genes involved in the synthesis of secondary metabolic biosynthetic gene clusters reflect a wealth of previously untapped enzymatic resources hidden in the bee bacteriophage community.

Novel Hendra Virus Variant Circulating in Black Flying Foxes and Grey-Headed Flying Foxes, Australia.

A novel Hendra virus variant, genotype 2, was recently discovered in a horse that died after acute illness and in Pteropus flying fox tissues in Australia. We detected the variant in flying fox urine, the pathway relevant for spillover, supporting an expanded geographic range of Hendra virus risk to horses and humans.

Nipah Virus Detection at Bat Roosts after Spillover Events, Bangladesh, 2012-2019.

Knowledge of the dynamics and genetic diversity of Nipah virus circulating in bats and at the human-animal interface is limited by current sampling efforts, which produce few detections of viral RNA. We report a series of investigations at Pteropus medius bat roosts identified near the locations of human Nipah cases in Bangladesh during 2012-2019. Pooled bat urine was collected from 23 roosts; 7 roosts (30%) had >1 sample in which Nipah RNA was detected from the first visit. In subsequent visits to these 7 roosts, RNA was detected in bat urine up to 52 days after the presumed exposure of the human case-patient, although the probability of detection declined rapidly with time. These results suggest that rapidly deployed investigations of Nipah virus shedding from bat roosts near human cases could increase the success of viral sequencing compared with background surveillance and could enhance understanding of Nipah virus ecology and evolution.

Heat-Treated Virus Inactivation Rate Depends Strongly on Treatment Procedure: Illustration with SARS-CoV-2.

Decontamination helps limit environmental transmission of infectious agents. It is required for the safe reuse of contaminated medical, laboratory, and personal protective equipment, and for the safe handling of biological samples. Heat treatment is a common decontamination method, notably used for viruses. We show that for liquid specimens (here, solution of SARS-CoV-2 in cell culture medium), the virus inactivation rate under heat treatment at 70°C can vary by almost two orders of magnitude depending on the treatment procedure, from a half-life of 0.86 min (95% credible interval [CI] 0.09, 1.77) in closed vials in a heat block to 37.04 min (95% CI 12.64, 869.82) in uncovered plates in a dry oven. These findings suggest a critical role of evaporation in virus inactivation via dry heat. Placing samples in open or uncovered containers may dramatically reduce the speed and efficacy of heat treatment for virus inactivation. Given these findings, we reviewed the literature on temperature-dependent coronavirus stability and found that specimen container types, along with whether they are closed, covered, or uncovered, are rarely reported in the scientific literature. Heat-treatment procedures must be fully specified when reporting experimental studies to facilitate result interpretation and reproducibility, and must be carefully considered when developing decontamination guidelines. IMPORTANCE Heat is a powerful weapon against most infectious agents. It is widely used for decontamination of medical, laboratory, and personal protective equipment, and for biological samples. There are many methods of heat treatment, and methodological details can affect speed and efficacy of decontamination. We applied four different heat-treatment procedures to liquid specimens containing SARS-CoV-2. Our results show that the container used to store specimens during decontamination can substantially affect inactivation rate; for a given initial level of contamination, decontamination time can vary from a few minutes in closed vials to several hours in uncovered plates. Reviewing the literature, we found that container choices and heat treatment methods are only rarely reported explicitly in methods sections. Our study shows that careful consideration of heat-treatment procedure-in particular the choice of specimen container and whether it is covered-can make results more consistent across studies, improve decontamination practice, and provide insight into the mechanisms of virus inactivation.

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.

Exploration of the virome of the European brown shrimp (Crangon crangon).

Crangon crangon is economically a very important species. Recently, promising culture attempts have been made, but a major problem is the uncontrollable mortality during the grow-out phase. As of yet, the life cycle of C. crangon is not closed in captivity so wild-caught individuals are used for further rearing. Therefore, it is important to investigate the virome of C. crangon both in wild-caught animals as in cultured animals. In recent years, next-generation-sequencing (NGS) technologies have been very important in the unravelling of the virome of a wide range of environments and matrices, such as soil, sea, potable water, but also of a wide range of animal species. This will be the first report of a virome study in C. crangon using NGS in combination with the NetoVIR protocol. The near complete genomes of 16 novel viruses were described, most of which were rather distantly related to unclassified viruses or viruses belonging to the Picornavirales, Bunyavirales Nudiviridae, Parvoviridae, Flaviviridae, Hepeviridae, Tombusviridae, Narnaviridae, Nodaviridae, Sobemovirus. A difference in virome composition was observed between muscle and hepatopancreatic tissue, suggesting a distinct tissue tropism of several of these viruses. Some differences in the viral composition were noted between the cultured and wild shrimp, which could indicate that in sub-optimal aquaculture conditions some viruses become more abundant. This research showed that a plethora of unknown viruses is present in C. crangon and that more research is needed to determine which virus is potentially dangerous for the culture of C. crangon.

Complete coding sequence of a novel picorna-like virus in a blackbird infected with Usutu virus.

Using random high-throughput RNA sequencing, the complete coding sequence of a novel picorna-like virus (a 9,228-nt contig containing 212,202 reads) was determined from a blackbird (Turdus merula) infected with Usutu virus. This sequence shares only 36% amino acid sequence identity with its closest homolog, arivirus 1, (an unclassified member of the order Picornavirales), and shares its dicistronic genome arrangement. The new virus was therefore tentatively named "blackbird arilivirus" (ari-like virus). The nearly complete genome sequence consists of at least 9,228 nt and contains two open reading frames (ORFs) encoding the nonstructural polyprotein (2235 amino acids) and structural polyprotein (769 amino acids). Two TaqMan RT-qPCR assays specific for ORF1 confirmed the presence of high levels of this novel virus in the original sample. Nucleotide composition analysis suggests that blackbird arilivirus is of dietary (plant) origin.

Highly diverse population of Picornaviridae and other members of the Picornavirales, in Cameroonian fruit bats.

BACKGROUND: The order Picornavirales represents a diverse group of positive-stranded RNA viruses with small non-enveloped icosahedral virions. Recently, bats have been identified as an important reservoir of several highly pathogenic human viruses. Since many members of the Picornaviridae family cause a wide range of diseases in humans and animals, this study aimed to characterize members of the order Picornavirales in fruit bat populations located in the Southwest region of Cameroon. These bat populations are frequently in close contact with humans due to hunting, selling and eating practices, which provides ample opportunity for interspecies transmissions. RESULTS: Fecal samples from 87 fruit bats (Eidolon helvum and Epomophorus gambianus), were combined into 25 pools and analyzed using viral metagenomics. In total, Picornavirales reads were found in 19 pools, and (near) complete genomes of 11 picorna-like viruses were obtained from 7 of these pools. The picorna-like viruses possessed varied genomic organizations (monocistronic or dicistronic), and arrangements of gene cassettes. Some of the viruses belonged to established families, including the Picornaviridae, whereas others clustered distantly from known viruses and most likely represent novel genera and families. Phylogenetic and nucleotide composition analyses suggested that mammals were the likely host species of bat sapelovirus, bat kunsagivirus and bat crohivirus, whereas the remaining viruses (named bat iflavirus, bat posalivirus, bat fisalivirus, bat cripavirus, bat felisavirus, bat dicibavirus and bat badiciviruses 1 and 2) were most likely diet-derived. CONCLUSION: The existence of a vast genetic variability of picorna-like viruses in fruit bats may increase the probability of spillover infections to humans especially when humans and bats have direct contact as the case in this study site. However, further screening for these viruses in humans will fully indicate their zoonotic potential.

Reassortment among picobirnaviruses found in wolves.

We conducted a viral metagenomics study in diarrheic free-ranging wolves in Portugal, revealing for the first time the presence of reassortant picobirnaviruses. These viruses shared identical capsid segments together with diverse RNA-dependent RNA polymerase segments. Even though causality between these picobirnaviruses and diarrhea could not be established, the study nonetheless confirms for the first time that wolves are a potential reservoir for picobirnaviruses, which might play a role as enteric pathogens.

Antigenic epitope analysis of Pakistani G3 and G9 rotavirus strains compared to vaccine strains revealed multiple amino acid differences.

Rotaviruses belong to genotype VP4-P[8] are a significant cause of severe loose diarrhea in infants and young children. In the present study, we characterised the complete genome of three of the Pakistani P[8]b RVA strains by Illumina HiSeq sequencing technology to determine the complete genotype constellation providing insight into the evolutionary dynamics of their genes using maximum likelihood analysis. The maximum genomic sequences of our study strains were similar to more recent human Wa-Like G1P[8]a, G3P[8]a, G4P[6], G4P[8], G9P[4], G9P[8]a, G11P[25],G12P[8]a and G12P[6] strains circulating around the world. Therefore, strains PAK274, PAK439 and PAK624 carry natively distinctive VP4 gene with universally common human Wa-Like genetic backbone. Comparing our study P[8]b strains with vaccines strains RotarixTM and RotaTeqTM, multiple amino acid differences were examined between vaccine virus antigenic epitopes and Pakistani isolates. Over time, these differences may result in the selection for strains that will escape the vaccine-induced RVA-neutralizing-antibody effect.