Search

Variant of concern (VOC) Omicron-BA.1 has achieved global predominance in early 2022. Therefore, surveillance and comprehensive characterization of Omicron-BA.1 in advanced primary cell culture systems and animal models are urgently needed. Here, we characterize Omicron-BA.1 and recombinant Omicron-BA.1 spike gene mutants in comparison with VOC Delta in well-differentiated primary human nasal and bronchial epithelial cells in vitro, followed by in vivo fitness characterization in hamsters, ferrets and hACE2-expressing mice, and immunized hACE2-mice. We demonstrate a spike-mediated enhancement of early replication of Omicron-BA.1 in nasal epithelial cultures, but limited replication in bronchial epithelial cultures. In hamsters, Delta shows dominance over Omicron-BA.1, and in ferrets Omicron-BA.1 infection is abortive. In hACE2-knock-in mice, Delta and a Delta spike clone also show dominance over Omicron-BA.1 and an Omicron-BA.1 spike clone, respectively. Interestingly, in naïve K18-hACE2 mice, we observe Delta spike-mediated increased replication and pathogenicity and Omicron-BA.1 spike-mediated reduced replication and pathogenicity, suggesting that the spike gene is a major determinant of replication and pathogenicity. Finally, the Omicron-BA.1 spike clone is less well-controlled by mRNA-vaccination in K18-hACE2-mice and becomes more competitive compared to the progenitor and Delta spike clones, suggesting that spike gene-mediated immune evasion is another important factor that led to Omicron-BA.1 dominance. A comprehensive characterisation of Omicron-BA.1 and VOC Delta in numerous in vitro and in vivo models uncovers the factors that led to its global dominance. [ABSTRACT FROM AUTHOR]

Emerging variants of concern (VOCs) are driving the COVID-19 pandemic1,2. Experimental assessments of replication and transmission of major VOCs and progenitors are needed to understand the mechanisms of replication and transmission of VOCs3. Here we show that the spike protein (S) from Alpha (also known as B.1.1.7) and Beta (B.1.351) VOCs had a greater affinity towards the human angiotensin-converting enzyme 2 (ACE2) receptor than that of the progenitor variant S(D614G) in vitro. Progenitor variant virus expressing S(D614G) (wt-S614G) and the Alpha variant showed similar replication kinetics in human nasal airway epithelial cultures, whereas the Beta variant was outcompeted by both. In vivo, competition experiments showed a clear fitness advantage of Alpha over wt-S614G in ferrets and two mouse models—the substitutions in S were major drivers of the fitness advantage. In hamsters, which support high viral replication levels, Alpha and wt-S614G showed similar fitness. By contrast, Beta was outcompeted by Alpha and wt-S614G in hamsters and in mice expressing human ACE2. Our study highlights the importance of using multiple models to characterize fitness of VOCs and demonstrates that Alpha is adapted for replication in the upper respiratory tract and shows enhanced transmission in vivo in restrictive models, whereas Beta does not overcome Alpha or wt-S614G in naive animals.The Alpha variant of SARS-CoV-2 outcompetes progenitor SARS-CoV-2 in upper respiratory tract replication competition in vivo. [ABSTRACT FROM AUTHOR]

During the evolution of SARS-CoV-2 in humans, a D614G substitution in the spike glycoprotein (S) has emerged; virus containing this substitution has become the predominant circulating variant in the COVID-19 pandemic1. However, whether the increasing prevalence of this variant reflects a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains unknown. Here we use isogenic SARS-CoV-2 variants to demonstrate that the variant that contains S(D614G) has enhanced binding to the human cell-surface receptor angiotensin-converting enzyme 2 (ACE2), increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a human ACE2 knock-in mouse model, and markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Our data show that the D614G substitution in S results in subtle increases in binding and replication in vitro, and provides a real competitive advantage in vivo—particularly during the transmission bottleneck. Our data therefore provide an explanation for the global predominance of the variant that contains S(D614G) among the SARS-CoV-2 viruses that are currently circulating. A SARS-CoV-2 variant containing a D614G substitution in the spike protein shows enhanced binding to human ACE2, increased replication in human cell cultures and a competitive advantage in animal models of infection. [ABSTRACT FROM AUTHOR]

Reverse genetics has been an indispensable tool to gain insights into viral pathogenesis and vaccine development. The genomes of large RNA viruses, such as those from coronaviruses, are cumbersome to clone and manipulate in Escherichia coli owing to the size and occasional instability of the genome1–3. Therefore, an alternative rapid and robust reverse-genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform to genetically reconstruct diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Pneumoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples or synthetic DNA, and these fragments were then reassembled in one step in Saccharomyces cerevisiae using transformation-associated recombination cloning to maintain the genome as a yeast artificial chromosome. T7 RNA polymerase was then used to generate infectious RNA to rescue viable virus. Using this platform, we were able to engineer and generate chemically synthesized clones of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)4, which has caused the recent pandemic of coronavirus disease (COVID-19), in only a week after receipt of the synthetic DNA fragments. The technical advance that we describe here facilitates rapid responses to emerging viruses as it enables the real-time generation and functional characterization of evolving RNA virus variants during an outbreak. A yeast-based synthetic genomics platform is used to reconstruct and characterize large RNA viruses from synthetic DNA fragments; this technique will facilitate the rapid analysis of RNA viruses, such as SARS-CoV-2, during an outbreak. [ABSTRACT FROM AUTHOR]

The Kv3.1b potassium channel subunit, which facilitates the fast-spiking phenotype characteristic of parvalbumin (PV)-expressing inhibitory interneurons, is also expressed by subpopulations of excitatory neurons in macaque cortex. We have previously shown that V1 neurons expressing Kv3.1b but not PV or GABA were largely concentrated within layers 4Cα and 4B of V1, suggesting laminar or pathway specificity. In the current study, the distribution and pattern of co-immunoreactivity of GABA, PV, and Kv3.1b across layers in extrastriate cortical areas V2 and MT of the macaque monkey were measured using the same triple immunofluorescence labeling, confocal microscopy, and partially automated cell-counting strategies used in V1. For comparison, densities of the overall cell and neuronal populations were also measured for each layer of V2 and MT using tissue sections immunofluorescence labeled for the pan-neuronal marker NeuN. GABAergic neurons accounted for 14% of the total neuronal population in V2 and 25% in MT. Neurons expressing Kv3.1b but neither GABA nor PV were present in both areas. This subpopulation was most prevalent in the lowest subcompartment of layer 3, comprising 5% of the total neuronal population in layer 3C of both areas, and 41% and 36% of all Kv3.1b+ neurons in this layer in V2 and MT, respectively. The prevalence and laminar distribution of this subpopulation were remarkably consistent between V2 and MT and showed a striking similarity to the patterns observed previously in V1, suggesting a common contribution to the cortical circuit across areas. [ABSTRACT FROM AUTHOR]

A new framework for measuring densities of immunolabeled neurons across cortical layers was implemented that combines a confocal microscopy sampling strategy with automated analysis of 3D image stacks. Its utility was demonstrated by quantifying neuronal density in macaque cortical areas V1 and V2. A series of overlapping confocal image stacks were acquired, each spanning from the pial surface to the white matter. DAPI channel images were automatically thresholded, and contiguous regions that included multiple clumped nuclear profiles were split using k-means clustering of image pixels for a set of candidate k values determined based on the clump's area; the most likely candidate segmentation was selected based on criteria that capture expected nuclear profile shape and size. The centroids of putative nuclear profiles estimated from 2D images were then grouped across z planes in an image stack to identify the positions of nuclei in x- y- z. 3D centroids falling outside user-specified exclusion boundaries were deleted, nuclei were classified by the presence or absence of signal in a channel corresponding to an immunolabeled antigen (e.g., the pan-neuronal marker NeuN) at the nuclear centroid location, and the set of classified cells was combined across image stacks to estimate density across cortical depth. The method was validated by comparison with conventional stereological methods. The average neuronal density across cortical layers was 230 × 10 neurons per mm in V1 and 130 × 10 neurons per mm in V2. The method is accurate, flexible, and general enough to measure densities of neurons of various molecularly identified types. [ABSTRACT FROM AUTHOR]