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Understanding how ligand binding influences protein flexibility is important, especially in rational drug design. Protein flexibility upon ligand binding is analyzed herein using 305 proteins with 2369 crystal structures with ligands (holo) and 1679 without (apo). Each protein has at least two apo and two holo structures for analysis. The inherent variation in structures with and without ligands is first established as a baseline. This baseline is then compared to the change in conformation in going from the apo to holo states to probe induced flexibility. The inherent backbone flexibility across the apo structures is roughly the same as the variation across holo structures. The induced backbone flexibility across apo-holo pairs is larger than that of the apo or holo states, but the increase in RMSD is less than 0.5 Å. Analysis of χ1 angles revealed a distinctly different pattern with significant influences seen for ligand binding on side-chain conformations in the binding site. Within the apo and holo states themselves, the variation of the χ1 angles is the same. However, the data combining both apo and holo states show significant displacements. Upon ligand binding, χ1 angles are frequently pushed to new orientations outside the range seen in the apo states. Influences on binding-site variation could not be easily attributed to features such as ligand size or x-ray structure resolution. By combining these findings, we find that most binding site flexibility is compatible with the common practice in flexible docking, where backbones are kept rigid and side chains are allowed some degree of flexibility.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) not only affects the respiratory tract but also causes neurological symptoms such as loss of smell and taste, headache, fatigue or severe cerebrovascular complications. Using transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2), we investigated the spatiotemporal distribution and pathomorphological features in the CNS following intranasal infection with SARS-CoV-2 variants, as well as after prior influenza A virus infection. Apart from Omicron, we found all variants to frequently spread to and within the CNS. Infection was restricted to neurons and appeared to spread from the olfactory bulb mainly in basally oriented regions in the brain and into the spinal cord, independent of ACE2 expression and without evidence of neuronal cell death, axonal damage or demyelination. However, microglial activation, microgliosis and a mild macrophage and T cell dominated inflammatory response was consistently observed, accompanied by apoptotic death of endothelial, microglial and immune cells, without their apparent infection. Microgliosis and immune cell apoptosis indicate a potential role of microglia for pathogenesis and viral effect in COVID-19 and the possible impairment of neurological functions, especially in long COVID. These data may also be informative for the selection of therapeutic candidates and broadly support the investigation of agents with adequate penetration into relevant regions of the CNS.

Puumala orthohantavirus (PUUV) is an emerging zoonotic virus that was first discovered in the Puumala region of Finland in the early 1980s and is the primary etiological agent of nephropathia epidemica (NE), a milder form of a life-threatening disease known as hemorrhagic fever with renal syndrome (HFRS). PUUV and other members of the Old World hantaviruses (OWHVs) predominantly circulate in rodents or insectivores across Eurasia, accounting for several thousand of reported HFRS cases every year (with many more unreported/misdiagnosed cases suspected). The rodent reservoir of PUUV is the common bank vole ( Myodes (M.) glareolus ), and transmission of the virus to humans occurs via inhalation of contagious aerosols and through contact with contaminated droppings or urine. Although PUUV is the subject of extensive research, due to its potential to cause severe disease outcomes in humans and its considerable economic and social impact, neither licensed vaccines nor specific antiviral treatments are available against PUUV. However, many important advancements have been made in terms of PUUV research over the last years. This included the elucidation of its glycoproteins, the discovery of broadly neutralizing hantavirus antibodies as therapeutic candidates and expanded research on the mRNA vaccine technology which will likely enable the development of strong PUUV vaccine candidates in the near future. Currently, there is still a lack of suitable animal models for the preclinical evaluation of experimental vaccines and antivirals, which hampers vaccine and antiviral development. Current attempts to decrease hantavirus-associated human infections rely primarily on prevention and countermeasures for rodent control, including reduced contact to droppings, saliva and urine, and disinfection of areas that are contaminated with rodent excreta. Here, we review these recent advances and other aspects including PUUV prevalence, virus biology, diagnosis and clinical features, and current animal models for vaccine and treatment development.

Orthohantaviruses are emerging zoonotic viruses that can infect humans via the respiratory tract. There is an unmet need for an in vivo model to study infection of different orthohantaviruses in physiologically relevant tissue and to assess the efficacy of novel pan-orthohantavirus countermeasures. Here, we describe the use of a human lung xenograft mouse model to study the permissiveness for different orthohantavirus species and to assess its utility for preclinical testing of therapeutics. Following infection of xenografted human lung tissues, distinct orthohantavirus species differentially replicated in the human lung and subsequently spread systemically. The different orthohantaviruses primarily targeted the endothelium, respiratory epithelium and macrophages in the human lung. A proof-of-concept preclinical study showed treatment of these mice with a virus neutralizing antibody could block Andes orthohantavirus infection and dissemination. This pan-orthohantavirus model will facilitate progress in the fundamental understanding of pathogenesis and virus-host interactions for orthohantaviruses. Furthermore, it is an invaluable tool for preclinical evaluation of novel candidate pan-orthohantavirus intervention strategies.

The emergence and spread of tick-borne arboviruses pose an increased challenge to human and animal health. In Europe this is demonstrated by the increasingly wide distribution of tick-borne encephalitis virus (TBEV, Flavivirus, Flaviviridae), which has recently been found in the United Kingdom (UK). However, much less is known about other tick-borne flaviviruses (TBFV), such as the closely related louping ill virus (LIV), an animal pathogen which is endemic to the UK and Ireland, but which has been detected in other parts of Europe including Scandinavia and Russia. The emergence and potential spatial overlap of these viruses necessitates improved understanding of LIV genomic diversity, geographic spread and evolutionary history. We sequenced a virus archive composed of 22 LIV isolates which had been sampled throughout the UK over a period of over 80 years. Combining this dataset with published virus sequences, we detected no sign of recombination and found low diversity and limited evidence for positive selection in the LIV genome. Phylogenetic analysis provided evidence of geographic clustering as well as long-distance movement, including movement events that appear recent. However, despite genomic data and an 80-year time span, we found that the data contained insufficient temporal signal to reliably estimate a molecular clock rate for LIV. Additional analyses revealed that this also applied to TBEV, albeit to a lesser extent, pointing to a general problem with phylogenetic dating for TBFV. The 22 LIV genomes generated during this study provide a more reliable LIV phylogeny, improving our knowledge of the evolution of tick-borne flaviviruses. Our inability to estimate a molecular clock rate for both LIV and TBEV suggests that temporal calibration of tick-borne flavivirus evolution should be interpreted with caution and highlight a unique aspect of these viruses which may be explained by their reliance on tick vectors.

The respiratory system is the main target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 19 (COVID-19) where acute respiratory distress syndrome is considered the leading cause of death. Changes in pulmonary blood vessels, among which an endothelialitis/endotheliitis has been particularly emphasized, have been suggested to play a central role in the development of acute lung injury. Similar vascular changes are also observed in animal models of COVID-19. The present study aimed to determine whether the latter are specific for SARS-CoV-2 infection, investigating the vascular response in the lungs of mice infected with SARS-CoV-2 and other respiratory viruses (influenza A and murine gammaherpesvirus) by in situ approaches (histology, immunohistology, morphometry) combined with RNA sequencing and bioinformatic analysis. Non-selective recruitment of monocytes and T and B cells from larger muscular veins and arteries was observed with all viruses, matched by a comparable transcriptional response. There was no evidence of endothelial cell infection in any of the models. Both the morphological investigation and the transcriptomics approach support the interpretation that the lung vasculature in mice mounts a stereotypic response to alveolar and respiratory epithelial damage. This may have implications for the treatment and management of respiratory disease in humans.

Neurological complications, including encephalopathy and stroke, occur in a significant proportion of COVID-19 cases but viral protein is seldom detected in the brain parenchyma. To model this situation, we developed a novel low-inoculum K18-hACE2 mouse model of SARS-CoV-2 infection during which active viral replication was consistently seen in mouse lungs but not in the brain. We found that several mediators previously associated with encephalopathy in clinical samples were upregulated in the lung, including CCL2, and IL-6. In addition, several inflammatory mediations, including CCL4, IFNγ, IL-17A, were upregulated in the brain, associated with microglial reactivity. Parallel in vitro experiments demonstrated that the filtered supernatant from SARS-CoV-2 virion exposed brain endothelial cells induced activation of uninfected microglia. This model successfully recreates SARS-CoV-2 virus-associated para-infectious brain inflammation which can be used to study the pathophysiology of the neurological complications and the identification of potential immune targets for treatment.

COVID-19 is a spectrum of clinical symptoms in humans caused by infection with SARS-CoV-2. The coalescence of SARS-CoV-2 with seasonal respiratory viruses, particularly influenza viruses, is a global health concern. To understand this, transgenic mice expressing the human ACE2 receptor (K18-hACE2) were infected with influenza A virus (IAV) followed by SARS-CoV-2 and the host response and effect on virus biology was compared to K18-hACE2 mice infected with IAV or SARS-CoV-2 alone. The sequentially infected mice showed reduced SARS-CoV-2 RNA synthesis, yet exhibited more rapid weight loss, more severe lung damage and a prolongation of the innate response compared to the singly infected or control mice. Sequential infection also exacerbated the extrapulmonary encephalitic manifestations associated with SARS-CoV-2 infection. Conversely, prior infection with a commercially available, multivalent live-attenuated influenza vaccine (Fluenz Tetra) elicited the same reduction in SARS-CoV-2 RNA synthesis, albeit without the associated increase in disease severity. This suggests that the innate immune response stimulated by IAV inhibits SARS-CoV-2. Interestingly, infection with an attenuated, apathogenic influenza vaccine does not result in an aberrant immune response and enhanced disease severity. Taken together, the data suggest coinfection (‘twinfection’) is deleterious and mitigation steps should be instituted as part of the comprehensive public health and management strategy of COVID-19.

Many different platforms have been used to develop highly protective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans. However, protection has eroded over time due to the emergence of antigenically diverse viral variants, especially the Omicron subvariants. One successful platform for the generation of SARS-CoV-2 vaccines are recombinant spike protein vaccines, of which two are licensed in the United States and Europe. Typically, purified recombinant protein antigens are poorly immunogenic and adjuvants must be included in the formulation. Here, we adjuvanted recombinant ancestral SARS-CoV-2 Wuhan-Hu-1 spike proteins with an emulsion formulation combined with synthetic Toll-like receptor (TLR) 4 and 7/8 agonists. This combination led to the induction of a Th1-skewed immune response that included high titers of antibodies against Wuhan-Hu-1 spike. These serum antibodies included neutralizing and cross-reactive antibodies that recognized the spike from multiple SARS-CoV-2 variants, as well as the receptor binding domain (RBD) from SARS-CoV-1. Despite an absence of robust cross-neutralization, vaccination against Wuhan-Hu-1 spike in the context of TLR-containing emulsions provided complete cross-protection against disease from a lethal challenge with XBB.1 in a stringent K18-hACE2 mouse model. We believe that the combination of recombinant spike antigens with TLR agonist-based emulsion formulations could lead to the development of next-generation SARS-CoV-2 vaccines that provide significant protection from future emerging variants.

The Omicron (B.1.1.529) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was initially identified in November 2021 in South Africa and Botswana, as well as in a sample from a traveller from South Africa in Hong Kong 1 , 2 . Since then, Omicron has been detected globally. This variant appears to be at least as infectious as Delta (B.1.617.2), has already caused superspreader events 3 , and has outcompeted Delta within weeks in several countries and metropolitan areas. Omicron hosts an unprecedented number of mutations in its spike gene and early reports have provided evidence for extensive immune escape and reduced vaccine effectiveness 2 , 4 – 6 . Here we investigated the virus-neutralizing and spike protein-binding activity of sera from convalescent, double mRNA-vaccinated, mRNA-boosted, convalescent double-vaccinated and convalescent boosted individuals against wild-type, Beta (B.1.351) and Omicron SARS-CoV-2 isolates and spike proteins. Neutralizing activity of sera from convalescent and double-vaccinated participants was undetectable or very low against Omicron compared with the wild-type virus, whereas neutralizing activity of sera from individuals who had been exposed to spike three or four times through infection and vaccination was maintained, although at significantly reduced levels. Binding to the receptor-binding and N-terminal domains of the Omicron spike protein was reduced compared with binding to the wild type in convalescent unvaccinated individuals, but was mostly retained in vaccinated individuals. Sera from unvaccinated, vaccinated, and previously infected and vaccinated individuals show reduced neutralizing and spike protein-binding activity towards the Omicron (B.1.1.529) variant of SARS-CoV-2 compared to other variants.