Accelerated evolution of SARS-CoV-2 in free-ranging white-tailed deer

The zoonotic origin of the COVID-19 pandemic virus highlights the need to fill the vast gaps in our knowledge of SARS-CoV-2 ecology and evolution in non-human hosts. Here, we detected that SARS-CoV-2 was introduced from humans into white-tailed deer more than 30 times in Ohio, USA during November 2021-March 2022. Subsequently, deer-to-deer transmission persisted for 2–8 months, disseminating across hundreds of kilometers. Newly developed Bayesian phylogenetic methods quantified how SARS-CoV-2 evolution is not only three-times faster in white-tailed deer compared to the rate observed in humans but also driven by different mutational biases and selection pressures. The long-term effect of this accelerated evolutionary rate remains to be seen as no critical phenotypic changes were observed in our animal models using white-tailed deer origin viruses. Still, SARS-CoV-2 has transmitted in white-tailed deer populations for a relatively short duration, and the risk of future changes may have serious consequences for humans and livestock

Immunogenicity and inflammatory properties of respiratory syncytial virus attachment G protein in cotton rats.

Human respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants and young children worldwide. The attachment (G) protein of RSV is synthesized by infected cells in both a membrane bound (mG) and secreted form (sG) and uses a CX3C motif for binding to its cellular receptor. Cell culture and mouse studies suggest that the G protein mimics the cytokine CX3CL1 by binding to CX3CR1 on immune cells, which is thought to cause increased pulmonary inflammation in vivo. However, because these studies have used RSV lacking its G protein gene or blockade of the G protein with a G protein specific monoclonal antibody, the observed reduction in inflammation may be due to reduced virus replication and spread, and not to a direct role for G protein as a viral chemokine. In order to more directly determine the influence of the soluble and the membrane-bound forms of G protein on the immune system independent of its attachment function for the virion, we expressed the G protein in cotton rat lungs using adeno-associated virus (AAV), a vector system which does not itself induce inflammation. We found no increase in pulmonary inflammation as determined by histology and bronchoalveolar lavage after inoculation of AAVs expressing the membrane bound G protein, the secreted G protein or the complete G protein gene which expresses both forms. The long-term low-level expression of AAV-G did, however, result in the induction of non-neutralizing antibodies, CD8 T cells and partial protection from challenge with RSV. Complete protection was accomplished through co-immunization with AAV-G and an AAV expressing cotton rat interferon α

Analysis of cotton rat CD150 expression and tissue distribution.

<p>The presence of cotton rat CD150 was determined on spleen cells and lymph node cells stimulated with Concanavalin A for 24 hours (B and D), and ex vivo leukocytes from spleen (A), thymus (C), Peyer’s patches (F) and lymph node (E) (anti-CD150 black line; grey area isotype control).</p

MV infection of CD150 expressing 293T cells.

<p>(A) HEK 293T and 293T-CrCD150 cells were infected with 50 pfu/well of wild type MV (Bilthoven strain) and plaques were counted 48 hours post-infection. The difference in the number of virus induced plaques was statistically significant between both cell types (p<0.001, ANOVA). (B) After infection of 293T-HuCD150 and 293T-CrCD150 with MV (Bilthoven strain) viral titers were measured at different time intervals. Infection with wild type MV (Bilthoven strain) caused the formation of numerous small viral plaques on 293T-CrCD150 cells (C) and large plaques on 293T-HuCD150 cells (D) (40x magnification).</p

Predicted amino acid sequence alignment of cotton rat, human and mouse CD150.

<p>Spaces (indicated by dashes) denote gaps generated during alignment for optimal sequence comparison. Dark shading represents fully conserved residues, while light shading indicates conserved changes. The predicted signal peptide and transmembrane domain of human CD150 are underlined. Potential N-linked glycosylation sites are circled, four cystine residues predicted to form disulfide bonds are denoted by asterisks and three tyrosine-based switch motifs are boxed.</p

Infectivity of VSV pseudotype viruses on 293T cells expressing cotton rat and human CD150.

<p>(A) HEK 293T cells were stably transfected with plasmids expressing cotton rat (293T-CrCD150) or human CD150 (293T-HuCD150). Expression of CD150 was verified with antibodies against the cotton rat or human CD150 molecule, respectively. (B) 293T, 293T-CrCD150 and 293T-HuCD150 cells were infected with VSVΔG*-Ed-H/F. The infectivity titer was calculated by counting the number of GFP expressing cells. There was a no statistically significant difference in titer between HEK 293T, 293T-CrCD150 and 293T-HuCD150 cells. (C) 293T, 293T-CrCD150 and 293T-HuCD150 cells were infected with VSVΔG*-WTF-H/F. The infectivity titer was calculated by counting the number of GFP expressing cells. There was a significant increase in titer between HEK 293T, 293T-CrCD150 and 293T-HuCD150 cells (p<0.001, ANOVA).</p

Features of Circulating Parainfluenza Virus Required for Growth in Human Airway

Respiratory paramyxoviruses, including the highly prevalent human parainfluenza viruses, cause the majority of childhood croup, bronchiolitis, and pneumonia, yet there are currently no vaccines or effective treatments. Paramyxovirus research has relied on the study of laboratory-adapted strains of virus in immortalized cultured cell lines. We show that findings made in such systems about the receptor interaction and viral fusion requirements for entry and fitness—mediated by the receptor binding protein and the fusion protein—can be drastically different from the requirements for infection in vivo. Here we carried out whole-genome sequencing and genomic analysis of circulating human parainfluenza virus field strains to define functional and structural properties of proteins of circulating strains and to identify the genetic basis for properties that confer fitness in the field. The analysis of clinical strains suggests that the receptor binding-fusion molecule pairs of circulating viruses maintain a balance of properties that result in an inverse correlation between fusion in cultured cells and growth in vivo. Future analysis of entry mechanisms and inhibitory strategies for paramyxoviruses will benefit from considering the properties of viruses that are fit to infect humans, since a focus on viruses that have adapted to laboratory work provides a distinctly different picture of the requirements for the entry step of infection

Features of Circulating Parainfluenza Virus Required for Growth in Human Airway

Respiratory paramyxoviruses, including the highly prevalent human parainfluenza viruses, cause the majority of childhood croup, bronchiolitis, and pneumonia, yet there are currently no vaccines or effective treatments. Paramyxovirus research has relied on the study of laboratory-adapted strains of virus in immortalized cultured cell lines. We show that findings made in such systems about the receptor interaction and viral fusion requirements for entry and fitness—mediated by the receptor binding protein and the fusion protein—can be drastically different from the requirements for infection in vivo. Here we carried out whole-genome sequencing and genomic analysis of circulating human parainfluenza virus field strains to define functional and structural properties of proteins of circulating strains and to identify the genetic basis for properties that confer fitness in the field. The analysis of clinical strains suggests that the receptor binding-fusion molecule pairs of circulating viruses maintain a balance of properties that result in an inverse correlation between fusion in cultured cells and growth in vivo. Future analysis of entry mechanisms and inhibitory strategies for paramyxoviruses will benefit from considering the properties of viruses that are fit to infect humans, since a focus on viruses that have adapted to laboratory work provides a distinctly different picture of the requirements for the entry step of infection

Broad spectrum antiviral activity for paramyxoviruses is modulated by biophysical properties of fusion inhibitory peptides

International audienceHuman paramyxoviruses include global causes of lower respiratory disease like the parainfluenza viruses, as well as agents of lethal encephalitis like Nipah virus. Infection is initiated by viral glycoprotein-mediated fusion between viral and host cell membranes. Paramyxovirus viral fusion proteins (F) insert into the target cell membrane, and form a transient intermediate that pulls the viral and cell membranes together as two heptad-repeat regions refold to form a six-helix bundle structure that can be specifically targeted by fusion-inhibitory peptides. Antiviral potency can be improved by sequence modification and lipid conjugation, and by adding linkers between the protein and lipid components. We exploit the uniquely broad spectrum antiviral activity of a parainfluenza F-derived peptide sequence that inhibits both parainfluenza and Nipah viruses, to investigate the influence of peptide orientation and intervening linker length on the peptides' interaction with transitional states of F, solubility, membrane insertion kinetics, and protease sensitivity. We assessed the impact of these features on biodistribution and antiviral efficacy in vitro and in vivo. The engineering approach based on biophysical parameters resulted in a peptide that is a highly effective inhibitor of both paramyxoviruses and a set of criteria to be used for engineering broad spectrum antivirals for emerging paramyxoviruses