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.

Lack of SARS-CoV-2 Viral RNA Detection among a Convenience Sampling of Ohio Wildlife, Companion, and Agricultural Animals, 2020-2021.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in humans in late 2019 and spread rapidly, becoming a global pandemic. A zoonotic spillover event from animal to human was identified as the presumed origin. Subsequently, reports began emerging regarding spillback events resulting in SARS-CoV-2 infections in multiple animal species. These events highlighted critical links between animal and human health while also raising concerns about the development of new reservoir hosts and potential viral mutations that could alter the virulence and transmission or evade immune responses. Characterizing susceptibility, prevalence, and transmission between animal species became a priority to help protect animal and human health. In this study, we coalesced a large team of investigators and community partners to surveil for SARS-CoV-2 in domestic and free-ranging animals around Ohio between May 2020 and August 2021. We focused on species with known or predicted susceptibility to SARS-CoV-2 infection, highly congregated or medically compromised animals (e.g., shelters, barns, veterinary hospitals), and animals that had frequent contact with humans (e.g., pets, agricultural animals, zoo animals, or animals in wildlife hospitals). This included free-ranging deer (n = 76 individuals), free-ranging mink (n = 57), multiple species of bats (n = 59), and other wildlife in addition to domestic cats (n = 275) and pigs (n = 184). In total, we tested 792 individual animals (34 species) via rRT-PCR for SARS-CoV-2 RNA. SARS-CoV-2 viral RNA was not detected in any of the tested animals despite a major peak in human SARS-CoV-2 cases that occurred in Ohio subsequent to the peak of animal samplings. Importantly, we did not test for SARS-CoV-2 antibodies in this study, which limited our ability to assess exposure. While the results of this study were negative, the surveillance effort was critical and remains key to understanding, predicting, and preventing the re-emergence of SARS-CoV-2 in humans or animals.

Production of Humanized Mice through Stem Cell Transfer.

The development of "humanized" mice has become a prominent tool for translational animal studies of human diseases. Immunodeficient mice can be humanized by injections of human umbilical cord stem cells. The engraftment of these cells and their development into human lymphocytes has been made possible by the development of novel severely immunodeficient mouse strains. Proven protocols for the generation and analysis of humanized mice in the NSG mouse background are presented here. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Human umbilical stem cell engraftment of neonatal immunodeficient mice Basic Protocol 2: Human umbilical stem cell engraftment of 4-week-old immunodeficient mice Support Protocol 1: Preparation of human umbilical stem cells Support Protocol 2: Submandibular blood collection from humanized mice and analysis of peripheral blood via flow cytometry.

Quantification of virus-infected cells using RNA FISH-Flow.

We present a protocol to detect cells that have been infected by RNA viruses. The method, RNA fluorescence in situ hybridization flow cytometry (RNA FISH-Flow), uses 48 fluorescently labeled DNA probes that hybridize in tandem to viral RNA. RNA FISH-Flow probes can be synthesized to match any RNA virus genome, in either sense or anti-sense, enabling detection of genomes or replication intermediates within cells. Flow cytometry enables high-throughput analysis of infection dynamics within a population at the single cell level. For complete details on the use and execution of this protocol, please refer to Warren et al. (2022).1.

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

While SARS-CoV-2 has sporadically infected a wide range of animal species worldwide1, the virus has been repeatedly and frequently detected in white-tailed deer in North America2â€"7. The zoonotic origins of this pandemic virus highlight the need to fill the vast gaps in our knowledge of SARS-CoV-2 ecology and evolution in non-human hosts. Here, we detected 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, which disseminated across hundreds of kilometers. We discovered that alpha and delta variants evolved in white-tailed deer at three-times the rate observed in humans. Newly developed Bayesian phylogenetic methods quantified how SARS-CoV-2 evolution is not only faster in white-tailed deer but driven by different mutational biases and selection pressures. White-tailed deer are not just short-term recipients of human viral diversity but serve as reservoirs for alpha and other variants to evolve in new directions after going extinct in humans. The long-term effect of this accelerated evolutionary rate remains to be seen as no critical phenotypic changes were observed in our animal model experiments using viruses isolated from white-tailed deer. Still, SARS-CoV-2 viruses have 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.

SARS-CoV-2 infection in free-ranging white-tailed deer.

Humans have infected a wide range of animals with SARS-CoV-21-5, but the establishment of a new natural animal reservoir has not been observed. Here we document that free-ranging white-tailed deer (Odocoileus virginianus) are highly susceptible to infection with SARS-CoV-2, are exposed to multiple SARS-CoV-2 variants from humans and are capable of sustaining transmission in nature. Using real-time PCR with reverse transcription, we detected SARS-CoV-2 in more than one-third (129 out of 360, 35.8%) of nasal swabs obtained from O. virginianus in northeast Ohio in the USA during January to March 2021. Deer in six locations were infected with three SARS-CoV-2 lineages (B.1.2, B.1.582 and B.1.596). The B.1.2 viruses, dominant in humans in Ohio at the time, infected deer in four locations. We detected probable deer-to-deer transmission of B.1.2, B.1.582 and B.1.596 viruses, enabling the virus to acquire amino acid substitutions in the spike protein (including the receptor-binding domain) and ORF1 that are observed infrequently in humans. No spillback to humans was observed, but these findings demonstrate that SARS-CoV-2 viruses have been transmitted in wildlife in the USA, potentially opening new pathways for evolution. There is an urgent need to establish comprehensive 'One Health' programmes to monitor the environment, deer and other wildlife hosts globally.

SARS-CoV-2 infection in free-ranging white-tailed deer ( Odocoileus virginianus ).

Human-to-animal spillover of SARS-CoV-2 virus has occurred in a wide range of animals, but thus far, the establishment of a new natural animal reservoir has not been detected. Here, we detected SARS-CoV-2 virus using rRT-PCR in 129 out of 360 (35.8%) free-ranging white-tailed deer ( Odocoileus virginianus ) from northeast Ohio (USA) sampled between January-March 2021. Deer in 6 locations were infected with at least 3 lineages of SARS-CoV-2 (B.1.2, B.1.596, B.1.582). The B.1.2 viruses, dominant in Ohio at the time, spilled over multiple times into deer populations in different locations. Deer-to-deer transmission may have occurred in three locations. The establishment of a natural reservoir of SARS-CoV-2 in white-tailed deer could facilitate divergent evolutionary trajectories and future spillback to humans, further complicating long-term COVID-19 control strategies. ONE-SENTENCE SUMMARY: A significant proportion of SARS-CoV-2 infection in free-ranging US white-tailed deer reveals a potential new reservoir.

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.

HTLV-1 viral oncogene HBZ drives bone destruction in adult T cell leukemia.

Osteolytic bone lesions and hypercalcemia are common, serious complications in adult T cell leukemia/lymphoma (ATL), an aggressive T cell malignancy associated with human T cell leukemia virus type 1 (HTLV-1) infection. The HTLV-1 viral oncogene HBZ has been implicated in ATL tumorigenesis and bone loss. In this study, we evaluated the role of HBZ on ATL-associated bone destruction using HTLV-1 infection and disease progression mouse models. Humanized mice infected with HTLV-1 developed lymphoproliferative disease and continuous, progressive osteolytic bone lesions. HTLV-1 lacking HBZ displayed only modest delays to lymphoproliferative disease but significantly decreased disease-associated bone loss compared with HTLV-1-infected mice. Gene expression array of acute ATL patient samples demonstrated increased expression of RANKL, a critical regulator of osteoclasts. We found that HBZ regulated RANKL in a c-Fos-dependent manner. Treatment of HTLV-1-infected humanized mice with denosumab, a monoclonal antibody against human RANKL, alleviated bone loss. Using patient-derived xenografts from primary human ATL cells to induce lymphoproliferative disease, we also observed profound tumor-induced bone destruction and increased c-Fos and RANKL gene expression. Together, these data show the critical role of HBZ in driving ATL-associated bone loss through RANKL and identify denosumab as a potential treatment to prevent bone complications in ATL patients.

Production of Humanized Mice through Stem Cell Transfer.

The development of humanized mice has become a prominent tool for translational animal studies of human diseases. Here we show how immune deficient mice can be "humanized" by injections of human umbilical cord stem cells. The engraftment of these cells and development into human lymphocytes has been possible because of the development of novel severely immune deficient mouse strains. Here we present proven protocols for the generation and analysis of humanized mice on the NSG mouse background.

Role of Wild-type and Recombinant Human T-cell Leukemia Viruses in Lymphoproliferative Disease in Humanized NSG Mice.

Chronic infection with human T-cell leukemia virus type 1 (HTLV1) can lead to adult T-cell leukemia (ATL). In contrast, infection with HTLV2 does not lead to leukemia, potentially because of distinct virus-host interactions and an active immune response that controls virus replication and, therefore, leukemia development. We created a humanized mouse model by injecting human umbilical-cord stem cells into the livers of immunodeficient neonatal NSG mice, resulting in the development of human lymphocytes that cannot mount an adaptive immune response. We used these mice to compare the ability of molecular clones of HTLV1, HTLV2, and select recombinant viruses to induce leukemia-lymphoma in vivo. Infection with HTLV1 strongly stimulated the proliferation of CD4+ T cells, whereas HTLV2 preferentially stimulated the proliferation of CD8+ T cells; both HTLV1 and HTLV2 induced lymphoproliferative disease. Uninfected and HTLV-infected humanized mice both showed granulomatous inflammation as a background lesion. Similarly, recombinant viruses that expressed the HTLV1 envelope protein (Env) on an HTLV2 background (HTLV2-Env1) or Env2 on an HTLV1 background (HTLV1-Env2) induced lymphoproliferative disease. HTLV2-Env1 stimulated the proliferation of CD4+ T cells, whereas HTLV1-Env2 stimulated both CD4+ and CD8+ T-cell subsets. Our results show that T-cell transformation in vivo is guided by the Env protein of the virus. Furthermore, our humanized mouse model is useful for exploring the preferred T-cell tropisms of HTLV1 and HTLV2.

HTLV-1 viral oncogene HBZ induces osteolytic bone disease in transgenic mice.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T cell malignancy that occurs in HTLV-1 infected patients. Most ATL patients develop osteolytic lesions and hypercalcemia of malignancy, causing severe skeletal related complications and reduced overall survival. The HTLV-1 virus encodes 2 viral oncogenes, Tax and HBZ. Tax, a transcriptional activator, is critical to ATL development, and has been implicated in pathologic osteolysis. HBZ, HTLV-1 basic leucine zipper transcription factor, promotes tumor cell proliferation and disrupts Wnt pathway modulators; however, its role in ATL induced osteolytic bone loss is unknown. To determine if HBZ is sufficient for the development of bone loss, we established a transgenic Granzyme B HBZ (Gzmb-HBZ) mouse model. Lymphoproliferative disease including tumors, enlarged spleens and/or abnormal white cell counts developed in two-thirds of Gzmb-HBZ mice at 18 months. HBZ positive cells were detected in tumors, spleen and bone marrow. Importantly, pathologic bone loss and hypercalcemia were present at 18 months. Bone-acting factors were present in serum and RANKL, PTHrP and DKK1, key mediators of hypercalcemia and bone loss, were upregulated in Gzmb-HBZ T cells. These data demonstrate that Gzmb-HBZ mice model ATL bone disease and express factors that are current therapeutic targets for metastatic and bone resident tumors.

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

Human 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.

Features of Circulating Parainfluenza Virus Required for Growth in Human Airway.

UNLABELLED: 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. IMPORTANCE: Mechanistic information about viral infection-information that impacts antiviral and vaccine development-is generally derived from viral strains grown under laboratory conditions in immortalized cells. This study uses whole-genome sequencing of clinical strains of human parainfluenza virus 3-a globally important respiratory paramyxovirus-in cell systems that mimic the natural human host and in animal models. By examining the differences between clinical isolates and laboratory-adapted strains, the sequence differences are correlated to mechanistic differences in viral entry. For this ubiquitous and pathogenic respiratory virus to infect the human lung, modulation of the processes of receptor engagement and fusion activation occur in a manner quite different from that carried out by the entry glycoprotein-expressing pair of laboratory strains. These marked contrasts in the viral properties necessary for infection in cultured immortalized cells and in natural host tissues and animals will influence future basic and clinical studies.

Traumatic spinal cord injury in mice with human immune systems.

Mouse models have provided key insight into the cellular and molecular control of human immune system function. However, recent data indicate that extrapolating the functional capabilities of the murine immune system into humans can be misleading. Since immune cells significantly affect neuron survival and axon growth and also are required to defend the body against infection, it is important to determine the pathophysiological significance of spinal cord injury (SCI)-induced changes in human immune system function. Research projects using monkeys or humans would be ideal; however, logistical and ethical barriers preclude detailed mechanistic studies in either species. Humanized mice, i.e., immunocompromised mice reconstituted with human immune cells, can help overcome these barriers and can be applied in various experimental conditions that are of interest to the SCI community. Specifically, newborn NOD-SCID-IL2rg(null) (NSG) mice engrafted with human CD34(+) hematopoietic stem cells develop normally without neurological impairment. In this report, new data show that when mice with human immune systems receive a clinically-relevant spinal contusion injury, spontaneous functional recovery is indistinguishable from that achieved after SCI using conventional inbred mouse strains. Moreover, using routine immunohistochemical and flow cytometry techniques, one can easily phenotype circulating human immune cells and document the composition and distribution of these cells in the injured spinal cord. Lesion pathology in humanized mice is typical of mouse contusion injuries, producing a centralized lesion epicenter that becomes occupied by phagocytic macrophages and lymphocytes and enclosed by a dense astrocytic scar. Specific human immune cell types, including three distinct subsets of human monocytes, were readily detected in the blood, spleen and liver. Future studies that aim to understand the functional consequences of manipulating the neuro-immune axis after SCI should consider using the humanized mouse model. Humanized mice represent a powerful tool for improving the translational value of pre-clinical SCI data.

Cotton rat (Sigmodon hispidus) signaling lymphocyte activation molecule (CD150) is an entry receptor for measles virus.

Cotton rats (Sigmodon hispidus) replicate measles virus (MV) after intranasal infection in the respiratory tract and lymphoid tissue. We have cloned the cotton rat signaling lymphocytic activation molecule (CD150, SLAM) in order to investigate its role as a potential receptor for MV. Cotton rat CD150 displays 58% and 78% amino acid homology with human and mouse CD150, respectively. By staining with a newly generated cotton rat CD150 specific monoclonal antibody expression of CD150 was confirmed in cotton rat lymphoid cells and in tissues with a pattern of expression similar to mouse and humans. Previously, binding of MV hemagglutinin has been shown to be dependent on amino acids 60, 61 and 63 in the V region of CD150. The human molecule contains isoleucine, histidine and valine at these positions and binds to MV-H whereas the mouse molecule contains valine, arginine and leucine and does not function as a receptor for MV. In the cotton rat molecule, amino acids 61 and 63 are identical with the mouse molecule and amino acid 60 with the human molecule. After transfection with cotton rat CD150 HEK 293 T cells became susceptible to infection with single cycle VSV pseudotype virus expressing wild type MV glycoproteins and with a MV wildtype virus. After infection, cells expressing cotton rat CD150 replicated virus to lower levels than cells expressing the human molecule and formed smaller plaques. These data might explain why the cotton rat is a semipermissive model for measles virus infection.

Success of measles virotherapy in ATL depends on type I interferon secretion and responsiveness.

Adult T cell leukemia/lymphoma (ATL) is a highly aggressive CD4+/CD25+ T-cell malignancy caused by human T cell lymphotropic virus type 1 (HTLV-1). Previous studies in the MET-1 cell/NOD/SCID mouse model of ATL demonstrated that MET-1 cells are very susceptible to measles virus (MV) oncolytic therapy. To further evaluate the potential of MV therapy in ATL, the susceptibility of several HTLV-1 transformed CD4+ T cell lines (MT-1, MT-2, MT-4 and C8166-45) as well as HTLV-1 negative CD4+ T cell lines (Jurkat and CCRF-CEM) to infection with MV was tested in vitro. All cell lines were permissive to MV infection and subsequent cell death, except MT-1 and CCRF-CEM cells which were susceptible and permissive to MV infection, but resistant to cell death. The resistance to MV-mediated cell death was associated with IFNß produced by MT-1 and CCRF-CEM cells. Inhibition of IFNß rendered MT-1 and CCRF-CEM cells susceptible to MV-mediated cell death. Cells susceptible to MV-induced cell death did not produce nor were responsive to IFNß. Upon infection with Newcastle Disease Virus (NDV), MT-1 and CCRF-CEM but not the susceptible cell lines up-regulated pSTAT-2. In vivo, treatment of tumors induced by MT-1 cell lines which produce IFNß demonstrated only small increases in mean survival time, while only two treatments prolonged mean survival time in mice with MET-1 tumors deficient in type I interferon production. These results indicate that type I interferon production is closely linked with the inability of tumor cells to respond to type I interferon. Screening of tumor cells for type I interferon could be a useful strategy to select candidate patients for MV virotherapy.

The cotton rat (Sigmodon hispidus) as an animal model for respiratory tract infections with human pathogens.

Respiratory viral infection is a great human health concern, resulting in disease, death and economic losses. Cotton rats (Sigmodon hispidus) have been particularly useful in the study of the pathogenesis of human respiratory virus infections, including the development and testing of antiviral compounds and vaccines. In this article, the authors outline the advantages of the cotton rat compared with the mouse as a model for infection with measles virus, respiratory syncytial virus, influenza virus, human parainfluenza virus and human metapneumovirus. From the literature and their own experience, the authors summarize guidelines for handling, maintaining and breeding cotton rats. In addition, they offer technical tips for carrying out infection experiments and provide information about the large array of immunological assays and reagents available for the study of immune responses (macrophages, dendritic cells, T cells, B cells, antibodies, chemokines and cytokines) in cotton rats.

Insights into the regulatory mechanism controlling the inhibition of vaccine-induced seroconversion by maternal antibodies.

The inhibition of vaccination by maternal antibodies is a widely observed phenomenon in human and veterinary medicine. Maternal antibodies are known to suppress the B-cell response. This is similar to antibody feedback mechanism studies where passively transferred antibody inhibits the B-cell response against particulate antigens because of epitope masking. In the absence of experimental data addressing the mechanism underlying inhibition by maternal antibodies, it has been suggested that epitope masking explains the inhibition by maternal antibodies, too. Here we report that in the cotton rat model of measles virus (MV) vaccination passively transferred MV-specific immunoglobulin G inhibit B-cell responses through cross-linking of the B-cell receptor with FcγRIIB. The extent of inhibition increases with the number of antibodies engaging FcγRIIB and depends on the Fc region of antibody and its isotype. This inhibition can be partially overcome by injection of MV-specific monoclonal IgM antibody. IgM stimulates the B-cell directly through cross-linking the B-cell receptor via complement protein 3d and antigen to the complement receptor 2 signaling complex. These data demonstrate that maternal antibodies inhibit B-cell responses by interaction with the inhibitory/regulatory FcγRIIB receptor and not through epitope masking.