SARS-CoV replication and pathogenesis in an in vitro model of the human conducting airway epithelium

SARS coronavirus (SARS-CoV) emerged in 2002 as an important cause of severe lower respiratory tract infection in humans and in vitro models of the lung are needed to elucidate cellular targets and the consequences of viral infection. The severe and sudden onset of symptoms, resulting in an atypical pneumonia with dry cough and persistent high fever in cases of severe acute respiratory virus brought to light the importance of coronaviruses as potentially lethal human pathogens and the identification of several zoonotic reservoirs has made the reemergence of new strains and future epidemics all the more possible. In this chapter, we describe the pathology of SARS-CoV infection in humans and explore the use of two models of the human conducting airway to develop a better understanding of the replication and pathogenesis of SARS-CoV in relevant in vitro systems. The first culture model is a human bronchial epithelial cell line Calu3 that can be inoculated by viruses either as a non-polarized monolayer of cells or polarized cells with tight junctions and microvilli. The second model system, derived from primary cells isolated from human airway epithelium and grown on Transwells, form a pseudostratified mucociliary epithelium that recapitulates the morphological and physiological features of the human conducting airway in vivo. Experimental results using these lung epithelial cell models demonstrate that in contrast to the pathology reported in late stage cases SARS-CoV replicates to high titers in epithelial cells of the conducting airway. The SARS-CoV receptor, human angiotensin 1 converting enzyme 2 (hACE2), was detected exclusively on the apical surface of cells in polarized Calu3 cells and human airway epithelial cultures (HAE), indicating that hACE2 was accessible by SARS-CoV after airway lumenal delivery. Furthermore, in HAE, hACE2 was exclusively localized to ciliated airway epithelial cells. In support of the hACE2 localization data, the most productive route of inoculation and progeny virion egress in both polarized Calu3 and ciliated cells of HAE was the apical surface suggesting mechanisms to release large quantities of virus into the lumen of the human lung. Preincubation of the apical surface of cultures with antisera directed against hACE2 reduced viral titers by 2 logs while antisera against DC-SIGN/DC-SIGNR did not reduce viral replication levels suggesting that hACE2 is the primary receptor for entry of SARS-CoV into the ciliated cells of HAE cultures. To assess infectivity in ciliated airway cultures derived from susceptible animal species we generated a recombinant SARS-CoV by deletion of open reading frame 7a/7b (ORF 7a/b) and insertion of the green fluorescent protein (GFP) resulting in SARS-CoV GFP. SARS-CoV GFP replicated to similar titers as wild type viruses in Vero E6, MA104, and CaCo2 cells. In addition, SARS-CoV replication in airway epithelial cultures generated from Golden Syrian hamster tracheas reached similar titers to the human cultures by 72 hours post infection. Efficient SARS-CoV infection of ciliated cell-types in HAE provides a useful in vitro model of human lung origin to study characteristics of SARS-CoV replication and pathogenesis

Reverse Genetic Analysis of the Transcription Regulatory Sequence of the Coronavirus Transmissible Gastroenteritis Virus

Coronavirus discontinuous transcription uses a highly conserved sequence (CS) in the joining of leader and body RNAs. Using a full-length infectious construct of transmissable gastroenteritis virus, the present study demonstrates that subgenomic transcription is heavily influenced by upstream flanking sequences and supports a mechanism of transcription attenuation that is regulated in part by a larger domain composed of primarily upstream flanking sequences which select appropriately positioned CS elements for synthesis of subgenomic RNAs

Humanized mice develop coronavirus respiratory disease

Coronavirus infections historically were associated with mild upper respiratory tract diseases in infants, children, and adults. Human coronavirus (HCoV)-OC43 and HCoV-229E were associated with 15–30% of common colds in winter and occasionally linked with lower respiratory tract disease in populations with chronic underlying diseases. HCoV research was complicated by the lack of a reverse genetic system or animal model. These viruses propagated poorly, and the number of reagents was limited. However, coronaviruses are capable of rapid host switching and evolution in changing ecologies (1), suggesting that their diversity and role in human disease were underappreciated. The 21st century heralded the arrival of the more pathogenic coronaviruses, like severe acute respiratory syndrome (SARS)-CoV. Then, HCoV-NL63 was identified as an important cause of severe lower respiratory tract infections in children and adults, including a tentative linkage with Kawasaki disease (2), and HCoV-HKU1 was identified in adults with pneumonia (3–5), renewing interest in the replication mechanisms and pathogenesis of HCoV-OC43 and HCoV-229E. In this issue of PNAS, Lassnig et al. (6) describe a transgenic mouse model to study HCoV-229E replication and pathogenesis, laying the groundwork for developing transgenic mouse models for other HCoVs

A Simulation Framework to Investigate in vitro Viral Infection Dynamics

AbstractVirus infection is a complex biological phenomenon for which in vitro experiments provide a uniquely concise view where data is often obtained from a single population of cells, under controlled environmental conditions. Nonetheless, data interpretation and real understanding of viral dynamics is still hampered by the sheer complexity of the various intertwined spatio-temporal processes. In this paper we present a tool to address these issues: a cellular automata model describing critical aspects of in vitro viral infections taking into account spatial characteristics of virus spreading within a culture well. The aim of the model is to understand the key mechanisms of SARS-CoV infection dynamics during the first 24hours post infection. We interrogate the model using a Latin Hypercube sensitivity analysis to identify which mechanisms are critical to the observed infection of host cells and the release of measured virus particles

SARS coronavirus replicase proteins in pathogenesis

Much progress has been made in understanding the role of structural and accessory proteins in the pathogenesis of severe acute respiratory syndrome coronavirus (SARS-CoV) infections. The SARS epidemic also brought new attention to the proteins translated from ORF1a and ORF1b of the input genome RNA, also known as the replicase/transcriptase gene. Evidence for change within the ORF1ab coding sequence during the SARS epidemic, as well as evidence from studies with other coronaviruses, indicates that it is likely that the ORF1ab proteins play roles in virus pathogenesis distinct from or in addition to functions directly involved in viral replication. Recent reverse genetic studies have confirmed that proteins of ORF1ab may be involved in cellular signaling and modification of cellular gene expression, as well as virulence by mechanisms yet to be determined. Thus, the evolution of the ORF1ab proteins may be determined as much by issues of host range and virulence as they are by specific requirements for intracellular replication

Altering SARS Coronavirus Frameshift Efficiency Affects Genomic and Subgenomic RNA Production

In previous studies, differences in the amount of genomic and subgenomic RNA produced by coronaviruses with mutations in the programmed ribosomal frameshift signal of ORF1a/b were observed. It was not clear if these differences were due to changes in genomic sequence, the protein sequence or the frequency of frameshifting. Here, viruses with synonymous codon changes are shown to produce different ratios of genomic and subgenomic RNA. These findings demonstrate that the protein sequence is not the primary cause of altered genomic and subgenomic RNA production. The synonymous codon changes affect both the structure of the frameshift signal and frameshifting efficiency. Small differences in frameshifting efficiency result in dramatic differences in genomic RNA production and TCID50 suggesting that the frameshifting frequency must stay above a certain threshold for optimal virus production. The data suggest that either the RNA sequence or the ratio of viral proteins resulting from different levels of frameshifting affects viral replication

Assessing Coastal Plain Risk Indices for Subsurface Phosphorus Loss

Phosphorus (P) Index evaluations are critical to advancing nutrient management planning in the United States. However, most assessments until now have focused on the risks of P losses in surface runoff. In artificially drained agroecosystems of the Atlantic Coastal Plain, subsurface flow is the predominant mode of P transport, but its representation in most P Indices is often inadequate. We explored methods to evaluate the subsurface P risk routines of five P Indices from Delaware, Maryland (two), Virginia, and North Carolina using available water quality and soils datasets. Relationships between subsurface P risk scores and published dissolved P loads in leachate (Delaware, Maryland, and North Carolina) and ditch drainage (Maryland) were directionally correct and often statistically significant, yet the brevity of the observation periods (weeks to several years) and the limited number of sampling locations precluded a more robust assessment of each P Index. Given the paucity of measured P loss data, we then showed that soil water extractable P concentrations at depths corresponding with the seasonal high water table (WEPWT) could serve as a realistic proxy for subsurface P losses in ditch drainage. The associations between WEPWT and subsurface P risk ratings reasonably mirrored those obtained with sparser water quality data. As such, WEPWT is seen as a valuable metric that offers interim insight into the directionality of subsurface P risk scores when water quality data are inaccessible. In the long term, improved monitoring and modeling of subsurface P losses clearly should enhance the rigor of future P Index appraisals

Participation in an Advanced Anatomy Capstone Project Facilitates Student Involvement in the Development of an Instructional Tool for Novel Dissection

Introduction. Student-driven design of instructional tools within basic sciences curricula in general, and in anatomy in particular,has been shown to be both a positive educational experience for the student developers and a viable way to create quality materials for future courses. We present here a description of a student collaboration arising from participation in an advanced anatomy capstone research project, resulting in the creation of a new dissection protocol for the thoracolumbar junction dorsal primary rami and their branches. Materials and Methods. This project was initiated by two third-year doctor of physical therapy (DPT)students and involved participation from faculty and other DPT students of varying experience levels, in order to pilot and refine the tool over a two-year period. We describe the process by which the tool was developed, from the genesis of the original idea through the piloting stage. Results and Discussion. This collaboration resulted in a new instructional tool to be launched within our first-year DPT gross anatomy labs in 2022. Evaluation of the project through qualitative interviews demonstrated the learning impact on student participants. Conclusions. The success of this project shows the potential for students to be meaningfully involved in instructional tool design. ,e complete dissection guide, along with photos, is included and will be of particular relevance for medical and health science educators with an interest in orthopedics, neurosurgery, pain management, or physical therapy

Genetic deletion of skeletal muscle iPLA2γ results in mitochondrial dysfunction, muscle atrophy and alterations in whole-body energy metabolism

Skeletal muscle is the major site of glucose utilization in mammals integrating serum glucose clearance with mitochondrial respiration. To mechanistically elucidate the roles of iPL

Middle East Respiratory Syndrome Coronavirus NS4b Protein Inhibits Host RNase L Activation

ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) is the first highly pathogenic human coronavirus to emerge since severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002. Like many coronaviruses, MERS-CoV carries genes that encode multiple accessory proteins that are not required for replication of the genome but are likely involved in pathogenesis. Evasion of host innate immunity through interferon (IFN) antagonism is a critical component of viral pathogenesis. The IFN-inducible oligoadenylate synthetase (OAS)-RNase L pathway activates upon sensing of viral double-stranded RNA (dsRNA). Activated RNase L cleaves viral and host single-stranded RNA (ssRNA), which leads to translational arrest and subsequent cell death, preventing viral replication and spread. Here we report that MERS-CoV, a lineage C Betacoronavirus , and related bat CoV NS4b accessory proteins have phosphodiesterase (PDE) activity and antagonize OAS-RNase L by enzymatically degrading 2′,5′-oligoadenylate (2-5A), activators of RNase L. This is a novel function for NS4b, which has previously been reported to antagonize IFN signaling. NS4b proteins are distinct from lineage A Betacoronavirus PDEs and rotavirus gene-encoded PDEs, in having an amino-terminal nuclear localization signal (NLS) and are localized mostly to the nucleus. However, the expression level of cytoplasmic MERS-CoV NS4b protein is sufficient to prevent activation of RNase L. Finally, this is the first report of an RNase L antagonist expressed by a human or bat coronavirus and provides a specific mechanism by which this occurs. Our findings provide a potential mechanism for evasion of innate immunity by MERS-CoV while also identifying a potential target for therapeutic intervention. IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is the first highly pathogenic human coronavirus to emerge since severe acute respiratory syndrome coronavirus (SARS-CoV). MERS-CoV, like other coronaviruses, carries genes that encode accessory proteins that antagonize the host antiviral response, often the type I interferon response, and contribute to virulence. We found that MERS-CoV NS4b and homologs from related lineage C bat betacoronaviruses BtCoV-SC2013 (SC2013) and BtCoV-HKU5 (HKU5) are members of the 2H-phosphoesterase (2H-PE) enzyme family with phosphodiesterase (PDE) activity. Like murine coronavirus NS2, a previously characterized PDE, MERS NS4b, can antagonize activation of the OAS-RNase L pathway, an interferon-induced potent antiviral activity. Furthermore, MERS-CoV mutants with deletion of genes encoding accessory proteins NS3 to NS5 or NS4b alone or inactivation of the PDE can activate RNase L during infection of Calu-3 cells. Our report may offer a potential target for therapeutic intervention if NS4b proves to be critical to pathogenesis in in vivo models of MERS-CoV infection