Genetic approaches to the study of coronavirus replication and pathogenesis

The recently developed coronavirus reverse genetic systems have been a tremendous asset for improving our understanding of the viruses' complex replication strategy, pathogenesis, mechanisms of host-range expansion, and in the development of anti-viral therapies. We completed two studies using coronavirus infectious clones. The first evaluated a severe acute respiratory syndrome coronavirus (SARS-CoV) vaccine to protect against an antigenically divergent strain. The second study determined the requirement for proteolytic processing of a highly conserved region of the replicase polyprotein for efficient replication. Ideally, a SARS-CoV vaccine should confer long-term protection, especially in vulnerable senescent populations, against both the 2003 epidemic strains and zoonotic strains that may yet emerge from animal reservoirs. Using Venezuelan equine encephalitis virus replicon particles (VRP) expressing the 2003 epidemic Urbani SARS-CoV strain spike (S) glycoprotein (VRP-S) or the nucleocapsid (N) protein from the same strain (VRP-N) as candidate vaccines, we tested their ability to protect young and senescent mice when challenged with homologous and heterologous SARS-CoV strains. The novel heterologous SARS-CoV strain (icGDO3-S) was constructed using synthetic biology and reverse genetics to generate a chimeric virus encoding a synthetic S glycoprotein gene of the most genetically divergent human strain, GDO3, which clusters among the zoonotic SARS-CoV, and represents a strain of SARS-CoV that emerged into the human population independently of the epidemic strain. VRP-S, but not VRP-N, provided protection for both young and senescent mice when challenged with the epidemic strain. When challenged with icGDO3-S, VRP-S protected young mice but only partially protected senescent animals. VRP-N vaccinated mice demonstrated enhanced pulmonary inflammation, which included eosinophils among the cellular infiltrates, following SARS-CoV or icGDO3-S challenge. The highly conserved region at the carboxy-terminus of the coronavirus replicase ORF1a polyprotein is processed by the main proteinase (Mpro) into mature products including nsp7, nsp8, nsp9 and nsp10, proteins with predicted or identified activities involved with RNA synthesis. Mpro continuous translation and processing of ORF1ab polyproteins is required for replication, but specific cleavage events may be dispensable. We determined the requirement for the nsp7-10 proteins and their proteolytic processing during the replication of murine hepatitis virus (MHV), which is phylogenetically grouped with the human coronaviruses OC43 and SARS-CoV. Using the MHV reverse genetics system, in frame deletions of the coding sequences for nsp7, 8, 9, and 10 were either deleted, or the flanking cleavage sites ablated, and the effect upon replication determined. Viable viruses were characterized through analysis of Mpro processing, subgenomic RNA transcription, and in vitro growth fitness. Deletion of any of the four regions encoding nsp7 through 10 was lethal. Disruption of the cleavage sites flanking the protein domains were lethal with the exception of the nsp9/10 cleavage site, which resulted in a mutant virus with severely attenuated replication. In order to determine if a distinct function could be attributed to preprocessed forms of the replicase polyprotein including nsp7-10, the genes encoding nsp7 and nsp8 were rearranged. The mutant virus MHV8/7 was not viable, suggesting that the noncleaved intermediate protein may be essential for replication or proteolytic processing

Vaccines to prevent severe acute respiratory syndrome coronavirus-induced disease

An important effort has been performed after the emergence of severe acute respiratory syndrome (SARS) epidemic in 2003 to diagnose and prevent virus spreading. Several types of vaccines have been developed including inactivated viruses, subunit vaccines, virus-like particles (VLPs), DNA vaccines, heterologous expression systems, and vaccines derived from SARS-CoV genome by reverse genetics. This review describes several aspects essential to develop SARS-CoV vaccines, such as the correlates of protection, virus serotypes, vaccination side effects, and bio-safeguards that can be engineered into recombinant vaccine approaches based on the SARS-CoV genome. The production of effective and safe vaccines to prevent SARS has led to the development of promising vaccine candidates, in contrast to the design of vaccines for other coronaviruses, that in general has been less successful. After preclinical trials in animal models, efficacy and safety evaluation of the most promising vaccine candidates described has to be performed in humans

Evaluation of a recombination-resistant coronavirus as a broadly applicable, rapidly implementable vaccine platform

Emerging and re-emerging zoonotic viral diseases are major threats to global health, economic stability, and national security. Vaccines are key for reducing coronaviral disease burden; however, the utility of live-attenuated vaccines is limited by risks of reversion or repair. Because of their history of emergence events due to their prevalence in zoonotic pools, designing live-attenuated coronavirus vaccines that can be rapidly and broadly implemented is essential for outbreak preparedness. Here, we show that coronaviruses with completely rewired transcription regulatory networks (TRNs) are effective vaccines against SARS-CoV. The TRN-rewired viruses are attenuated and protect against lethal SARS-CoV challenge. While a 3-nt rewired TRN reverts via second-site mutation upon serial passage, a 7-nt rewired TRN is more stable, suggesting that a more extensively rewired TRN might be essential for avoiding growth selection. In summary, rewiring the TRN is a feasible strategy for limiting reversion in an effective live-attenuated coronavirus vaccine candidate that is potentially portable across the Nidovirales order

Modelling height in adolescence: a comparison of methods for estimating the age at peak height velocity

Background: Controlling for maturational status and timing is crucial in lifecourse epidemiology. One popular non-invasive measure of maturity is the age at peak height velocity (PHV). There are several ways to estimate age at PHV, but it is unclear which of these to use in practice. Aim: To find the optimal approach for estimating age at PHV. Subjects and methods: Methods included the Preece & Baines non-linear growth model, multi-level models with fractional polynomials, SuperImposition by Translation And Rotation (SITAR) and functional data analysis. These were compared through a simulation study and using data from a large cohort of adolescent boys from the Christ’s Hospital School. Results: The SITAR model gave close to unbiased estimates of age at PHV, but convergence issues arose when measurement error was large. Preece & Baines achieved close to unbiased estimates, but shares similarity with the data generation model for our simulation study and was also computationally inefficient, taking 24 hours to fit the data from Christ’s Hospital School. Functional data analysis consistently converged, but had higher mean bias than SITAR. Almost all methods demonstrated strong correlations (r > 0.9) between true and estimated age at PHV. Conclusions: Both SITAR or the PBGM are useful models for adolescent growth and provide unbiased estimates of age at peak height velocity. Care should be taken as substantial bias and variance can occur with large measurement error

A Mouse-Adapted SARS-Coronavirus Causes Disease and Mortality in BALB/c Mice

No single animal model for severe acute respiratory syndrome (SARS) reproduces all aspects of the human disease. Young inbred mice support SARS-coronavirus (SARS-CoV) replication in the respiratory tract and are available in sufficient numbers for statistical evaluation. They are relatively inexpensive and easily accessible, but their use in SARS research is limited because they do not develop illness following infection. Older (12- to 14-mo-old) BALB/c mice develop clinical illness and pneumonitis, but they can be hard to procure, and immune senescence complicates pathogenesis studies. We adapted the SARS-CoV (Urbani strain) by serial passage in the respiratory tract of young BALB/c mice. Fifteen passages resulted in a virus (MA15) that is lethal for mice following intranasal inoculation. Lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by lymphopenia, neutrophilia, and pathological changes in the lungs. Abundant viral antigen is extensively distributed in bronchial epithelial cells and alveolar pneumocytes, and necrotic cellular debris is present in airways and alveoli, with only mild and focal pneumonitis. These observations suggest that mice infected with MA15 die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes and ciliated epithelial cells. The MA15 virus has six coding mutations associated with adaptation and increased virulence; when introduced into a recombinant SARS-CoV, these mutations result in a highly virulent and lethal virus (rMA15), duplicating the phenotype of the biologically derived MA15 virus. Intranasal inoculation with MA15 reproduces many aspects of disease seen in severe human cases of SARS. The availability of the MA15 virus will enhance the use of the mouse model for SARS because infection with MA15 causes morbidity, mortality, and pulmonary pathology. This virus will be of value as a stringent challenge in evaluation of the efficacy of vaccines and antivirals

Vaccine Efficacy in Senescent Mice Challenged with Recombinant SARS-CoV Bearing Epidemic and Zoonotic Spike Variants

BACKGROUND: In 2003, severe acute respiratory syndrome coronavirus (SARS-CoV) was identified as the etiological agent of severe acute respiratory syndrome, a disease characterized by severe pneumonia that sometimes results in death. SARS-CoV is a zoonotic virus that crossed the species barrier, most likely originating from bats or from other species including civets, raccoon dogs, domestic cats, swine, and rodents. A SARS-CoV vaccine should confer long-term protection, especially in vulnerable senescent populations, against both the 2003 epidemic strains and zoonotic strains that may yet emerge from animal reservoirs. We report the comprehensive investigation of SARS vaccine efficacy in young and senescent mice following homologous and heterologous challenge. METHODS AND FINDINGS: Using Venezuelan equine encephalitis virus replicon particles (VRP) expressing the 2003 epidemic Urbani SARS-CoV strain spike (S) glycoprotein (VRP-S) or the nucleocapsid (N) protein from the same strain (VRP-N), we demonstrate that VRP-S, but not VRP-N vaccines provide complete short- and long-term protection against homologous strain challenge in young and senescent mice. To test VRP vaccine efficacy against a heterologous SARS-CoV, we used phylogenetic analyses, synthetic biology, and reverse genetics to construct a chimeric virus (icGDO3-S) encoding a synthetic S glycoprotein gene of the most genetically divergent human strain, GDO3, which clusters among the zoonotic SARS-CoV. icGD03-S replicated efficiently in human airway epithelial cells and in the lungs of young and senescent mice, and was highly resistant to neutralization with antisera directed against the Urbani strain. Although VRP-S vaccines provided complete short-term protection against heterologous icGD03-S challenge in young mice, only limited protection was seen in vaccinated senescent animals. VRP-N vaccines not only failed to protect from homologous or heterologous challenge, but resulted in enhanced immunopathology with eosinophilic infiltrates within the lungs of SARS-CoV–challenged mice. VRP-N–induced pathology presented at day 4, peaked around day 7, and persisted through day 14, and was likely mediated by cellular immune responses. CONCLUSIONS: This study identifies gaps and challenges in vaccine design for controlling future SARS-CoV zoonosis, especially in vulnerable elderly populations. The availability of a SARS-CoV virus bearing heterologous S glycoproteins provides a robust challenge inoculum for evaluating vaccine efficacy against zoonotic strains, the most likely source of future outbreaks

Processing of Open Reading Frame 1a Replicase Proteins nsp7 to nsp10 in Murine Hepatitis Virus Strain A59 Replication▿

Coronaviruses express open reading frame 1a (ORF1a) and ORF1b polyproteins from which 16 nonstructural proteins (nsp) are derived. The highly conserved region at the carboxy terminus of ORF1a is processed by the nsp5 proteinase (Mpro) into mature products, including nsp7, nsp8, nsp9, and nsp10, proteins with predicted or identified activities involved in RNA synthesis. Although continuous translation and proteolytic processing of ORF1ab by Mpro is required for replication, it is unknown whether specific cleavage events within the polyprotein are dispensable. We determined the requirement for the nsp7 to nsp10 proteins and their processing during murine hepatitis virus (MHV) replication. Through use of an MHV reverse genetics system, in-frame deletions of the coding sequences for nsp7 to nsp10, or ablation of their flanking Mpro cleavage sites, were made and the effects upon replication were determined. Viable viruses were characterized by analysis of Mpro processing, RNA transcription, and growth fitness. Deletion of any of the regions encoding nsp7 to nsp10 was lethal. Disruption of the cleavage sites was lethal with the exception of that of the nsp9-nsp10 site, which resulted in a mutant virus with attenuated replication. Passage of the attenuated nsp9-nsp10 cleavage mutant increased fitness to near-wild-type kinetics without reversion to a virus capable of processing nsp9-nsp10. We also confirmed the presence of a second cleavage site between nsp7 and nsp8. In order to determine whether a distinct function could be attributed to preprocessed forms of the polyprotein, including nsp7 to nsp10, the genes encoding nsp7 and nsp8 were rearranged. The mutant virus was not viable, suggesting that the uncleaved protein may be essential for replication or proteolytic processing