Recombinant Subgroup B Human Respiratory Syncytial Virus Expressing Enhanced Green Fluorescent Protein Efficiently Replicates in Primary Human Cells and Is Virulent in Cotton Rats

Abstract

Human respiratory syncytial virus (HRSV) is IMPORTANCEVirology as a discipline has depended on monitoring cytopathic effects following virus culture in vitro. However, wild-type viruses isolated from patients often do not cause significant changes to infected cells, necessitating blind passage. This can lead to genetic and phenotypic changes and the generation of high-titer, laboratory-adapted viruses with diminished virulence in animal models of disease. To address this, we determined the genome sequence of an unpassaged human respiratory syncytial virus from a sample obtained directly from an infected infant, assembled a molecular clone, and recovered a wild-type recombinant virus. Addition of a gene encoding enhanced green fluorescent protein allowed this wild-type virus to be tracked in primary human cells and living animals in the absence of significant cytopathic effects. Imaging of fluorescent cells proved to be a highly valuable tool for monitoring the spread of virus and may help improve assays for evaluating novel intervention strategies. H uman respiratory syncytial virus (HRSV) is the most important viral cause of respiratory tract disease in infants (1). HRSV infections are observed during seasonal outbreaks in winter or during the rainy season in the tropics (2). The virus usually causes a self-limiting upper respiratory tract (URT) infection, resulting in rhinorrhea and other common cold-like clinical signs (3). However, in a minority of cases the infection can also spread to the lower respiratory tract (LRT), resulting in severe pneumonia or bronchiolitis. Risk factors for developing severe LRT infections include prematurity, pulmonary or cardiac disease, compromised immunity, and old age (4). Current treatment options are limited, although a monoclonal antibody directed against the fusion (F) glycoprotein has been developed for prophylactic use (5). Despite significant efforts in vaccine development over the past 50 years, no HRSV vaccines are currently licensed (6). Limited availability of natural animal models of disease adds to the challenge of developing vaccines and antivirals.HRSV is a member of the family Paramyxoviridae, subfamily Pneumovirinae, genus Pneumovirus (1). It is an enveloped virus with a negative-sense, single-stranded RNA genome containing 10 transcription units. The glyco-(G) proteins facilitate virus attachment and entry (1, 7), and the F glycoprotein is an important target of virus neutralizing antibodies (8). Molecular epidemiological studies have identified two HRSV subgroups (A and B),

Functional Analysis of the 60-Nucleotide Duplication in the Respiratory Syncytial Virus Buenos Aires Strain Attachment Glycoprotein

“…Further studies specifically on the duplication in subgroup A strains are needed to confirm this but could easily be performed based on the results and reagents established here. Further, a recombinant B strain of RSV was recently described (37) and would provide an additional tool to investigate the role of the duplication in RSV spread and pathogenesis. …”

Animal models of respiratory syncytial virus infection

“…Cotton rats are susceptible to infection throughout life, but virus replication is greater and persists for longer in the nasal passages of 3 day-old rats than in older animals. Viral antigen can be detected in ciliated epithelial cells of the nose and lungs, but not trachea [68], [69], [70], and induces an increase in IFNγ, IL-10, IL-6, MCP-1 and growth-regulated oncogene (GRO) (IL-8 homologue) mRNA in the lungs [71], and histopathological changes characterised by a desquamative, exudative rhinitis, and a mild proliferative bronchiolitis (Fig. 1I) [68], [72].…”

“…HRSV antigen-positive cells are brown; (B) section of lung from a child with an acute hRSV infection showing acute bronchiolitis in a medium-sized airway with intraluminal cellular debris and inflammatory cells (A and B are adapted from [12], reprinted by permission from Macmillan Publishers Ltd: Modern Pathology, 20:108–119, Johnson et al, copyright 2007); (C) localisation of bovine (b)RSV antigen in bronchial, bronchiolar, and alveolar cells of a calf experimentally infected with bRSV, 6 days previously. BRSV antigen-positive cells are red (adapted from [114], reprinted from The American Journal of Pathology, 161:2195–2207, copyright 2002, with permission from Elsevier); (D) section of lung from a calf, inoculated intranasally and intracheally 6 days previously with bRSV, showing bronchiolitis with intraluminal cellular debris and inflammatory cells, and interstitial pneumonia; (E) section of chimpanzee lung, naturally infected with hRSV, showing alveolar and interstitial pneumonitis and bronchitis (reprinted from [25], J Com Pathol, 110:207–212, copyright 1994, with permission from Elsevier; (F) section of lung from an infant rhesus monkey infected by aerosol with hRSV, 7 days previously, showing mild lymphocytic and histiocytic inflammatory cell infiltrates in the walls and lumens of the terminal conducting airways, and in the septa and lumens of adjacent alveoli (adapted from [45], reprinted from Vaccine, 23:2928–2942, copyright 2005, with permission from Elsevier); (G) section of lung from a lamb infected by aerosol with hRSV, 8 days previously, showing viral antigen within epithelial cells lining the bronchioles (brown cells); (H) section of lung from a lamb infected by aerosol with hRSV, 8 days previously, showing bronchiolitis with degenerate/necrotic individual epithelial cells (thin arrow), occasional syncytial cells (long arrow), accumulation of degenerate neutrophils (short arrow), and occasional macrophages (G and H adapted from [59]; (I) section of lung from a cotton rat infected 5 days previously with hRSV, showing mild peribronchiolitis (adapted from [70], reprinted from Vaccine, 31:306–312, copyright 2013, with permission from Elsevier).…”

Mapping the evolutionary trajectories of morbilliviruses: what, where and whither

“…This highlights a major challenge in virus discovery, the gap between the identification of genomic fragments in clinical samples and understanding the biological properties of pathogens which could be impossible to isolate [89]. Synthetic biology offers an opportunity to bridge this gap and given the success with assembling reverse genetics systems for paramyxoviruses directly from unpassaged clinical material [90] it should be possible to develop completely synthetic reverse genetics systems as have been generated for non-segmented positive strand RNA viruses [91 ] and segmented negative strand RNA viruses [92 ]. For putative morbilliviruses such as DrMV this might be the only tractable option to obtain a cultivatable isolate.…”