Reverse Genetics and Rhinovirus—A New Approach to an Old Problem?

(See the major Article by Gern et al on pages 187–94) Human rhinovirus (HRV) infections not only are the predominant cause of the common cold but are major triggers for acute exacerbations of a number of lower-airway diseases, including asthma, chronic obstructive pulmonary disease, and cystic fibrosis [1]. HRV is a member of the Picornaviridae family of viruses, with an approximately 7.2-kb single-strand, positive-sense RNA genome enclosed in a protein capsid roughly 27 nm in diameter. Over 160 strains of HRV have been identified and are classified into 3 genetic clades based on sequence homology. Seventy-four strains are classified as HRV-A, while HRV clade B comprises 25 members. The remaining strains belong to the recently discovered HRV clade C [2]. The human airway epithelial cell is the primary site of HRV infection, and cell culture studies have provided several insights into epithelial cell responses to infection. It is essential, however, to extend studies performed in cell culture, to ensure that the key pathways identified from such studies are also activated in vivo. Because there is no animal model that fully recapitulates the sustained viral replication and symptomatic and inflammatory responses observed in humans, experimental HRV infections in human volunteers have proven invaluable in examining both the transmission and pathogenesis of HRV infections, as well as the immune response to such infections [3–6]. Historically, HRV preparations used for experimental infection have been derived from nasal secretions from an otherwise healthy young volunteer, using a laborious and time-consuming protocol. Because of concerns that repeated passaging through diploid tissue culture cell lines may yield virus stocks with altered functional characteristics that are less effective at causing infections in humans, prior stocks are typically used to infect a small number of healthy young volunteers who do not have any neutralizing antibodies to the strain of HRV used for infection. These volunteers also must be screened, to ensure the absence of other major pathogens, including Mycobacterium tuberculosis, herpes simplex virus, human immunodeficiency virus, human T-cell leukemia virus types 1 and 2, and hepatitis A, B, and C viruses. Infected volunteers are monitored for about 1 week after infection, with daily recording of symptom scores and measurement of viral titers in nasal lavage samples. A nasal lavage specimen obtained during the day of peak symptoms and viral titers from a subject with a pronounced cold is then chosen to prepare a new inoculum. This nasal lavage specimen is passaged twice, initially in roller tubes and then in roller bottles, to generate a large volume of an inoculum that is frozen in aliquots. The volunteer selected must return up to approximately 1 year after the initial infection to again be screened, to ensure that there has been no subsequent emergence of any of the pathogens initially determined to be absent [7]. The final inoculum is then also screened, to ensure the absence of a long list of potentially contaminating pathogens. Only then can permission be requested for use in humans. Although this general approach is still used, the introduction of standards requiring the use of good manufacturing procedures in the production of viral inocula has added an additional layer of complexity. Because of the complex, time-consuming, and expensive nature of this process, only a small number of HRV strains have been produced for experimental infection studies. In the current issue of The Journal of Infectious Diseases, Gern et al describe a new approach using reverse genetics (RG) that represents a significant advance for the generation of HRV strains for experimental infection [8]. In this approach, viral RNA was isolated from a previous human inoculum of HRV-A16, reverse transcribed into complementary DNA (cDNA), and then amplified in fragments by polymerase chain reaction using multiple pairs of primers. After the sequence of each fragment is confirmed, fragments are assembled in a stepwise fashion to create a full-length cDNA encoding the full viral genome. This cDNA was cloned into a plasmid (pR16.939) and amplified, and the full-length sequence was confirmed. To produce a viral inoculum, the plasmid was linearized, and the HRV-A16 full-length RNA genome was generated by in vitro transcription. Transcripts were then transfected into a “clean” pretested cell line, where viral replication occurred. The virus was then purified from cell lysates and stored in aliquots. It was then subjected to characterization and safety testing according to regulatory guidelines, to ensure the lack of any other adventitious agents or contaminants. A potential concern with this new approach to generating an HRV inoculum was whether the inoculum produced via RG would produce symptomatic colds and show comparable effectiveness to that of a conventional inoculum when used in human volunteers. Gern et al directly addressed this issue by performing a dose-ranging study in which subjects were exposed either to placebo or to varying doses (100, 500, or 1000 median tissue culture infective doses [TCID50]) of the RG inoculum. For each group, 5 subjects were initially infected, and the dose for the next group of 5 subjects was based on clinical symptoms observed in the previous 5 subjects. The data clearly show that the RG inoculum induced symptomatic colds that were associated with viral shedding and the generation of neutralizing antibodies. The percentage of subjects with symptoms that were defined as moderate in severity was dependent on infectious dose, with 87.5% of subjects having colds of moderate severity when infected with 1000 TCID50. Other dose-dependent outcomes were also observed, including level of viral RNA shedding, peak symptom scores, and numbers of leukocytes in nasal lavage specimens. When compared to responses from a historical study that used a conventional inoculum, a dose of 1000 TCID50 was needed to cause moderately severe colds in at least 75% of subjects in both studies, and mean peak symptom scores were similar in each study. Thus, an HRV inoculum produced using RG functions comparably to a conventional inoculum of the same HRV strain. A limitation acknowledged by the authors is that these initial studies were conducted only in healthy volunteers. Additional studies will be needed to confirm that the inoculum produced by RG also behaves similarly to a conventional inoculum in individuals who have chronic lower airway diseases, such as asthma. A number of previously published studies are available to use for such a comparison [9, 10]. Establishing the ability of RG to produce effective HRV inocula for experimental human infection studies represents a significant advance in the field. Although all HRV inocula still must be screened for adventitious agents according to regulatory guidelines, a major advantage of the RG approach is that the risk of potential contaminating pathogens from the initial seed stock used to create conventional inocula is removed. This is particularly important because the relatively recent emergence of several “new” respiratory pathogens always raises concern that a seed stock may contain an as yet unrecognized virus. Most importantly, removing the need to create new inocula by infecting healthy volunteers also removes the potential for unknown pathogens in these subjects, as well as the requirement for a 1-year follow-up period, greatly reducing the time needed to generate a new HRV inoculum. Another advantage of using RG is the sequence stability of a cDNA clone. Because the clone is amplified using Escherichia coli DNA polymerase, which has very high fidelity, sequence mutations are unlikely to occur, providing a stable source of the viral genomic sequence. By contrast, natural HRV genomes are subject to higher mutation rates, as the viral RNA polymerase does not have proofreading capacity to permit error corrections. Finally, having successfully negotiated a regulatory pathway to approval for HRV preparations prepared by RG, the authors have established a template that can be used to produce other strains of HRV for infection studies. Since several clinical strains of HRV have already been cloned, with more likely to be cloned in the future, this will help increase the number of highly stable HRV strains available for experimental infection studies, permitting studies of differential virulence among strains. Financialsupport. This work was supported by the Government of Canada (Tier 1 Canada Research Chair in Inflammatory Airway Diseases), the Canadian Institutes of Health Research (grant PJT-159635), and the National Sciences and Engineering Research Council of Canada (grant RGPIN-2018-03861). Potential conflicts of interest. Author certifies no potential conflicts of interest. The author has submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

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