Development of a Rhinovirus Inoculum Using a Reverse Genetics Approach

Abstract

Background: Experimental inoculation is an important tool for common cold and asthma research. Producing rhinovirus (RV) inocula from nasal secretions has required prolonged observation of the virus donor to exclude extraneous pathogens. We produced a RV-A16 inoculum using reverse genetics and determined the dose necessary to cause moderate colds in seronegative volunteers.

Methods: The consensus sequence of RV-A16 from a previous inoculum was cloned, and inoculum virus was produced using reverse genetics techniques. After safety testing, volunteers were inoculated with either RV-A16 (n = 26) or placebo (n = 10), Jackson cold scores were recorded, and nasal secretions were tested for shedding of RV-A16 ribonucleic acid.

Results: The reverse genetics process produced infectious virus that was neutralized by specific antisera and had a mutation rate similar to conventional virus growth techniques. The 1000 median tissue culture infectious dose (TCID50) dose produced moderate colds in most individuals with effects similar to that of a previously tested conventional RV-A16 inoculum.

Conclusions: Reverse genetics techniques produced a RV-A16 inoculum that can cause clinical colds in seronegative volunteers, and they also serve as a stable source of virus for laboratory use. The recombinant production procedures eliminate the need to derive seed virus from nasal secretions, thus precluding introduction of extraneous pathogens through this route.

Keywords: common cold; inoculation; reverse genetics; rhinovirus.

Figure 1.

Overview of conventional and reverse genetics approaches to producing a rhinovirus (RV) inoculum. RNA, ribonucleic acid.

Figure 2.

Comparisons of gene sequences of rhinovirus (RV)-A16 inocula. RG, reverse genetics; UTR, untranslated region; VP, viral protein.

Figure 3.

Selection of study subjects. TCID₅₀, median tissue culture infectious dose.

Figure 4.

Effects of inoculation with reverse genetics-rhinovirus (RG-RV)-A16. After inoculation with placebo or RG-RV-A16 (100 or 1000 median tissue culture infectious dose [TCID₅₀]), clinical symptoms (daily symptom score), viral shedding (RV ribonucleic acid [RNA]), and leukocytes in nasal secretions were measured during the acute cold and during recovery. *, P ≤ .05 compared with placebo; †, P ≤ .05 compared with 100 TCID₅₀ dose.