Transmission of Aerosolized Seasonal H1N1 Influenza A to Ferrets

Influenza virus is a major cause of morbidity and mortality worldwide, yet little quantitative understanding of transmission is available to guide evidence-based public health practice. Recent studies of influenza non-contact transmission between ferrets and guinea pigs have provided insights into the relative transmission efficiencies of pandemic and seasonal strains, but the infecting dose and subsequent contagion has not been quantified for most strains. In order to measure the aerosol infectious dose for 50% (aID50) of seronegative ferrets, seasonal influenza virus was nebulized into an exposure chamber with controlled airflow limiting inhalation to airborne particles less than 5 µm diameter. Airborne virus was collected by liquid impinger and Teflon filters during nebulization of varying doses of aerosolized virus. Since culturable virus was accurately captured on filters only up to 20 minutes, airborne viral RNA collected during 1-hour exposures was quantified by two assays, a high-throughput RT-PCR/mass spectrometry assay detecting 6 genome segments (Ibis T5000™ Biosensor system) and a standard real time RT-qPCR assay. Using the more sensitive T5000 assay, the aID50 for A/New Caledonia/20/99 (H1N1) was approximately 4 infectious virus particles under the exposure conditions used. Although seroconversion and sustained levels of viral RNA in upper airway secretions suggested established mucosal infection, viral cultures were almost always negative. Thus after inhalation, this seasonal H1N1 virus may replicate less efficiently than H3N2 virus after mucosal deposition and exhibit less contagion after aerosol exposure

Exhaled Aerosol Transmission of Pandemic and Seasonal H1N1 Influenza Viruses in the Ferret

Person-to-person transmission of influenza viruses occurs by contact (direct and fomites) and non-contact (droplet and small particle aerosol) routes, but the quantitative dynamics and relative contributions of these routes are incompletely understood. The transmissibility of influenza strains estimated from secondary attack rates in closed human populations is confounded by large variations in population susceptibilities. An experimental method to phenotype strains for transmissibility in an animal model could provide relative efficiencies of transmission. We developed an experimental method to detect exhaled viral aerosol transmission between unanesthetized infected and susceptible ferrets, measured aerosol particle size and number, and quantified the viral genomic RNA in the exhaled aerosol. During brief 3-hour exposures to exhaled viral aerosols in airflow-controlled chambers, three strains of pandemic 2009 H1N1 strains were frequently transmitted to susceptible ferrets. In contrast one seasonal H1N1 strain was not transmitted in spite of higher levels of viral RNA in the exhaled aerosol. Among three pandemic strains, the two strains causing weight loss and illness in the intranasally infected ‘donor’ ferrets were transmitted less efficiently from the donor than the strain causing no detectable illness, suggesting that the mucosal inflammatory response may attenuate viable exhaled virus. Although exhaled viral RNA remained constant, transmission efficiency diminished from day 1 to day 5 after donor infection. Thus, aerosol transmission between ferrets may be dependent on at least four characteristics of virus-host relationships including the level of exhaled virus, infectious particle size, mucosal inflammation, and viral replication efficiency in susceptible mucosa

Evidence of Infection among Ferrets exposed to high levels of nebulized NC99.

a<p>For exposures to ferrets 2-C and 2-D, the diffusion dryer was inserted into the line exiting the Collison generator, reducing the humidity at the end of the hour nebulization.</p>b<p>Calculation of inhaled virus based on measured viral RNA in aerosol inhaled during 60 min of aerosol exposure.</p>c<p>Nasal wash was collected on day 2 post exposure (pe) in Exp 1, and daily in Exp 2; culture results are reported for the entire collection as FFU/mL; viral RNA is the peak titer on day 3 pe and is reported as GEq/collection.</p

Comparison of sensitivity of the T5000 and RT-PCR Assays.

<p>Comparison of sensitivity of the assays for samples expected to contain relatively higher concentrations (3.0 to 7.0 log₁₀ Geq) of viral RNA (throat swab and nasal wash) and relatively lower concentrations (1.0 to 4.0 log₁₀ Geq) of viral RNA. The Ibis T5000 Influenza assay was not significantly more sensitive for detecting viral RNA in the airway specimens under the conditions of this experimental infection, but was significantly more sensitive in detection viral RNA in filter and impinger aerosol collection devices (Chi-Squared test, p<0.01).</p

Comparison of impinger and PTFE filter efficiencies for culturable virus and viral RNA.

a<p>Virus NC99 was nebulized in 6 experiments with impinger alone and 9 experiments with impinger and PTFE filter collections in parallel. The mean (range) total dose nebulized was 1.8 (1.1–2.2)×10⁷ FFU, calculated by virus concentration in Collison at beginning of aerosol generation times fluid volume nebulized for each run.</p>b<p>Impinger collection measured for 5.4 (range 5.3–5.5) L/min, reported as total FFU collected during interval of nebulization. N = 5 at each time point as one outlier value was removed from each group.</p>c<p>PTFE filter collection combines two filters in parallel for total flow of 4.0 L/min, reported as total FFU and total genome equivalents (GEq) of RNA measured by T5000 assay.</p>d<p>Ratio of group means of FFU and GEq RNA collected respectively.</p

Characteristics of the aerosol exposure system.

<p><b>a.</b> Photograph of exposure apparatus during nebulization with Collison generator inside the origin chamber shows the visible cloud of airborne particles in both the left (origin) and right (recipient) chambers. Placement of the Collison generator outside of the origin chamber, connected with 20 cm tubing, resulted in no visible suspended airborne particles in the chambers, and all subsequent experiments reported here had this configuration. The Grimm particle spectrometer is placed on top of the left chamber and the sampling port is located on top of the tunnel. <b>b.</b> The number particle size distribution with GRIMM optical counter. The fitted bimodal distribution has the median diameters of 0.44 and 1.70 µm, and geometric standard deviation of 1.25 and 1.46, for the two size modes, respectively. <b>c.</b> Particle number concentrations detected by Grimm laser-based particle counter sampled during four 30–60 sec intervals of the continuous one-hour nebulization.</p

Evidence of viral replication in ferrets exposed to low doses of aerosolized NC99.

<p>a. Actual dose calculated was within 30% of target dose.</p><p>b. Calculations based on viral RNA collected on PTFE filter during 60 min of aerosol exposure.</p