Influenza viruses responsible for epidemics and pandemics have the ability to spread rapidly among susceptible individuals by contact (direct or indirect) or through the air via close-range or long-range transmission (1
). The relative predominances of these modes of transmission have been debated for some time. A key determinant of whether a particular virus can successfully transmit through the air from one host to cause infection in another is whether the virus maintains viability as it passes through the air. Here, we selected a panel of influenza viruses that exhibit a range of transmissibility phenotypes and, using the ferret model, evaluated morbidity and the aerosol shedding profiles during normal breathing and sneezing of animals infected IN or by AR. We found that overall, ferrets infected by transmissible human influenza viruses exhale more aerosolized respiratory secretions and higher levels of infectious virus in aerosols than animals with avian influenza virus infections. Although there are additional determinants of influenza virus transmission, these results show that highly transmissible influenza viruses maintain viability in exhaled aerosols of the size capable of prolonged suspension in the air.
Comparison of morbidities observed in animals inoculated IN or by AR inhalation revealed no significant differences in weight loss in ferrets regardless of the method of inoculation; a similar finding was previously reported for PN99 virus (12
). MV of respiration values in AR-inoculated animals were higher (with the exception of MX09 virus) than those in i.n. inoculated animals, with a high degree of variability among animals. Increases in MV of respiration due to lower airway obstruction after i.n. inoculation of influenza virus have also been reported in cotton rats, but only in young animals, which exhibit greater lung elasticity than their older counterparts (22
). Others have shown increased morbidity in mice after AR inoculation of an H3N2 influenza virus than after IN delivery of virus, but the MV of respiration was not measured (23
). We also observed differences between the virus shedding kinetics in nasal washes of animals based on the method of inoculation; peak virus shedding was delayed in AR-inoculated compared to IN inoculated ferrets. Similar kinetics were previously shown in AR-inoculated ferrets and in those naturally infected by exposure to other infected ferrets (12
). We also observed a ≥1-log drop in virus nasal wash titers at 1 day after inoculation but only with the avian influenza viruses. Nasal wash sampling in these animals may represent secretions primarily from the upper respiratory tract, which has been shown to better support the replication of human influenza viruses than that of avian influenza viruses, and this may account for the drop in virus titers observed on the first day after infection (24
). Collectively, our findings highlight differences in morbidity and virus shedding kinetics in nasal washes between IN and AR-inoculated ferrets, but characterization of the aerosols exhaled from infected animals is essential to understanding the factors affecting influenza virus transmission.
A number of studies have characterized the aerosol shedding patterns of people during aerosol-generating activities such as breathing, coughing, sneezing, and talking (26
), but until recently, little was known about the effect of influenza virus infection on aerosol production in people. Lindsley et al. (7
) reported increases in the number and volume of aerosol particles exhaled by infected patients during coughing, with a great degree of variability among samples and with the majority of particles in the respirable size range. Similarly, we found that ferrets also shed more respirable aerosols after infection with human influenza viruses, and with substantial variability among samples. These findings are corroborated by previous studies showing a greater incidence of rhinorrhea among ferrets during the first few days after infection by highly transmissible influenza viruses than after infection by poorly transmissible strains and correlations with local proinflammatory cytokine responses (21
). The majority of the aerosols measured in the current study during normal breathing were in the respirable size range, but when the volumes were compared, a greater difference between the two groups of infected animals was revealed at the submicrometer particle size range (<1 μm), albeit with substantial variability. Others have measured the size distribution of aerosols in air exhausted from cages housing ferrets infected by influenza virus and have reported the greatest concentrations of particles in the submicrometer size range (13
). Submicrometer aerosols may remain suspended in still air for 12 hours or more, increasing the opportunity for aerosols of this size to be inhaled (27
The potential of respirable aerosols to carry infectious influenza virus from one host to another has remained uncertain, primarily because published reports are, for the most part, limited to the detection of influenza virus RNA and not infectious virus. Multiple laboratories have reported the detection of the majority of influenza virus RNA in exhaled aerosols from humans in the respirable size range (8
) and that infected patients exhaled up to 20 copies/min (32
). Infectious virus has been detected in aerosol particles exhaled by humans (8
), but, with limited preservation of virus viability, quantitative analyses have not been reported. Infectious virus has been detected in air samples collected from guinea pigs infected by human influenza virus, and although droplets in air samples were not segregated by size, an average of 40 PFU/liter of air sampled was measured (33
). Using ferrets infected by human influenza viruses in the current study, we measured not only viral RNA in exhaled aerosols during normal breathing (up to 26 copies/min) but also infectious virus (up to 4 PFU/min). When similar experiments were performed with poorly transmissible, avian influenza viruses, <1 PFU/min and up to 18 copies/min were detected, with the most striking differences observed in the respirable size range of aerosols. The 50% ferret infectious dose (FID50
) for aerosol delivery of human influenza virus (PN99) is 1.9 PFU, while the FID50
for avian influenza virus (TH04) is 4 PFU (12
). For comparison, the 50% human infectious dose of influenza virus delivered in 1- to 3-μm-size aerosols is reported as 0.6 to 3 50% tissue culture infectious doses (34
). Additional factors affecting transmission are certainly involved here, including differences between human and avian influenza virus receptor binding preferences and tissue tropisms (24
), but the amount of infectious virus in aerosols being exhaled by infected hosts is also an important consideration.
Because the ferret transmission model is such a valuable tool in the risk assessment of emerging influenza viruses, it is important that we have a good understanding of how transmission is occurring for each virus evaluated using this model. During a DCT experiment, transmission may occur by direct or indirect contact, large droplets, or aerosols, while during an RDT experiment, transmission may occur by large droplets or aerosols. The detection of infectious influenza virus in respirable aerosols highlights the potential for a role of respirable aerosols in transmission events occurring during RDT experiments. Others have shown that influenza virus present in particles <15 μm is responsible for transmission of virus between ferrets, although only viral RNA was detected in these studies (13
). It is also plausible that aerosols play a role in transmission experiments in which animals are housed in direct contact; the contribution of close-range aerosol transmission cannot be ruled out in these scenarios (36
). The improved techniques and information derived from our current study will enhance the utility of the ferret influenza transmission model by providing a more comprehensive understanding of the virus and aerosol shedding profiles of infected animals.
There are a number of limitations to this study that warrant mention. Many of the data reported are dependent on instrumentation that has certain functional restraints. Size distribution data included only particles of 0.5 to 20 μm in size, and infectious virus in aerosols of <0.65 μm was not collected. The rates of recovery of infectious influenza virus were 7 to 35%, depending on the virus and collection time. Similar recovery rates (15 to 34%) were reported using a different collection device (37
), but nevertheless, improvements in recovery rates would provide a more accurate account of the amounts of infectious virus present in exhaled aerosols and would allow us to better represent the kinetics of viable virus shedding in aerosols, which may not be possible with the sensitivity of the current assay. All experiments reported here were conducted under ambient laboratory conditions. The aerosol and virus shedding profiles observed under diverse environmental conditions (temperature and humidity) are not known, but such knowledge may improve our understanding of the seasonality of influenza viruses. Lastly, data extrapolated from animal model studies will never exactly represent the human situation, but the amount of information that can be obtained from animal studies, most of which could never be performed in human volunteers, makes the ferret model an invaluable tool in influenza research and in our mission to improve global public health. The findings reported here using the ferret model do not unequivocally prove that transmission occurs via respirable aerosols; however, they do confirm the need for a continued focus on the potential for short-range and long-range aerosol transmission of influenza viruses. Data obtained from further research using the ferret model as well as epidemiological reports and, when possible, human challenge studies will bring us closer to fully understanding the mechanisms of influenza virus transmission.