Peer review of: Influenza A virus is transmissible via aerosolized fomites (2020)

Peer Review File 

REVIEWER COMMENTS, first round: 
 
Reviewer #1 (Remarks to the Author): 
 
In this manuscript, Sima Asadi and colleagues investigated the presence of infectious influenza A 
virus in aerosolized fomites and the potential of such fomites to result in virus transmission 
between guinea pigs via the air. This is an important and interesting area of research, but the 
manuscript has a few problems: 
 
1. The quantitative aspects of the work are not detailed properly. 
2. Some observations in animal studies are not discussed well. 
 
With respect to the first point: 
A. For all particle measurements, an APS is used with detection range of 500-20000 nm. As a 
consequence, the lower range of particle sizes that may contain influenza virus (100-500 nm) is 
missed. This is particularly problematic given that (at least in humans) the vast majority of 
exhaled aerosols appear to be <500 nm, see eg Fabian et al., Plos ONE 2008. Thus, the 
quantitation of exhaled particles (e.g. in figure 1E) is potentially incorrect for a major fraction of 
the aerosols of importance (<500nm). The number of counted particles is mentioned in the text 
(around line 75) but not the corresponding particle sizes and the fact that small particles are 
missed. The number of exhaled small particles is not known for guinea pigs, as far as I am aware. 
With the use of the Biospot collector, ALL infectious particles are quantified (large plus small), 
making it very difficult, if not impossible, to allow quantitative conclusions. 
B. The experimental procedures need to be much better identified. For instance, the authors show 
that the cage bedding determines – in part – the generation of aerosolized fomites, but the cage 
bedding is not specified (e.g. in figure 1) and as a consequence, the experiment can not be 
interpreted. 
C. The scale bars in the different figures are not the same, making interpretation difficult, if not 
impossible. Fig 1B shows that up 1000 particles are detected per second (60000/min) but then Fig 
1C shows a scalebar no larger than 10000/min (~166/sec) for the same(?) data and Fig 1E shows 
a scalebar with 0.1-0.5 particles per second (6-30/min). In Fig S1, which supposedly matches Fig 
1B/1C, I see 0-70 particles/sec, orders of magnitude different between the two figures. The unit of 
measure should be the same throughout and an explanation should be provided for the difference 
between Fig S1 and 1. 
D. In Fig 4, 500-1000 particles per second are generated (30000-60000/min), which yield 1-5/min 
infectious units, again with different units of measure. 
E. In Fig S5, more infectious virus appears to have been loaded on the guinea pig fur than 
detected upon grooming, pushing the likelihood of virus transmission. 
F. Although infectious aerosolized fomites can be created with tissues and towels, the fraction of 
infectious particles (1-4 per minute) is miniscule compared to 30,000-60,000 particles per minute 
that are created with relatively fresh (30-45 min) large amount (3.6 x 10E5) of virus. 
On the whole, these quantitative aspects of the study require attention and a more thorough 
discussion. 
 
With respect to the second point, the authors did not discuss the fact that many influenza viruses 
are NOT transmitted in animal models (e.g. ferrets) although the viruses clearly replicate in these 
hosts. H5N1 viruses and other zoonotic viruses replicate extensively in the ferret model and would 
thus contaminate the ferret (by grooming) and the cage without evidence of transmission via 
aerosol or respiratory droplets, whereas seasonal and pandemic influenza viruses are readily 
transmitted via this route. This validation of the model (e.g. summarized in Linster et al., Cell 
2014), copying what is generally observed in real life, is not discussed in the manuscript. The 
authors challenge the validity of the ferret model (L164-167) but without providing an explanation 
for the lack of transmission of replication-competent viruses in this model. Some research groups 
even go as far as demonstrating contact transmission between ferrets or guinea pigs (through 
contaminated fomites, grooming, etc) without evidence of transmission via aerosol or respiratory 
droplets. This appears to contradict the importance of transmission via aerosolized fomites. 
 

Additional comments: 
In Fig 2A, LOD lines are misplaced (at least in my copy). 
 
In L227-233, the authors highlight the background particle counts that are well known in the field 
and describe a “wash out” that is commonly used to deal with this problem in aerosol science. Was 
this wash out only done here, and not in other experiments? 
 
 
 
Reviewer #2 (Remarks to the Author): 
 
A. Summary of the key results 
1. Virus-contaminated resuspended particles can transmit influenza in the guinea pig model. 
 
B. Originality and significance 
This work is highly original and significant because it shows that a previously underappreciated 
route of transmission—deposition of virus onto surfaces followed by resuspension and subsequent 
exposure to these—is possible. 
 
C. Data, methodology, statistics 
The methods are valid. 
 
D. Conclusions 
The conclusions are justified. The researchers showed, through multiple lines of evidence, that 
virus-containing particles are resuspended by physical motion and that exposure to them can 
initiate infection. 
 
E. Clarity and context 
The writing, figures, and overall presentation are very clear. The authors cite prior work 
appropriately to place this study in context. 
 
1. line 45: “Uncertainty surrounding the modes by which influenza virus transmits among humans 
under different conditions hinders the development of interventions, like vaccines, designed to 
prevent influenza’s spread.” I can see how uncertainty hinders the development of non-
pharmaceutical interventions, but the link to vaccine development is not obvious to this reader. 
2. Figure 4: Images of the plaque assay plates are not needed.