New research from the University of California, Davis and the Icahn School of Medicine at Mt. Sinai published on August 18, 2020 in Nature Communications reveals that influenza virus can spread through the air on dust, fibres and other microscopic particles. Researchers produced sound, robust experimental evidence which supports this conclusion. The possibility that airborne dust can carry the virus may have implications for coronavirus transmission as well.

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In the present study, the researchers provided evidence of a mode of transmission seldom considered, for influenza: airborne virus transport on microscopic particles called “aerosolised fomites.” In the guinea pig model of influenza virus transmission, they demonstrated that the airborne particulates produced by infected animals are mainly non-respiratory in origin. This was an unexpected result.

“Surprisingly, we find that an uninfected, virus-immune guinea pig whose body is contaminated with influenza virus can transmit the virus through the air to a susceptible partner in a separate cage”, they said. They showed it through well-designed experiments.

Researchers also demonstrated that inanimate objects such as paper tissue contaminated with influenza virus can generate aerosolised fomites, by manual rubbing. “Our data suggest that aerosolised fomites may contribute to influenza virus transmission in animal models of human influenza, if not among humans themselves, with important but understudied implications for public health,” they asserted.

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Review of information on influenza virus

During every season, influenza causes hundreds of thousands of deaths worldwide annually. Occasional pandemics led to millions of deaths. The precise way the disease spreads has long been controversial. However, there is broad consensus on the possible ways in which the disease spreads among humans.

“Direct or indirect contact modes require a susceptible person to self-inoculate by, for instance, touching one’s nose with a virus contaminated hand; “direct” indicates that person-to-person contact transfers the virus between infected and susceptible hosts, whereas “indirect” implies transmission via a fomite, which is an object like a doorknob or toy that has been contaminated with infectious virus," they suggested.

Airborne transmission may be either by sprays of virus-laden respiratory droplets, such as from a cough or sneeze, impacting immediately onto the respiratory mucosa of a susceptible individual, or by the eventual inhalation of droplet nuclei, microscopic aerosol particles consisting of the residual solid cores of evaporated respiratory droplets.

Until very recently researchers were thinking that spreading the disease is by expiratory droplets "It's really shocking to most virologists and epidemiologists that airborne dust, rather than expiratory droplets, can carry influenza virus capable of infecting animals," said Professor William Ristenpart of the UC Davis Department of Chemical Engineering, who lead the research.

"The implicit assumption is always that airborne transmission occurs because of respiratory droplets emitted by coughing, sneezing, or talking. Transmission via dust opens up whole new areas of investigation and has profound implications for how we interpret laboratory experiments as well as epidemiological investigations of outbreaks." the researchers clarified.

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Fomites and influenza virus

Influenza virus is thought to spread by several different routes, including in droplets exhaled from the respiratory tract or on secondary objects such as door handles or used tissues. Investigators called these secondary objects “fomites”. During the earlier period, these ideas were in the realm of educated guesswork; not many experimental studies were there to support the conclusions. Researchers did not know which routes are the most important.

In the new study the researchers examined whether the tiny, non-respiratory particles they call "aerosolised fomites" could carry influenza virus between guinea pigs.

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Experiments

In the first experiment, the researchers showed how guinea pigs aerosolise non-respiratory particles. In order to measure the airborne particulates emanating from the cages of uninfected guinea pigs, they sampled air from a HEPA-filtered guinea pig cage with an aerodynamic particle sizer (APS) that enumerates particles in the size range of 0.3–20 μm.

A camera placed above the cage simultaneously captured guinea pig movement over time. The researchers measured airborne particle production by uninfected guinea pigs, placed individually, awake and unrestrained, in the cage and found that airborne particulates were generated primarily as irregular, sharp spikes, up to 1000 particles/s−1, and observed that the particle count spikes were almost entirely coincident with guinea pig motion.

Based on an elegant experiment, the researchers could show that a sizeable portion of the particulate matter might be dust aerosolised by animal movement rather than respiratory droplets. Interestingly, the mobile guinea pigs emitted orders of magnitude more particles than stationary guinea pigs, regardless of whether their standard bedding was replaced with a polar fleece-covered absorbent pad (PF) or removed completely.

In the next experiment, they measured airborne particulates exhaled directly from the respiratory tracts of three guinea pigs. For this, they anesthetised each guinea pig, placed them in a closed aluminium sleeve, with only a small aperture for its nose. This arrangement helped to contain non-respiratory particulates like dander, fur, and dust emanating from the bodies of the animals themselves. In this instance, the dynamics of particle emission for the stationary animals were qualitatively different from that of the mobile animals; there were no spikes and the emission rates overall were considerably smaller.

Guinea pigs within the aluminium sleeve emitted 0.10–0.18 particles/s−1 prior to inoculation with influenza virus (day 0 post-inoculation), a 10-to-100-fold reduction in the average number of particles/s−1 produced by the same guinea pigs while awake and moving around in the cage.

“Intranasal inoculation with influenza A/Panama/2007/1999 (H3N2) (Pan99) virus only slightly increased particle emission by anesthetized, stationary guinea pigs, with up to 0.5 particles/s−1 measured on days 2–3 post-inoculation from two of the three animals”, the researchers noted.

In contrast to mobile guinea pigs, the size distribution of the particles emitted by stationary animals was weighted toward the smallest size range. Approximately 11% of the particles emitted from a cage containing an awake, mobile guinea pig were 0.3–0.5 μm in diameter, but in contrast ~58% of the particles emitted by the anesthetised animals were in the same size range, similar to the proportion of 0.3–0.5 μm particles in the exhaled breath of humans.

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Transmission of influenza A virus via aerosolised fomites

Since the environmental dust comprised such a large fraction of the total airborne particulates emitted by experimental guinea pigs, the researchers hypothesised that, if these airborne environmental dust particulates were to become contaminated with influenza virus, they could serve as vehicles on which influenza virus might transmit through the air. We call these virus-contaminated dust particles “aerosolised fomites, ” to differentiate them not only from virus-laden respiratory droplets that are exhaled, coughed, or sneezed into the air by an infectious person or animal, but also from the macroscopic virus-contaminated objects that are traditionally thought of as fomites.

To investigate this hypothesis, the researchers first infected guinea pigs with Pan99 influenza virus by intranasal inoculation using a standard protocol, and then assessed the degree of environmental contamination in their cages over time. They found that swab samples from their fur, ears, paws, and cages all yielded viable virus by 2 days post-inoculation (dpi), and virus remained cultivable from swabs until at least 3 dpi.

Researchers could not culture measurable virus from swabs taken either on the day of inoculation or on 1 dpi, demonstrating that virus replicating within the respiratory tract, rather than the initial inoculum, was being spread to and persisting on their bodies and environment. Grooming and nose rubbing might have contributed to the spread of the virus to the animal’s body and environment.

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Crucial experiment

The researchers determined whether airborne influenza virus transmission could occur from a virus-contaminated environment, in the absence of viral replication in the donor animal’s respiratory tract. To mimic the self-contamination that they had observed in intranasally inoculated animals, they applied Pan99 stock virus with a paintbrush to the bodies of guinea pigs that had been previously infected with Pan99 and thus were immune to re-infection.

They then paired the contaminated virus-donor animals with susceptible virus-recipient animals in cages that only permit transmission by airborne routes. They did not detect any virus in nasal washes from any of the immune donor guinea pigs; however, they observed influenza virus transmission in 3 of 12 animal pairs (25% transmission rate). Swab samples from the bodies of the immune, virus-contaminated guinea pigs and their environment confirmed the presence of viable virus at days 2 and 4 post-contamination (Thus, the researchers conclude that airborne particulate matter from a non-respiratory source is able to transmit influenza virus through the air to a susceptible host.

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Generation of aerosolised fomites from an inanimate source

Finally, they explored the generation of infectious aerosolised fomites from a virus-contaminated but inanimate dust source. They applied stock Pan99 virus in liquid solution to various commercially available paper tissues and towels and let them dry thoroughly in a bio-safety cabinet. Crumpling, folding, and rubbing the dried paper tissues by hand released up to ~900 particles/ s−1 as measured by the Aerodynamic Particle Sizer (APS). The size distribution of the tissue-generated airborne particulates was in the respirable range, with 99.8% of the particles in the range 0.3 to 10 µm, similar to those generated by guinea pigs moving in their cages.

After 8 minutes of crumpling paper tissues by hand and collecting the aerosols with a bio-aerosol sampler, plaque assay titration of the collection media from the air sampler demonstrated that these aerosolised fomites contained cultivable influenza virus, which was captured at a rate of 1–5 pfu/min−1 of air sampling (pfu means Plaque-Forming Unit. It is a measure of the quantity of viruses that are capable of lysing host cells and forming a plaque).

They conservatively estimate that only 0.03% of the mass of the contaminated paper tissue was actually aerosolised into the bio-aerosol collector, yielding a maximum estimated virus release rate of 14 pfu/min − 1 of tissue manipulation, similar to that observed experimentally.

Because the bio-aerosol sampler is designed to preserve virus infectivity rather than discriminate by size, the researchers concluded that any particles within the range of 0.3 to 10 µm may have carried viable virus; and they were unable to draw any further conclusions about particle size and viral payload. Nevertheless, their experiments do demonstrate that an influenza virus contaminated tissue, dried under typical indoor environmental conditions, retained its infectiousness and, upon handling, released viable influenza virus into the air, carried on airborne particulates in the respirable range.

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Discussion

“These results show that dried influenza virus remains viable in the environment, on materials like paper tissues and on the bodies of living animals, long enough to be aerosolised on non-respiratory dust particles that can transmit infection through the air to new mammalian hosts”, the researchers concluded.

They demonstrated that an influenza A virus, dried on paper tissues for 30 to 45 minutes at ambient room temperature and humidity, retained infectiousness on aerosolised fomites that were collected from the air with a bio-aerosol sampler and subsequently grown in cell culture.

In vivo, they showed that guinea pigs painted with influenza virus harboured viable virus on their bodies for up to 4 days post-contamination, which was subsequently transmitted through the air to infect 3 of 12 virus-susceptible partner animals housed in separate cages (25% transmission rate, 95% credible interval 8–52%).

“To our knowledge, no experimental evidence exists to establish that the airborne transmission of influenza viruses between experimental animals, or even between humans, occurs entirely via exhaled respiratory particles, as is commonly presumed. Our experimental data confirm that influenza virus transmission by aerosolised fomites is, at minimum, biologically plausible, and possibly generalisable to other respiratory viruses that transmit preferentially or opportunistically by the airborne route”, the researchers clarified.

Disclaimer- The views and opinions expressed in this article are those of the author's and do not necessarily reflect the official policy or position of M3 India.

Dr. K S Parthasarathy is a freelance science journalist and a former Secretary of the Atomic Energy Regulatory Board.