The amount of virus per amount of snot, air, monkey or whatever is a way to describe the concentration of virus present in that thing. It might seem intuitive to you that the more virus you get (the dose), the greater the chance you will be infected. Perhaps also, the greater the chance you will get sick, or really sick, as a result. After all, the dose makes the poison, right? This post will have a look at what some of the scientific literature has to say about infectious dose and SARS-CoV-2 (the virus that causes COVID-19) and why you should care.
The amount of virus we receive can determine the severity of the disease that results. This “infectious dose” is affected by how much virus is in the particles we inhale as well as how long we inhale them. Its impact is modified by our pre-existing immunity. The physical distance between us and the infectious source reduces the risk of us getting a dose. Masks also reduce the size of and even prevent us from getting a dose altogether. Cleaning or refreshing the air we breathe also reduces the infectious dose. We can exert some control over our risk of infection.
What is viral load?
This phrase is generally used to describe virus concentration. In other words, the amount of virus in a quantity of something. That something could be water, air, mucous or tissue. It could be per human or hamster or monkey. It could also be used to discuss the concentration of virus in a ‘something’ deposited somewhere – like on a benchtop, light switch, toilet bowl or door handle.
Viral loads have been used to express how much virus is inside your system right now – for example, how much HIV is present in a patient’s blood. It can also be used to define how much virus is in what you emit – for example, the viral load in your nasal swab.
Neither of those examples reflects the viral load in every part of your body nor in every fluid you produce. For example – a patient may have infectious Ebola virus present in their semen but no detectable viral RNA in their blood.
A viral load can be related to the presence of infectious virus through the use of a virus culture method. It may also be related to the amount of nucleic acid detected using a PCR method. For example, comparing a PCR test result on a patient sample to the PCR result from a standard included in the same test lets the lab relate the patient result back to a clinically relevant, internationally recognised standard concentration or amount of infectious virus.[1,2]
The amount of virus that is required to create a detectable infection that leads to disease is the minimum infectious dose. Nonhuman primate studies have reported between 10s of SARS-CoV-2 viral particles required for the body to recognise infection (mount an immune response) to 100s of viral particles for the body to develop a fever in response to an infection after being inhaled.
A virus gaining a successful foothold and going on to replicate and cause disease is related to the size of the dose. Dose can be affected by the length of exposure to an infected source, the proximity to that source and how much virus the source is emitting. Successful infection is also related to:
- immune response (strength, whether pre-existing)
- nature of the viral variant (for example, the D614G impact )
Vaccination can help prepare our immune response – moreso when it is a good match or the specific viral variant circulating. But the dose is also important, and it may be an element that we can exert some control over, even if we are still ultimately infected.
What is TCID50?
The 50% tissue culture infectious (TCID50) dose is a way to describe the concentration of virus present in a sample. Replicates of a number of dilutions of the unknown sample are added to cells usually cultured in microwell plate wells. The dilution at which 50% of the cells are infected is the TCID50 (there is maths involved in this bit). Infection of the cells is determined by looking down a microscope to see if they are damaged due to virus replication, or we use a virus-specific label to tell us which cells have the virus replicating in them and which don’t, or we can test which wells still have living versus dead cells (a “viability assay”). [11,12,13]
The plaque-forming assay
Another tool is the plaque-forming assay – it relies on counting the zones (plaques) of virus-induced damage after a diluted preparation of virus is added onto a cell monolayer for a time, and then virus diffusion is limited by the addition of a gel (agarose or carboxymethyl cellulose). Cells are incubated, and plaques are then counted. Each plaque is presumed to be initiated by a single virion infecting a cell and spreading outwards from it.
The focus-forming assay
Not all viruses form plaques or kill their cells. To measure the number of such viruses, an antibody specific to a viral protein is used. It’s tagged with something that can be used to identify the labelled virus inside a cluster, or “focus”, of infected cells. The same process of diluting the virus, incubating with cells then restricting virus movement with a gel is used.
What does the literature tell us about infectious doses?
A few reviews have pulled together others’ data, and they generally point to hundreds of viable SARS-CoV-2 virus particles (virions) being enough to start an infection in an animal/human.[9,28] Some reviews also point to the importance of viral dose to disease.
Multiple monkey studies have used high doses (105-106 TCID50; mostly 2020 variant of SARS-CoV-2) of virus usually applied to the nose and throat of cynomolgus or rhesus macaques resulting in high viral loads, mild to moderate disease similar to that seen in many humans and indications of lung inflammation.[49,50,52]
Using a 2020 variant of SARS-CoV-2, a cynomolgus-macaque monkey model was developed, which delivered the virus using aerosols, resulting in a disease similar to mild human COVID-19. This model showed that measurable disease was dependent on dose and that a higher dose (100s of TCID50) resulted in symptomatic disease (fever), a spike in interleukin 6 (a pro-inflammatory molecule), shedding of nasopharyngeal and oropharyngeal viral RNA and development of neutralising antibodies (called seroconversion).
A lower dose (10s of TCID50) only produced seroconversion and intermittent viral RNA shedding without fever or signs of illness – an indication that the body had “seen” enough virus to respond to it but not have its defences overwhelmed.
Less than 10 TCID50 did not result in seroconversion.
A study infecting golden Syrian hamsters used detection of the infectious virus rather than just viral RNA. It exposed uninfected hamsters to the droplets/aerosols of an inoculated hamster which led to successful infection after as little as an hour when the infected hamster’s viral loads were high. Interestingly, the timing was more important than temperature or relative humidity. But those two factors did matter if there was a lower viral load exposure; in that case, high relative humidity and temperature were better for transmission then.
The key takeaway for this blog is that the higher the dose received by the hamster contacts from the infected hamster, the more likely that obvious transmission of the infectious virus would happen. Yes, hamsters aren’t humans, but until we disprove such studies using humans, these remain reliable data.
In a study that infected cats with 105 TCID50 of a Delta (B.1.617.2) variant via the nose and throat, it appeared that what may cause little harm to one species can in a lower dose cause much more severe disease in another.
The infectious dose had clear implications for mice infected with SARS-CoV (the original) – more was worse. To make things interesting, the same dose of different variants of SARS-CoV in hamsters resulted in different amounts of virus being produced in their lungs. So viral variant also plays a role in disease outcome – as we’ve seen with SARS-CoV-2.
Early in the pandemic, three clusters of SARS-CoV-2 infections hinted that exposure to less virus (better social distancing, more spacious rooms) produced fewer infections and milder infections. Close, repeated and prolonged exposures in smaller indoor spaces – presumably equating to a higher infectious dose – tended to result in more serious disease.
In a study of non-hospitalised children with and without COVID-19, there was no difference in peak viral load compared to adults, but the viral load in children reduced more quickly. Its likely children would be infectious for a shorter period (Figure 2).
In the most relevant study to date, 18 of 34 (53%) human volunteers were infected using intranasal drops containing 10 TCID50 (a low dose). Virus load peaked in the nose but was first detected in the throat, and infectious virus could be detected for approximately 10 days after the volunteers were inoculated; 8 days after symptoms started (Figure 3).
Only two of the patients (6%) in this small human volunteer group remained truly asymptomatic throughout.
On an aside, rapid antigen tests worked least effectively early on. More worryingly, a rapid antigen test (RAT) took one or more days to detect positivity compared to both PCR and virus culture (less sensitive than PCR) among 9 of the 18 volunteers. Viral culture is the gold standard for detecting infectious viruses, and it’s expected that any diagnostic test can detect a viral load at least equal to it. If you want to be sure of detecting early infection (when you start to feel sick) and the earliest infectious period (e.g. prior to you being admitted to a hospital, visiting a high-risk setting or travelling), or identifying RNA persistence (useful in future long COVID studies?) then PCR was more useful.
In a study of household contacts, the viral load in the first or “index” case was a factor in onward transmission to others in the household; those with a higher viral load were more likely to infect others.
A study collecting breath emissions from COVID-19 patients while they sang, talked or breathed found more virus in fine aerosols (particles sizes <5 μm) than in coarse. Two infected patients sharing a hospital room shed 6-47 TCID50 units of infectious virus per litre of air which was collected 2 – 5 m away from them, showing SARS-CoV-2 can be airborne. This is a distance beyond what droplets (>100 μm) are thought to travel. Also, this is in the ballpark of that human volunteer study mentioned earlier, so we know that this amount of emission into aerosol in a room is a relevant dose.
It’s also worth considering the impact of prior SARS-CoV-2 infection and vaccination when reading through human studies. Immunity is likely to raise the threshold for an infectious dose (you are more resistant after having COVID-19 or a vaccine) and can make it more difficult to culture virus from immune patients as antibody-covered virus particles may struggle to enter a cell and start an infection.
Protection from infection
We know that COVID-19 vaccination has so far prevented a lot of severe disease, hospitalisation and death, even if given after a previous SARS-CoV-2 infection.[21-23]
We could also think about dose the other way around – the amount of exposure to a virus it takes to protect from disease rather than cause it. This is what we look at in studies seeking to determine the minimum dosage packaged into a future vaccine. This “protective dose” effect can be seen in early studies of COVID-19 vaccines.[53,54] There was a stronger immune response when more active ingredient was used (Figure 4), but in human Phase I trials, there were also more reactions, so a lower dose was ultimately used.
Vaccination also has a role in reducing viral loads for some weeks after vaccination.[18,20,29] We know that when a breakthrough infection does occur – that is, infection of a vaccinated person – the amount of virus present in the newly infected but vaccinated person doesn’t really differ from the amount in an unvaccinated infected person.[18,19] Breakthrough infections are likely to lead to infectious infections. So, as others have said, we need more than a vaccine-only strategy to reduce the likelihood of transmitting an infectious dose of SARS-CoV-2 (or influenza virus, or respiratory syncytial virus, or a rhinovirus…)
Adding other layers to prevent infection
Distance from an infected person helps reduce the risk that person will infect us because close-up aerosols are at higher concentrations (Figure 5).
Preventing virus-laden aerosols from building up indoors by increasing air exchange (introducing more “fresh air”) or using filtration to return particle-free air to a room is another helpful layer to consider if aiming to reduce any infectious dose.
Masks – they do work, but humans don’t
Wearing a mask – any mask at all – can reduce the risks of infection and disease from airborne pathogens.[17,24] It is the only method that can protect against short-range (up close) transmission. I used “can” here because no disposable mask is perfect.
Masks are worn by humans who can be random in so many ways.
Here are some things to think about if you wear a mask or read studies looking at the benefits of masks, or you are comparing cases between a mask-wearing country and a non-mask-wearing country:
- Even the best quality mask is only as good as its fit 
- A face covering is not useful if not worn when there is a risk
- For example, wearing a mask on a bus but taking it off when seated for hours in an office/classroom/restaurant with inadequate air change or filtration means it isn’t useful as personal protective equipment (PPE) to protect you from others
- Households remain a risky environment because we think they are safe when they are better thought of as social network hubs for virus interchange. We likely don’t mask at home, right?
- A surgical/procedure mask deflects air and isn’t great as PPE
- It can prevent the person in front of you from getting a full exhaled breath, but it doesn’t trap much of the exhalation – it deflects it up and sideways, filling the room with aerosol and potentially putting those sitting next to you at risk of your newly directed exhalation
- We turn our heads or bend over, and these actions disturb mask fit
- Some people wear masks with a beard or stubble – this makes a seal impossible
- Some masks aren’t worn properly to cover the nose, just the mouth
- Some people constantly pick at the front of their mask, which breaks the seal
- Cloth face coverings are not as good at filtering out small particles as surgical/procedure masks, which are not as good as respirators. And where there is a hole, inhaled/exhaled air carrying particles will find it as they seek the easiest way in/out. Such holes, or pores, can occur between the organised threads of cloth masks – especially if the material is stretched. Commercial masks have a much more random weave (“meltblown” fabric layer), are less stretchable and sometimes include a charged filter layer.
But one thing we can be 100% certain of is that not wearing a mask at all means zero per cent risk reduction.
All masks provide some protection by filtering our virus-laden particles. The benefit is affected by how long you are exposed to the aerosols and how much of a viral dose si in them (Figure 7). And remember, purified individual virus particles aren’t what travels from our infected cells; the viruses we emit are complexed with the mostly dried-down gel-like mucous into which they emerge from our infected cells.
The virus-laden particles we’re most worried about are those we expel while infected. In particular, the ones less than about 100 millionths of a meter (100 μm) in diameter because they are emitted in large numbers and can stay aloft and can be moved around by as little as the breeze of a door opening. Bigger particles fall to the ground around two meters from their source, depending on how much power was behind their propulsion.[34,35]
Particles are generated in different parts of our airways.[41,44,45] Most particles we breathe or emit by speech are 10 μm in diameter or smaller (Figure 8; ) Interestingly, it’s the smaller diameter droplets that contain the highest concentration of virus.
P2 or N95 respirators are tight-fitting, often fluid-resistant, minimally leaking masks that filter out/exclude passage of ≥94% or ≥95% of particles that are 0.3 μm in diameter or greater, respectively (see some particle sizes in Figure 9) These generally have two over-the-head loops instead of the earloop surgical/procedure mask use. This helps form a tight seal with your face. P2/N95 masks are generally thought of as being for healthcare worker use but they are the best disposable mask if you are super-serious about reducing the risk of inhaling an infectious dose of virus.
But we know there is also a real outgoing filtration benefit from the use of surgical/procedure masks. One example shows them preventing the release of coronaviruses (229E, NL63, OC43 or HKU1) and influenza viruses into the surrounding air (Figure 10). Even a surgical/procedure mask that leaks works to reduce the release of infectious aerosols (virus was able to be cultured from aerosols in this study).
INTERESTING ASIDE: In this study, the non-enveloped rhinoviruses were not as significantly stopped by the 3-ply pleated, cellulose polypropylene polyester surgical/procedure mask. Perhaps the nature of the material in the masks fails to achieve the same level of capture of non-enveloped viruses as it does the enveloped SARS-CoV-2 and influenza viruses?
Effective mask use needs education and communication of the kind provided by strong support from public health experts. That should include advice on the correct choice of face coverings, how to wear them and good hygiene practices in relation to their use. Ideally, this should precede the use of masks so everyone is using them properly and aware of why and what to do/not to do.
Disappointingly, that didn’t happen in many parts of the world before masks were used and then mandated. And, of course, as soon as mandates dropped, so mostly did masks. And who is surprised to see that?
Can we avoid all infections forever?
For the majority of the planet, the answer is a hard no because we’re just…too human. We could wear elastomeric masks and avoid human contact, of course, but I don’t think that’s the preferred option of most humans.
However, we each can take steps to reduce the amount of virus we get exposed to. Masks matter. A mask can help. So can clean or fresh air. As can distance. And these measures work against multiple very different airborne viruses and the diseases they cause. Reducing the infectious dose also means we may get a milder form of illness and still develop an immune response.
I know this amounts to personal responsibility but let’s be practical; that’s all we’re left with now.
If you want to avoid getting sick from respiratory viruses -it’s up to you. The help from professional expert bodies is largely gone now.
So you can:
- Wear a good mask well – reduce the particles you inhale. Masks matter.
- Don’t spend long inside crowded stuffy spaces – limited time = limited dose
- Advocate for cleaner air. The purity of our air is as important to our health as the cleanliness of our food and water
- World Health Organization collaborative study to calibrate the 3rd International Standard for Hepatitis C virus RNA nucleic acid amplification technology (NAT)-based assays
- Evaluation of CMV viral load using TaqMan CMV quantitative PCR and comparison with CMV antigenemia in heart and lung transplant recipients
- Timing of exposure is critical in a highly sensitive model of SARS-CoV-2 transmission
- The role of face coverings in mitigating the transmission of SARS-CoV-2 virus: statement from the Respiratory Evidence Panel
- Understanding transmission of SARS-CoV-2 in the ongoing COVID-19 pandemic
- Persistence and clearance of Ebola virus RNA from seminal fluid of Ebola virus disease survivors: a longitudinal analysis and modelling study
- Upper respiratory tract SARS-CoV-2 RNA loads in symptomatic and asymptomatic children and adults
- Safety, tolerability and viral kinetics during SARS-CoV-2 human challenge in young adults
- Review: What is the infectious dose of SARS-CoV-2?
- Seroconversion and fever are dose-dependent in a nonhuman primate model of inhalational COVID-19
- Household Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 in the United States: Living Density, Viral Load, and Disproportionate Impact on Communities of Color
- SARS-CoV-2 D614G variant exhibits efficient replication ex vivo and transmission in vivo
- Viral Load of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Respiratory Aerosols Emitted by Patients With Coronavirus Disease 2019 (COVID-19) While Breathing, Talking, and Singing
- Impact of community masking on COVID-19: A cluster-randomized trial in Bangladesh
- SARS-CoV-2 delta (B.1.617.2) variant in vaccinated and unvaccinated individuals in the UK: a prospective, longitudinal, cohort study
- No Significant Difference in Viral Load Between Vaccinated and Unvaccinated, Asymptomatic and Symptomatic Groups Infected with SARS-CoV-2 Delta Variant
- Initial report of decreased SARS-CoV-2 viral load after inoculation with the BNT162b2 vaccine
- Effectiveness of CoronaVac, ChAdOx1 nCoV-19, BNT162b2, and Ad26.COV2.S among individuals with previous SARS-CoV-2 infection in Brazil: a test-negative, case-control study
- Living Evidence – COVID-19 vaccines [COVID-19 Critical Intelligence Unit]
- Global impact of the first year of COVID-19 vaccination: a mathematical modelling study
- SARS-CoV-2 aerosol transmission in schools: the effectiveness of different interventions
- Viable SARS-CoV-2 in the air of a hospital room with COVID-19 patients
- Detecting viruses: the plaque assay
- Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems
- Review of infective dose, routes of transmission and outcome of COVID-19 caused by the SARS-COV-2: comparison with other respiratory viruses.
- Effect of Covid-19 Vaccination on Transmission of Alpha and Delta Variants
- The role of face coverings in mitigating the transmission of SARS-CoV-2
- COMMENTARY: What can masks do? Part 1: The science behind COVID-19 protection
- Increased close proximity airborne transmission of the SARS-CoV-2 Delta variant
- ACGIH. COVID-19: Workers need respirators
- A Paradigm Shift to Align Transmission Routes With Mechanisms
- Airborne transmission of SARS-CoV-2
- Zooming In: Visualizing the Relative Size of Particles, Visual Capitalist
- Morphometry of SARS-CoV and SARS-CoV-2 particles in ultrathin plastic sections of infected Vero cell cultures
- SARS-CoV-2: preliminary study of infected human nasopharyngeal tissue by high resolution microscopy
- Nanometer-resolution in situ structure of the SARS-CoV-2 postfusion spike protein
- SARS-CoV-2 – Remarks for Diagnostic EM
- Airborne Transmission of SARS-CoV-2: A Virtual Workshop, National Academies of Science. Engineering and Medicine
Prof Lidia Morawska, QUT, Size characteristics of particles generated by people
SLIDE DECK: https://www.nationalacademies.org/event/08-26-2020/docs/DCC780CE8E9FB66682E3F58D6F23F1D9820EB117BFAB
WORKSHOP (8th presentation in the video): https://www.nationalacademies.org/event/08-26-2020/airborne-transmission-of-sars-cov-2-a-virtual-workshop
- Respiratory virus shedding in exhaled breath and efficacy of face masks
- How can airborne transmission of COVID-19 indoors be minimised?
- Modality of human expired aerosol size distributions
- Size distribution and sites of origin of droplets expelled from the human respiratory tract during expiratory activities
- Comparing the fit of N95, KN95, surgical, and cloth face masks and assessing the accuracy of fit checking
- Modeling the filtration efficiency of a woven fabric: The role of multiple lengthscales
- Inoculum at the time of SARS-CoV-2 exposure and risk of disease severity
- Respiratory disease in rhesus macaques inoculated with SARS-CoV-2
- Comparison of rhesus and cynomolgus macaques as an infection model for COVID-19
- Bronchoalveolar lavage affects thorax computed tomography of healthy and SARS-CoV-2 infected rhesus macaques (Macaca mulatta)
- SARS CoV-2 (Delta Variant) Infection Kinetics and Immunopathogenesis in Domestic Cats
- A single dose of an adenovirus-vectored vaccine provides protection against SARS-CoV-2 challenge
- Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial
- A Mouse-Adapted SARS-Coronavirus Causes Disease and Mortality in BALB/c Mice
- Animal models and vaccines for SARS-CoV infection
- Reducing risks from coronavirus transmission in the home—the role of viral load