Transmission of Ebola viruses: What we know and jumping the black swan

Last week a review was released entitled Transmission of Ebola Viruses: What We Know and What We Do Not Know. The review, which is listed in the Opinion / Hypothesis section of mBio, was penned by by Michael T Osterholm and a large team of Ebola experts. You may know him from such articles as What We’re Afraid to Say About Ebola and What we should — and shouldn't — be worried about regarding Ebola or his entertaining seminar at the Johns Hopkins Bloomberg School of Public Health Ebola forum.

First up a few random points from me...

• This is basically a good review of the historical literature on transmission of Ebola virus and some other ebolaviruses. By the way, this literature is based on real experience, observation and experimentation, defining our understanding of how Ebola virus has transmitted among humans.
  It's worth noting that there have been no concerns made public, nor any new transmission data from the epidemic in West Africa, which indicate changes in the way Ebola virus spreads from person to person
• This review is an opinion/hypothesis piece so it has a lot of room to move. The language fairly clearly defines where the thinking strays into areas without any actual data to support them. Look for phrases with words like "possible", "could", "postulated", "may", "suggesting" and "can"

A quote from the new review by Osterholm
and experts. Highlighting is mine.

• Does anyone actually care whether we use words like aerosol, airborne, droplet, droplet nuclei, wet droplets or propelled to mean "not by touch"? I'm not sure any more, but I think they should. Words have meaning and slightly different words carry subtley, yet importantly different meanings. It's important to keep in mind who the messages relating to public health should ultimately try and reach - that would be the public. Experts, comparatively few in number, already have an innate sense of the differences between the words above, right? Right?! Well, many do anyway. Trying to change language or redefine a target in the midst of an epidemic, is at best bad timing and at worst it seems self-serving (although to what end I cannot guess). 

Suggested ways in which an ebolavirus can spread from a known EVD case to a new person. The most likely route is suggested by the thickest arrows with solid outlines while the least likely or most improbable route is indicated by the thinnest arrow with a dashed outline.
Click on image to enlarge.

• Ebolaviruses are not just blood-borne viruses in humans like HIV is for example; they are not just gastrointestinal viruses like norovirus (although droplets play a role here too); they are not considered by anyone to be airborne viruses like influenza virus
  What they are, in a transmission sense, is a hybrid of the first two - reaching high loads in the blood and the gastrointestinal system. To me, these shared features make it more clear why a different level of personal protective equipment (PPE) is needed than would be considered essential for caring with patients with just one or other type of virus
• One distinct viral group may remain infectious for a longer period, shorter period, or not at all compared to another distinct viral group, in droplet nuclei - the air-dried (gel-like mix of proteins and salts..and infectious or non-infectious virus) form of droplets that have not yet hit an object or the ground.
• The figure of just 1-10 viruses being required for an infection to take hold has generously been bandied about during the Zaire ebolavirus (the EBOV|Mak variant) epidemic of 2014/2015. But some overlook a simple component of this apparently easy infection process; 1-10 viruses landing on a cell is not the same as 1-10 intact infectious viruses being emitted from an infected host, travelling out of the host's infected cells in a drop of blood, semen, urine, sweat, saliva, diarrhoea or vomit, retaining infectivity while passing through various environmental conditions, onto a new host's mucosal surface, perhaps indirectly via a hard surface, getting past that body's innate immune defences and eventually attaching onto and gaining entry into that new host's cell, successfully replicating within it and then infecting neighbouring cells to establish a new infection. It may take thousands or hundreds of thousands of viruses in that initial drop of infection material to get those 1-10 infectious virus particles to start a productive, symptomatic human infection.
  We know little about this part of the story outside of the laboratory
• On that theme, there is much in general we still do not know about the ebolaviruses and Ebola virus disease (EVD). Direct contact with virus-laden fluids or a needle-stick injury are still considered to be major routes for acquiring an ebolavirus infection but direct mucosal contact with propelled droplets may occur at distance further away than the old 3ft/1m rule.(6)
  Truly airborne dried or semi-dried droplets that contain sufficient infectious Ebola virus, can be inspired and can result in an upper or lower respiratory tract infection that progresses to become systemic EVD in humans....have yet to be found. They may contribute to infections, but it will be very hard to prove that this is a transmission pathway that exists as a thing separate from droplet transmission. The authors sort of note this difficulty too; both droplets and droplet nuclei result from coughs, sneezes and explosive vomiting and diarrhoea.
  The suggestion that droplets are produced from the respiratory tract and then inhaled by another person (6) resulting in EVD is really straining the use of "improbable"
• I've written about some of this stuff before - on the distinct issue of Ebola virus and pigs here, on droplets and droplet nuclei here, the complexities of contact here, on seeking some better words here and on previous versions of this theme by some authors of this latest review here and here and I'm not going to rehash all of that here! I invite you to read those posts

Mixed messages..

A problems I have with this review is this line in the abstract..

We also hypothesize that Ebola viruses have the potential to be respiratory pathogens with primary respiratory spread.

While Osterholm and expert colleagues round off the review by clearly stating that airborne ebolavirus transmission is an "improbable scenario", and that droplet transmission is plausible (I don't disagree with the latter statement), parts of the rest of the review struggle to tow that line quite so clearly. The media seemed to have struggled to find that message too..

It is 'very likely' that the Ebola virus will spread through airborne particles, experts say
Daly Mail

Limited airborne transmission of Ebola is ‘very likely,’ new analysis says
Washington Post

..although one bright light in the gloom managed extremely well..

No, A New Scientific Report Does Not Say That Ebola Is Now Airborne
Vice News

Prof Vincent Racaniello noted in his blog post about the review that we understand what viruses do now, by what we have observed them to be capable of doing in the past. 

Do ebolaviruses actually have the potential to shift to a primary method of spread that occurs via droplets or droplet nuclei and spread like a rhinovirus, influenza virus or the measles..to name a few? If no virus which we humans have ever watched has changed its method of spread so dramatically before, why would this particular one do it now? Well, why wouldn't it, you may well ask? Because it takes more than some genomic mutations and drift to do this. At some point we need to remember that each virus comes with its own toolset and it doesn't usually have a lot of replacement parts or upgrades in a satchel over its capsid. It can only tweak its component parts so much and so far before it reaches the limit of what it "is". Could one virus become another virus? Maybe it could. I look forward to becoming Superman myself. What would it take to overcome whatever biological throttles have existed on the ebolaviruses prior to so much human spread, for a virus to stop spreading primarily by fluids resulting from certain host disease processes, to being spread mostly by inspiration of respirable droplets? Certainly something we've never seen before and something in need of a utility belt and  can of bat EBOla repellent. Again, we're not just talking about some "genotypic changes"; the ebolaviruses would need to accumulate a plethora of stable genetic changes to make that sort of transition, possibly in combination with changes in the disease processes within its host...us.

An opinion by any other title...

Despite the review being an opinion piece, it seems to have some trouble owning up to its own real opinion; that Ebola viruses can spread by a new route and cause new disease. The title really should have reflected the content better in this regard. In approximate number 5,800 words included 440 (8%) on animal transmission studies which are mostly about aerosol spread; 925 (16%) devoted to defining aerosols and droplets and trying to change the paradigm; 670 (12%) about what we need to learn, which includes some content on aerosol transmission; 670 (12%) on a respiratory transmission hypothesis. So a sizable chunk, nearly half of the content, is heavily focussed on educating us abut Ebola and aerosol transmission. The topic is additionally reinforced within every other section as well. So why hide what the article was really focussed on; not the general transmission of ebolaviruses, but transmission via an as yet unproven-route? The authors note that an "aerosol" contains all the different droplet sizes and degrees of droplet wetness and that this entire range is propelled out of us via cough, vomit, diarrhoea and by  aerosol generating mechanical procedures. We agree on that bit. But once the bigger wetter droplets fall away and one is not standing unprotected within their range, is there an infectious virus left in the drier smaller droplet nuclei which are held aloft by air currents until they impact with something or someone? 

Have we ever seen Ebola virus infections caught by people walking into a room after an infected case has left it...as is the case for measles or rhinovirus, truly airborne transmissible viruses? Or is droplet spread only occurring in close proximity to the source? This is another point the authors raise-that being close to someone who just vomited may result in breathing in larger droplets that are infectious but have not yet fallen to the ground. How will we ever know that this is not a propelled droplet instead? Explosively coughed or vomited material can travel a sizable distance as well? So we still await some evidence to support the musing that inhaled droplets carry infectious ebolavirus in them, and that they are distinct from the more likely impacting of propelled droplets. Propelled droplets are likely a key reason that updated PPE guidelines recommend against any exposed skin and the use of eye protection, gloves, boots and a respirator; the yellow suits that will forever be linked to EVD in West Africa. But even those suits don't support that Ebola virus has been, or is showing new signs of, spreading primarily via a respiratory route?

What I could not find in this new review was a more thorough discussion - and some hypothesis and opinion - of the risk associated with how healthcare workers acquired their infections when outside of Ebola treatment units or in western hospitals. Also absent was opinion on the practical risks of semen remaining infectious, or harbouring viral RNA as was found in 2014 in a returning asymptomatic convalescent man [3] (sexual transmission has not been documented [11]). I would very much have liked to read some hypotheses on the role infectious urine might play in urban settings lacking no sewers and with densely co-located populations, since urine has been shown to remain infectious for longer than blood, in a detailed case study from Germany in 2014.[3]

Wrap up...

So to summarize, coming into contact with virus-laden body fluids either by touch, perhaps via an intermediate surface (a fomite; unproven) or by having these fluids propelled onto you (as yet unproven), are considered the main risk factors that comprise the overwhelming majority of human-acquired ebolavirus infections. Current PPE guidelines are designed to combat these and if western hospitals are any guide, they work well - although it's a tough comparison given the different carer-to-patient ratios in western hospitals compared to outbreak conditions in west Africa. 

What role "respirable droplets" or droplet nuclei play in transmitting ebolaviruses between humans awaits evidence but nothing points to a role for an airborne route of infection in west Africa.[6] Hopefully some studies will be looking very hard at this question. Nothing hints at any changes in  EBOV/Mak that could result in it becoming a "respiratory pathogen with primary respiratory spread" capability.

I recommend reading a few other recent reviews and articles to get a more rounded view [7,8,10,12] and if you want to see droplet, aerosol and airborne get smooshed together into an undifferentiated mess, that's in print too.[9]

References...

1. Transmission of Ebola Viruses: What We Know and What We Do Not Knowhttp://mbio.asm.org/content/6/2/e00137-15
2. Experts suspect Ebola virus sometimes spreads by air
   http://www.cidrap.umn.edu/news-perspective/2015/02/experts-suspect-ebola-virus-sometimes-spreads-air
3. A Case of Severe Ebola Virus Infection Complicated by Gram-Negative Septicemia.
   Kreuels B, Wichmann D, Emmerich P et al.  N Engl J Med. 2014 Dec 18;371(25):2394-401
   http://www.nejm.org/doi/full/10.1056/NEJMoa1411677
4. Ethical issues in isolating people treated for Ebola
   http://www.ncbi.nlm.nih.gov/pubmed/25588871
5. 2007 guideline for isolation precautions: preventing transmission of infectious agents in health care settingshttp://www.ajicjournal.org/article/S0196-6553(07)00740-7/pdf
6. Ebola virus disease in Africa: epidemiology and nosocomial transmissionhttp://www.ncbi.nlm.nih.gov/pubmed/25655197
7. Understanding Ebola Virus Transmission
   http://www.mdpi.com/1999-4915/7/2/511
8. Chains of transmission and control of Ebola virus disease in Conakry, Guinea, in 2014: an observational study
   http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(14)71075-8/abstract
9. Ebola, through air or not through air: that is the question
   http://www.ncbi.nlm.nih.gov/pubmed/25646157
10. Review of Human-to-Human Transmission of Ebola Virus from the US CDC
    http://www.cdc.gov/vhf/ebola/transmission/human-transmission.html
11. Sexual transmission of the Ebola Virus : evidence and knowledge gaps
    http://www.who.int/reproductivehealth/topics/rtis/ebola-virus-semen/en/
12. What we know about transmission of the Ebola virus among humans from the WHO
    http://www.who.int/mediacentre/news/ebola/06-october-2014/en/

What words would you use to separate influenza spread from Ebola virus disease spread?

I need your help.

I have spent umpteen hours on trying to make this message simple. None of that has been aided by the way that the CDC, the WHO and now the UN use the terms and words confusingly to convey messages to the public. The message is often delivered as if they were sitting around their meeting rooms talking to other health and science professionals. In my opinion, we all look to these guys for simple clear and consistent messages. Right now they need to do much better to convey complex concepts, simply, quickly and more often. Education helps prevent panic, mistakes and conspiracy theories (well-as much as anything can anyway).

So here is another attempt by me to get this wording into line with what the rest of world can make sense of. 

I could also really use your input to make this work - so leave a comment below, or Tweet me @MackayIM or email me or send me a carrier pigeon - with how to make this message simpler for you and your kids and your grandparents and that weird uncle you stay clear of at Christmas, to understand. 

Let's crowdsource a solution to this confusion, help out others and then see if the major public health bodies can come on board.

Propelled droplets versus a cloud of suspended.

This post and issue have been fuelled most recently by the Ebola virus disease (EVD) epidemic but is also fuelled by my experiences in talking to people about the MERS-CoV and influenza A(H7N9) virus outbreaks. They are respiratory viruses while ebolaviruses are not. Different viruses yes, but common concerns for people and to the issues around trying to understand overly technical terms when they are used differently in everyday life. 

Public health speaking is very public.

Public health issues are spoken about on a global stage, more now than ever. It is up to us to better define the right words and use them consistently. That has definitely not happened for "aerosol" and "airborne". 

We professionals can't just sit back and expect our stakeholders to come along with us for the ride - they will get confused when imagery conflicts with lingo and official statements, and when different public organizations disagree with each other or use tiny but significant differences in their language to communicate risks. 

People are not stupid and deserve more respect than they are currently getting from those who should know much, much better about how to work alongside the public (public health and all).  

So what is the problem here? 

Droplets would probably be an ideal word to differentiate from airborne - and it has been used to differentiate the level of precautions of personal protective equipment (PPE) to prevent infections - droplet precautions and airborne precautions - but the evil physicist types have ruined the use of that word for us by introducing droplet nuclei (the part of the aerosol that lingers in the air and can convey those viruses that survive in it, to a new person to infect them). Physicists like technicalities.

So the problem is trying to define a name for that other process that can simply and clearly describe infectious disease transmission of viruses & bacteria that are propelled from/by the sick person, across the gap between them and an uninfected person, measurably infecting the recipient. The name should make clear that it is a different process to the one that sees a person get sick by inhaling infectious viruses or bacteria held aloft by the air, in a cloud, made by a previously ill person, that has been hanging around for perhaps an hour or more. That one is an airborne route of transmission. 

Some people have berated me for talking technicalities and semantics in recent days while I try to better define this. Tough! Water off an influenza-host's back. Words have meaning and impact and useful words are needed. Especially when everyone is freaking out over a disease they have only read about in dramatized books or seen in Hollywood blockbusters. The two processes listed above are distinct and different for some viruses & bacteria. But it is biology and nothing is 100%, except death. 

Some infections, like those leading to influenza, could result from both processes. Some, like Ebola virus disease have never been observed in humans via one route (airborne), whereas there is a defined risk of them occurring by the other (direct contact between a range of virus-laden body fluids propelled onto a mucous membrane). Yes, coughing a tiny barely visible droplet onto someone else's mouth is direct contact between the wet fluids and the mucous membrane.

They two processes are battled differently. We protect ourselves from them differently. And names can tell us about the different levels of risk. But what is that other route to be called? 

I have an idea. First some perspective.

Ways to think of the differences.

A word cloud of ways to think of the
two different processes of spreading viruses
or bacteria that result in infection and disease
in humans.
v2 Thanks to Nina West for good analogy (Fog/Rain)

The idea.

How about we call the process of relatively short (up to about 3m) distance, coughed/sneezed/vomited wet droplet transfer of disease-causing doses of viruses or bacteria, "Propelled"?

Over to you, world.

Some greats from the comments below...

• "void the spray and live another day"
• Only touched by air, no need to care. Where it splatters, that's where it matters

It's what falls out of the aerosol that matters....

v2 031014

"Aerosol" is a messy word. It means different things to different people. So does "airborne".

What's in an aerosol?
Here we're talking about a mixture of different sized stuff. Think  of the size range in a handful the sand from a shelly beach.

A cough/sneeze includes big, wet, heavy propelled droplets that quickly fall to the ground or hit your windscreen (hate it when that happens) or your friend's face (they hate it when that happens) down to dried or gel-like "droplet nuclei" that can float in the air for hours, travelling where the wind blows them; and every size in between.

I've also talked about this before, here.

The public rightly get confused about aerosols. And science and physics and medicine have their own defined meanings - sometimes at odds with each other - that may well be out of step with what the public think.

I do wish the the big public health entities would settle on some definitions for these and other words. It would make everyone's life a lot easier.

Direct contact.

When we talk about "direct contact" and Ebola virus transmission, we do include the bigger wetter heavier droplets that might be propelled from of a sick person during vomiting, or coughing as a risk for transmitting virus. 

Even though that is not physical direct contact, and even though the droplets travel across a gap between people - through the air - it is still a direct line from person A (red in the graphic below) to B (blue). If B is too far away, then those droplets fall to the ground before they hit B. The droplets may remain infectious on the ground. That depends on temperature, humidity, surface type and the type and amount of virus.

The airborne route.

Even though it involves a short period of travel through the air, coughing wet droplets directly onto someone's mucous membranes is not an airborne thing. The term "airborne" is reserved for floaty clouds of droplet nuclei. In humans droplet nuclei have not, to the very best of our knowledge and observations and tests, been found to contain doses of Ebola virus that cause disease in humans. Too little virus coughed into the cloud perhaps or too little that survives..it's not known why, but it is pretty clear that in households where a case of Ebola virus disease was residing, only those household members who had direct contact developed disease, and those that breathed the same air but did not have direct contact, did not develop disease. 

While Ebola viruses may be present in floaty clouds of droplet nuclei, or forced to be in a floaty clouds of droplet nuclei under lab conditions with lab viruses at lab virus concentrations, a floaty cloud of droplet nuclei has not been shown to act as a source of acquisition for Ebola virus and resulting disease among humans. Sorry, did I just repeat myself?

Rest in peace.

Please don't say Reston ebolavirus or the Hot Zone. That (by all accounts riveting) book was not a scientific work, it is a dramatized work and the language is colourful and emotive and scary. The Reston ebolavirus event in non-human primates was never proven to be airborne.

Lastly and most recently, an airborne route was not found to play any role in causing disease or infection when Ebola virus infected and uninfected non-human primates were caged near each other. I've written about this and other non-human primate studies here.

To summarize.

Healthcare workers wear face protection(masks and goggles) to prevent their eyes and mouth being hit by wet droplets of virus-laden body fluids while they are in close contact with ill Ebola virus diseases patients. The also wear all-over gowns so that they don't have to sterilize their clothes between each room they move between. Use of protective equipment doesn't need to convey confusing messages about the type of route Ebola virus uses to spread but it's just lacking in enough public discussion via forums the public attend/view. Knowledge is a bit like vaccination - when coverage reaches a certain level, the community is safe (or it's understanding is complete anyway).

And why wouldn't healthcare workers protect themselves from ill patient fluids-however they come into contact with them? For a healthcare worker, body fluids from ill people they are in close and often prolonged contact with, should generally be considered infectious. This is the case whether we're talking about Ebola virus disease, HIV, measles, influenza or something else. Some of those are caused by airborne viruses, some, like Ebola virus and HIV, not.

Below is my latest attempt at trying to make all those words into a picture. 

If you have ways that can help me make this even simpler - please pass them along (thanks @chrisfharvey).

Ebola, pigs, primates and people

This is a companion piece to my collaborative article, Ebola virus may be spread by droplets, but not by an airborne route: what that means, posted a couple of days ago. I suggest you read the both together.

In this post, I'd like to make sure we all understand that an airborne route of Ebola virus infection has been used to deliberately infect non-human primates (NHPs). It is possible and it can be done. Okay? I'm not covering up any secret knowledge or trying to conceal facts that only we few evil-society-of-science types know. I don't secretly work for an agency aiming to delude you dear readers into feeling falsely safe about the risks associated with being near an Ebola virus infected person (which most reading this will likely never be). Frankly, I'm learning this as I go.

Don't expect perfection from risk mitigation advice.

Like all things that involve biology, there are hardly ever clear-cut lines and yes or  no definitions and explanations. Sometimes that's because things vary...because biology! Sometimes that's because we haven't yet done enough science to know those answers. I'm not an expert on ebolaviruses nor on Ebola virus disease (EVD) - but in my time learning about the viruses and the disease, its clear that this is (yet another) area that is lacking in all sorts of information. So risks are judged using what we do know and can support and verify, with softer language used when decision makers don't know for certain; less so when they think they do. 

When that message of risk gets passed to the public, it is important to be accurate, clear, concise but not to over-simplify things because that may degrade trust in the body(s) sending the message if things change later. That's a very tough balance when dealing with biological risks.

So having said that, let's talk pigs.

Pigs are not primates.

In ebolaville - the virtual world created by social and mainstream media stories and discussion about ebolaviruses - a lot of people have been throwing the 2012 pig to macaque study (8) around as an argument for why we should admit that ebolaviruses spread by an airborne route and run for the hills. This is why that is not a good comparison:

• Pigs have a different disease and replication process to humans. 
• Pigs tend to have much more virus growing in their lungs.(12) 
• Pigs tend to cough and sneeze and generally propel more of said pathogen from their lungs.(11) 
• Pigs may eject more infectious viruses in their droplets than do primates

If we look at the study of disease occurrence and spread in previous outbreaks, that epidemiology does not suggest an airborne spread - the numbers and nature of human-to-human spread don;t show it as any sort of major contributor to spread. Might it be a minor contributor? Possibly. We don;t know either way with 100% surety. But we do  know some other things. One of these is that it takes very little virus to infect pigs and NHPs. If there are not obvious signs of an airborne spread in humans, we just not have detected it yet or, it may have a biological basis. It is possible that the infectious dose (amount of virus needed to get a foothold and start an infection) may be much higher for humans; infected and severely ill human cases may not breathe out infectious virus or ebolaviruses may not survive for long in the aerosols expired by humans,(19) even if they can survive on hard surfaces or in generated aerosols under laboratory conditions.(20)

Non-human primates can be infected with ebolaviruses via a lab-made aerosol with lots of lab virus at lab temperatures and lab humidity and other lab conditions in a lab.

You get that this is done in a lab? Cool.

It apparently does not take much virus to infect a human via an aerosol according to the Public Health Agency of Canada's (PHAC) Pathogen Safety Data Sheet (PSDS) on ebolavirus.(1) Only 1-10 infectious organisms (see above). But one problem with that PSDS is that it cites only 1 paper to support that range. Ref 21 from the PSDS is entitled Clinical recognition and management of patients exposed to biological warfare agents.(2) It is 1997 review that does not specify if this range is specific to any 1 or more of the ebolaviruses, just "viral hemorrhagic fevers". The PSDS seems to rely on that 1 line. In that reference, there are no further links to studies that define this range for humans, ebolaviruses or an Ebola virus (EBOV) of the species Zaire ebolavirus. I've sent a couple of emails in the past week, seeking further clarification from ebolavirus experts, but have yet to hear anything back.

In a laboratory experiment reported in 1995, transmission of an EBOV from one set NHPs infected by injection, to another set  resulted in 2 of 3 NHPs (1 with a heavy load of virus in the lung) becoming infected and that seemed to have occurred through some kind of airborne route as the 2 groups of animals were separated by 3m and care was taken to avoid creating bigger droplets and splashes during cage cleaning.(15) While the authors noted that fomites (contaminated objects and surfaces) or contact droplet transmission of virus was unlikely, the exact mode of transmission to the second group of NHPs could not be determined. In a follow-up study, the authors were able to prove that conjunctival and oral exposure to an EBOV could indeed result in infection in NHPs.(18) Thus we have plenty of reason for the use of masks, goggles and face shields that are already part of the recommended personal protective equipment (PPE) items for dealing with infected humans.

However, there are a number of issues related to forced aerosol infection of NHPs, many of which can be found in a massive and detailed 2008 review by Dr. Jens Kuhn.(3) These include:

• Often unrealistically high viral loads - the exact amount of infectious virus humans are exposed to during outbreaks has not been defined.
• Temperature and humidity conditions that were unlikely to reflect conditions during outbreaks in Africa - but may reflect conditions in hospitals.
• An initially lung-focussed pattern of viral replication (7) results from direct aerosol delivery of virus to NHP airways which seems different to infection of humans via the more frequent natural direct contact route. Systemic spread to multiple organs then follows via infected dendritic cells and macrophages and blood monocytes.
• Different routes of virus acquisition can lead to different incubation periods.
• Different virus isolates, sources and preparations may affect the course of infection and disease
• Because of the small and enclosed space and air throughput in head-only chambers, droplets rather than droplet nuclei may be the vehicle carrying infectious virus. This is important because, as you can read in the companion piece, droplet nuclei are the component of a lingering "airborne route" of acquisition and if NHPs are in fact infected by the droplets, that may be more indicative of direct fluid contact than true airborne travel.

A head-only inhalation chamber of the sort used in NHP
aerosol inoculation studies. Biaera Technologies.
Image from http://www.biaera.com/our-technology/peripheral-
aerosol-instruments/head-only-chamber/. See also (17)
Click on image to enlarge.

Some NHP studies that have successfully caused initial respiratory infection using an airborne route to infect NHPs under controlled experimental conditions include the following:

• 1,000 plaque forming units (PFU; a measure of how much virus is in a preparation using cell culture methods in the lab) of either a Kikwit EBOV isolate or a Boniface isolate of Sudan virus (SUDV; species Sudan ebolavirus) isolate were delivered using a Collison nebulizer (producing small droplets) after intramuscular immunization with a recombinant adenovirus vaccine.(5) 
• 1,000 PFU of a Kikwit EBOV isolate was delivered with a Collision nebulizer via a head-only aerosol chamber, after intramuscular immunization with a recombinant vesicular stomatitis virus (VSV) vaccine.(6)
• 743-274,000 PFU of a Kikwit EBOV isolate was delivered to with a Collision nebulizer via a head-only aerosol chamber, to examine aerosol-related pathology.(7)
• ~50 or ~500 PFU of a Boniface SUDV isolate were delivered to 3 different NHP species using a Collison nebulizer via a head-only chamber to compare species-specific effects.(14) 
• 0.8-128 PFU of a Kikwit EBOV isolate was delivered to 3 different NHP species using a Collision nebulizer via a head-only aerosol chamber, to examine disease course between species.(9)
• ~300-50,000 PFU of an EBOV isolate was delivered to with a Collision nebulizer (0.8-1.2um droplets) via a head-only aerosol chamber, to examine aerosol-related pathology.(16)

Are primates humans?

Judging by the effort we put into getting rid of our fur compared to an NHP, I'd say we're not! 

But on the topic of EVD, some NHPs that we infect with an ebolavirus, show very similar disease signs, symptoms and disease progression to those of EVD in humans; especially rhesus macaques [Macaca mulatta] although oneo f the studies above showed that 3 different NHP species were not that different in the way they responded to infection (rhesus macaques as well as cynomolgous macaques [Macaca fascicularis] and African green monkeys [Chlorocebus aethiops]).(9) 

Rhesus macaques become febrile, anorexic, lethargic, viraemic, develop a rash and sometimes develop diarrhoea and melena (gastrointestinal bleeding).(3)

But no animal model seems to completely capture other components of human disease which have historically included conjunctivitis, diarrhoea and vomiting and coughing up blood. Vomiting up blood and having bleeding gums occurs more often in fatal cases than in survivors.(3) 

Bleeding only occurred in 41% of 103 observed human patients during the 1995 Kikwit outbreak of an EBOV.(3)

So the answer is, primates are not humans when it comes to EVD, but they are pretty close. Yet within that "pretty close" lies an immeasurable amount of variation that may mislead when trying to map the course of NHP disease onto that of humans.

Where does that leave us?

I admit to being very uneasy saying that there is no risk at all of an airborne route of ebolavirus infection. Clearly it can be forced to happen, but we have no evidence that it has ever happened in humans in an outbreak. But let's put that into context. An absence of evidence is not evidence of absence. Outbreaks of ebolaviruses are not particularly conducive to large careful research projects measuring infectious droplet nuclei around critically ill people, especially when the occur in exotic locations in someone else's back yard.

So have I deserted by position from yesterday's post stating no airborne role for ebolavirus transmission between humans? No, not at all. What we know is that the overwhelming majority of human EVD cases acquire their infection during the time they are in direct contact with the fluids of a very ill EVD case; be that through physical contact or wet droplet spray impact. Beyond that fact, it may just be a discussion based on academic musings and hand-waving. But it is a discussion we should be having a little more I feel. A back-and-forth rather than messages with guarantees and statements dealing in black and white absolutes. I'm not sure the public believe in or feel safer with such absolutes today. We're all a bit too cynical for that.

If infection can happen between primates via the air, it is a very, very inefficient process as a study of 78 people from 27 households with EVD cases during the 1995 Kikwit  revealed.(10) Those 78 household members had no physical contact with the cases, and they did not get sick. Others who had physical contact, got EVD. 

In a recent study by the authors of the 2012 pig/macaque study we started this post with, infected NHPs did not pass EBOV to uninfected NHPs only 30cm away.[21] Not only was there no disease in the inoculated animals but no antibodies were detectable in the uninfected NHPs 4-weeks later. There had been no infection at all.

While at some point we'll need to be more sure of all this for humans than we are now, we can say that pigs aren't primates and airborne route has not been shown to be a risk for human acquisition of an EBOV.

References..

1. http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/ebola-eng.php#note21
2. http://www.ncbi.nlm.nih.gov/pubmed/9244332
3. http://www.ncbi.nlm.nih.gov/pubmed/18637412
4. http://www.ncbi.nlm.nih.gov/pubmed/9988155
5. http://www.ncbi.nlm.nih.gov/pubmed/20181765
6. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3398796/pdf/nihms390624.pdf
7. http://vet.sagepub.com/content/50/3/514.long
8. http://www.nature.com/srep/2012/121115/srep00811/full/srep00811.html
9. http://www.ncbi.nlm.nih.gov/pubmed/21651988
10. http://jid.oxfordjournals.org/content/179/Supplement_1/S87.long
11. https://www.sciencenews.org/article/airborne-transmission-ebola-unlikely-monkey-study-shows
12. http://www.vox.com/2014/8/10/5980553/ebola-outbreak-virus-aerosol-airborne-pigs-monkeys/in/5712456
13. http://www.nature.com/srep/2014/140725/srep05824/full/srep05824.html
14. http://www.ncbi.nlm.nih.gov/pubmed/23202456
15. http://www.ncbi.nlm.nih.gov/pubmed/8551825
16. http://www.ncbi.nlm.nih.gov/pubmed/7547435
17. http://www.mdpi.com/1999-4915/4/8/1305/htm
18. http://www.ncbi.nlm.nih.gov/pubmed/8712894
19. http://www.ncbi.nlm.nih.gov/pubmed/15588056
20. http://www.ncbi.nlm.nih.gov/pubmed/20553340
21. http://www.ncbi.nlm.nih.gov/pubmed/25059478

Ebola virus may be spread by droplets, but not by an airborne route: what that means

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Rhinovirus transmission by aerosol and lower respiratory tract disease after inoculation

In the next instalment to answer the question posed in last week's post, we also find that rhinovirus can a lower respiratory tract infection (LRTI), if it is delivered directly to the site; several issues around this topic are contentious in current age of PCR diagnosis of lower respiratory tract disease using specimens from the upper respiratory tract (URT).

From Thomas R. Cate et al, Am J Epidemiol.

Author: Thomas R Cate et al
Journal:  Am J Epidemiol 81(1):95-105
Year: 1965
RV type used: NIH 1734 (RV-A15¹)
RV receptor type: major group; ICAM-I

This study set out to investigate the impact of RV on the lower respiratory tract.

Key features of the study layout..

• 16 healthy adult male inmate volunteers
• Safety-tested preparation of RV-15
• 6 volunteers given 1ml nasopharyngeal serum-inactivated virus via a hand atomizer (coarse droplets expected to mainly deposit in the upper respiratory tract), and 1ml instilled intransally by pipette with subject lying on back
• 8, RV-15-antibody-free volunteers were exposed to 10l of air (16, 20 or 66 TCID₅₀ RV15), via a mask, containing 15-second-old 0.2-3.0um particles generated from a Collison atomizer (see Figure)
• A number of re-inoculations were also performed on each virus-delivery group
• Aerosols was also sampled using a Shipe impinger (this device contained cell culture medium onto which some aerosol was impacted) for virus isolation, after storage at -70°C. These data determined the dose that had been used
• Prior (2-days) to inoculation, nasal, pharyngeal and anal swab specimens and 10ml of nasopharyngeal wash (NPW) were collected, frozen at -20°C for testing to identify pre-existing viruses or bacteria (all culture based). The same specimen types were collected after inoculation (minus the anal swab). RV culture was conducted on human embryonic fibroblast cultures, with rotation at 33°C)

Key results included...

• Only 1 other virus, apart form RV-15, was found in the subjects. Culture may have missed fastidious or unculturable respiratory viruses (like the RV-Cs) however.
• During the 1st week after inoculation, usually starting from day-2..
• NPWs contained culturable virus in at least 1 specimen from 8/8 subjects
• During the 2nd week after inoculation..
• 7/8 subjects gave virus-positive NPWs
• During the 3rd week after inoculation..
• 5/8 subjects gave intermittent virus-positive NPWs
• Maximal virus titre aligned in time with most severe illness
• Nasal and pharyngeal swabs specimens did not yield virus as often as NPWs
• All subjects had a rise in antibody titre of 4-fold or greater, indicating infection, by 3-weeks with a further bump after 4-5-weeks
• Tracheobronchitis was diagnosed in 6/8 antibody-free aerosol-inoculated volunteers. This is a lower respiratory tract disease.
• Signs and symptoms included cough (sometimes in fits), substernal chest pain,, wheezing, tender trachea.
• 3 had a primary diagnosis of tracheobronchitis , the other 3 also had a prominent coryzal illness (nasal obstruction/discharge, sneezing, sore throat, swollen neck lymph nodes). 
• Fever was determined in 5/8, within the 1st 1-2-days.
• Signs and symptoms lasted for 1-4 days, a little longer for a rhinitis-alone
• No tracheobronchitis developed among 31 antibody-free volunteers inoculated through a course spray/drop method into the nasopharynx
• No infection (no suitable rise in antibody) or illness was detected among 6 volunteers inoculated with a preparation of virus that had first been inactivated by incubation with an antibody-positive serum. This identified that there were no other viruses/bacteria in the preparation that could have caused the disease. This had been, infrequently, found in other preparations by the authors so this step was important part of their comprehensive approach.
• 4-weeks later, 2 volunteers from the aerosol infection group, 2 from the inactivated virus group, and 2 new volunteers, were (re-)inoculated
• No infection, illness or virus shedding resulted in the aerosol pair
• No illness but infection and shedding occurred in the pair previously inoculated with inactivated virus
• Infection, illness and shedding were apparent in the new volunteer pair
• Neutrophil counts were significantly raised in aerosol-inoculated volunteers at illness onset and also, but to a lesser extent, in the 6 volunteers given inactivated virus. This explains to me why in those with a predisposition to severe RV outcomes, including those with asthma, a symptomatic RV infection is not necessary to trigger an attack.

The authors concluded...

• The aerosols generated here, which carried relatively small amounts of virus, would likely travel beyond the nasopharynx and tracheobronchial tree and be carried into the lungs, probably with <50% deposited and the remainder exhaled
• No evidence of pneumonia was found
• If RV is suitably aerosolized in sufficiently small particles, inhalation can result in lower respiratory tract disease while site-specific installation into the upper respiratory tract usually results a typical URTI or "common cold"

How do these findings translate to everyday exposures to RV coughs and sneezes and in children? In the general community we are constantly exposed to virus and have a complex, person-specific panoply of antibodies resulting from different infections beginning in childhood. This is probably why we are incapacitated by bad colds and LRTIs all the time! An addendum in the discussion of Cate's paper highlights how symptoms resulting from RV infection are best considered as part of the entire spectrum of possible outcomes. 

Previous symptomatic infection, as shown above, protects from lower respiratory tract disease hence adults are less likely to have LRTIs than children who see these viruses for the first time. Also, there is literature showing that the antibody to some RVs can protect against, or moderate, disease due to infection by other RVs. If you are antibody-free, then disease can potentially be more severe.

Cate's studies are all conducted without knowledge of the 50+ RV-Cs because they could not be grown (detected) using the cells employed by the culture methods of the day. Why is that relevant? Because some consider RV-Cs to be more asthmagenic/pathogenic and because we don't know the receptor or natural tissue tropism/distribution of the RV-Cs in humans. How the RV-Cs perform in human volunteer infections is unknown.

Certainly room remains for some new research building upon excellent studies like this one by Cate et al and highlighting (a) that RV can infect the lungs and cause disease if an aerosol is encountered and (b), that one outcome from RV infection does not fit all.

Further reading and references...

1. First HRV nomenclature assignment publication
   http://www.nature.com/nature/journal/v213/n5078/pdf/213761a0.pdf

Rhinovirus (RV) transmission by aerosol: does it happen or is transmission solely by hand-contact and self-inoculation?

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