Evidence that Reston ebolavirus resides in live bats in the Philippines...

Update #1 18JUN2015

Jayme and colleagues find some
"smoking bats"-possible bat reservoir
species for Reston ebolavirus
in the Philippines.

In what I think is only the second example of this, a new collaborative study from Jayme and a team of eminent researchers in the Philippines, Australia, Vietnam and the United States, have reported the finding of Reston ebolavirus (RESTV) viral RNA and antibodies to viral infection in a range of different bat species....some more "smoking bats" - bats with more than just past evidence, sometimes considered vague and unreliable, of an ebolavirus being hosted by the animal.

The finding of RNA is not the same as actual infectious virus, but RNA is a very specific marker for the virus nonetheless. And the authors note that they didn't want to kill the bats so only a small volume of sample was available-not enough for culture.

Leroy and colleagues had previously reported finding Zaire ebolavirus RNA and antibodies against this species of virus in Hypsignathus monstrosus, Epomops franqueti and Myonycteris torquatebats, all fruit-eating megabats of the family Pteropodidae. These are considered to be important reservoir hosts, yet they do not show signs of disease.[2] 

According to one of the authors on the latest study, bats in the Philippines also seemed clinically well...

@MackayIM all bats we sampled appeared clinically healthy.

� Jonathan Epstein (@EpsteinJon) July 17, 2015

Locating the Philippine RESTV sequences
on the ebolavirus phylogenetic tree.
Jayme et al. Virology J. (2105) 12:107.[1]

Jayme's findings are important to the story of RESTV importations to animal facilities in the United States from the Philippines which occurred multiple times between 1989 to 1996. These fed into the dramatized retelling we know of as The Hot Zone. There were also signs of antibodies to the virus in humans working with infected non human primates in the Philippines in 1994, 1996 and 2008.

The amount of viral RNA in most of the bats was quite low - but was usually repeatably detectable. I'm a firm believer in PCR giving a specific signal when there is something specific present to detect (assuming it was done in a professional laboratory setting that reduces the risk of false positives-which it was in this instance). So low viral loads are not no viral loads.

RESTV RNA was repeatably found in oropharyngeal swabs taken from bats assigned to the following species:

• Cynopterus brachyotis (Lesser dog-faced fruit bat; range; fruit bat)
• Miniopterus australis (Little long-fingered or little bent-wing bat; range; insectivorous bat)
• Miniopterus schreibersii (Schreiber's Bent-winged Bat; range; insectivorous bat)

...and in one sample from:

• Chaerephon plicata (Wrinkle-lipped Free-tailed Bat; range; insectivorous bats)

What's particularly interesting to me is that some of these bat species are found in Australia. However, keep in mind that the range of some (?many) bats may be underestimated. The example here is using the IUCN Red List's described range for M. schreibersii-apparently it's a bat that inhabits an area around the Mediterranean.[4] Last I looked, the Philippines is a bit south of there. In the past, as Wikipedia lists, a much bigger range was ascribed to this bat, also including Australia,[5] Guinea, Liberia and Sierra Leone - among many others. Looks like there may be lots of work to do in the area of bat census.

Jayme and colleagues also sampled the blood of 61 flying foxes (of the fruit-eating bat family Pteropodidae) and antibodies were found by ELISA and Western blot in 3 Acerodon jubatus (giant golden crowned flying fox; range) bats and by ELISA alone in a Pteropus vampyrus (Large flying fox; range). If you trust the test, then this indicates past exposure.

Superman and the Joker know very well - Bats can be very tricky. But at least this finding helps to further address the Riddle(r) of the reservoir. Now, if only we could only nail down the specific culprit(s) in West Africa.

References...

1. Molecular evidence of Ebola Reston virus infection in Philippine bats
   http://www.virologyj.com/content/12/1/107?utm_content=bufferf022a&utm_medium=social&utm_source=twitter.com&utm_campaign=buffer
2. Fruit bats as reservoirs of Ebola virus
   http://www.nature.com/nature/journal/v438/n7068/abs/438575a.html
3. Many details about bats to be found at the excellent IUCN Red List
   http://www.iucnredlist.org/
4. Population Structure of a Cave-Dwelling Bat, Miniopterus schreibersii: Does It Reflect History and Social Organization?
   http://jhered.oxfordjournals.org/content/100/5/533.full
5. Seasonal movements of the Schreibers� bat, Miniopterus schreibersii, in the northern Iberian Peninsulahttp://www.tandfonline.com/doi/abs/10.1080/11250000801927850#.Vamrtvnzp1M

Updates...

1. Added bat specie range data (and discussion) from IUCN Red List and Wikipedia.

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Ebola virus: wild and domestic animals, plants and insects...

Initial Ebola virus (EBOV) infection of humans is a rare zoonotic spillover event.  

Hypsignathus monstrosus, Epomops franqueti and Myonycteris torquatebats, all fruit-eating megabats of the familyPteropodidae, are considered to be important reservoir hosts, yet they do not show signs of disease.[1] 

While a great deal remains unknown about the identity and spectrum of natural ebolavirus hosts,[1] zoonoses appear to co-occur with bat pregnancy.[2]

Animals that have died from ebolavirus infections include:[3,4]

• Duiker (Cephalophus sp.; an antelope) 
• Gorilla (Gorilla gorilla) 
•  Chimpanzee (Pan troglodytes)

Living animals found to harbour ebolavirus RNA include:[1,4,23]

• Cynomolgus macaque monkey (Macaca fascicularis; RESTV) 
• Franquet�s epauletted fruit bat (Epomops franqueti; EBOV) 
• Hammer-headed bat (Hypsignathus monstrosus; EBOV) 
• Little collared fruit bat (Myonycteris torquata; EBOV)

Those animals with only antibodies to EBOV in the absence of infectious virus, suggesting past exposure include:[5,6]

• Domestic dogs (Canis lupus familiaris) 
• Peter�s lesser epauletted fruit bat (Micropterus pusillus; fruit-eating) 
• Angolan free-tailed bat (Mops condylurus; insect-eating) 
• Giant roundleaf bat (Hipposideros gigas; insect-eating) 
• Egyptian fruit bat (Roussetus aegyptiacus; fruit-eating) 
• Geoffrey�s rousette (Rousettus amplexicaudatus; a bat species; fruit-eating) 
• Lord Derby�s scaly-tailed squirrel (Anomalurus derbianus)

Porcupines (Hystrix cristata) have been implicated as a source for human EBOV exposure but virus-positive animals have not been documented.[4] 

Between nine and 25% of 337 domestic dogs from various towns and villages in Gabon during an EBOV outbreak in 2001-2002 were identified as possible hosts for EBOV when found to be seropositive.[7,8] It was not known when they became seropositive nor has it been experimentally determined that dogs are able to host an active EBOV infection.[9,10] Dogs were observed in contact with suspected virus-laden fluids and with other animals during the Gabon outbreak but seropositive dog specimens did not contain EBOV antigen or viral RNA. Three specimens from these seropositive dogs did not yield infectious virus in cell culture either and thus there remains no documented evidence for a canine source of human EBOV infection. In 2014, two dogs owned by human cases of EBOV/Mak in Spain (euthanized without testing [11]) and the United States of America (tested negative for EBOV[12,13]) did not exhibit any signs of disease. 

Domestic pigs have been found to be a natural host for the Reston ebolavirus[9,14] and antibodies to EBOV have also been found in guinea pigs, an animal that can also be experimentally infected.[15] Domestic dogs and guinea pigs appear to become infected without symptoms.[6,7] Horses, mice, guinea pigs and goats have been experimentally inoculated with EBOV to produce antisera or test therapeutic preparations.[16,17] 

Pigs experimentally infected with a member of the Zaire ebolavirus become symptomatic.[8] NHP, guinea pigs and mice have been used to examine aspects of disease progression and exhibit various degrees of disease when experimentally infected.[18,19] 

On a few occasions in one study into possible hosts, a low viral load of EBOV could be sporadically recovered after inoculation of a snake (up to 11 days post inoculation), a mouse (up to nine days later) and a spider (21 days later) but the authors of this study concluded that these results could have represented residual inoculum.[21]

Plants, arthropods, cows, cats and sheep have not been found to naturally carry or host ebolavirus infection but only small numbers of some species have been examined.[3,20-22]

References...

1. Leroy EM, Kumulungui B, Pourrut X, et al. Fruit bats as reservoirs of Ebola virus. Nature 2005;438:575-6. 
2. Plowright RK, Eby P, Hudson PJ, et al. Ecological dynamics of emerging bat virus spillover. Proc Biol Sci 2015;282:20142124.
3. Olson SH, Reed P, Cameron KN, et al. Dead or alive: animal sampling during Ebola hemorrhagic fever outbreaks in humans. Emerg Health Threats J 2012;5
4. Lahm SA, Kombila M, Swanepoel R, Barnes RF. Morbidity and mortality of wild animals in relation to outbreaks of Ebola haemorrhagic fever in Gabon, 1994-2003. Trans R Soc Trop Med Hyg 2007;101:64-78.
5. Marsh GA, Haining J, Robinson R, et al. Ebola Reston virus infection of pigs: clinical significance and transmission potential. J Infect Dis 2011;204 Suppl 3:S804-9.
6. Gonzalez JP, Herbreteau V, Morvan J, Leroy EM. Ebola virus circulation in Africa: a balance between clinical expression and epidemiological silence. Bull Soc Pathol Exot 2005;98:210-7.
7. Allela L, Boury O, Pouillot R, et al. Ebola virus antibody prevalence in dogs and human risk. Emerg Infect Dis 2005;11:385-90.
8. Weingartl HM, Nfon C, Kobinger G. Review of Ebola virus infections in domestic animals. Dev Biol (Basel) 2013;135:211-8.
9. Stansfield SK, Scribner CL, Kaminski RM, Cairns T, McCormick JB, Johnson KM. Antibody to Ebola virus in guinea pigs: Tandala, Zaire. J Infect Dis 1982;146:483-6.
10. Connolly BM, Steele KE, Davis KJ, et al. Pathogenesis of experimental Ebola virus infection in guinea pigs. J Infect Dis 1999;179 Suppl 1:S203-17.
11. Why Dallas Won't Kill The Dog Of The Texas Nurse With Ebola. Business Insider, 2014. (Accessed 27/4/2015, at http://www.businessinsider.com.au/what-will-happen-to-dallas-nurses-dog-2014-10 )
12. Starting today, Dallas Animal Services will begin testing Nina Pham�s year-old dog Bentley for Ebola. The Dallas Morning News, 2014. (Accessed 17/4/2015, at http://thescoopblog.dallasnews.com/2014/10/starting-today-dallas-animal-services-will-begin-testing-nina-phams-year-old-dog-bentley-for-ebola.html/.)
13. EBOLAVIRUS, ANIMAL RESERVOIR (05): USA, DOG, NOT. 2014. (Accessed 01/05/2015, at http://promedmail.org/direct.php?id=20141026.2901733 )
14. Barrette RW, Metwally SA, Rowland JM, et al. Discovery of swine as a host for the Reston ebolavirus. Science 2009;325:204-6.
15. Rouquet P, Froment JM, Bermejo M, et al. Wild animal mortality monitoring and human Ebola outbreaks, Gabon and Republic of Congo, 2001-2003. Emerg Infect Dis 2005;11:283-90.
16. Kudoyarova-Zubavichene NM, Sergeyev NN, Chepurnov AA, Netesov SV. Preparation and use of hyperimmune serum for prophylaxis and therapy of Ebola virus infections. J Infect Dis 1999;179 Suppl 1:S218-23.
17. Bray M, Davis K, Geisbert T, Schmaljohn C, Huggins J. A mouse model for evaluation of prophylaxis and therapy of Ebola hemorrhagic fever. J Infect Dis 1998;178:651-61.
18. Ebihara H, Takada A, Kobasa D, et al. Molecular determinants of Ebola virus virulence in mice. PLoS Pathog 2006;2:e73.
19. Geisbert TW, Young HA, Jahrling PB, Davis KJ, Kagan E, Hensley LE. Mechanisms underlying coagulation abnormalities in ebola hemorrhagic fever: overexpression of tissue factor in primate monocytes/macrophages is a key event. J Infect Dis 2003;188:1618-29.
20. Turell MJ, Bressler DS, Rossi CA. Short report: lack of virus replication in arthropods after intrathoracic inoculation of Ebola Reston virus. Am J Trop Med Hyg 1996;55:89-90.
21. Swanepoel R, Leman PA, Burt FJ, et al. Experimental inoculation of plants and animals with Ebola virus. Emerg Infect Dis 1996;2:321-5.
22. Ebola haemorrhagic fever in Sudan, 1976. Report of a WHO/International Study Team. Bull World Health Organ 1978;56:247-70.
23. Miranda ME, Ksiazek TG, Retuya TJ, Khan AS, Sanchez A, Fulhorst CF, Rollin PE, Calaor AB, Manalo DL, Roces MC, Dayrit MM, Peters CJ. Epidemiology of Ebola (subtype Reston) virus in the Philippines. J Infect Dis. 1999 Feb;179 Suppl 1:S115-9.

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The wind beneath my Ebola virus.... [UPDATED]

Only a couple of weeks ago the report in Science presented 99 genomes representing some of the thousands of those circulating in Sierra Leone this year.[1] I say thousands because each infected person has a range of subtley different viral variants among the billions of viruses per millilitre of blood that all compete to be the champion. The words "mutant" and "ebolavirus" are now hard to avoid. And of course as soon as you talk mutations, you can only see one endgame - a virus that is easily transmissible and turns us all into zombies. spreads across the world in a pandemic and kills as many as 80% of those it infects. Yes, its seems the proportion of fatal cases (PFC) in West Africa may not be as simply calculated as most of us were thinking these past months. When we take into account that Ebola virus disease deaths occur in people that were part of a case tally days earlier (if they were counted of course) when the total case numbers were smaller, the PFC inflates. How much, we don't really know.

And so the story of mutants was brought full circle today thanks to Dr. Michael T. Osterholm. In a very nicely written piece for the New York Times,[2] Dr. Osterholm, ventured behind the scenes to crack the door into the world of whispered discussions, shadowy frapp� meetings by chino and beige blazer-wearing figures, many of whom were men with with thinning hair. Yes, he found where the real virologists hang out and what were they discussing at length? Why they were talking about how soon it would be until Zaire ebolavirus was going to mutate and become an airborne killer virus identifiable only through watching big wall-mounted LCD screens as they are rendered in red because of the fusion of rapidly growing dots, spreading across a map of a world filling rapidly with infected hosts. Or red dots. Or something. Okay, some of that was from me.

Only problem is, I think he may have entered the tinfoil hat room next door to the (but very similarly attired) room full of virologists. Maybe not. Hard to tell sometimes. But seriously.

For sure, a virus changes over time. It will change randomly through mutations that happen because viruses, especially those with genes/a genome made of RNA, are always making errors in their gene/genome copying and sometimes those errors make the virus better at something. Viruses may hold on to those changes in response to all sorts of pressures on them. These genetic sequence changes sometimes results in change to the proteins that make their structures and enzymes. Sometimes the changes may revert back as pressures go away or new ones come to be. It's a constant micro-environment of change; evolution on fast forward.

We should also keep in mind ebolaviruses didn't come down in the last shower. They are viruses that are happy in their own envelopes...and natural host(s). But mutational changes can impact on how the viral "bits" assemble and release from the cell and perhaps on how the virus causes disease, where virus replicates in the body, how it interferes with the host immune system's attempts to interfere with it, how hardy it is, how well it replicates in response to temperature and so on. 

A virus doesn't "think" about any this of course. It doesn't plan to do the nastiest thing to us that we can imagine when it jumps into us from an animal (a zoonosis). Headlines might make you think otherwise. These changes happen because, in a new host species producing many subtley different viral variants all vying for supremacy, the virus with the mutation(s) that allow it to get out from under some sort of controlling pressure or to do something better than the earlier viral versions, wins the day. The winner thrives, makes more of itself or does it better, and passes to new hosts.

A virus may keep more of these mutational changes while it is "settling in" to a new animal host species if they help that process. It may be under more pressure to adapt to slightly different environments, different receptor structures, temperatures, immune responses - all sorts of things may created a different environment from the one the virus came from and so it may need to make use of more mutations in order to "find its footing". Or fail and not find a home in the new host.

There can be all sorts of new and negative pressures to try and avoid or adapt to for a virus. So ebolaviruses seem to naturally infect bats, not us, and in bats the infection does not seem to cause a whole lot of disease. Of course we don't know a whole lot about how bats spread virus among themselves. Perhaps they do it via an airborne route. The theory then goes that humans or other forest animals including chimpanzees, gorillas, porcupines and antelopes may eat the bats or bat/virus-contaminated fruit. We, and those animals, do get sick.


Another unsure thing, a sizable knowledge gap you might call it, is whether an ebolavirus would actually be under any pressure at all to keep the mutations that change its proteins, site of replication and disease course which result in it being:

• More stable in dried droplets
• Shed in higher concentrations from the upper respiratory tract
• Able to trigger more coughing or sneezing.

Each and all of these major changes might be necessary to create the mythical airborne Ebola virus. The outcome? Creation and propulsion of more droplets from an infected human, that dry down and linger in the air (the airborne part) while still containing infectious ebolavirus, and enough of it to result in human infection and disease. Phew. That is an unbelievable series of huge changes, even for a "sloppy" replicating RNA virus. 

I think we all understand that a virus doesn't "know" that these changes would provide better spreading outcomes and we now know that Ebola virus already spreads very well between bats and in humans (see the West Africa outbreak numbers which have not at any time been linked to a different or unusual spread of virus compared to any earlier outbreak[11]). To date, airborne spread has never been found to happen naturally in the dissemination of Ebola virus disease in humans. That is some kind of significant considering it does not take a lot of virus to start an infection through direct contact and considering there have been non-human primate transmission chains in the forests for a long time.

Each of those changes to the virus and the host's disease might happen by a series of accumulating mutations over time. But is their pressure to keep each of them? And really to be airborne, these changes would need to co-occur and do so without any trade-offs that meant the 'new airborne virus' was negatively impacted in some other area of its attachment, cell entry, replication, interference with the immune response enzymatic efficiencies etc. 

We do already know that in the lab, under laboratory conditions, with lab animals, lab equipment, plenty of lab-grown virus and a closed space with a lot of aerosol (probably some of which is wet droplets, not just droplet nuclei, meaning not truly airborne conditions), an Ebola virus can be forced to infect non-human primates. I've written about that previously.[3] 

And yet even when it was sought, no sign of such airborne infection has been found to occur naturally among humans. Direct. Contact. 

So I think it was a stretch to expend so many words on the chances of an airborne virus emerging rather than one that causes more bleeding, or less diarrhoea, or more vomiting, or more shedding in sweat, or having lower viral loads, more rash, more hiccups etc. 

Many additional things could result from mutational changes. We know next to nothing about the mutations recorded from the 99 genomes in Sierra Leone.[1] So why all this focus on one specific yet really quite complex outcome of viral air travel instead of many/any others? I don't know. But hey, now we have indeed been able to talk about this aspect some more, so good one Dr O! 

Others have come out to comment too. 

• Dr Anthony Fauci, director of the National Institute of Allergy and Infectious Diseases in the US noted that "it�s fundamentally unlikely"[9]
• Dr Amesh Adalja, University Pittsburgh said "it may not be the best path for the virus to take"[10]
• Dr Derek Gatherer of Lancaster University in the UK noted an airborne Ebola virus would need a "conjunction of coincidental, unlikely events" [10] 
• Dr David Heymann, Chair of the Health Protection Agency in the UK stated "No one can predict what will happen with the mutation of the virus", reminding us that "The virus's epidemiology is consistent with transmission via bodily secretions and excretions, which is exactly the same as other past epidemics".[11]
• Prof Vincent Racaniello, a virologist at the College of Physicians and Surgeons at Columbia University stated "We have been studying viruses for over 100 years, and we've never seen a human virus change the way it is transmitted."[12]
• Story at Scientific American by Dina Fine Maron
  "Is it, for example, growing faster or at higher viral concentrations than previous strains? But the jury is still out on this and other questions"[13]
  

Dr Osterholm did hit some other nails flush with the timber though. West Africa needs fewer promises to defeat Ebola virus and more plans that include actual rapid mobilization and on the ground experienced leadership to make inroads into getting beds for sick people and tracing contacts. Two key items on a long list of things to do better (in my opinion).

Thankfully some promising signs are appearing. The scope of this outbreak has now been guesstimated - 20k-100k cases.[4,5,6,7] I list this range rather than extending it to much higher levels [8] because I do still have hope that things will improve and interventions will turn the exponential case growth curves away from the sky and back to the horizon, sooner rather than before entire nations are destroyed. Because that's what is coming without successful intervention.

Money is being freed up and arriving from all over and more resources are slowly moving in to the region. Resources needed just to keep the people safe who have come from all over the world, including the nations of Africa, to care for the overwhelming numbers of sick and sickening people. Not to mention the money needed to prevent more infections. More specific and insistent pleas for defined numbers of healthcare professionals are also being broadcast. Drugs and vaccines are closer than they have ever been to use in humans. They may be our only hope to stop this virus.

If the many thousands of people predicted to die from a virus that is killing 4 in 5 confirmed cases (see the Medicins Sans Frontieres tweet below) is not enough reason for stopping the spread of Ebola virus, then stopping this particular evolving variant before it does change into something worse or more ingrained to the communities all over Africa and beyond, really is. 

While this Ebola virus variant may never make the changes necessary for it to go airborne, it has shown signs of relatively rapid change and that was relatively early in what looks to be a very long chain of human-to-human-to-human-to-human... transmission. Each person allowing the virus to adapt further, if that's what it needs to do.

Such a long transmission chain, from 1 animal>human infection, has never been recorded before and so we are indeed in new territory when it comes to the ebolaviruses. For now at least, the Ebola virus in 5 countries in and around West Africa has the upper hand. This tiny self-assembling unthinking, randomly mutating thing is totally dependent on our cells to replicate itself - and we are not doing enough to starve it of those cells. 

It clearly doesn't "need" to be airborne to spread efficiently.

#Ebola #MSF operates 5 Centres, 457 hospital beds. 2,615 patients were admitted, 1,408 confirmed & 342 survived �MSF pic.twitter.com/sADmn8Zdm3
� Stefano Zannini (@StefZannini) September 12, 2014

References...

1. http://www.sciencemag.org/content/345/6202/1369
2. http://www.nytimes.com/2014/09/12/opinion/what-were-afraid-to-say-about-ebola.html?smid=tw-share
3. http://newsmedicalnet.blogspot.com.au/2014/08/ebola-pigs-primates-and-people.html
4. http://currents.plos.org/outbreaks/article/estimating-the-reproduction-number-of-zaire-ebolavirus-ebov-during-the-2014-outbreak-in-west-africa/
5. http://currents.plos.org/outbreaks/article/obk-14-0036-early-epidemic-dynamics-of-the-west-african-2014-ebola-outbreak-estimates-derived-with-a-simple-two-parameter-model/
6. http://healthintelligence.drupalgardens.com/content/predicting-number-cases-ebola-virus-disease-outbreak-countries-widespread-and-intense
7. http://news.sciencemag.org/health/2014/08/disease-modelers-project-rapidly-rising-toll-ebola
8. http://www.dw.de/ebola-threatens-to-destroy-sierra-leone-and-liberia/a-17915090
9. http://time.com/3342305/airbone-ebola-not-happening/
10. http://news.yahoo.com/could-ebola-become-airborne-153701091.html
11. http://www.dailymail.co.uk/health/article-2753421/Real-risk-Ebola-virus-mutate-AIRBORNE-disease-expert-warns.html
12. http://www.vox.com/2014/9/19/6543157/ebola-is-unlikely-to-go-airborne
13. http://www.scientificamerican.com/article/fact-or-fiction-the-ebola-virus-will-go-airborne/

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Ebola virus in semen is the real deal.... [UPDATED]

The World Health Organization (WHO) Ebola virus disease factsheet notes that ebolaviruses may be transmitted via the semen of a male who is getting over an ebolavirus infection, for a period of 7-weeks (~49-days).[1] 

The European fact sheet for health professionals and a Public Health Agency of Canada Pathogen Safety Data Sheet both note the 7-week figure, the latter also adds a 61-day figure.[2,3] 

The United States Army Medical Research Institute of Infectious Diseases (USAMRIID) Medical Managements of Biological Casualties Handbook (7th edition) notes a 3-month (~80-days) period, during which one should probably avoid sexual relations so as not to deliver virus directly to a mucosal surface.[4]

Semen is therefore listed as one of the body fluids from which Ebola virus disease may be contracted. 

While convalescent patients seem to be discharged before 7-weeks have elapsed, I presume the men are made very aware of this risk. This was specifically noted in one of the studies below. [8] 

But I find it hard to just accept things. 

As a scientist I'm used to looking for the little bracketed or superscripted numbers or perhaps "(Scientist et al)", at the end of sentences. Then I can check out the information source for myself. So here, I thought I'd try and add those and pt it altogether in one place here - and you can do your own checking out if you feel the need. 

Here are the research papers I've found for EBOV so far (there are also Marburg virus studies) - by all means send me any others I've missed and I'll add them.

• Bausch and colleagues [5] were able to isolate, in cell culture in the laboratory, infectious Ebola virus (EBOV) from the semen of 1 of 2 samples from a single recovering patient who had EVD. 
• The sample was collected 40-days after disease onset; at 45-days he was no longer positive for EBOV
• No acute phase (active infections) samples were tested.
• 1 of 2 samples were also positive for EBOV RNA by RT-PCR (detecting a portion of the virus's RNA genome)
• Rodriguez and colleagues [6] could isolate infectious EBOV from seminal fluid 82 days after disease onset from a 27-year old male (also RT-PCR positive then). A sample at 51-days after onset was RT-PCR positive, but did not yield infectious virus.
• EBOV RNA , but not virus, in 3 other convalescent cases (33, 29 and 25-years of age) at times ranging from 57 to 101-days after disease onset.
• Rowe and colleagues [7], who examined the same patients, detected EBOV RNA by RT-PCR from 4 convalescent cases (27, 25, 29 and 33-years of age as above) at times ranging from 47 to 91-days after disease onset 
• No infectious virus could be isolated and no viral antigens were found
• Emond and colleagues [8] were able to isolate infectious EBOV from seminal fluid collected 39 and 61 days after disease onset
• No EBOV was isolated 76, 92 or 110-days later

So if you are a man who has been diagnosed with an Ebola virus infection and survived, please, seriously, take extra care to practice safe sex. Use a condom. Or, even safer, just wait.

References...

1. http://www.who.int/mediacentre/factsheets/fs103/en/
2. http://ecdc.europa.eu/en/healthtopics/ebola_marburg_fevers/factsheet-for-health-professionals/Pages/factsheet_health_professionals.aspx
3. http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/ebola-eng.php
4. http://www.usamriid.army.mil/education/bluebookpdf/USAMRIID%20BlueBook%207th%20Edition%20-%20Sep%202011.pdf
5. http://jid.oxfordjournals.org/content/196/Supplement_2/S142.full
6. http://jid.oxfordjournals.org/content/179/Supplement_1/S170.long
7. http://www.ncbi.nlm.nih.gov/pubmed/9988162
8. http://www.ncbi.nlm.nih.gov/pubmed/890413

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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

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Behind the naming of ebolaviruses... [UPDATE 2]

Just don't call me a taxi

Virus taxonomy is the classification of viruses into groups based on similarities. 

Today classification is supported by viral gene and genome sequence information.

The International Committee on Taxonomy of Viruses (ICTV) takes care of the official virus taxonomy. It has a pretty friendly website with a good search engine and the latest (2013 at writing) virus taxonomy can be found here. [1]

So what does it, and its tome, Virus Taxonomy, Ninth Report of the International Committee on Taxonomy of Viruses [2], have to say about ebolaviruses? Well, not as much as you might like, if you want to be able to name them, talk conversationally about them and discuss the issues around the disease resulting from infection by most of them. Sure, you can just call it all "Ebola" (which is a river in Africa by the way) and be done with it, but you'd be wrong. And some smart alec will correct you for sure. Here is where the ICTV Filoviridae Study Group fills in a lot of blanks.

One thing to get on top of first up. When talking or writing about these    looking little pathogens, we can just call lump them together under the conversational term "ebolaviruses" thus..."Hey Jeanette, did you hear about the latest ebolavirus infection numbers over the weekend?" is a question that could refer to any of the 5 very different viruses.

So, let's try and make that tearoom conversation a little more accurate.

The very dry detailed stuff down to the level of a species.

The italicization and the capitalization below are really important to taxonomy guys - so, ya know, care. 

Also, the ICTV reminds us that the name of the species is not the name of a virus - they are 2 different things. The species is a broad term for all the measurably different viruses out in the wild, that it contains. Here, species is to viruses what Mitsubishi Sigma is to identifying my old car. A virus name,

e.g. Ebola virus (see below), is like identifying my car as "a silver 1988 Mitsubishi Sigma". 

What if we used taxonomy on cars? A different way to explain how to name ebolaviruses.
Click to enlarge

The viruses we are talking about here belong to the order Mononegavirales, family Filoviridae, genus Ebolavirus. 

There are 5 species within the genus. The species names are in italics below. Underneath is the name of the virus (the virus belongs to the species container) and its abbreviation. The viruses in bold have caused outbreaks of human disease.

• Ta� Forest ebolavirus
• Ta� Forest virus (TAFV)
• Reston ebolavirus
• Reston virus (RESTV)
• Sudan ebolavirus
• Sudan virus (SUDV)
• Zaire ebolavirus
• Ebola virus (EBOV)
• Bundibugyo ebolavirus
• Bundibugyo virus (BDBV)

But what should I call the latest ebolavirus strain, variant, genotype, subtype, serotype, isolate thingy?

In the case of the current outbreak, that latest virus is an Ebola virus (EBOV), which we can now say belongs to the species Zaire ebolavirus (a Mitsubishi Sigma). 

But back to the car analogy. The silver 1989 Mitsubishi Sigma name is still not enough to tell it apart from any other silver Mitsubishi Sigma parked at the same shopping centre. How do you choose yours in a way that won't get you arrested for breaking into someone else's car? They both look like silver Mitsubishi Sigmas. But the silver Mitsubishi Sigma with license plate ABC 321 is yours and your alone, and that code differentiates your car from any other anywhere in the world.

We know from genome sequencing studies that the virus circulating in Guinea is an EBOV (a silver Mitsubishi Sigma) and is not identical to the one in (what was called) Zaire in 1976 (this silver Mitsubishi Sigma car has a different license plate). They couldn't be the same physical virus anyway, because each person hosts millions and millions of virions, each cell has a varied population of viruses in it, and each virion has a relatively short life. 

There is no universal definition for classification of viruses below the level of a species. But there are lots of terms that are used - most are listed in the heading to this section. In filovirus-land, the Study Group has sought to impart some order upon the chaos [3]. 

A virus strain needs to have 1 or more observable, genetically stable and unique differences compared to other viruses in the same species. For instance, one might cause a disease that is different from the one we know. So apart from a different license plate, it might also have an Awesome Mix #1 CD in the tray. 

From Kuhn et al.[5]
Viruses  2014, 6(11), 4760-4799Click on image to enlarge.

A variant has some genetic sequence or other differences that may result in a slightly different observable change. The West African EBOV is a variant and not a strain of Zaire ebolavirus and was named after the Makona river (see figure) which makes contact with all three countries that have had widespread and intense transmission.[5] For example, these from Guinea and Sierra Leone:

• Ebola virus H.sapiens-wt/GIN/2014/Makona-Kissidougou-C15, complete genome
• Ebola virus H.sapiens-wt/GIN/2014/Makona-Gueckedou-C07, complete genome
• Ebola virus H.sapiens-wt/SLE/2014/Makona-EM104

A virus isolate is a virus sample resulting from growing or culturing it in cells or tissues. Variants can therefore be represented by isolates. These isolates can be identical or slightly different (your neighbour could order the exact same car as you did-but he would still have a different license plate and no bobble-heads and fluffy dice).

The naming schemes do go into further detail, but you can read that in [3].

The disease.

The disease caused by EBOV, SUDV, TAFV and BDBV is called Ebola virus disease (EVD). Frankly, that is a tough one to explain after all of the above. It reads as though we are talking about just 1 virus causing disease (EBOV). But not so. Viruses from 4 species cause EVD - EBOV, SUDV, TAFV and BDBV. Diseases are named by World Health Organization's International Classification of Diseases (ICD) site, (4) and the name of this disease goes back many years and has not been updated yet. The disease has been called Ebola haemorrhagic fever, but is not now. Ebola virus disease is, by itself, a proper noun - that is its name - so it always gets the capital 'E'. And to continue from the taxonomy above, EVD is caused by a virus that can be ascribed to a species. In West Africa right now, EVD is due to infection by an Ebola virus variant classified in the species  Zaire ebolavirus.

Navigating a tree in the ebolavirus jungle.

Lastly, I've cobbled together a tree of genomes from each of the 5 ebolavirus species. It may help. Or not.

A phylogenetic tree of some genome sequences of the 5 species of ebolavirus, each indicated with a specific coloured dot.

References...

1. ICTV Virus Taxonomy: 2013 Release
   http://www.ictvonline.org/virusTaxonomy.asp
2. Ebola virus disease World Helath Organization fact sheet
   http://www.who.int/mediacentre/factsheets/fs103/en/
3. Virus nomenclature below the species level: a standardized nomenclature for natural variants of viruses assigned to the family Filoviridae.Arch Virol (2013) 158:301�311.
   http://download.springer.com/static/pdf/619/art%253A10.1007%252Fs00705-012-1454-0.pdf?auth66=1408093824_af9d701ab93574066469a6f6745c11d7&ext=.pdf
4. International Classification of Diseases (ICD)
   http://www.who.int/classifications/icd/en/
5. Nomenclature- and Database-Compatible Names for the Two Ebola Virus Variants that Emerged in Guinea and the Democratic Republic of the Congo in 2014
   http://www.mdpi.com/1999-4915/6/11/4760

Updates...

1. 05AUG2015: Added comment about Ebola virus disease being a proper noun

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