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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Dromedary camels are a host of MERS-CoV...

Yes. Not a "MERS-CoV-like" virus or "something very closely related to but slightly different" from MERS-CoV. Camels. Are. A. Host. 

There was already plenty of evidence to suggest this (see some of my previous posts on this linked below), and none to really dissuade me from thinking otherwise. And yesterday we saw a new paper by Ian Lipkin and his collaborating crew from King Saud University in Saudi Arabia that make this issue more obvious than ever. 

So let's stop messing around. There is an elephant in the MERS-room...and its a camel! 

The Middle East respiratory syndrome coronavirus, does in fact look to be a camel virus that causes few symptoms in that host, is acquired by young camels and has been for at least 22-years, and then people somehow get infected, probably from proximity to camels or due to habits involving camels. the keeping of camels or at gatherings in which grumpy slavering camels are congregating. 

Yes, there is little evidence for any contact with camels among the 186 human cases as Dr Ziad Memish, deputy health minister of the Kingdom of Saudi Arabia (KSA) points out, but as Prof Marion Koopmans noted to NPR, "few people [with MERS] have had the kind of follow-up you would want". So we take denial of any contact with a grain of salt.

There is definitely some contact however; some that is pretty solid. For example, the owner of a camel in Jeddah (4) that both tested positive for MERS-CoV or the Qatari farm camels and owner and an employee that were all MERS-CoV positive (5,7). 

And there is far less evidence against camels as a source of  some/many/most human cases and for anything else. 

So in the new paper in mBio published online 25-Feb, we read of the most comprehensive KSA camel study to date. Camels from 2013 were sampled and camel sera collected and frozen since 1992-2010 were tested and many were found to be MERS-CoV antibody positive. From the more recent dromedary camels, nasal swabs were also found to be viral RNA positive; RNA that is definitely from MERS-CoV.

Some key findings...

• No sheep or goats were MERS-CoV antibody or RNA positive; a routine finding now. Bovine CoV antibody reactivity was identified in these animals however, and in 17% of camels
• Antibody was detected using infected cells and also using a method that employs a specific portion of a MERS-CoV protein (part of the nucleoprotein)
• 150/203 (74%) of camels from all over the KSA had MERS-CoV antibodies in a pattern reminiscent of any endemic human respiratory virus
• 95% in camels older then 2-years (adults)
• 55% in those =2-years (juveniles)
• The south west had the lowest proportion of positive camels (5%)
• Higher proportions were found in central KSA (Riyadh)
• 3 rectal swabs were positive for MERS-CoV RNA using real-time reverse transcriptase polymerase chain reaction (RT-rtPCR). 2/3 camels were also nasal swab POS
• 36/104 juvenile camels were nasal swab POS
• 15/98 adult camels were POS
• 66% of samples from the west (Taif) were POS but none from the south west
• no RNA was detected in a sampling of camel blood/sera and so the archived samples from earlier years could not be sequenced to verify that they had MERS-CoV sequences in them
• Amplification and sequencing of a 1,044nt portion of the Spike gene, 2,004nt ORF1ab region found that less than 1% difference from previous published MERS-CoV sequences and the nucleocapsid gene region was identical. Great to see a move away from recent reliance on complete genome sequences and a more practical and rapid subgenomic, multi-target molecular epidemiology approach used. 
• 11/13 higher viral load samples could be amplified and sequenced

It was interesting hear the TWiV (this week in vrilgy) podcast interview with Prof Lipkin and Assoc. Prof Thomas Briese in which they noted:

• MERS-CoV is a "puny" virus causing little overt disease on camels
• The MERS-CoV genome seems to be fairly stable; its not influenza virus and does not seem likely to evolve rapidly
• Baboons, dogs, cats, rodents are on the list to test when the team return to KSA
• There has been a previous report on limited human antibody levels to MERS-CoV in at least the east of the KSA

Sources and previous posts on camels and MERS-CoV...

1. mBio paper by Alagaili and colleagues
   http://mbio.asm.org/content/5/2/e00884-14.full.pdf+html
2. NPR's Richard Knox: excellent story
   http://www.npr.org/blogs/health/2014/02/25/282136478/deadly-mers-virus-circulates-among-arabian-camels
3. CIDRAP Story
   http://www.cidrap.umn.edu/news-perspective/2014/02/study-mers-cov-may-have-been-saudi-camels-22-years-ago
4. Camels owner in Jeddah
   http://newsmedicalnet.blogspot.com.au/2013/11/camel-cough-coronavirus-caught.html
5. Two Eurosurveillance studies reporting MERS-CoV antibodies in camels
   http://newsmedicalnet.blogspot.com.au/2013/12/middle-east-respiratory-syndrome.html
6. MERS-CoV antibodies in 10-year old UAE camel sera
   http://newsmedicalnet.blogspot.com.au/2014/01/antibodies-in-10-year-old-uae-camel.html
7. More on the Qatari camels and some MERS-CoV sequencing and social media chatting
   http://newsmedicalnet.blogspot.com.au/2013/11/dutch-researchers-in-collaboraion-with.html
8. MERS-CoV antibodies in camels from the Canary islands and Oman
   http://newsmedicalnet.blogspot.com.au/2013/08/camels-carry-signs-of-coronavirus.html
9. Early cautionary thoughts from the WHO
   http://newsmedicalnet.blogspot.com.au/2013/08/who-urges-dont-put-camel-before-cart.html
10. Thoughts about MERS-CoV acquisition
    http://newsmedicalnet.blogspot.com.au/2013/09/most-mers-may-not-have-met-camel-but.html
11. Querying whether there is a better possible source for human cases
    http://newsmedicalnet.blogspot.com.au/2013/09/is-there-better-smoking-bat-or-camel.html
12. Summing up the first 100-days of (human) MERS-CoV infections
    http://newsmedicalnet.blogspot.com.au/2014/02/middle-east-respiratory-syndrome.html
13. MERS-CoV antibodies in camel sera dating back to 2005 in the UAE
    http://newsmedicalnet.blogspot.com.au/2014/01/mers-cov-antibodies-in-dromedary-camels.html
14. Gatherings and acquisition/transmission of MERS-CoV between animals and humans
    http://newsmedicalnet.blogspot.com.au/2014/01/a-date-with-middle-east-respiratory.html
15. Qatari camels clear the MERS-CoV from their systems
    http://newsmedicalnet.blogspot.com.au/2013/12/qatari-camels-clear-coronavirus.html
16. TWiV podcast
    http://www.twiv.tv/2014/02/25/twiv-special-mers-coronavirus-in-dromedary-camels/

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MERS-CoV genetic sequences found in Taphozous perforatus bat

Profs Ziad Memish and Ian Lipkin, and a team of collaborators including researchers from the EcoHelath Alliance, have published, in Emerging Infectious Diseases, their discovery of viral sequences in the faecal pellet of an Egyptian tomb bat.

Photo courtesy of Dr Jonathan H. Epstein.

Copyright EcoHealth Alliance 2013

MERS-CoV was only found in 1 of 29 Taphozous perforatus (Egyptian tomb bat, see some more detail on these in my next post) animals. These and 67 other bats captured in mist nets for this study, were observed nesting in abandoned ruins.

Samples from Bisha, Unaizah and Riyadh (Kingdom of Saudi Arabia) were snap-frozen on site, collected during October 2012 and April 2013. The October shipment was opened and thawed by US customs. Samples included wing biopsy, blood, throat swab, rectal swab and faecal pellets were collected for testing. Apart from RNA virus testing  bats were speciated by DNA analysis (cytochrome B gene). The T. perforatus bat identity could not be confirmed genetically because there was no reference sequence on GenBank - but it was similar to another member of the genus.

Helicase, RNA-dependent RNA polymerase (RdRp) and nucleocapsid or envelope regions were targeted for amplification and sequencing. 227/1003  samples (22.6%) were positive for an alpha or beta-CoV. 

The find, represented by a phylogenetic tree using on a 181nt RNA sequence fragment from the RNA-dependent RNA polymerase gene (100% identical to a sequence from the index case in Bisha, betaCoV 2c EMC/2012 over this region), secures bats as the/a primary animal source. So long as there was no contamination at customs or that the sequence actually came from a food source. Not too likely for either of those. 

Obviously more work will need to be done to find more instances, complete the genome (or at least sequence larger genetic fragments to make everyone happy) and isolate infectious virus - but this finding is a significant step in confirming a starting point for understanding how humans get infected by the MERS-CoV.

It's a shame this new fragment of the RdRp does not overlap with that sequence from the recent South African "nearest match" to MERS-CoV. In adjacent regions of the RdRp though, the South African virus does seems more genetically distant than this T. perforatus find.
 Perhaps we can re-visit the transmission chain issue with a view to how bats might infect a (probable) secondary host - say the camel for now - I'd suggest that palm trees might have a role in this as well as a possible role in direct human infections if sap/dates/drinks were consumed by the most at risk groups; elderly men with underlying conditions. Perhaps this consumption even has a role in them developing a chronic kidney-related disease? I previously wrote a little about this 19th June and on risk in a post 28th July.

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Camels carry signs of coronavirus contagion

Reusken and a European collaborative team have this morning described the first study looking for evidence of prior infection with the MERS-CoV, in animals. This evidence take the form of antibodies (immunoglobulin G or IgG ) made after the animal's immune system recognizes and then defends against future infection by that invader. 

The study used a very specific piece of the MERS-CoV Spike (S) protein. S is the bit of a CoV that sticks out and gives it the characteristic crown-like appearance under electron microscopy. The small piece of S acts as bait to detect the antibodies in serum samples and this interaction is identified by a fluorescent signal in the protein microarray system they used. 

These antibodies were found in the sera of 50 of 50 retired racing dromedary camels from Oman and from 1 in 7 Spanish (14 of 105) dromedary camels from the Canary islands. 

No antibodies were found in 80 cattle, 40 sheep, 40 goats or 34 other camelids.

The antibodies retained an ability to stop MERS-CoV infection in test that diluted the sera between 1:320-1:2560 for the Omani camels, and 1:20-1:320 for the Spanish camels 

Camels also had some signs of antibody reactivity to bovine coronavirus (BCoV), but the authors, after additional testing, concluded that the MERS-CoV reactivity was specific to that virus and not to BCoV. Sera from 2 human cases of infection by with the BCoV relative (both betacoronaviruses), HC0V-OC43, did not stop MERS-CoV from infecting cells - infection was not neutralized by the patient's HCoV-OC43 antibodies.

Camels were implicated earlier during the outbreak, in the death from MERS-CoV (then the "novel coronavirus" or nCoV) of a 73-year old male from Abu Dhabi, capital of the United Arab Emirates. 

"The patient owned racing camels. One of them got ill and was very weak; the patient was in close contact with that camel, and on the evening the camel got very sick, the patient developed flu-like symptoms. Three days later, he was in a medical unit in Abu Dhabi. There is another family member who also had close contact with the camel; he also got ill, but we could not follow up with that gentleman."

So this article points a finger at camels as some sort of host, possibly as an intermediate host between Pipistrellus spp and Rousettus aegypticus bats and humans. Perhaps the MERS-CoV story is akin to the Hendra virus story - bats contaminate horses and from there, close contact with horses can, on occasion  result in disease in humans. 

At the very least - there may be other animals involved yet and we still don't have viral RNA or a viral isolate from within a camel  - we now have a specific animal contact to track and trace for each human case. Perhaps specific risk avoidance measures can also be implemented, and the hotzones can communicate to their populations that close, perhaps any, contact with camels carries with it some risk of MERS-CoV infection  especially to be if you are in a category that places you at higher risk of severe outcomes from a MERS-CoV infection-older male, underlying conditions. 

Housing camels away from bats and areas known to be bat flyovers or frequented by feeding or birthing bats, or keeping camels under cover may all be helpful reduce transmission of the virus between these animals.

Jennifer Yang and Helen Branswell have breakdowns of this story as well.

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MERS-CoV numbers-where are we at?

As the dust settles from several days of new cases, and deaths and retrospective case identifications, I sit waiting for some caped crusader (no capes!) to step from the shadows and announce "I have the numbers you seek!" Okay, I'm a sucker for a caped crusader.
Alas, there are no such wonderful heroes to help fill the data gaps we lack among the MERS-CoV case data. There are plenty trying though. And so we watch the numbers climb, the cases spread, then contract (depending on which reliable source of information is speaking) and we wait for the likely spike in new cases due to the upcoming Hajj which, even with calls to reduce numbers, will likely go ahead as a mass gathering that puts MERS-CoV transmissibility to the test.

Sometimes we seem to hear a proposed new case or a death, and then we hear no more. 

Where is this virus coming from - animals, are older males with underlying conditions (and what precisely are all these conditions?) getting it from Pipistrellus sp. or perhaps Rousettus aegyptiacus bats via contamination of dates, date products of palm sap-derived drinks/alcohol? How can the world prepare, or understand whether it needs to prepare, for a novel virus when the region of its apparent origin (we don't know that either) has trouble sorting out whether members of its own populace are positive or not? A rough - what else can there be - count shows at least 23 dates of onset missing, 9 dates of death, 10 ages, 67 dates of hospitalisation and 11 sexes undefined for around 72 cases.

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That (don't call me novel) coronavirus is back!

Media reports, FluTrackers and Avian Flu Diary describe five recent deaths and two other critically ill cases under close watch in the Al-Ahasa region of the Kingdom of Saudi Arabia, linked to infection with the newly identified human coronavirus HCoV-EMC. This virus was first isolated in September 2012 from a 60M (60-year-old male) with pneumonia and renal failure in Jeddah, KSA. 

Further evidence for bats as a major source of CoVs came in a recent study in Emerging Infectious Diseases. Yang and colleagues identified a novel CoV from each of 2 bat species. 

The newly identified betacoronaviruses (betaCoVs), Bat Rp-coronavirus/Shaanxi2011 and Bat Cp-coronavirus/Yunnan2011 (rolls of the tongue doesn't it?) were not that closely related to human betaCoVs but resided in the bat verison of the SARS-like COVs.

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