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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Liberia gave Ebola the boot...and a virus may soon be removed from the wild

The people of Liberia have earned our respect, some time for national celebrations and frankly any other rewards that may flow from denying the Makona variant of Ebola virus any hosts among their community. 

The world considered this viral species to be one of the list-toppers when it came to ranking the causes of the most scary acute infectious diseases. Ebola virus has been the basis for all sorts of 'end-of 'the-world' mutating virus horror movies, books, and TV shows. It's not at all surprising that the public view of an Ebola virus infection had long been one of blood, fear and terror.

Figure 1. The decline of the Makona variant of
Ebola virus in Guinea, Sierra Leone and Liberia
(now free of EVD transmission).
Click on image to enlarge.

Behavioural change was a major factor in reducing virus transmission in Liberia. Alongside that was a broad range of aid given from within and beyond Africa's nations. By working together, a widespread outbreak that was not initially thought likely to happen at all, was routed. 

For now. 

Liberia is not immune to new cases of Ebola virus disease (EVD) crossing its borders or popping up due to a new animal-to-human jump (a zoonosis). That could happen any day - it might be happening now. But those who are still on watch will be searching out new cases while the remaining sites of transmission - Guinea and Liberia - do their best to deny Ebola virus a chance to replicate and spread. The people of Liberia will keep watch help because they have learned very tough lessons about viruses, epidemiology and communication. At least 10,604 suspect, probable and confirmed EVD cases, 4,769 deaths and way too many stories of sadness and families destroyed are a very strict teacher. 

Figure 2. The number of confirmed EVD
cases (yellow) grinds to a standstill. Only
9 cases in the week to 10th May 2015.
Click on image to enlarge.

The crude prediction in Figure 1 suggests that zero cases across all three countries could happen at the end of May, but many stars must align for that to be a real event. 

Human factors - the causal and sustaining variables of any outbreak of infectious disease in humans and sometimes animals - remain very much in play. But once that tri-country zero case value is attained, we have 42 days of watching and waiting - from the time the final case tests negative. 

New cases may arise from sources as-yet-unknown. But even if they do keep popping up, it seems very unlikely that widespread transmission will amplify to earlier levels (see the steep slopes in Figure 2) unless a major lapse in attention occurs. Hence,the need for continued vigilance - and Liberia remains on alert for a further 90 days. That more recent figure comes about because we know that infectious Ebola virus can persist in some body sites for many weeks after signs of disease have passed. Whether that virus reservoir is present in every person and whether it actually does cause new Ebola virus infections remain unproven. When you consider what can happen when one person gets infected by an Ebola virus in a tiny remote village in a country that is ill prepared to cope with it and has traditions that lend themselves to its spread...even minor risks rightly come under more intense scrutiny.

What next for this particular virus though? The only place where the Makona variant of this member of the Zaire ebolavirus species will soon exist, is in the freezer of (hopefully) very biosecure laboratories in the US, UK, Africa, Russia, China and probably other laboratories in countries that hosted, evacuated or repatriated cases of EVD. 

There is no sign at all - and this is because of the continued efforts and focus of many currently working throughout west Africa - of the fabled "endemic Ebola" becoming a reality. Unless you mean enzootic 'Ebola'- in which case , it already is, I suspect. It seems very, very likely that the forests of west Africa continue to shelter animal hosts with less mutated versions of this and other ebolaviruses (and filoviruses and who-knows-what else). The host species and route(s) of transmission to humans are yet to be confirmed but for now, we are not too far off eradicating one unwanted viral scourge from the wild. Impressive what we can do when we pull together.

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Bats in a tree...

Meliandou and the burnt tree that
once housed a bat colony (from Fig 3, [1]).

While not snakes on a plane, I'm fairly sure the level of swearing has at times been at least as bad among those suffering from and dealing with the possible fall-out from these bats - if in fact they were the source for the biggest Ebola virus disease (EVD) epidemic on record.

A recent animal counting, trapping and testing study in Guinea included sampling in and around the village of Meliandou.[1] This village is, to the best of our knowledge, the site of the first animal-to-human, or zoonotic, transmission of the Ebola virus variant called Makona.[2]

The study team, made up of researchers affiliated with Germany, Sweden, Core d'Ivoire and Canada, did not find any decline in numbers of usually susceptible larger mammals around the index village; a sign during other outbreaks, of active local ebolavirus "activity". The team also found that primate hunting was not a big thing in this region, which is rather devoid of these and other Ebola virus mammalian host animals (including few of the Duiker, or forest antelope). Fruit bat hunting was common though.

The team captured 169 bats representing at least 13 different species and 6 families. But in the house of the 2-year old boy considered the epidemic's index case, fruits bats were not eaten and no bat hunters resided there. No Ebola virus RNA was detected in any bats and antibody screening results from bat blood were inconclusive. 

These findings led the authors to study Meliandou, resulting in an hypothesis that a nearby hollow tree that once housed a large colony of free-tailed bats [locally described as lolibelo - small and smelly bats - otherwise known to belong to the species of insectivorous bat, Mops condylurus of the family Molossidea; [3], may have been the source of  infection. Why only one child was infected this way when the tree was a site of frequent play by many children is not known. The tree was burned out in March 2014 which caused many bat deaths, some of which were collected for consumption. Sequencing of a PCR-amplified mitochondrial DNA segment found that in 5 of 11 ash and soil samples from around the tree, contained traces of Mops condylurus genetic material. So that species was at least there.

So, this is all quite far from a conclusive link between the 2-year old boy and these bats. But it does read as though every avenue has been tested in this village, perhaps apart from better animal antibody testing (serology), and some serology on the blood of those villagers who remain alive in Meliandou. 

Serology testing is going to be very important for answering many questions around EVD and this outbreak and epidemic. 

Of course this will raise the usual question of whether we cull all bats to prevent this from ever happening again. Don't be ignorant! Bats have very important roles in pollinating and thus in keeping our ecosystem going. Should we kill all bees because they sting us? I'm pretty sure I've been stung by a bee more times than I've had Ebola/Hendra/SARS/Nipah/MERS/Lyssavirus or any other bat-hosted virus infection. Killing off everything to prevent a very rare zoonotic event when better knowledge can resolve the problem is just a typically short-sighted and knee-jerk human reaction (not a fan-can you guess?).

One question that does still remain, and one that is of extreme interest to me, is how often mild disease results from an Ebola virus infection? Good, robust serology methods to the rescue.


References...

1. Investigating the zoonotic origin of the West African Ebola epidemic. EMBO Molecular Medicine(2014). http://embomolmed.embopress.org/content/embomm/early/2014/12/29/emmm.201404792.full.pdf
2. Nomenclature- and Database-Compatible Names for the Two Ebola Virus Variants that Emerged in Guinea and the Democratic Republic of the Congo in 2014. Viruses 2014, 6(11), 4760-4799.
   http://www.mdpi.com/1999-4915/6/11/4760
3. Mops condylurus via the IUCN Red List of threatened species (listed as of least concern)
   http://www.iucnredlist.org/details/full/13838/0

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Stuff from the literature: very SARS-like coronavirus in Chinese horsehoe bats...

The smoking bat for SARS-CoV?

Xing-Yi Ge and colleagues from China, USA, Australia and Singapore described some new severe acute respiratory syndrome (SARS)-like coronaviruses (SL-CoVs) in bats, publishing in Nature last month.

These discoveries were especially notable (not that any new virus discovery isn't) because they displayed more "SARS-like" properties than many earlier so-called SARS-like CoVs. One could grow in the same line of lab cells and also in human cells, it could be visualized by electron microscopy and it could use the same receptor as the SARS-CoV (angiotensin converting enzyme II; ACE2) . Plus, they were genetically very similar.

The bat species was confirmed by gene sequencing to be Rhinolophus sinicus, family Rhinolophidae; the Chinese rufous horseshoe bat.

Throat and faecal samples (anal swabs and faeces) were screened using RT-PCR with primers towards the conserved RNA-dependent RNA polymerase region (RdRp) and new primers were designed to detect other regions of any discoveries. 27 of 117 samples were CoV POS and had sequences determined.

Two novel (and 5 previously identified) SL-CoVs, each with a 29,787+ base pair RNA genome and sharing 95% nucleotide identity with the Tor2 strain of the SARS-CoV which is higher than previous SL-CoVs from China. The receptor binding domain (RBD) of the new CoVs shared 85-96% amino acid identify with the SARS-CoV. These were called:

1. RsSHC014
2. Rs3367

Vero cells were used to attempt growth of SL-CoV virions that were first concentrated from samples. This succeeded for one sample, a variant of Rs2267 (99.9% nucleotide identity with Rs3367) and they named this isolate SL-CoV-WIV1. This success is something that hasn't been achieved with the majority of recently identified bat CoVs.

WIV1 also grew, although less efficiently, in:

• human alveolar basal epithelial (A549) cells
• pig kidney (PK-15) cells
• R.sinicus kidney (RSKT) cells

...but not in...

• Human cervix (HeLa) cells
• Syrian golden hamster kidney (BHK21) cells
• Myotis davidii kidney (BK) cells
• Myotis chinensis kidney (MCKT) cells
• Rousettus leschenaulti kidney (RLK) cells
• Pteropus alecto kidney (PaKi) cells

So we have much more convincing evidence that the SARS-CoV is likely to have originated from a bat.

h/t to @MERS_inSAUDI

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Prof Lipkin: There is no more sequence coming from that bat sample

Many thanks to Prof Ian Lipkin's indulgence of my eMail questions.
Also, check out the TWiV webcast by Prof Lipkin.

So, I guess to carry on from last night's post....I stand surprised. 

Not even next-generation sequencing could pull any more sequence from the MERS-CoV-positive T.perforatus bat samples that thawed after the dry shipper (not a box+dry ice as I previously guessed, but a vacuum sealed vessel previously brought to -150�C then all free liquid nitrogen removed for transport; shipped by FedEx) was opened and the cold chain interrupted after arriving from the Kingdom of Saudi Arabia (KSA). 

According to Prof Lipkin, in an email exchange we had last night, the group also tried a couple of runs of next-gen sequencing.

..we tried two separate ion torrent runs with no joy.

So why was only 1 October 2012 sample positive for the MERS-CoV strain? Prof Lipkin concludes that..

..the concentration of template was already extremely low in the sample at the time of field collection and lower still at the time of arrival in our Center. I would not be surprised if two aliquots of the same sample yielded different results in different labs. However, we will never have an opportunity to know because there is no more sample to test.

At the time of receiving the October samples (no MERS-CoV was found in the April samples), no viral gene/gene fragment/genome cloning had been done in Prof Lipkin's lab. A common potential source of PCR contamination ruled out. 

How does your group know that this 182 basepair nucleotide sequence was not a contaminant from somewhere else? 

The one sample came up positive repeatedly with the same assay. No other sample did so. We have recovered no other fragments that correlate with a MERS-like CoV in samples collected in the October 2012 or in the subsequent April 2013 field collections.

Antibodies were not sought in the massive 10�l of bat blood obtained per bat (the bats were released after sampling). But do these findings exclude the possibility that other bats, like those from genus Pipistrellus and Neoromicia (both from the family Vespertilionidae), or genus Nycteris, family Nycteridae, may be a host for MERS-CoV? At a World Health Organisation meeting in Cairo, Prof Lipkin told the the audience that..

...our findings don't exclude the presence of virus in a Vesper bat and that we were doing everything anyone suggested to test alternative explanations, including reagent contamination. We went back to the original materials using every specific and consensus primer set we and others had designed until all of the original material was exhausted. The results were the same. I sat on these data for months hoping to find another positive bat in subsequent field expeditions where we could report more sequence.

The decision to report it now was multifactorial. First and foremost, we tested every possible alternative explanation for the sample coming up positive other than that this fragment is bona fide-we can't find an alternative explanation. Second, there are no other reports from animals in KSA-I discussed phylogenetic analyses with several people in light of what was found elsewhere in Africa in Vesper bats. This fragment, although short and located in the RdRp is informative....Third, the scientists who did the work in the US and the Ministry of Health of KSA wanted to see it reported. There is a point where one has to get the work out in the public domain.

What's next in the search for animals hosting this virus and in trying to confirm what the group has just reported? There will also be a new European collaborative report (UK and KSA) coming out very soon that has new human MERS-CoV sequences suggesting multiple human introductions (animal to human?) with much more sequence variation in the MERS-CoV genome than we have seen thus far. This will further support the conclusion that the T.perforatus CoV is one and the same virus as that which infects humans.

...field expeditions should begin in the next few months and we will look again. The amount of time and resource invested already is far more than intended. I've never put in so much to recover so little.

Thank you to Prof Lipkin. This gives a some valuable insight into his careful efforts to deduce what animals may host a MERS-CoV strain,m as the first step in tracking how humans in the KSA are getting infected. It also highlights that finding even a basic piece of information requires many steps, lots of people, much effort and some luck. But if virus hunting was easy, everyone would do it right?

Some slight editing for brevity, and to account for mobile phone thumbs, was undertaken by VDU.

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MERS-CoVs: South African bats vs Saudi Arabian bats

The latest sign of MERS-CoV in an animal, the Taphozous perforatus bat, is based on a 181 basepair (bp) fragment amplified from the viral RNA collected from a bat's droppings. 

The sequence is not yet available on the public sequence database, GenBank, and I haven't asked Prof Lipkin et al. for it. In the meantime though, I've aligned the primers mentioned in the new Emerging Infectious Diseases article by Memish and et al., against a full genome of MERS-CoV (EMC, the Munich strain). Sorry the image doesn't come out perfectly-if you click on it it will expand to the size of your browser.

Click then expand browser for full size. The expected position of the Memish et al. Taphozous perforatus bat MERS-CoV sequence is shown as a grey box. Primer locations for the nested RT-PCR are shown as red (outer primers) and orange (inner primers; the sequence region depicted in the phylogenetic tree in the recent EID paper) boxes. The recent South African bat CoV relative of MERS-CoV is show in pink (not overlapping) and the same region of full length CoV genomes are shown in blue (MERS-CoV EMC Munich) and green (HKU5 bat CoV)

For fun (yeah, I should get out more) I wanted to see just how close the "Close Relative of Human Middle East Respiratory Syndrome Coronavirus in Bat, South Africa" was, as described from another recent EID paper, to the new bat CoV. '

Unfortunately, as you can see above, the two fragments don't overlap. So my fun is ruined! 
We do know from yesterdays article however, that the 181bp fragment was 100% identical to human MERS-CoV over this short span (about 0.6% of the length of the entire MERS-CoV EMC genome). 

As Prof Andrew Rambaut noted to Helen Branswell in the Vancouver Sun, we need a whole genome to get more information that will better place the T. perforatus into the clade of viruses that seem related to MERS-CoV.

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Taphozous perforatus - The Egyptian Tomb Bat

Rage of Taphozous perforatus.
Image from the IUCN Red List, via Wikipedia

This furry little fella '(~10cm long, 6cm forearm, 34cm wingspan and weighing in at 28g) occurs  throughout northern and sub-Saharan Africa, the Arabian peninsula and Asia, east to India. 

It's name, "tomb bat" comes from the genus name Taphozous which is derived from the Greek word for tomb/grave (Taphos). Also, males don't have beards like T. hildegardeae males do...apparently...just in case you meet one in a dark alley.

It is an insectivorous bat (moths and beetles) found in small colonies that avoid forest and preferring open woodland along rivers and wooded savanna. It roosts under rocks (e.g. sea caverns, deep caverns, old wells, tunnels,) or in buildings (e.g. old disused structures, castles, forts,mosques) during the day.

This bat is a member of the Order Chiropetera, Family Emballonuridae, Genus Taphozous, Species T.perforatus.

Specific countries where the bat has been found include: Benin, Botswana, Burkina Faso, The Democratic Republic of the Congo, Djibouti, Egypt, Ethiopia, Gambia, Ghana, Guinea-Bissau, India, Iran, Israel, Kenya, Mali, Mauritania, Niger, Nigeria, Oman, Pakistan, Saudi Arabia, Senegal, Somalia, Sudan, Tanzania, Togo, Uganda, Yemen, Zimbabwe

This bat is a threatened species.

Some more information, and the references:

• http://www.iucnredlist.org/details/21463/0
• http://books.google.com.au/books?id=sdY_sa1FPw0C&pg=PA50&lpg=PA50&dq=egyptian+tomb+bat&source=bl&ots=Jm_q9QKssF&sig=0hB56REa0KnXTsZWDhv_MZp8Qlc&hl=en&sa=X&ei=TUcVUvzXCY2jkQW0qIDQCA&ved=0CGwQ6AEwCg#v=onepage&q=egyptian%20tomb%20bat&f=false
• http://www.iucnredlist.org/details/21463/0
• South Asian Mammals,Their Diversity, Distribution, and Status Srinivasulu and Srinivasulu. http://link.springer.com/book/10.1007/978-1-4614-3449-8/page/1
• http://en.wikipedia.org/wiki/Mauritian_tomb_bat
• Dengis, Carol A. (May 17 1996). "Taphozous mauritianus". Mammalian Species (American Society of Mammalogists) 522: 1�5
• Colket and Wilson. Taphozous hildegardeae, Mammalian species, No. 597. p1-3. 

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Time for the bat signal? The need for an animal model for Middle East respiratory syndrome coronavirus.

Elizabeth Devitt notes in Nature Medicine, that unlike its cousin, the severe acute respiratory syndrome coronavirus (SARS-CoV), some important features of MERS-CoV including its transmission, incubation period, and ability to spread systemically within the host, have not been able to be defined for the MERS-CoV using non-human models, because the virus does not like to infect the same animals. 

When the MERS-CoV infects a larger non-human animal, the rhesus macaque monkey, the disease it produces, while still defined as pneumonia and proving the casual link between MERS-CoV infection and disease, resolved faster and was not as severe as that in humans. These animals are also not easy to work with. I wonder if older monkeys with comorbidities have been looked at in particular? [UPDATE: The macaques above live to about 25-years]. It is this population in which MERS is most severe. Nonetheless, the monkey studies provide an excellent vehicle on which to test the usefulness of 2-drug an antiviral approach (Falzano et al, described earlier) that can clear MERS-CoV infections in vitro.

While cell/tissue culture methods using primary human airway cells have proven extremely useful for looking at cellular biology, antiviral effects, and immunobiology related to MERS-CoV infection, something with legs will be needed for future vaccines and to address the list above. We've seen many examples of how animal models massively improve our understanding of influenza virus pathogenesis, if an example is needed.

Also according to Devitt, Ian Lipkin is still wading through the data from samples collected from a range of animals that may be the natural hosts for the MERS-CoV in Saudi Arabia. Meanwhile, we recently learned of another CoV (PML/2011) found in the fecal pellets from a South African Neoromicia cf. zuluensis bat in 2011. PML/2011's nearest CoV relative was the MERS-CoV - its closest viral relative found to date (at least in the conserved RdRp region used by the authors).

This all begs the question, is there a bat animal model? CoVs, but also studies of other viruses like Hendra and Nipah, would benefit from a well-defined model based on these critters. That is, if they can be worked with and if they show any signs of these diseases - which they may not. My very quick skim of the literature found that bats used for neurological studies and for Hendra virus.

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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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