How many viruses on Earth?

EarthHow many different viruses are there on planet Earth? Twenty years ago Stephen Morse suggested that there were about one million viruses of vertebrates (he arrived at this calculation by assuming ~20 different viruses in each of the 50,000 vertebrates on the planet). The results of a new study suggest that at least 320,000 different viruses infect mammals.

To estimate unknown viral diversity in mammals, 1,897 samples (urine, throat swabs, feces, roost urine) were collected from the Indian flying fox, Pteropus giganteus, and analyzed for viral sequences by consensus polymerase chain reaction. This bat species was selected for the study because it is known to harbor zoonotic pathogens such as Nipah virus. PCR assays were designed to detect viruses from nine viral families. A total of 985 viral sequences from members of 7 viral families were obtained. These included 11 paramyxoviruses (including Nipah virus and 10 new viruses), 14 adenoviruses (13 novel), 8 novel astroviruses, 4 distinct coronaviruses, 3 novel polyomaviruses, 2 bocaviruses, and many new herpesviruses.

Statistical methods were then used to estimate that P. giganteus likely harbor 58 different viruses, of which 55 were identified in this study. If the 5,486 known mammalian species each harbor 58 viruses, there would be ~320,000 unknown viruses that infect mammals. This is likely to be un under-estimate as only 9 viral families were targeted by the study. In addition, the PCR approach only detects viruses similar to those that we already know. Unbiased approaches, such as deep DNA sequencing, would likely detect more.

Let’s extend this analysis to additional species, even though it might not be correct to do so. If we assume that the 62,305 known vertebrate species each harbor 58 viruses, the number of unknown viruses rises to 3,613,690 – over three times more than Dr. Morse’s estimate. The number rises to 100,939,140 viruses if we include the 1,740,330 known species of vertebrates, invertebrates, plants, lichens, mushrooms, and brown algae. This number does not include viruses of bacteria, archaea, and other single-celled organisms. Considering that there are 1031 virus particles in the oceans – mostly bacteriophages – the number is likely to be substantially higher.

Based on the cost to study viruses in P. giganteus ($1.2 million), it would require $6.4 billion to discover all mammalian viruses, or $1.4 billion to discover 85% of them. I believe this would be money well spent, as the information would allow unprecedented study on the diversity and origins of viruses and their evolution. The authors justify this expenditure solely in terms of human health; they note that the cost “would represent a small fraction of the cost of many pandemic zoonoses”. However it is not at all clear that knowing all the viruses that could potentially infect humans would have an impact on our ability to prevent disease. Even the authors note that “these programs will not themselves prevent the emergence of new zoonotic viruses”. We have known for some time that P. giganteus harbors Nipah virus, yet outbreaks of infection continue to occur each year. While it is not inconceivable that such information could be useful in responding to zoonotic outbreaks, the knowledge of all the viruses on Earth would likely impact human health in ways that cannot be currently imagined.

Update 1: I neglected to point out an assumption made in this study, that detection of a PCR product in a bat indicates that the virus is replicating in that animal. As discussed for MERS-CoV, conclusive evidence that a virus is present in a given host requires isolation of infectious virus, or if that is not possible, isolation of full length viral genomes from multiple hosts, together with detection of anti-viral antibodies. Obviously these measures cannot be taken for a study such as the one described above whose aim is to estimate the number of unknown viruses.

Update 2: We discussed this estimate of mammalian viruses on TWiV #249.

TWiV 247: Today’s weather in virology

On episode #247 of the science show This Week in Virology, Ian Lipkin joins Vincent, Alan, Rich, and Kathy to describe how his laboratory is searching for the origin of MERS-coronavirus.

You can find TWiV #247 at www.microbe.tv/twiv.

Part of MERS-CoV nucleotide sequence found in a bat

What is wrong with this paragraph from today’s New York Times that describes the origin of the Middle East Respiratory Syndrome (MERS) coronavirus:

Health officials confirmed Wednesday that bats in Saudi Arabia were the source of the mysterious virus that has sickened 96 people in the Middle East, killing 47 of them.

Here is the science behind that statement, which has been published in Emerging Infectious Diseases. Samples (fecal, fecal swab, throat swab, blood) were collected from bats in an area of Saudi Arabia where human infections with MERS-CoV have been identified. Total nucleic acids were extracted and analyzed for the presence of coronavirus sequences by polymerase chain reaction. Coronavirus sequences were amplified from 220 of 732 fecal samples and 7 of 91 rectal swab samples or fecal pellets. One PCR product obtained from a single bat sample (fecal pellet of a T. perforatus bat captured in October 2012 in Bisha) had 100% nucleotide identity to a human MERS-CoV isolate.

A single PCR product 190 nucleotides in length from one bat was a perfect match with the genome sequence of a MERS-CoV isolate.

No infectious MERS-CoV has yet been isolated from this single bat. Therefore it is not yet possible to say that bats are the source of virus causing the MERS-CoV outbreak. As I have written previously, a virus is very different from a viral sequence.

It is certainly possible that MERS-CoV originated in a bat. Bats are known to harbor many viruses, and of course the SARS coronavirus originated in bats. But there is more than one explanation for the presence of this short viral sequence in bats. Perhaps the virus (or viral sequence) was obtained when the bat ingested a meal. Perhaps the 190 nucleotides are from a recombinant virus that is not MERS-CoV. I can think of other reasons why bats might not be the source of MERS-CoV.

For these reasons I believe that it is inaccurate for ‘health officials’ and the New York Times to confirm that bats are the source of MERS-CoV. Additional work is clearly needed to show that T. perforatus is the source of MERS-CoV, including isolation of infectious virus from bats and demonstrating infection of bats by the presence of antibodies to the virus. The work is in clearly progress; indeed the results might even be known, but they are not included in the Emerging Infectious Diseases article on which the NY Times piece was based.

Update 1: The term ‘frag-virus’ was proposed in 2008 to indicate viruses known only from sequence data. Although the term never caught on, the short article points out the problems that arise when genomic fragments are used to identify new viruses :

Although unintentional, these reports may mislead the readership of scientific journals and the general press. Having no distinction between preliminary genome-based evidence and conclusive proof by biological isolation and characterization of a replication-competent virus blurs the meaning of new virus.

Update 2: A phylogenetic analysis of the DNA fragment amplified from T. perforatus has been carried out. The author writes that “although this fragment means a very close relative of the human MERS-CoV is found in a bat geographically close to the first case, the fact it is identical in this short region doesn’t mean that these bats are the direct source of the human case.”  I would add even more uncertainty because we have no evidence that the virus was replicating in this single bat.

TWiV 239 – Filterable camels

On episode #239 of the science show This Week in Virology, Matt joins Vincent, Alan, and Rich to summarize what we know and what we do not know about the MERS coronavirus.

You can find TWiV #239 at www.microbe.tv/twiv.

TWiV 231: Hepaciviruses and pegiviruses in bats and rodents

On episode #231 of the science show This Week in Virology, Vincent meets up with Amit, Lan, and Ian to discuss their discovery of hepaciviruses and pegiviruses in bats and rodents.

You can find TWiV #231 at www.microbe.tv/twiv.

No further evidence of novel coronavirus

disease timelineThere is no evidence for further spread among humans of a novel coronavirus recently isolated from two individuals with severe respiratory illness. This conclusion has been drawn after scrutinizing the travels (figure) and contacts of a Qatari adult who was transferred to intensive care in London.

While in Saudi Arabia the 49 year old male patient developed mild respiratory illness (rhinorrhea and fever). These symptoms resolved several days after his return to Qatar on 18 August. At the beginning of September he developed another respiratory illness which worsened and required his transport to London. Later that month the novel coronavirus was detected in his respiratory tract.

This timeline suggests that the patient acquired the viral infection in Qatar, as he was there for 16 days prior to illness. How he obtained the infection is not known. He did spend time on a Qatari farm where sheep and camels are kept. The SARS coronavirus is believed to have originated in bats and spread to humans either directly or through animals in meat markets, and the new coronavirus is related to bat coronaviruses.

Sixty-four close contacts were identified among the patient’s health care workers, friends, and family during his stay in the United Kingdom. None of these have developed severe disease, while 13 have displayed mild respiratory symptoms, and the new coronavirus was not detected 10 of these individuals.

These results show that no human to human transmission of the novel coronavirus has taken place that resulted in mild or severe disease. Serological testing for anti-viral antibodies must be done to determine if asymptomatic infections have occurred. It will also be important to conduct serological surveys to determine whether there is evidence for infection in the general populations of Qatar and Saudi Arabia. It is also likely that animal surveys will be done to identify potential reservoirs for the virus.

RG Pebody et al. 2012. The United Kingdom public health response to an imported laboratory confirmed case of a novel coronavirus in September 2012. Eurosurveillance, Volume 17, Issue 40.

Update: A third human infection with the novel coronavirus was confirmed on 4 November 2012 in Saudi Arabia.

A new coronavirus isolated from humans

Coronavirus virionA new coronavirus has been isolated from two individuals with severe respiratory illness. It is different from the SARS coronavirus, but health officials are nonetheless preparing for a rapid response should the virus be detected elsewhere.

The novel coronavirus was first reported by Ali Mohamed Zaki on ProMED-mail on 15 September 2012, from a 60 year old male patient in Saudi Arabia with pneumonia and acute renal failure who died in July. The virus was isolated by culturing sputum on Vero and LLC-MK2 cells, and identified as a coronavirus by polymerase chain reaction. Dr. Zaki sent the virus to Ron Fouchier in the Netherlands who sequenced its genome and confirmed that it is a beta-coronavirus closely related to bat coronaviruses.

At the beginning of September 2012 a 49 year old male Qatari national who had previously traveled to Saudi Arabia was admitted to an intensive care unit in Doha with severe respiratory illness. He was moved to the United Kingdom where laboratory tests confirmed the presence of the novel coronavirus. Comparison of a 200 nucleotide genome sequence with that from the Saudi national revealed 99.5% identity (one mismatch). Alignment of this sequence with that of other coronaviruses shows that the new virus is related to bat coronaviruses.

This new virus is not the SARS coronavirus, but because it is related to bat coronaviruses there is concern that it could spread rapidly among humans and cause serious respiratory disease. This is why WHO has placed health officials in its six regions on alert, and has issued a case definition so that the disease may be readily detected. The definition comprises: acute respiratory syndrome which may include fever (≥ 38°C , 100.4°F) and cough requiring hospitalization or with suspicion of lower airway involvement (clinical or radiological evidence of consolidation) not explained by any other infection or any other aetiology; and close contact within the last 10 days before onset of illness with a probable or confirmed case of novel coronavirus infection while the case-contact was ill, or travel to or residence in an area where infection with novel coronavirus has recently been reported or where transmission could have occurred.

Ron Fouchier doesn’t believe that we should become overly worried about these cases:

There are now six known human coronaviruses; one of them is SARS, but four cause the common cold and are quite innocuous. So let’s keep both feet on the ground and not blow this out of proportion.

The fact that the virus has been isolated from individuals with severe respiratory disease does not mean that it is the causative agent. To prove this requires additional work, as Fouchier notes:

 For starters, we’ll find out whether animals get sick from this virus. You can isolate a virus from a patient, but that does not mean they died from it; to show that it causes disease you need to fulfill Koch’s postulates. That’s what we did for SARS, and it’s what we hope to do here; we’ve applied for emergency ethical approval. The most obvious animal species to put this virus in are mice, ferrets, and perhaps monkeys.

Proof that the new coronavirus is an agent of respiratory disease would come from its isolation from additional patients with the disease. An outbreak of severe respiratory disease in Jordan in April of 2012 is now being reviewed for evidence of the novel coronavirus.

Coronaviruses are composed of enveloped virions that contain a positive strand RNA genome. Human coronaviruses may cause the common cold or severe respiratory illness. In 2002 the SARS coronavirus emerged in China and spread globally, infecting over 8000 individuals and killing more than 900. The SARS coronavirus is believed to have originated in bats and spread to humans either directly or through animals in meat markets. Because the new coronavirus isolated from two patients is related to bat coronaviruses, there is concern that a scenario similar to the SARS outbreak is in the making. Whether or not this is true will be revealed in the coming weeks.

Update. Eurosurveillance has published communications on how to detect the novel coronavirus by real-time polymerase chain reaction; and the case definition and public health measures. The authors conclude:

There is strong evidence that a novel virus caused the severe disease in the two patients. Based on this assumption it can be concluded that the virus poses an as yet poorly defined level of threat to people’s health. There may have been other cases in the past that were missed and serological testing of stored sera and other specimens from such cases will be important. […] Our assessment, based on the limited information currently available, is that the risk of wide spread transmission resulting in severe disease is low. However, the emergence of a novel coronavirus requires a thorough assessment which is currently being coordinated at international level.

Update 2. CDC has published a travel advisory:

At this time CDC, does not recommend that travelers change their travel plans.

TWiV 183: Bats out of hell

On episode #183 of the science show This Week in Virology, Connor Bamford joins the TWiV team to discuss bats as hosts for major mammalian paramyxoviruses.

You can find TWiV #183 at www.microbe.tv/twiv.

Bats harbor many viral sequences

How large is the zoonotic pool – all the animal viruses that could one day infect humans? Assuming that there are 50,000 vertebrates on earth, each with 20 viruses, the number is one million – probably a vast underestimate. Determining just how many viruses exist in a variety of animal species is technically feasible, limited only by the number of hosts that can be sampled. A study of the virome of several North America bats reveals that these animals – which constitute 25% of all the known mammalian species – harbor a very large collection of viral sequences.

Advances in nucleotide sequencing technologies (deep sequencing) have made it possible in recent years to study the virome – the genomes of all viruses in a host – in human blood, diarrhea, and respiratory secretions; grapevines, and feces of horses and bats. The latter mammals are a particularly important subject because it is known that they harbor the predecessors of several important human viruses, including SARS coronavirus, ebolavirus, marburgvirus, Nipah, Hendra, and rabies viruses. Since there are about 1200 known species of bats, the potential for future human zoonoses is significant.

A multicenter group comprising virologists* and chiropterists (scientists who study bats) has examined the virome of three North American bat species: big brown bats (Eptesicus fuscus), tri-colored bats (Perimyotis subflavus), and little brown myotis (Myotis lucifugus). Deep sequencing was used to analyze fecal and oral samples from 41 bats captured on one night in Western Maryland. The results provide a comprehensive glimpse of the bat virome.

The 576,624 sequence ‘reads’ (a read is the result of a single sequence reaction, in this study ~250 nucleotides) on six pools of fecal samples revealed an amazing diversity of viral sequences (figure), with representatives of human, other mammals, insect, bacteriophage, fish, shrimp, protist, reptile, plant, avian, fungi, algae, and marine viruses. Among the interesting findings are sequences of three novel coronaviruses from big brown bats. Many coronaviruses have previously been found in bats; the results of this study provide more evidence that these mammals are likely to be sources of future human infections.

Novel viruses of plants and insects were also identified in fecal samples from all bats. This observation can be explained by the bats’ prodigious appetite for insects, which harbor both insect or plant viruses (the latter transmitted to plants by insect vectors). It seems unlikely that these viruses replicate in bats, but pass through the gastrointestinal tract into the feces.

Perhaps not surprisingly (given the diversity of bacteria that colonize mammalian intestinal tracts), sequences of novel bacteriophages were also identified in fecal samples. One appears to have high sequence identity with a bacteriophage that infects the plague bacterium Yersina pestis. Curiously, such bacteria are not known to colonies animals in the northeastern United States. A second bacteriophage infects strains of E. coli associated with human illnesses. These findings suggest that bats could be involved in the spread of human bacterial pathogens.

The main viral sequences identified in pooled oral samples were of a novel cytomegalovirus. These viruses appear to be common in bats, and have been been detected in many previous studies.

One question that arises from these findings is whether bats are unique in harboring a large collection of diverse viruses. The answer to this question awaits studies of the viromes of other wild animals.

Viral discovery by massive sequencing will no doubt identify many new viruses in a wide range of species. However, this technology cannot answer some of the more intriguing biological questions, such as which hosts support viral replication, and whether it is associated with disease. Answers to these questions will require construction of complete DNA copies of viral genomes and recovery of infectious viruses by transfection of cells in culture.

The title of this post is ‘Bats harbor many viral sequences’, not ‘many viruses’. That’s because no infectious viruses were identified – only parts of their genomes. If you wish to conclude that a certain virus infects bats, you must either isolate the virus in cell culture, or show that the entire viral genome is present in tissues or fluids.

*including Eric F. Donaldson and Matt Frieman, who spoke about this work on TWiV 90 and 65.

Donaldson EF, Haskew AN, Gates JE, Huynh J, Moore CJ, & Frieman MB (2010). Metagenomic Analysis of the Virome of three North American Bat Species: Viral Diversity Between Different Bat Species that Share a Common Habitat. Journal of virology PMID: 20926577

TWiV 90: Guano happens

Hosts: Vincent RacanielloAlan Dove, Rich Condit, and Eric F. Donaldson

On episode #90 of the podcast This Week in Virology, Vincent, Alan, Rich and Eric discuss identification of viruses in Northeastern American bats, vaccinia virus infection after sexual contact with a military vaccinee, and identification of a new flavivirus from an Old World bat in Bangladesh.

Click the arrow above to play, or right-click to download TWiV #90 (64 MB .mp3, 89 minutes)

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