TWiV 249: An inordinate fondness for viruses

On episode #249 of the science show This Week in Virology, Vincent, Dickson, Alan and Rich discuss an estimate of the number of different mammalian viruses on Earth.

You can find TWiV #249 at www.microbe.tv/twiv.

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 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.

Avian influenza H7N9 viruses isolated from humans: What do the gene sequences mean?

Influenza A virionThere have been over 60 human infections with avian influenza virus H7N9 in China, and cases have been detected outside of Shanghai, including Beijing, Zhejiang, Henan, and Anhui Provinces. Information on the first three cases has now been published, allowing a more detailed consideration of the properties of the viral isolates.

The first genome sequences reported were from the initial three H7N9 isolates: A/Shanghai/1/2013, A/Shanghai/2/2013, and A/Anhui/1/2013. These were followed by genome sequences from A/Hongzhou/1/2013 (from a male patient), A/pigeon/Shanghai/S1069/2013), A/chicken/Shanghai/S1053/2013), and A/environment/Shanghai/S1088/2013, the latter three from a Shanghai market.

Analysis of the viral genome sequences reveals that all 8 RNA segments of influenza A/Shanghai/1/2013 virus are phylogenetically distinct from A/Anhui/1/2013 and A/Shanghai/2/2013, suggesting that the virus passed from an animal into humans at least twice. Similar viruses have been isolated from pigeons and chickens, but the source of the human infections is not known. There is as yet no evidence for human to human transmission of the H7N9 viruses, and it seems likely that all of the human infections are zoonotic – transmission of animal viruses to humans. Since the H7N9 viruses are of low pathogenicity in poultry, infected animals may not display disease symptoms, further facilitating transmission to humans.

The RNA sequences reveal that the H7N9 viruses isolated from humans are all triple reassortants, which means that they contain RNA segments derived from three parental viruses. The gene encoding the hemagglutinin protein (HA) is most closely related to the HA from A/duck/Zhejiang/12/2011 (H7N3), while the NA gene is most similar to the NA gene from A/wild bird/Korea/A14/2011 (H7N9). The remaining 6 RNA segments are most related to genes from A/brambling/Beijing/16/2012-like viruses (H9N2). The type of animal(s) in which the mixed infections took place is unknown.

Some observations on the relatedness of these sequences:

  • A/Shanghai/2/2013, A/Anhui/1/2013, and A/Hangzhou/1/2013 were isolated in distant cities yet have over 99% identity. The pigeon, chicken, and environmental isolates are also very similar except for one gene of A/pigeon/Shanghai/S1069/2013. Long-range shipping of infected poultry might explain these similarities.
  • There are 53 nucleotide differences between A/Shanghai/1/2013 and A/Shanghai/2/2013. Perhaps A/Shanghai/1/2013 and the remaining viruses originated from different sources.

When the gene sequences of these human viral isolates are compared with closely related avian strains, numerous differences are revealed. The locations of the proteins in the influenza virion are shown on the diagram; click for a larger version (figure credit: ViralZone).

  • All seven H7N9 viruses do not have multiple basic amino acids at the HA cleavage site. The presence of a basic peptide in this location allows the viral HA to be cleaved by proteases that are present in most cells, enabling the virus to replicate in many organs. Without this basic peptide, the HA is cleaved only by proteases present in the respiratory tract, limiting replication to that site. This is one reason why the H7N9 viruses  have low pathogenicity in poultry.
  • All seven viruses have a change at HA amino acid 226 (Q226L) which could improve binding of the viruses to alpha-2,6 sialic receptors, which are found throughout the human respiratory tract. Avian influenza viruses prefer to bind to alpha-2,3 sialic acid receptors. This observation suggests that the H7N9 isolates should be able to infect the human upper respiratory tract (alpha-2,3 sialic acid receptors are mainly located in the lower tract of humans). However, viruses which bind better to alpha-2,3 sialic acids still bind to alpha-2,6 receptors and can infect humans.
  • All seven viruses have a change at HA amino acid 160 from threonine to alanine (T160A). This change, which has been identified in other circulating H7N9 viruses, prevents attachment of a sugar to the HA protein and could lead to better recognition of human (alpha-2,6 sialic acid) receptors.
  • Five amino acids are deleted from the neuraminidase (NA), the second viral glycoprotein, in all seven viruses. In avian H5N1 influenza virus this change may influence tropism for the respiratory tract and enhance viral replication, and might regulate transmission in domestic poultry. This change is believed to be selected upon viral replication in terrestrial birds.
  • One of the viruses (A/Shanghai/1/2013) has an amino acid change in the NA glycoprotein associated with oseltamivir resistance (R294K).
  • An amino acid change in the PB1 gene, I368V, is known to confer aerosol transmission to H5N1 virus in ferrets.
  • An amino acid change in the PB2 gene, E627K, is associated with increased virulence in mice, higher replication of avian influenza viruses in mammals, and respiratory droplet transmission in ferrets.
  • Changes of P42S in NS1 protein, and N30D and T215A in M1 are associated with increased virulence in mice, but these changes are also observed in circulating avian viruses.
  • All seven viruses have an amino acid change in the M2 protein known to confer resistance to the antiviral drug amantadine.
  • All seven viruses lack a C-terminal PDZ domain-binding motif which may reduce the virulence of these viruses in mammals.

For the most part we do not know the significance of any of the amino acid changes for viral replication and virulence in humans.

I believe that these H7N9 viruses might take one of two pathways. If they are widespread in birds, they could spread globally and cause sporadic zoonotic infections, as does avian influenza H5N1 virus. Alternatively, the H7N9 viruses could cause a pandemic. Influenza H7N9 virus infections have not occurred before in humans, so nearly everyone on the planet is likely susceptible to infection. Global spread of the virus would require human to human transmission, which has not been observed so far. Some human to human transmission of avian H7N7 influenza viruses was observed during an outbreak in 2003 in the Netherlands, but those viruses were different from the ones isolated recently in China. Whether or not these viruses will acquire the ability to transmit among humans by aerosol is unknown and cannot be predicted. If a variant of H7N9 virus that can spread among humans arises during replication in birds or humans, it might not have a chance encounter with a human, or if it did, it might not have the fitness to spread extensively.

What also tempers my concern about these H7N9 viruses is the fact that the last influenza pandemic (H1N1 virus) took place in 2009.  No influenza pandemics in modern history are known to have taken place 4 years apart, although only 11 years separated the 1957 (H2N2) and 1968 (H3N2) pandemics. I suppose that is not much consolation, as there are always exceptions, especially when it comes to viruses.

Meanwhile a vaccine against this H7N9 strain is being prepared (it will be months before it is ready), surveillance for the virus continues in China and elsewhere, and health agencies ready for a more extensive outbreak. These are not objectionable courses of action. But should this be our response to every zoonotic influenza virus infection of less than 100 cases?

Sources

Human Infection with a Novel Avian-Origin Influenza A (H7N9) Virus.

Genetic analysis of novel avian A(H7N9) influenza viruses isolated from patients in China, February to April 2013.

Did hepatitis C virus originate in horses?

Dog and horseAbout 2% of the world’s population is chronically infected with hepatitis C virus (HCV). This enveloped, positive-strand RNA virus was discovered in 1989, but serological and phylogenetic evidence indicates that it has been infecting humans for hundreds of years, perhaps as long ago as the 14th century. All human viral infections most likely originated in non-human species, but the progenitor of HCV is not known. Recent evidence suggests that horses might have been the source of HCV in humans.

For many years there were no known non-human relatives of HCV until canine hepacivirus was discovered in dogs (we discussed this virus on TWiV #137). However two subsequent studies failed to reveal additional evidence for CHV infection of dogs. In one study, no antibodies to CHV were found in sera from 80 dogs in New York State, and in a second study, PCR failed to detect CHV nucleic acid sequences in 190 samples from dogs in Scotland. Samples from rabbits, deer, cows, cats, mice, and pigs were also negative for CHV. However both groups found evidence for infection of horses. These viruses have been called non-primate hepaciviruses (NPHV).

In one study carried out on horses in New York State, 8 of 103 samples were found to contain antibodies to NPHV. Complete viral genomes were identified from all 8 horses. Most are genetically distinct from CHV, but one viral sequence, obtained from a pool of sera from New Zealand horses, is nearly identical to CHV. NPHV was also detected by PCR in sera from 3 of 175 Scottish horses. Separate serum samples obtained from one horse 5 months apart were positive for viral RNA, indicating persistent infection. None of the horses had any evidence of clinical hepatitis or any other illness.

These results from geographically distinct areas suggest that horses are a reservoir of NPHV. It seems likely that dogs might acquire NPHV infection from horses, as there are opportunities for contact between the two animals on farms or in kennels. Additional NPHV isolates from horses must be studied to confirm this hypothesis.

It will be important to determine if horse NPHV was the source of human HCV. This is theoretically possible because horse products, such as serum containing antibodies to pathogens or toxins, have been injected into humans. There are six genotypes of HCV, each of which is believed to have emerged at different times and geographic locations. Whether their emergence represent different cross-species transmissions, as is the case with the different groups of HIV-1, remains to be determined.

I also wonder how horses originally acquired NPHV. Perhaps it was transmitted to them from another species via a vector bite, such as a mosquito – but from what species?

Spillover and science communication

Spillover by David QuammenDavid Quammen, whose book Spillover was recently published, has been the recipient of a good deal of publicity in the past week. Last Wednesday he participated in a New York Academy of Sciences Symposium called ‘Wrath Goes Viral‘; on Saturday he was profiled in the New York Times (The Subject is Science, the Style is Faulkner), and yesterday Spillover was reviewed in the Sunday Book Review by Sonia Shah. Publicity for science is always good, but Shah identifies a key shortcoming of the book.

Shah notes that Spillover describes the “unfolding convergence between veterinary science and human medicine, and how veterinary-­minded medical experts discover and track diseases that spread across species”, detailing “Quammen’s prodigious, globe-trotting adventures with microbe hunters in the field, trapping bats in southern China and hysterical monkeys in Bangladesh”. But Quammen shies away from explanation, saying that he “would rather dazzle us with the difficulty of the science than help us comprehend it”:

He practically apologizes for having to describe fundamental concepts like the basic reproduction rate, or “R0” (the number of new infections caused by an initial case), critical community size (the number of susceptible individuals required to sustain transmission of an infectious disease) and the high mutation rate of RNA viruses. C’mon. Kate Winslet explained R0 in Steven Soderbergh’s film “Contagion” in 20 seconds. As “Spillover” so richly details, we’re talking about the potential end of the human race here. We can take it.

On page 305, before presenting an equation for R0, Quammen writes “There will be no math questions in the quiz at the end of this book, but I thought you might like to cast your eyes upon it. Ready? Don’t flinch, don’t worry, don’t blink”. I’m not fond of this approach. If you have read this blog or listened to any of my science podcasts, you know that I don’t believe that science needs to be dumbed down for a lay audience.

I’m not sure why Quammen shies away from the details. Perhaps he doesn’t feel qualified to explain science (he was an English major in college), or did not believe it was within the scope of the book (Walter Isaacson calls it a ‘masterpiece of science reporting’.) Fortunately, there are many other places online where you can learn the details of virology (see the sidebar of this blog for some examples), or for that matter, any type of science. Sadly, Quammen does not appear to be aware of any of these sources of good science.

I have a copy of Spillover on my desk and when I’m finished reading I’ll have more to say here, and perhaps also on TWiV. The good news is that he had a number of scientists read over the manuscript. At least Quammen doesn’t shy away from fact checking.

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.

Canine hepacivirus, a relative of hepatitis C virus

yellow labradorContemporary human viruses most likely originated by cross-species transmission from non-human animals. Examples include HIV-1, which crossed from chimpanzees to humans, and SARS coronavirus, which originated in bats. Since the 1989 discovery of hepatitis C virus (classified as a hepacivirus in the family Flaviviridae) the origin of the virus been obscure. During the characterization of respiratory infections of domestic dogs, a virus was discovered that is the most genetically similar animal virus homolog of HCV.

HCV is a substantial human pathogen: 200 million people worldwide are chronically infected and are at risk for the development of hepatocellular carcinoma. The source of HCV is unknown because there are no closely related animal virus homologs, but the hunt for related viruses has focused mainly on nonhuman primates. The identification of a related virus was fortuitous, and came about during a study of respiratory viruses that infect dogs. Nasal swabs were obtained from dogs with respiratory illness in shelters in Texas, Utah, and Pennsylvania. Sequence analysis of viral nucleic acids revealed the presence of a virus related to HCV, which was named canine hepacivirus (CHV). The virus was found in respiratory samples from 6 of 9 and 3 of 5 dogs in two separate outbreaks of respiratory disease, but not in 60 healthy pet dogs.

CHV was present in liver, but not lung, of 5 dogs that had died from unexplained gastrointestinal illness. The amount of CHV RNA in respiratory samples was substantially higher than in liver. Viral RNA was detected in the cytoplasm of hepatocytes in canine liver, but whether CHV is hepatotropic (replicates in liver cells) in dogs is not known. In humans, the amount of HCV in respiratory samples is typically very low. CHV may therefore infect different cells and tissues in dogs than does HCV in humans.

Bioinformatic analysis of CHV revealed that it is the genetically more related to HCV than any other known virus. HCV and CHV probably shared a common ancestor that circulated between 500 and 1,000 years ago – many years after dogs were domesticated. It is possible that hepaciviruses are mainly dog viruses, and that HCV arose by transmission of the virus from a dog to a human. An alternative scenario that cannot be excluded is that hepaciviruses infect many animal species. Screening of other animals for the presence of hepaciviruses must be done to determine which hypothesis is correct.

It was not possible to infect canine cultured cells with CHV, using clinical specimens from dogs.  The reason for this failure is not known, but could mean that the cells used are not susceptible and/or permissive for viral replication. Furthermore, a full-length DNA copy of the viral genome, which could be used to produce infectious viral RNA, was not reported. Propagation of the virus in cell cultures will be essential for enabling research on CHV replication and pathogenesis.

The discovery of CHV is exciting because the virus provides clues about the origins of HCV and will likely stimulate a search for related viruses in other animals. It is possible that CHV infection of dogs might be a model for understanding the pathogenesis of HCV, which currently is only possible in chimpanzees. A convenient animal model would be valuable for devising new ways to prevent and treat HCV infections.

A. Kapoor, P. Simmonds, G. Gerold, N. Qaisar, K. Jain, J.A. Henriquez, C. Firth, D.L. Hirschberg, C. Rice, S. Shields, & W.I. Lipkin. (2011). Characterization of a canine homolog of hepatitis C virus Proc. Natl. Acad. Sci. USA

TWiV 128: Virologists in the mist

gorillaHosts: Vincent Racaniello, Alan Dove, Dickson Despommier, and Welkin Johnson

Vincent, Alan, Dickson and Welkin review how a virus regulates the severity of mucocutaneous leishmaniasis, virophage control of antarctic algal host-virus dynamics, and human metapneumovirus infection in gorillas.

Click the arrow above to play, or right-click to download TWiV #128 (67 MB .mp3, 92 minutes).

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Weekly Science Picks

Welkin – Walter Reed Collection, University of Virginia
Dickson – Bacteria-phage antagonistic coevolution in soil (Science)
Alan –
The Artful Amoeba
Vincent – Potential bacteriophage applications (Microbe)

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