Receptor for new coronavirus-EMC identified

Coronavirus virionViruses are obligate intracellular parasites, which means that they must enter a cell to reproduce. As virions are too large to diffuse passively across the plasma membrane, cellular pathways for uptake of extracellular materials provide entry routes. The first step in entry is adherence of virus particles to the membrane, an interaction mediated by binding to one or more receptor molecules on the cell surface. Identification of cell receptors for viruses is an important objective because their study may lead to information about how the virus enters the cell, how it is targeted to specific tissues, and how it causes disease. The cell receptor for the recently identified coronavirus-EMC, which has so far infected 15 humans with 9 deaths, has been identified as dipeptidyl peptidase 4 (DPP4).

Enveloped viruses such as CoVs typically attach to cell receptors via spike glycoproteins embedded in the viral envelope. To identify the CoV-EMC receptor, part of the viral spike (S) glycoprotein was expressed as a fusion protein with the Fc domain of IgG antibody. The protein was mixed with lysates of cells known to be infected with CoV-EMC, and bound proteins were isolated by using agarose beads bound to protein A (which binds the Fc domain). A single polypeptide bound to the CoV-EMC spike protein was identified as DPP4. Four different lines of evidence indicate that DPP4 is a bona fide receptor for CoV-EMC:

  • Soluble DPP4 blocks infection of susceptible cells with CoV-EMC
  • Expression of DPP4 in non-susceptible cells renders them susceptible to infection
  • Antibody to DPP4 blocks infection of cells with CoV-EMC
  • Purified DPP4 protein binds CoV-EMC and inhibits infection

Considering that CoV-EMC was isolated in November 2012 from a sick patient, the identification of the cell receptor is indeed rapid progress.

DPP4 protein is expressed in primary human bronchiolar lung tissue and on primary bronchiolar epithelial cell cultures, consistent with the ability of the virus to infect the respiratory tract. The protein is also present on the epithelium of kidney, small intestine, liver and prostate. CoV-EMC has been detected in the respiratory tract and in urine. Whether the virus replicates in the respiratory tract, and then disseminates and replicates elsewhere (e.g. kidney) remains to be determined.

CoV-EMC is believed to have originated in bats. Consistent with this hypothesis, expression of bat DPP4 confers susceptibility to the virus, although not to the same extent as human DPP4. Once the bat precursor of CoV-EMC is identified, it will be interesting to determine how the viral spike glycoprotein has evolved to enable more efficient usage of human DPP4.

DPP4 is a transmembrane protein that regulates the activity of hormones and chemokines through proteolytic cleavage. The cell receptors for two other CoVs are also membrane-bound peptidases, but proteolytic activity is not needed for infection. A soluble form of DPP4 is also present in blood. The authors speculate that reduction of DPP4 protein levels by CoV-EMC infection could result in higher virus-induced disease. If DPP4 is important in regulating the activity of cytokines – major components of immune responses – their removal from the circulation could result in greater virus replication and more tissue damage. It will be important to study the levels of DPP4 in humans infected with CoV-EMC, and to determine whether levels of the receptor affect viral disease.

TWiV 215: Illuminating rabies and unwrapping a SARI

On episode #215 of the science show This Week in Virology, Vincent, Alan, and Kathy review the finding that rabies virus infection alters but does not kill neurons, and provide an update on the novel coronavirus in the Middle East.

You can find TWiV #215 at www.microbe.tv/twiv.

TWiV 212: Apocalypse TWiV 122112 212

On episode #212 of the science show This Week in Virology, the TWiVerers answer listener email about genetically modified chickens, a hendra vaccine for horses, online education, curing color blindness, Roosevelt and polio, Th cells, and much more.

You can find TWiV #212 at www.microbe.tv/twiv.

TWiV 207: Silk sheets and viral infidelity

On episode #207 of the science show This Week in Virology, Vincent, Alan, Matt, and Kathy review the use of silk to stabilize antibiotics and a viral vaccine, and an impaired-fidelity vaccine against SARS coronavirus.

You can find TWiV #207 at www.microbe.tv/twiv.

TWiV 204: M m m my corona

On episode #204 of the science show This Week in Virology, Vincent, Alan, Matt and Kathy review isolation of a new coronavirus from two patients in the Middle East, and expansion of the enteric virome during simian AIDS.

You can find TWiV #204 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 155: XXII Brazilian National Virology Meeting

sbv_logoenv2011Hosts: Vincent Racaniello, Grant McFadden, Eurico de Arruda Neto, Paulo Eduardo Brandão, Francisco Murilo Zerbini, and Janice Reis Ciacci Zanella

Vincent, Grant, Eurico, Paulo, Francisco and Janice discuss their work on bocavirus, infectious bronchitis virus, begomoviruses, and circoviruses at the Brazilian Virology Society meeting in Atibaia, São Paulo, Brazil.

Click the arrow above to play, or right-click to download TWiV 155 (56 MB .mp3, 93 minutes).

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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 83: An hour with Dr. Kiki

Hosts: Vincent Racaniello, Alan Dove, Rich Condit, and Kirsten Sanford

On episode #83 of the podcast This Week in Virology, Vincent, Alan, Rich, and special guest Dr. Kirsten Sanford talk about her career in science media, then consider whether smallpox eradication led to the AIDS pandemic, high fidelity RNA synthesis, and a new Ebola virus vaccine.

This episode is sponsored by Data Robotics Inc. Use the promotion code TWIVPOD to receive $75-$500 off a Drobo.

Win a free Drobo S! Contest rules here.

Click the arrow above to play, or right-click to download TWiV #83 (66 MB .mp3, 91 minutes)

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Send your virology questions and comments (email or mp3 file) to twiv@microbe.tv or leave voicemail at Skype: twivpodcast. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.