Genome recombination across viral families

coronavirus and reovirus A novel coronavirus isolated from bats in China is unusual because the genome contains a gene from a virus in a completely different family, the Reoviridae (link to paper). The finding suggests that recombination occurred between a (+) strand RNA virus and a virus with a segmented, double-stranded RNA genome.

The unusual recombinant virus was identified in rectal swabs from Rousettus leschenaulti bats in Yunnan Province, China. Sequence analysis revealed a typical coronavirus genome with the exception of a small region near the 3′-end of the viral RNA with homology to a bat reovirus. This sequence, called p10, was also detected in viral mRNAs from infected bats, further demonstrating the presence of the reovirus-like gene in the coronavirus genome.

In bat reoviruses, the p10 gene is known to encode a protein that causes cell fusion. When the p10 gene from the bat coronavirus was expressed in cells, formation of syncytia (fused cells) was observed. Furthemore, the p10 protein was detected by western blot analysis of feces from infected bats. These results indicate that the p10 protein is produced from the viral genome and that the protein is functional.

This report is not the first suggesting recombination between viruses of different families – we discussed one example here previously (link to article), and there are a handful of other examples. The important question is how such inter-family recombinants arise. It must begin with co-infection of a host with two different viruses – in this case, likely a bat – but the precise molecular events are unknown. It might be useful to attempt to isolate such recombinants in cell culture to understand the underlying mechanisms.

Moving beyond metagenomics to find the next pandemic virus

I was asked to write a commentary for the Proceedings of the National Academy of Sciences to accompany an article entitled SARS-like WIV1-CoV poised for human emergence. I’d like to explain why I wrote it and why I spent the last five paragraphs railing against regulating gain-of-function experiments.

Towards the end of 2014 the US government announced a pause of gain-of-function research involving research on influenza virus, SARS virus, and MERS virus that “may be reasonably anticipated to confer attributes to influenza, MERS, or SARS viruses such that the virus would have enhanced pathogenicity and/or transmissibility in mammals via the respiratory route.”

From the start I have opposed the gain-of-function pause. It’s a bad idea fostered by individuals who continue to believe, among other things, that influenza H5N1 virus adapted to transmit by aerosol among ferrets can also infect humans by the same route. Instead of stopping important research, a debate on the merits and risks of gain-of-function experiments should have been conducted while experiments were allowed to proceed.

Towards the end of last year a paper was published a paper on the potential of SARS-virus-like bat coronaviruses to cause human disease. The paper reawakened the debate on the risks and benefits of engineering viruses. Opponents of gain-of-function research began to make incorrect statements about this work. Richard Ebright said that ‘The only impact of this work is the creation, in a lab, of a new, non-natural risk”. Simon Wain-Hobson wrote that a novel virus was created that “grows remarkably well” in human cells; “if the virus escaped, nobody could predict the trajectory”. I have written extensively about why these are other similar statements ignore the value of the work. In my opinion these critics either did not read the paper, or if they did, did not understand it.

Several months later I was asked to write the commentary on a second paper examining the potential of SARS like viruses in bats to cause human disease. I agreed to write it because the science is excellent, the conclusions are important, and it would provide me with another venue for criticizing the gain-of-function pause.

In the PNAS paper, Menachery et al. describe a platform comprising metagenomics data, synthetic virology, transgenic mouse models, and monoclonal antibody therapy to assess the ability of SARS-CoV–like viruses to infect human cells and cause disease in mouse models. The results indicate that a bat SARS-like virus, WIV1-CoV, can infect human cells but is attenuated in mice. Additional changes in the WIV1-CoV genome are likely required to increase the pathogenesis of the virus for mice. The same experimental approaches could be used to examine the potential to infect humans of other animal viruses identified by metagenomics surveys. Unfortunately my commentary is behind a paywall, so for those who cannot read it, I’d like to quote from my final paragraphs on the gain-of-function issue:

The current government pause on these gain-of-function experiments was brought about in part by several vocal critics who feel that the risks of this work outweigh potential benefits. On multiple occasions these individuals have indicated that some of the SARS-CoV work discussed in the Menachery et al. article is of no merit. … These findings provide clear experimental paths for developing monoclonal antibodies and vaccines that could be used should another CoV begin to infect humans. The critics of gain-of-function experiments frequently cite apocalyptic scenarios involving the release of altered viruses and subsequent catastrophic effects on humans. Such statements represent personal opinions that are simply meant to scare the public and push us toward unneeded regulation. Virologists have been manipulating viruses for years—this author was the first to produce, 35 y ago, an infectious DNA clone of an animal virus—and no altered virus has gone on to cause an epidemic in humans. Although there have been recent lapses in high-containment biological facilities, none have resulted in harm, and work has gone on for years in many other facilities without incident. I understand that none of these arguments tell us what will happen in the future, but these are the data that we have to calculate risk, and it appears to be very low. As shown by Menacherry et al. in PNAS, the benefits are considerable.

A major goal of life science research is to improve human health, and prohibiting experiments because they may have some risk is contrary to this goal. Being overly cautious is not without its own risks, as we may not develop the advances needed to not only identify future pandemic viruses and develop methods to prevent and control disease, but to develop a basic understand- ing of pathogenesis that guides prevention. These are just some of the beneficial outcomes that we can predict. There are many examples of how science has progressed in areas that were never anticipated, the so-called serendipity of science. Examples abound, including the discovery of restriction enzymes that helped fuel the biotechnology revolution, and the development of the powerful CRISPR/Cas9 gene-editing technology from its obscure origins as a bacterial defense system.

Banning certain types of potentially risky experiments is short sighted and impedes the potential of science to improve human health. Rather than banning experiments, such as those described by Menachery et al., measures should be put in place to allow their safe conduct. In this way science’s full benefits for society can be realized, unfettered by artificial boundaries.

TWiV 364: It’s not SARS 2.0

On episode #364 of the science show This Week in Virology, Vincent, Rich, and Kathy speak with Ralph Baric and Vineet Menachery about their research on the potential of SARS-like bat coronaviruses  to infect human cells and cause disease in mice.

You can find TWiV #364 at www.microbe.tv/twiv.

Bat SARS-like coronavirus: It’s not SARS 2.0!

SARSA study on the potential of SARS-virus-like bat coronaviruses to cause human disease has reawakened the debate on the risks and benefits of engineering viruses. Let’s go over the science and then see if any of the criticisms have merit.

The SARS epidemic of 2003 was caused by a novel coronavirus (CoV) that originated in bats. Results of sequence analyses have shown that viruses related to SARS-CoV continue to circulate in bats, but their potential for infecting humans is not known. One can learn only so much from looking at viral sequences – eventually experiments need to be done.

To answer the question ‘do the SARS-CoV like viruses circulating in bats have the potential to infect humans’, a recombinant virus was created in which the gene encoding the spike glycoprotein of SARS virus was swapped with the gene from a bat virus called SHC014. The SARS-CoV that was used (called SARS-MA15) had been previously passaged from mouse to mouse until it was able to replicate in that host. The use of this mouse-adapted virus allows studies on viral disease and its prevention in a mammalian host.

The recombinant virus, called SHC014-MA15, replicated well in a human epithelial airway cell line and in primary human airway epithelial cell cultures. The recombinant virus replicated just as well as the Urbani strain of human SARS-CoV. This result was surprising because the part of the spike protein of SCH014 that binds the cell receptor, ACE2, is sufficiently different from the SARS-CoV spike, suggesting that the virus might not infect human cells.

First lesson learned: looking at a viral genome sequence alone does not answer all questions. The spike glycoprotein of a bat coronavirus can mediate virus entry into human cells.

Next the authors wanted to know if SHC014-MA15 could infect mice and cause respiratory disease. Ten week old mice were infected intranasally with either SCH014-MA15 or SARS-MA15. Animals infected with SARS-MA15 lost weight rapidly and died within 4 days. Mice infected with SCH014-MA15 lost weight but did not die. When older (12 month) mice were used (these are more susceptible to SARS-MA15 infection), both viruses caused weight loss, but SARS-MA15 killed all the mice while SCH014-MA15 was less virulent (20% of mice died).

Second lesson learned: a human SARS-CoV with a bat glycoprotein can infect mice but is attenuated compared to a human, mouse adapted strain.

The next question asked was whether monoclonal antibodies (think ZMAPP, used in some Ebolavirus infected patients) against SARS-CoV could protect cells from infection with SCH014-MA15. The answer is no.

Third lesson learned: anti-SARS-CoV monoclonal antibodies do not protect from infection with SCH014-MA15.

Could an inactivated SARS-CoV vaccine protect mice from infection with SCH014-MA15?  An inactivated SARS-CoV vaccine provided no protection against infection SCH-014-MA15. When mice were first infected with a high dose of SCH014-MA15, there was some protection against challenge with the same virus, but protection did not last. And the side effects, weight loss and some death, would not be acceptable for a vaccine.

Fourth lesson learned: an inactivated SARS-CoV vaccine does not protect against infection with SCH014-MA15, and the recombinant virus itself is barely protective but not a safe vaccine.

In the final experiment of the paper, the SCH014 virus was recovered from an infectious DNA clone made from the genome sequence. This virus infected primary human airway epithelial cell cultures but not as well as did SARS-CoV Urbani. In mice SCH014 did not cause weight loss and it replicated to lower titers than SARS-CoV Urbani.

Fifth lesson learned: At least one circulating SARS-like bat CoV can infect human cells, but causes only mild disease in mice. Additional changes in the viral genome would likely be needed to cause a SARS-like epidemic.

Let’s now take a look at some of the public statements that have been made about this work.

Richard Ebright says that ‘The only impact of this work is the creation, in a lab, of a new, non-natural risk”. He could not be more wrong. For Ebright’s benefit, I submit my summary above of what we have learned from this work. Furthermore, I suggest that Ebright has not read the paper, or if he had, he has not put it in the context of the gaps in our knowledge of bat coronavirus potential to infect humans. This type of negative quote is easily picked up by the press, but it’s completely inaccurate.

Simon Wain-Hobson says that a novel virus was created that ‘grows remarkably well’ in human cells; ‘if the virus escaped, nobody could predict the trajectory’.

I do agree that we cannot predict what would happen if SCH014-MA15 were released into the human population. In my opinion the risk of release and spread of this virus in humans is very low. The attenuated virulence of the SCH014-MA15 virus in mice suggests (but does not prove) that the recombinant virus is not optimized for replication in mammals. Recall that the virus used to produce the recombinant, SARS-MA15 is mouse-adapted and may very well have lost some virulence for humans. In a broader sense, virologists have been manipulating viruses for years and none have gone on to cause an epidemic in humans. While there have been recent lapses in high-containment biological facilities, none have resulted in harm, and work has gone on for years in many other facilities without harm. I understand that none of these arguments tell us what will happen in the future, but these are the data that we have to calculate risk. Bottom line: the risk of these experiments is very low.

I think the statements by Ebright and Wain-Hobson are simply meant to scare the public and push us towards regulation of what they believe are ‘dangerous’ experiments. They are misleading because they ignore the substantial advances of the work. The experiments in this paper were well thought out, and the conclusions (listed above) are substantial. Creation of the recombinant virus SCH014-MA15 was needed to show that the spike glycoprotein could mediate entry into human cells. Only after that result was obtained did it make sense to recover the SCH014 bat virus. We now understand that at least one circulating bat SARS-like CoV can infect human cells and the mouse respiratory tract. More importantly, infection cannot be prevented with current SARS monoclonal antibodies or vaccines.

This information means that we should embark on a program to understand the different SARS-like spike glyocoproteins on bat CoVs, and try to develop therapeutics to prevent a possible second spillover into humans. This work will require further studies of the type reported in this paper.

My conclusion: these are low risk, high benefit experiments. You may disagree with my assessment of risk, but you cannot deny the benefits of this work. If you do, you simply haven’t read and understood the paper.

As you might imagine, the press has had a field day with this work. But many of these articles are misleading. For example, the headline of the Motherboard article touts “Ethical Questions Arise After Scientists Brew Super Powerful ‘SARS 2.0’ Virus”. As I pointed out above, both SCH014-MA15 and SCH104 are less virulent in mice than SARS-CoV, so this headline is completely wrong. An article in Sputnik International has the headline “Uncaging the Animal: Concerns Rise Over Scientists Tests on SARS 2.0” and the sub-headline is “‘SARS 2.0’ is closer than you might think as scientists are continuing medical tests that could create a whole new virus outbreak.” The article claims that the experiments are ‘science for the sake of science’. If the author had read the Nature article, he or she could not have reached that conclusion. Both articles feature scary quotations by Ebright and Wain-Hobson. The most egregious may be an article in the Daily Mail, which claims that “New SARS-like virus can jump directly from bats to humans without mutating, sparking fears of a future epidemic”. This statement is also wrong – there are no data in the paper which show that the virus can jump from bats to humans!

Perhaps at fault for much of this hyperbole is the press release on this work issued by the University of North Carolina, the home of the paper’s authors. The headline of the press release is: “New SARS-like virus can jump directly from bats to humans, no treatment available”. Exactly the same as the Daily Mail! Other errors in the press release emphasize that researchers need to work more closely with publicity departments to ensure that the correct message is conveyed to writers.

TWiV 332: Vanderbilt virology

On episode #332 of the science show This Week in Virology, Vincent visits Vanderbilt University and meets up with Seth, Jim, and Mark to talk about their work on a virus of Wolbachia, anti-viral antibodies, and coronaviruses.

You can find TWiV #332 at www.microbe.tv/twiv.

TWiM 90: Think globally, act locally

I usually don’t post TWiM episodes here, but #90 has a lot of virology. In this episode, recorded in La Jolla, CA at the annual meeting of the Southern California Branch of the American Society for Microbiology, I first speak with Laurene Mascola, Chief of Acute Communicable Diseases at the Los Angeles County Department of Public Health. Dr. Mascola talks about how Los Angeles county has prepared for an outbreak of Ebola virus. Next up is David Persing, Executive Vice President and Chief Medical and Technology Officer at Cepheid. His company has developed an amazing, modular PCR machine that is brining rapid diagnosis everywhere, including the United States Post Office. And it might even be available on your refrigerator one day.

Watch TWiM #90 below, or listen at microbeworld.org/twim or iTunes.

 

TWiV 287: A potentially pandemic podcast

On episode #287 of the science show This Week in Virology, Matt Frieman updates the TWiV team on MERS-coronavirus, and joins in a discussion of whether we should further regulate research on potentially pandemic pathogens.

You can find TWiV #287 at www.microbe.tv/twiv.

TWiV 276: Ramblers go viral

On episode #276 of the science show This Week in Virology, Vincent meets up with Susan Baker and Tom Gallagher at Loyola University to talk about their work on coronaviruses.

You can find TWiV #276 at www.microbe.tv/twiv.

Bat SARS-like coronavirus that infects human cells

Rhinolophus sinicusThe SARS pandemic of 2002-2003 is believed to have been caused by a bat coronavirus (CoV) that first infected a civet and then was passed on to humans. The isolation of a new SARS-like coronavirus from bats suggests that the virus could have directly infected humans.

A single colony of horseshoe bats (Rhinolophus sinicus) in Kunming, Yunnan Province, China, was sampled for CoV sequences over a one year period. Of a total of 117 anal swabs or fecal samples collected, 27 (23%) were positive for CoV sequences by polymerase chain reaction (PCR). Seven different SARS-like CoV sequences were identified, including two new ones. For the latter the complete genome sequence was determined, which showed a higher nucleotide sequence identity (95%) with SARS-CoV than had been previously observed before among bat viruses.

One of these new viruses was recovered by infecting monkey cell cultures with one of the PCR-positive samples. This virus could infect human cells and could utilize human angiotensin converting enzyme 2 (ACE2) as an entry receptor. The infectivity of this virus could also be neutralized with sera collected from seven different SARS patients.

None of the SARS-like coronaviruses previously isolated from bats are able to infect human cells. The reason for this block in replication is that the spike glycoprotein of these bat viruses do not recognize ACE2, the cell receptor for SARS-CoV. SARs-like CoVs isolated from palm civets during the 2002-2003 outbreak have amino acid changes in the viral spike glycoprotein that improve its interaction with ACE2. The civet was therefore believed to be an intermediate host for adaptation of SARS-CoV to humans. The isolation of bat SARS-like CoVs that can bind human ACE2 and replicate in human cells suggests that the virus might have spread directly from bats to humans.

This finding has implications for public health: if SARS-like CoVs that can infect human cells are currently circulating in bats, they have the potential to infect humans and cause another outbreak of disease. The authors believe that the diversity of bat CoVs is higher than we previously knew:

It would therefore not be surprising if further surveillance reveals a broad diversity of bat SL-CoVs that are able to use ACE2, some of which may have even closer homology to SARS-CoV than SL-CoV-WIV1.

Is there any implication of this work for the recently emerged MERS-CoV? Sequences related to MERS-CoV have been found in bats, and given that bats are known to be hosts of a number of viruses that infect humans, it is reasonable to postulate that MERS-CoV originated in bats. So far a 190 fragment of MERS-CoV nucleic acid has been found in a single bat from Saudi Arabia. Identification of the reservoir of MERS-CoV will require duplicating the methods reported in this paper: finding the complete viral genome, and infectious virus, in bats.

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.