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.

TWiEVO 5: Looking at straw colored fruit bats through a straw

TWiEVOOn episode #5 of the science show This Week in Evolution, Sara Sawyer and Kartik Chandran join Nels and Vincent to talk about how the filovirus receptor NPC1 regulates Ebolavirus susceptibility in bats.

You can find TWiEVO #5 at microbe.tv/twievo, or you can listen below.

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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.

An epidemic of porcine diarrhea in North America

pig farmPorcine epidemic diarrhea arrived in the United States in the spring of 2013. The disease, caused by a coronavirus, was first identified in the United Kingdom in 1971, and has subsequently spread throughout Europe and Asia. The disease is a concern for the swine industry because it is associated with high case fatality ratios in suckling pigs.

Porcine epidemic diarrhea virus is a member of the coronavirus family, which also includes the SARS and MERS coronaviruses (CoV). Before 2013 the virus had not been isolated in North America. It was detected on a farm in Iowa in May and subsequently spread to 22 states. It is estimated that between 1-4 million pigs have died of the disease in the US.  Recently the virus was found on a Canadian pig farm in Middlesex County, where it most likely arrived from the US.

PEDV can infect pigs of all ages, but is most serious in nursing pigs in which the clinical symptoms are the most severe. The disease is characterized by acute vomiting and watery diarrhea which in nursing pigs leads to dehydration and frequently death. There is no treatment for the disease other than rehydration; no antiviral drugs are available, but a vaccine was developed in 2013. The virus does not infect humans.

Sequence analysis of genomes from three US isolates of PEDV indicate that they are most closely related to a virus isolated in 2012 in Anhui, China. These data suggest, but do not prove, that the US PEDV originated from China. How the virus might have arrived from that country is a matter of speculation. The virus is believed to move from farm to farm on trucks that are used to carry pigs, as well as on contaminated boots and clothing. Many farms observe strict biosecurity procedures in which trucks are properly washed, disinfected, and heated to inactivate the virus. However these procedures cost money and take time, and may be bypassed in some cases. If older pigs are asymptomatically infected, they might be transported to other farms and spread the virus. Once in a farm, stopping spread of the virus is difficult: it is spread by fecal-oral contamination.

The coronavirus family is divided into four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus. Several human coronaviruses that cause common cold-like illness, as well as PEDV, are alphacoronaviruses. SARS-CoV and MERS-CoV are betacoronaviruses. Viruses in the alphacoronavirus and betacoronavirus genera are believed to have originated from bats, while birds might have been the origin of viruses in the other two genera. Analysis of the PEDV genome sequence indicate that the 5’-untranslated region is similar to that of a bat coronavirus. Based on this information it has been suggested that PEDV originated from a cross-species transfer of a bat alphacoronavirus into pigs.

National Hog Farmer is not usually on my reading list, but it has a good summary of porcine epidemic diarrhea.

TWiV 268: Transmission is inevitable

On episode #268 of the science show This Week in Virology, Vincent, Alan, Kathy, and Ashlee discuss fomites in physicians offices, plant virus factories involved in aphid transmission, and clues from the bat genome about flight and immunity.

You can find TWiV #268 at www.microbe.tv/twiv.

Influenza A viruses in bats

A/bat/Peru/10 H18It is well known that aquatic birds are a major reservoir of influenza A viruses, and that pandemic human influenza virus strains of the past century derive viral genes from this pool. The recent discovery of two new influenza A viruses in bats suggests that this species may constitute another reservoir with even greater genetic diversity.

A new influenza virus had previously been isolated from little yellow-shouldered bats (Sturnira lilium) in Guatemala. Three of 316 rectal swabs were positive when tested by a pan-influenza polymerase chain reaction assay. Viral sequences were also detected in liver, intestine, lung, and kidney tissues, suggestive of viral replication and not passage of ingested material through the intestinal tract. Analysis of the viral genome sequence revealed that A/little yellow-shouldered bat/Guatemala/164/2009 (H17N10) is significantly diverged from all known influenza viruses.

When the same PCR approach was used to screen 114 rectal swabs from 18 different species of bats captured in Peru, a single flat-faced fruit bat (Artibeus planirostris) was positive. Viral sequences were also detected in liver, intestine, and spleen tissues from the same bat. Comparison of the sequences of all 8 genome RNA segments with those of the H17N10 Guatemalan isolate revealed sufficient divergence to justify naming it a new HA and NA subtype, A/flat-faced bat/Peru/033/2010 (H18N11).

Comparison of the nucleotide sequences of bat influenza A viruses from Peru and Guatemala with other influenza viruses leads to two amazing conclusions. First, 7 of the 8 viral RNAs of the bat influenza A viruses group separately from the RNAs of all other known influenza viruses. Second, the RNA sequences encoding four proteins, PB2, PB1, PA and NA, display greater genetic diversity than in all non-bat influenza virus sequences combined. The implication is that New World bats harbor a diverse pool of influenza viruses.

The H17 and H18 HA RNA sequences are, in contrast, far more related to known influenza virus HA and NA sequences. The implication of this observation is clear: some time after the bat and non-bat influenza A viruses diverged, a reassortment event occurred that introduced the HA of a non-bat influenza A virus into the genome of a bat influenza A virus.

Serological studies have revealed widespread circulation of these two new influenza viruses in bats. Sera from 55 of 110 (50%) Peruvian bats representing 13 different species were positive for antibodies against the viral HA or NA proteins. Twenty-one of these samples were positive for antibodies against both viral glycoproteins, while 30 were positive only for anti-HA18 antibodies and 4 were positive for only anti-N11 antibodies. These observations suggest that some bats are infected with reassortant viruses carrying the H18 or N11 genes. A study of sera from 8 different species of Guatemalan bats revealed antibodies to the H17 HA protein in 86 of 228 sera (38%).

A number of human viruses, such as SARS-coronavirus and Nipah and Hendra viruses, are known to have originated in bats. Can bat influenza A viruses infect humans and serve as a source of future pandemic strains? The answer to this question is not known, but the two new bat viruses cannot infect human cell lines in culture. However, it is possible that acquisition of other (e.g. avian or swine) influenza virus genes by reassortment could produce a virus with bat influenza virus genes that is capable of infection humans. The pathogenic and pandemic potential of such viruses is unknown. A first step to answering this question would be to determine if human populations with contact with bats have antibodies to the two new bat influenza A viruses.

The cell receptor for all known influenza A viruses is the carbohydrate molecule known as sialic acid  The cell receptor for the two new bat influenza A viruses is not known, but it is clearly not sialic acid, a conclusion reached by studying the crystal structures and binding properties of the H17 (paper one and two) and H18 HA (illustrated) molecules. Furthermore, the crystal structures of the N10 (paper one and two) and N11 proteins reveal that their substrate cannot be sialic acid (the function of the influenza A virus NA is to remove sialic acids from the cell surface, allowing newly synthesized virions to move away from the cell). For this reason the N10 and N11 proteins are called ‘NA-like’.

Bats also harbor many other kinds of viruses, including hepatitis B viruses, Marburg virus, hepaciviruses, pegiviruses, paramyxoviruses, coronaviruses, and many more. They also contain parasites – specifically, malaria parasites. For more information, listen to these podcast episodes:

Hedging our bats (TWiV 258)
More bats out of hell (TWiP 62)
Hepaciviruses and pegiviruses in bats and rodents (TWiV 231)
Bats out of hell (TWiV 183)
Going to bat for flu research (TWiV 173)
Matt’s bats (TWiV 65)

TWiV 258: Hedging our bats

On episode #258 of the science show This Week in Virology, Matt joins the TWiV team to discuss the discovery of a SARS-like coronavirus in bats that can infect human cells, and what is going on with MERS-coronavirus.

You can find TWiV #258 at www.microbe.tv/twiv.

Hepatitis B viruses in bats

hepadnaviridae virionHepatitis B virus (HBV, illustrated) is a substantial human pathogen. WHO estimates that there are now 240,000,000 individuals chronically infected with HBV worldwide, of which 25% will die from chronic liver disease or hepatocellular carcinoma. The hepatitis B virus vaccine is highly effective at preventing infection. Because there are no known animal reservoirs of the virus, it is believed that HBV could be globally eradicated. The recent finding of HBV in bats raises the possibility of zoonotic introduction of the virus.

Serum and liver samples from 3,080 bats from Panama, Brazil, Gabon, Ghana, Germany, Papua New Guinea, and Australia were screened for HBV-like sequences by polymerase chain reaction (PCR). Ten positive specimens were found from three bat species: Uroderma bilobatum from Panama, and Hipposideros cf. ruber and Rhinolophus alcyone from Gabon. The complete viral genome sequence was determined for 9 of the positive specimens. Phylogenetic analysis revealed that the bat viruses form three different lineages, and that each virus differs by at least 35% from known hepadnaviruses.

The virus from Hipposideros cf. ruber has been named roundleaf bat HBV, while those from Rhinolophus and Uroderma have been named horshoe bat HBV, and tent-making bat HBV.

Viral DNA in the liver of Hipposideros bats was found to be higher than in other organs or serum. Some lymphocyte infiltration was observed in the liver of these animals, as well as deposits of viral DNA within hepatocytes. These observations indicate that the bat HBV viruses likely replicate in the bat liver and cause hepatitis.

Serological studies revealed that hepadnaviruses are widespread in Old World bats: antibodies against bat hepadnaviruses were detected in 18% of hipposiderid bats and 6.3% of rhinolophid bats.

An important question is whether these three bat hepadnaviruses can infect human cells. Only tent-making bat HBV could infect primary human hepatocytes, which occurred via the human HBV cell receptor, sodium taurocholate cotransporting polypeptide. However serum from humans that had been immunized with HBV vaccine did not block infection of human hepatocytes with this virus.

These observations show that viruses related to human HBV are replicating in the liver of bats. Earlier this year another hepadnavirus was identified in long-fingered bats (Miniopterus fuliginosus) in Myanmar. The complete genome sequence was obtained and virus particles were observed in bat liver tissues.

The finding of hepadnaviruses in bats raise many interesting questions. The first is whether human HBV originated by infection with bat HBV, either by consumption of bat meat or another mode of transmission. How long ago this occurred is not known. It has been suggested that HBV has been in humans for at least 15,000 years. Some avian species contain avihepadnaviral sequences integrated into their genome, indicating that these viruses originated at least 19 million years ago.

These findings also raise many questions about the pathogenesis of hepadnaviral infection in bats, including the mode of transmission (in humans, the virus is transmitted by exposure to blood, e.g. by injection or during childbirth), and whether chronic infections can occur as they do in humans.

Finally it is interesting to consider the zoonotic potential of tent-making bat HBV, which can infect human cells. Because bat hepadnaviruses are genetically distinct from HBV, current serological and nucleic acid screening programs would not detect human infections. The authors suggest that human and non-human primate sera from areas in which these bat viruses were isolated should be screened using assays that detect the bat hepadnaviruses. Without such information we do not know if these viruses currently infect humans.

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.