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 191: When two rights make a wrong

On episode #191 of the science show This Week in Virology, Nissin joins the TWiV crew to discuss an outbreak of lethal disease among Cambodian children, and recombination among attenuated herpesvirus vaccines leading to pathogenic viruses.

You can find TWiV #191 at

Murine gammaretroviruses in prostate cancer cell lines

nude mouseThe retrovirus XMRV arose during passage of a human prostate tumor in nude mice. The genomes of these mice contain two different proviral DNAs related to XMRV, pre-XMRV-1 and pre-XMRV-2, that recombined to produce XMRV that has been isolated from humans. Two other prostate cancer cell lines also contain mouse gammaretroviruses that are not XMRV. These viruses may have entered the prostate tumor cells during xenograft passage in immunocompromised mice.

The discovery of infectious XMRV in the prostate tumor cell line 22Rv1 prompted the examination of other prostate tumor cell lines for the presence of murine gammaretroviruses. Antisera against Moloney murine leukemia virus were used to screen 72 cell lines by immunohistochemistry for the presence of murine gammaretroviruses. Three human prostate tumor cell lines (22Rv1, LAPC4, and VCaP) and one lung carcinoma cell line (EKVX) reacted with the antisera.

Polymerase chain reaction and nucleotide sequencing analysis revealed that these viruses are not XMRV. The virus in the EKVX cell line is a xenotropic MLV similar to a virus previously isolated from a human B-lymphoblastoid cell line. The virus from the LAPC4 and VCaP cell lines is the murine xenotropic retrovirus Bxv-1. A different sample of VCaP cells obtained from the ATCC were also positive for Bxv-1, as were LAPC4 cells obtained from a different laboratory. Replication-competent viruses were detected in all three cell lines.

How did these human prostate cancer cell lines become contaminated with murine gammaretroviruses? The authors believe this is a consequence of passage of the tumors through immunocompromised mice. In support of this hypothesis, the retrovirus Bxv-1 is present in nude mice, and passage of tumors in these mice can lead to infection with xenotropic MLVs. In contrast to the origin of XMRV, recombination was not needed to produce these viruses.

These findings led the authors to examine two other prostate cancer cell lines, present in their laboratory, DU145 and LNCaP, for the presence of gammaretroviruses. They found these cell lines to be contaminated with XMRV, likely obtained from 22RV1 cells in use in the laboratory. Fresh aliquots of the DU145 and LNCaP cells obtained from other sources were not contaminated. The authors conclude that if

CWR22Rv1 cells are routinely cultured in a typical biomedical research laboratory setting (e.g. using standard Class II biosafety cabinets and procedures for cell culture in which two different cell lines are never present under the hood at the same time), that XMRV can infect and contaminate other cell lines.

Karen Sandell Sfanos, Amanda L. Aloia, Jessica L. Hicks, David M. Esopi, Jared P. Steranka, Wei Shao, Silvia Sanchez-Martinez, Srinivasan Yegnasubramanian, Kathleen H. Burns, Alan Rein, & Angelo M. De Marzo (2011). Identification of Replication Competent Murine Gammaretroviruses in Commonly Used Prostate Cancer Cell Lines PLoS One : 10.1371/journal.pone.0020874

XMRV is a recombinant virus from mice

recombinant xmrvThe novel human retrovirus XMRV has been associated with prostate cancer and chronic fatigue syndrome. The nucleotide sequence of XMRV isolated from humans indicates that the virus is nearly identical with XMRV produced from a human prostate tumor cell line called 22Rv1. This cell line was derived by passage of human prostate tumor tissue in nude mice. Sequence analyses reveal that the genomes of these mouse strains contain two different proviral DNAs related to XMRV. These viral genomes recombined to produce XMRV that has been isolated from humans.

XMRV was originally isolated from a human prostate cancer in 2006, and subsequently associated with ME/CFS. The human cell line 22Rv1, which was established from a human prostate tumor (CWR22), produces infectious XMRV. An important question is whether XMRV was present in the original prostate tumor, or was obtained by passage through nude mice. To answer this question, DNA from various passages of the prostate tumor in nude mice (called xenografts), and the mouse strains used to passage the tumor, were analyzed for the presence of XMRV proviral DNA.

Early-passage xenografts did not contain XMRV, but mouse cells found in them did contain two related proviruses called PreXMRV-1 and PreXMRV-2. The 3’-3211 nucleotides of PreXMRV-1, and both LTRs, are identical to XMRV save for two nucleotide differences. The genomic 5’-half of XMRV and PreXMRV-1 differs by 9-10%. PreXMRV-1 is defective for replication due to mutations in genes encoding the gag and pol proteins. PreXMRV-2 does not contain obvious mutations that would prevent the production of infectious viruses. The gag-pro-pol and a part of the env region of this viral genome is identical to that of XMRV save for two base differences; the LTRs and the remainder of the genome differ by 6-12% from XMRV.

Comparison of the sequences of PreXMRV-1 and PreXMRV-2 indicates that recombination between the two viral genomes led to the formation of XMRV. When the sequences of PreXMRV-1 and −2 are used to construct the recombinant XMRV, the resulting virus differs by only 4 nucleotides from the consensus XMRV sequence derived from all human isolates reported to date.

The nude mice used for passage of the original prostate tumor were likely the NU/NU and Hsd strains. Neither mouse strain contains XMRV proviral DNA, but both contain PreXMRV-1 and PreXMRV-2 proviral DNA.

These data demonstrate that XMRV was not present in the original CWR22 prostate tumor, but arose by recombination of PreXMRV-1 and PreXMRV-2 between 1993-1996. When the original prostate tumor was implanted into nude mice, some of the mice harbored both pre-XMRV-1 and −2 endogenous proviruses, which recombined to form XMRV. The authors believe that XMRV originating from the CRWR22 xenografts, the22Rv1 cell line, or other related cell lines has contaminated all human samples positive for the virus. In addition, they suggest that PCR assays for XMRV may actually detect PreXMRV-1 and −2 or other endogenous viral DNA from contaminating mouse DNA.

Another possibility to explain the origin of XMRV is that it arose in mice and can infect humans. If this is true, then XMRV would have to be present in the nude mice used to passage the CWR22 human prostate tumor. No evidence for an XMRV provirus was found in 12 different nude mouse strains, including two used to passage the CWR22 tumor. Furthermore, a screen of 89 inbred and wild mice failed to reveal the presence of proviral XMRV DNA. Hence the authors conclude:

…that XMRV arose from a recombination event between two endogenous MLVs that took place around 1993-1996 in a nude mouse carrying the CWR22 PC xenograft, and that all of the XMRV isolates reported to date are descended from this one event.

It is possible that XMRV produced during passage of CWR22 in nude mice subsequently infected humans. Because XMRV arose between 1993-1996, this scenario could not explain cases of prostate cancer and chronic fatigue syndrome that arose prior to that date.

How can these findings be reconciled with the published evidence that sera of ME/CFS patients from the 1980s contain antibodies to XMRV? Those antibodies were not shown to be directed specifically against XMRV, and therefore cannot be used to prove that XMRV circulated in humans prior to 1993-96. Furthermore, in the absence of clear isolation of an infectious virus, antibody tests alone have proven highly unreliable for identification of new viruses.

Where do these findings leave the hypothesis that XMRV is the etiologic agent of prostate cancer and ME/CFS? All published sequences of human XMRV isolates are clearly derived by recombination of PreXMRV-1 and −2. The finding of human XMRV isolates that are not derived from PreXMRV-1 and −2 would leave a role for XMRV in human disease. As of this writing, no such XMRV isolates have been reported in the scientific literature.

Update: A second paper has also been published in Science Express today entitled “No evidence of murine-like gammaretroviruses in CFS patients previously identified as XMRV-infected”. Editors of the journal Science have asked the authors to retract their 2009 paper linking XMRV infection with chronic fatigue syndrome. The authors have refused.

T. Paprotka, K. A. Delviks-Frankenberry, O. Cingoz, A. Martinez, H.-J. Kung, C.G. Tepper, W-S Hu, M. J. Fivash, J.M. Coffin, & V.K. Pathak (2011). Recombinant origin of the retrovirus XMRV. Science Express