Zika Zoo

When we decided to work on Zika virus in February 2016, experiments in mice were certainly part of our plans. However, one does not simply walk into a mouse facility and start inoculating animals with viruses! Carrying out animal experiments requires approval of a detailed protocol by the Institutional Animal Care and Use Committee (IACUC). I have filed many IACUC protocols in the past 30 years, and to work on Zika virus in mice, we had to file a new one. Here is how the process works.

Read the remainder of this article at Zika Diaries.

TWiM 30: Unraveling melioidosis and insulin resistance

On episode #30 of the science show This Week in Microbiology, Vincent, Elio, and Michael review how a toxin from Burkholderia pseudomallei inhibits protein synthesis, and the role of the gut microbiome in modulating insulin resistance in mice lacking an innate immune sensor.

You can find TWiM #30 at microbeworld.org/twim.

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

Is the 2009 H1N1 influenza virus more dangerous than we think?

Mustela_putorius_furoThe results of experiments comparing the virulence in animals of the 2009 H1N1 influenza virus with seasonal strains have spawned the headline Study Suggests H1N1 Virus More Dangerous Than Suspected. In my view, the best experiment is now being done in humans: infection of millions with the pandemic virus. The results show that the virus is no more virulent than last season’s H1N1 strain.

In mice, ferrets, and non-human primates, the 2009 H1N1 swine-origin influenza virus (S-OIV) replicated more efficiently, and caused more severe lesions in the lungs than a seasonal H1N1 virus. These findings lead the lead author of the study to comment:

There is a misunderstanding about this virus. People think this pathogen may be similar to seasonal influenza. This study shows that is not the case. There is clear evidence the virus is different than seasonal influenza.

I’m puzzled by this statement. As far as I know, the 2009 H1N1 strain has so far likely infected millions of people, and most have concluded that the disease is no more severe than seasonal influenza. Are mice, ferrets, and non-human primates more reliable indicators of influenza virus virulence than humans?

I agree that the 2009 H1N1 influenza virus does seem to multiply more extensively in the respiratory tract than a seasonal H1N1 strain, as does the 1918 virus. But how many influenza virus strains have been studied in such animals? There are probably others that can replicate in the lower tract of experimental animals but are not very pathogenic in humans.

Two other research groups published the results of similar experiments in ferrets. They both found that the 2009 H1N1 virus replicated to higher titers, and more extensively in the lower respiratory tract, than seasonal H1N1 influenza virus. One of the groups concluded:

Our results indicated that the 2009 A(H1N1) influenza virus replicates efficiently in the upper and lower respiratory tract of ferrets, is associated with mild or moderate clinical signs and pathological changes and is transmitted efficiently between ferrets via aerosols or respiratory droplets. These results are in agreement with observations in humans, where generally mild disease but relatively efficient human-to human transmission has been observed

In other words, although the 2009 H1N1 replicated more efficiently, and in different parts of the lungs than seasonal H1N1 virus, these authors felt the observations were consistent with the low virulence of the virus humans.

It’s worth noting that when the authors of the Nature paper infected miniature pigs with the 2009 H1N1 influenza virus, the virus replicated without clinical symptoms. This result emphasizes the importance of remembering that animals are models for studying virus infections; they rarely duplicate the effects of a virus infection in humans. Furthermore, the virulence of a virus strain may vary dramatically depending on the dose and the route of infection, as well as on the species, age, gender, and susceptibility of the host. Virulence is a relative property. Consequently, when the degree of virulence of two very similar viruses are compared, the assays must be identical.

Do we really want to conclude that the new H1N1 strains is ‘more dangerous’ than we think based on tests in animal models which may or may not accurately reflect what occurs in humans? The ongoing infection of millions of humans with the new H1N1 virus seems a better source of data – and so far the results indicate that the new pandemic strain is no more dangerous than seasonal influenza.

Of course, it is possible that a more virulent version of the 2009 H1N1 virus could emerge in the coming months – what will happen as this virus evolves in humans is anyone’s guess – but who knows if it will retain the ability to multiply in the lower tract? The only thing that is certain is that it will be a different virus from the one that has been studied in mice, ferrets, and small pigs.

Y. Itoh1 et al. (2009). In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses Nature adv. online pub 13 July 2009.

Maines, T., Jayaraman, A., Belser, J., Wadford, D., Pappas, C., Zeng, H., Gustin, K., Pearce, M., Viswanathan, K., Shriver, Z., Raman, R., Cox, N., Sasisekharan, R., Katz, J., & Tumpey, T. (2009). Transmission and Pathogenesis of Swine-Origin 2009 A(H1N1) Influenza Viruses in Ferrets and Mice Science DOI: 10.1126/science.1177238

Munster, V., de Wit, E., van den Brand, J., Herfst, S., Schrauwen, E., Bestebroer, T., van de Vijver, D., Boucher, C., Koopmans, M., Rimmelzwaan, G., Kuiken, T., Osterhaus, A., & Fouchier, R. (2009). Pathogenesis and Transmission of Swine-Origin 2009 A(H1N1) Influenza Virus in Ferrets Science DOI: 10.1126/science.1177127