Some extracts I found interesting from the main paper under discussion (no minutiae as such):
Restricted Replication of Xenotropic Murine Leukemia Virus-Related Virus in Pigtailed Macaques
Gregory Q. Del Prete1, Mary F. Kearney2, Jon Spindler2, Ann Wiegand2, Elena Chertova1, James D. Roser1, Jacob D. Estes1, Xing Pei Hao1, Charles M. Trubey1, Abigail Lara1, KyeongEun Lee2, Chawaree Chaipan2, Julian W. Bess, Jr.1, Kunio Nagashima3, Brandon F. Keele1, Rhonda Pung4, Jeremy Smedley4, Vinay K. Pathak2, Vineet N. KewalRamani2, John M. Coffin2,6, and Jeffrey D. Lifson1*
Abstract1. ...These findings indicate that XMRV replication and spread were limited in pigtailed macaques, predominantly by APOBEC-mediated hypermutation. Given that human APOBEC proteins restrict XMRV infection in vitro, human XMRV infection, if it occurred, would be expected to be characterized by similarly limited viral replication and spread...
Introduction2. ‘In 2006, Urisman and coworkers identified sequences from a novel gammaretrovirus in an analysis of human prostate tumor tissues using a Virochip DNA microarray and named the new virus xenotropic murine leukemia virus-related virus (XMRV) due to its high sequence identity with xenotropic murine leukemia viruses (X-MLV) (64). Although several follow up studies described findings interpreted as evidence of XMRV infection in up to 27% of prostate cancer patients and up to 4% of healthy prostate controls using PCR (3, 9, 11, 54), immunohistochemistry (54), and fluorescence in situ hybridization (3) on prostate tissues, as well as anti-XMRV serum neutralization assays (3), the majority of studies detected little or no evidence for XMRV infection in either prostatic tumors or healthy controls, raising questions about the authenticity of human XMRV infection and what role, if any, XMRV might play in prostate cancer (1, 2, 15, 23, 50, 56, 61)…’
3. ‘…Although there is a growing consensus that evidence for XMRV infection in human samples is more likely the result of contamination than genuine in vivo infection, the fact remains that XMRV is a novel replication-competent retrovirus of unknown pathogenic potential once implicated in the etiology of several human diseases...'
4. '...Given uncertainties about the capacity of XMRV to cause human disease as well as the potential target cell tropism, tissue distribution, in vivo replication capacity and sequence evolution of the virus, and elicited antiviral immune responses, we infected two pigtailed macaques (Macaca nemestrina) with a well-characterized 22Rv1-produced XMRV stock. We conducted these studies with two primary goals.
First, we sought to generate bona fide in vivo derived positive control samples for PCR, RT-PCR, and serology assays performed on clinical samples (10, 28).
Second, we aimed to comprehensively examine the natural history of in vivo XMRV infection in a primate host, evaluating levels and kinetics of replication, viral sequence changes, cell and tissue tropism, and cellular and humoral antiviral immune responses.'
5. 'We show here that XMRV replication in pigtailed macaques is restricted, with limited, transient viremia associated with the accumulation of extensive G-to-A hypermutation in cell-associated viral DNA. In spite of limited viral replication, humoral immune responses to the virus were relatively robust and stable, while innate immune responses were transient and adaptive cellular immune responses were negligible.’
Discussion6. 'Although based on accumulating results, the proposed association of XMRV infection with human disease as well as evidence of any authentic human XMRV infection appear increasingly unlikely, the virus itself is a replication-competent retrovirus of unknown pathogenic potential that is capable of infecting human cells (7, 11, 47).'
7. 'Given the lack of any confirmed positive cases of human XMRV infection (29, 58), the in vivo replication capacity, sequence evolution, tissue tropism, and elicited immune responses associated with XMRV infection are unknown and it is unclear what results using a variety of direct and indirect detection methods might be expected in the setting of authentic XMRV infection.'
8. 'Information about the natural history of XMRV infection and host immune responses to the virus would provide a framework to help interpret results suggesting evidence of human XMRV infection. In the absence of any known human infection, animal models must be relied upon to provide this information.'
9. 'To address these questions in a non-human primate species, we intravenously infected two adult male pigtailed macaques with >1010 XMRV virions, an inoculum which likely exceeds by many orders of magnitude any viral inoculum that might be involved in a physiological human transmission.'
10. 'Despite this large viral inoculum, XMRV replicated only transiently to relatively low peak levels in both animals, achieving peak plasma viral loads ≤ 2,200 RNA copies/ml that declined to undetectable levels within 4 weeks of infection.'
11. 'This decline in viremia was associated with striking levels of G-to-A hypermutation in PBMC-associated vDNA, likely reflective of APOBEC-mediated viral restriction.'
12. 'Although plasma viremia was brief, within the first 2-4 weeks of infection both animals raised robust anti-XMRV antibody responses primarily directed towards p15ETM, p30CA, and gp70SU that were largely maintained up 550 to 119 days post-infection.'
13. 'In addition to these binding antibody responses, neutralizing antibodies were also elicited within the first two weeks of infection and maintained through the study’s conclusion.'
14. 'In contrast, innate immune responses in
lymph nodes were only transiently upregulated in the first week of infection and rapidly diminished to baseline levels by two weeks post-infection, while adaptive T cell responses were essentially negligible in ICS format assays using whole XMRV virions or purified recombinant XMRV proteins as stimuli.'
'
Our findings differ markedly from some of those made in a previous study by Onlamoon and coworkers, which examined XMRV infection of rhesus macaques (Macaca mulatta).Although Onlamoon et al. also reported low, transient levels of viremia that declined to undetectable levels within 3 weeks of infection (albeit with a less sensitive qRT-PCR assay) associated with the induction of a relatively stable antibody response with neutralizing activity, there were several key differences between results for the rhesus macaque infection study and those reported here for pigtailed macaques.
15. 'First, while we observed a relatively stable pool of PBMC-associated XMRV DNA composed predominantly of G-to-A hypermutated viral genomes that were detectable through 119 dpi, Onlamoon et al. reported a complete loss of PBMC-associated vDNA within the first month of infection (44). Although a recent follow-up study has shown G-to-A hypermutation in PBMC-associated vDNA sequences in these infected rhesus monkeys (67), the disappearance of vDNA in PBMCs seems to suggest that factors other than APOBEC-mediated restriction may contribute importantly to control of XMRV infection in rhesus macaques.
It is not clear, however, if the PCR assay used here and that used by Onlamoon and coworkers are equally capable of detecting hypermutated vDNA, which are likely less efficiently amplified due to inefficient priming, and it is possible that the apparent loss of vDNA-positive PBMCs in rhesus macaques simply reflects the accumulation of hypermutated genomes.'
16. 'Other key differences between this and the Onlamoon et al. study were noted when comparing analyses of various host tissues. In our pigtailed macaques, we identified vDNA in LNMCs by X-SCA at early and late post-infection time points, however longitudinal LN sections were vRNA negative when probed with our ISH riboprobe cocktail, the specificity of which was verified using in vitro infected pigtailed macaque cells (see Figs. 2B and S1).
These results were consistent with our observations in PBMCs vis-à-vis plasma viremia and suggest that the vDNA positive cells detected by X-SCA in LNs were not productively infected.
Conversely, the Onlamoon et al. study reported that LNs from infected rhesus macaques contained productively infected cells detectable by immunohistochemistry (IHC) at both early and late (>140 dpi) post infection time points (44).'
17. 'Since XMRV was initially linked to human prostate cancers, we also evaluated viral replication in
prostate tissue. By X-SCA, we detected very low levels (<15 copies/million cells) of vDNA in snap frozen prostate tissue pieces collected at necropsy, and no vRNA positive cells were detectable in prostate tissue sections by ISH.'
Since a small number of contaminating blood cells could be the source of this low level vDNA, these data indicate very limited or no XMRV infection in prostate with no productive infection at 119 dpi. Although Onlamoon and coworkers noted fewer productively infected cells in prostate tissue at late post infection time points than at early post-infection time points, they reported that vRNA+ cells could still be detected by ISH in prostate tissue >140 dpi (44).'
Concluding remarks18. 'These results in
rhesus macaques [from Onlamoon] suggest the establishment of a chronic viral infection, characterized by the stable presence of productively infected cells in LNs and other tissues, concomitant with the apparent loss of vDNA+ PBMCs and undetectable plasma viremia, suggesting that once the virus makes it into LNs and other tissues it continues to replicate but becomes trapped and does not reseed the peripheral blood compartment despite the apparent presence of suitable target cells.
19. 'This conclusion
is in stark contrast to the results we report here for pigtailed macaques, wherein early rounds of viral replication lead to the seeding of PBMCs and LNMCs with a stable pool of archived, hypermutated,
likely non-functional viral genomes. In addition to the difference in macaque species, differences in the sensitivity and specificity of the assays employed could also contribute to the discordant results.'
20. 'In the absence of any confirmed human XMRV infection cases, it is unclear whether infection of pigtailed macaques accurately models human XMRV infection. There is, however, evidence to suggest that several features of pigtailed macaque infection may mirror what would occur in an XMRV infected human.'
Previous work has shown that XMRV infection of human PBMCs in vitro results in a non-spreading, restricted infection with viral production showing a dose-dependent plateau effect with extensive G-to-A hypermutation in cell associated vDNA, likely mediated by APOBEC proteins (7).
Infection of pigtailed macaque PBMCs in vitro showed strikingly similar kinetics and replication patterns to those reported for human cells (see 610 Fig. 2A), and we observed extensive G-to-A hypermutation in vivo (see Fig. 5). It therefore might be expected that XMRV would be similarly hypermutated and restricted during any in vivo human infection, leading to comparably transient, low level viremia.'
21. 'The potential importance of
APOBEC-mediated hypermutation in limiting viral replication is underscored by the rapid decline of plasma viral loads to undetectable levels in our infected animals in the face of persistent vDNA positive cells in blood and LNs,
demonstrating that a total elimination of infected cells, either by immunological or viral lytic mechanisms, was not responsible for the disappearance of viremia, but rather that these cells contained defective viral genomes that did not express viral gene products.
In agreement with this notion, vDNA in LNMCs was first detected by X-SCA just after peak viremia and was still detectable at necropsy, however longitudinal LN sections were vRNA negative by ISH throughout the study.
Although these vDNA positive cells in blood and LNs persisted at 119 dpi, innate immune responses in LN were only transiently detectable in the first week of infection, in stark contrast to the continual LN immune activation associated with chronic SIV infection (see Fig.
, and cellular adaptive immune responses were negligible, consistent with the view that the hypermutated proviral genomes were rendered non-functional and did not express viral gene products.
Taken together, these results suggest that APOBEC-mediated hypermutation is likely largely responsible for the limited viral replication we observed in our infected animals.
22. 'These results may have broader implications concerning the potential for gammaretroviral infection of humans, where such concerns have been raised in particular regarding transfer of porcine gammaretroviruses to immunosuppressed humans in the setting of xenotransplantation (66).
Because gammaretroviruses lack the accessory genes possessed by other retroviruses, such as HIV and SIV, they are ill-equipped to counteract intrinsic host restriction factors like the APOBEC3 proteins.
23. '
As shown here, these primate host restriction factors can be remarkably effective at inhibiting gammaretroviral replication in vivo, and may explain, at least in part, why gammaretroviruses have not been significant pathogens in humans, including immunosuppressed individuals, despite close human association with their animal hosts.'
24. 'Hypermutation of viral genomes also likely explains our unsuccessful efforts to rescue virus from differentially stimulated PBMCs collected at the study’s conclusion using a LNCaP based reporter cell line (DERSE.LiG-puro) (data not shown).
Given the extensive G-to-A hypermutation identified by X-SGS in cell associated vDNA (Fig. 4) it is likely that the proviral genomes were rendered incapable of producing infectious progeny.'
Humans25. 'These results suggest that the isolation of replication-competent XMRV from human blood samples may be difficult or unlikely, particularly when working with samples from unknown post-infection time points that are not likely to represent peak viremia, and are in contrast to previous studies that reported an ability to isolate infectious XMRV from human PBMCs and plasmas similarly using LNCaP target cells (34, 40).
While these disparities might be explained by methodological differences, it seems that XMRV would have to be less susceptible to human APOBEC3-mediated hypermutation in order for virus to be isolatable from PBMCs; however, previous work has shown that XMRV is highly susceptible to APOBEC3-mediated hypermutation in human cells (7, 19, 47).
To successfully rescue virus from human plasma, particularly without the ability to select peak viremia time points, XMRV would likely have to replicate to higher sustained levels in humans than those observed in our pigtailed macaques or exist in human plasma in immune-complexes that have a less detrimental effect on viral infectivity.
If the course of XMRV infection in pigtailed macaques does reflect the natural history of XMRV infection in humans, we would expect serological assays and PCR assays to detect vDNA in PBMCs to have the greatest likelihood of detecting XMRV infection in clinical samples, due to the greater stability of these parameters following XMRV infection in vivo.
In contrast, we would expect assays to detect vRNA in plasma and assays to rescue culturable virus to be less likely to detect XMRV infection due to transient positivity and lack of sensitivity, respectively.
One of the potential shortcomings of previous studies designed to identify XMRV infection in human samples using PCR, serology, immunohistochemistry, in situ hybridization, virus rescue, or other means has been the lack of bona fide in vivo derived positive control samples...
...Therefore, in addition to characterizing the natural history of XMRV infection in vivo, we initiated these studies in pigtailed macaques to generate and make available positive control samples for future analyses...
The reagents we have generated here should facilitate future studies aimed at examining XMRV infection in in vivo derived samples (a). Though XMRV infection of pigtailed macaques doubtless does not perfectly model human XMRV infection, these results suggest that a small, physiological viral inoculum, in the setting of functional APOBEC proteins, will at best likely replicate only briefly and to extremely low levels, and might therefore be very unlikely to induce any significant pathogenesis.'
