Dr J. Blomberg
Transcription
Dr J. Blomberg
Jonas XMRV Blomberg 1 Section of Clinical Virology Department of Medical Sciences Xenotropic Murine Email Farid. [email protected] RetroVirus a blood transfusion risk? a murine endogenous virus which became exogenous and zoonotically infected humans? Paper 1 Identification of a Novel Gammaretrovirus in Prostate Tumors of Patients Homozygous for R462Q RNASEL Variant Anatoly Urisman1[, Ross J. Molinaro2,3[, Nicole Fischer4[, Sarah J. Plummer2, Graham Casey2, Eric A. Klein5, Krishnamurthy Malathi2, Cristina Magi-Galluzzi6, Raymond R. Tubbs6, Don Ganem4,7,8, Robert H. Silverman2*, Joseph L. DeRisi1,8* Nature medicine Aug 2010 Chronic controversy continues over mysterious XMRV virus Jern, Sperber and Blomberg Retrovirology 2005 Jern, Sperber and Blomberg Retrovirology 2005 Simplified overview of gammaretroviruses ERVIP (ape) MERV G2 (mouse, rat) MERV G1 (MmERV, MdERV, GaLV, KoRV)(mouse, rat, gibbon, koala) PERV ABC (pig, mouse) MERV G3 (ampho-, eco-, xeno-, polytropic MLV)(mouse, rat) cat (FeLV), rabbit, baboon, chimpanzee, rhesus lemur, chimpanzee, rhesus opossum MLLV ERVT (primate) opossum REV (bird) ERV3 (OWM) ERVE (primate, cow, pig) ERV9W (ape, cow) ERVHF ERVS jb 2011/04/01 With RetroTector©, we find 8000 murine ERVs of which 2000 are gammaretoviral However, around 400 are MLV-like, of which 100 modified polytropic 1-3 xenotropic (according to sequence similarity) gp70 (SU) p15E (TM) p15 (MA) p12 p30 (CA) p10 (NC) Envelope with attachment protein Capsid LTR gag MA p12 CA NC pol PR RT viral genomic RNA RH IN env SU TM reverse transcriptase LTR How well does XMRV/HMRV fulfill the expected properties of an MLV spread to humans? Property Expected Observe d Dis covery/followu p Viral n ucleic acid Hybrid iz ation array w. conserved viral 70-mers. Seren dipitous. PCR (positive and n egative results) Integration into host genome. Clonin g of XMRV into infectious clon e (PC, ME/CFS) Epidemiology, early ph ase Epidemiology, late phase Virus isolation Virus isolation (ME/CFS patients ) Viral antibod ies SFFV FACS (ME/CFS), p ositive outcome ELISA (PC, ME/CFS), positive and negative outcomes Western blot (ME/CFS), negative outcome CMIA (ME/CFS), negative outcome Not observed No clear case (occasion al ME/CFS outb reaks) No known overrep resen tation in STD and in IVDU Contact with Mouse Human-Human sp read, via saliva, sex, moth er-child Conclusion, for or against XMRV/HMRV in humans? For + For, but p roven or sus pected contamination w 22Rv1 like viru s an d mouse DNA For, but s uspected contamination with DNA from XMRV producing cell line For, but s uspicion of contamination with DNA from XMRV producing cell line For, but s uspicion of contamination from XMRV produ cing cell lin e Und ecided ? Agains t Agains t? - How well does XMRV/HMRV fulfill the expected properties of an MLV spread to humans? Property Expected Observe d Conclusion, for or against XMRV/HMRV in humans? Pathogenesis Leukemia, lymphoma Immunodeficiency Encephalitis Enteritis (MAIDS, FAIDS) Autoimmunity Relatively easy to detect in blood Strong B cell respons e, positive WB Prostate cancer? Immunodeficiency? Myalgic Encephalomyelitis? Irritable Bowel Syndrome? No counterpart? Hard to detect in blood Agains t? Weak or absent B cell respons e Agains t? Replication Immune response Agains t? ? ? Epidemiology and comparative pathobiology do not clearly favour presence of XMRV in humans ”Stealth” retroviral infection, with low replication and minute immune response HTLV-2 Studies of Swedish blood donors (Krook et al 1994, 1997) demonstrated 9% HTLV-2 positivity. PCR signal + Immune response Low PCR copy numbers, weak and few bands in WB ? Samples Detection limit Alternative explanation 1 XMRV is a virus which occurs in monoclonal antibody preparations, and certain cell lines Evidence: For: The cell culture virus, 22rv1, is highly similar to XMRV Vp62. The same gag-env recombination occurs in both. Against: -How to explain the FISH results of Silverman et al? - How did a 22rv1 retrovirus get intto Mikovits´cell cultures? - Mikovits reports concordance of PCR, virus isolation and serology? Alternative explanation 2 XMRV/HMRV are endogenous retroviruses of mice. A slight mouse DNA contamination could explain some PCR results, especially those of Lo and Alter Evidence: For: -Amplification from mouse DNA with Lo/Alter primers gives similar HMRV sequences as Lo and Alter from ME patients (our resullts).. Against: -How did mouse DNA get into ME (and prostate cancer?) samples? -Mouse DNA does not grow -Why did this happen ten times more often in ME samples than in controls? This is a difficult scientific and analytical situation The HRV5 story At the end of the 1990ies, Robin Weiss´group published several papers on a novel retrovirus in some of rheumatoid arthritis, systemic lupus and lymphoma patients. They called in human retrovirus 5 (HRV5) My group also found it in a few cases, in low copy number 1 blood donor 1 SLE 2 NHL 1 RA My group also found it in a few cases, in low copy number 1 blood donor 1 SLE 2 NHL 1 RA Then RW published that it was a rabbit endogenous retrovirus present in 700 copies/ rabbit genome. My group also found it in a few cases, in low copy number 1 blood donor 1 SLE 2 NHL 1 RA Then RW published that it was a rabbit endogenous retrovirus present in 700 copies/ rabbit genome. Where did the rabbit DNA come from? My group also found it in a few cases, in low copy number 1 blood donor 1 SLE 2 NHL 1 RA Then RW published that it was a rabbit endogenous retrovirus (RERV-H) present in 700 copies/ rabbit genome. Where did the rabbit DNA come from? Rabbit serum contains much RERV-H DNA. Why did we see it selectively in patient samples? Own work with XMRV/HMRV Samples: 85+100+12 PBMC and plasma samples from ME patients 404+5 prostate samples Methods: Three sensitive real time PCRs (gag, INT, env) One nested gag PCR (same as Lo/Alter) 1 mouse mitochondrial PCR, 1 IAP PCR 1 His 3.3 amplifiability control 48 plex multiepitope serology Own work with XMRV/HMRV Samples: 85+100+12 PBMC and plasma samples from ME patients 0 positive 404+5 prostate samples 1 positive? Methods: Three sensitive real time PCRs (gag, INT, env) One nested gag PCR (same as Lo/Alter) 1 mouse mitochondrial PCR, 1 IAP PCR 1 His 3.3 amplifiability control 48 plex multiepitope serology XMRV/MLV serology in Uppsala 39 (25-30mers) synthetic peptides selected from known MLV epitopes, modelled on XMRV and other MLVs. Multiplex indirect antibody detection test with Luminex (Sjösten, Sheikholvaezin, Hessel, Öhrmalm et al) Y Y B B Y SMIA Suspension Multiplex Immuno Assay B protein G B High throughput multiepitope antibody test 2500 2000 MFI ME BD ME BD 1500 ME BD ME BD ME BD ME BD ME BD ME BD ME BD ME BD 1000 500 0 G1 G1deg MA0deg MA0 MA1 MA2 CA0a Ca0adeg CA0b CA0bdeg CA1 CA2 SU1 SU2 SU3 SU3b SU4 SU5 SU6 SU7 SU8 SU9 SU9b SU9c SU10 SU11 SU12 SU12b SU13 SU14 TM0a TM0b TM0c TM1 TM2 TM3 TM4 TM5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Sera diluted 1/100, peptides bound to Luminex beads. 1h incubation with serum, wash, 30 min incubation with biotinylated protein G, incubation with streptavidin-phycoerythrin, reading in Luminex 200 workstation. No tendency to higher reactivity in ME sera Characterization of antibodies elicited by XMRV infection and development of immunoassays useful for epidemiologic studies Xiaoxing Qiu1*, Priscilla Swanson1, Ka-Cheung Luk1, Bailin Tu1, Francois Villinger2, Jaydip Das Gupta3, Robert H Silverman3, Eric A Klein4, Sushil Devare1, Gerald Schochetman1, John Hackett Jr1 Retrovirology July 2010 One prostate tissue sample positive with three MLV/HMRV/XMRV PCRs CHR11 60402203 CHR1 133470452 CHR4 15169963 Blomberg, Elfaitouri, Danielsson, et al 23Balb c CHR10 22424071 CHR13 21819875 CHR6 73223771 CHR7 64005512 CHR11 102900091 CHR11 76365341 CHR11 86698849 CHR5 24741102 CHR5 43496527 CHR2 15946952 CHR3 67184346 CHR10 4628507 CHR12 70465730 CHR5 122453776 CHR3 152260864 CFS BD28 HM630557 CHR9 62237522 CHR8 44818939 CHR19 60988354 CHR5 110148316 22Balb c CFS type2 HM630558 CFS type1 HM630562 CFS BD26 HM630561 CFS type3 HM630559 CFS BD22 HM630560 24Balb c 12Balb c 9Balb c 5Balb c 1Balb c 2Balb c Black=Gammaretroviral Mouse ERV sequences (mm8, C57Black) predicted to be amplifiable by nested Lo/Alter primers 3Balb c 4Balb c 7Balb c Blue=Amplimers from Balb/c DNA using nest Lo/Alter primers 10Balb c 11Balb c 13Balb c Orange=Amplimers from Lo/Alter paper 15Balb c 14Balb c Magenta=Amplimer from prostate cancer sample 20Balb c CHR4 33126333 18Balb c 19Balb c CHRX 14631230 CHR5 78005469 CHR19 38440157 CHR5 144923629 CHR16 93594754 CHR4 101369892 CHRX 51501683 CHR4 107652725 CHR13 100095189 CHR5 44422900 CHR2 57038510 CHR10 8241473 CHR11 8820301 CHR7 29324348 CHR11 6658083 CHR1 184094018 21Balb c CHR1 193634451 PC1.67 CHR16 76026481 CHR10 41125197 CHR7 30397623 XMRV 0.01 One prostate tissue sample positive with three MLV/HMRV/XMRV PCRs CHR11 60402203 CHR1 133470452 CHR4 15169963 Blomberg, Elfaitouri, Danielsson, et al 23Balb c CHR10 22424071 CHR13 21819875 CHR6 73223771 CHR7 64005512 CHR11 102900091 CHR11 76365341 CHR11 86698849 CHR5 24741102 CHR5 43496527 CHR2 15946952 CHR3 67184346 CHR10 4628507 CHR12 70465730 CHR5 122453776 CHR3 152260864 CFS BD28 HM630557 CHR9 62237522 CHR8 44818939 CHR19 60988354 CHR5 110148316 22Balb c CFS type2 HM630558 CFS type1 HM630562 CFS BD26 HM630561 CFS type3 HM630559 CFS BD22 HM630560 24Balb c 12Balb c 9Balb c 5Balb c 1Balb c 2Balb c Black=Gammaretroviral Mouse ERV sequences (mm8, C57Black) predicted to be amplifiable by nested Lo/Alter primers 3Balb c 4Balb c 7Balb c Blue=Amplimers from Balb/c DNA using nest Lo/Alter primers 10Balb c 11Balb c 13Balb c Orange=Amplimers from Lo/Alter paper 15Balb c 14Balb c Magenta=Amplimer from prostate cancer sample 20Balb c CHR4 33126333 18Balb c 19Balb c CHRX 14631230 CHR5 78005469 CHR19 38440157 CHR5 144923629 No clear separation between CHR16 93594754 CHR4 101369892 CHRX 51501683 CHR4 107652725 CHR13 100095189 CHR5 44422900 CHR2 57038510 CHR10 8241473 ME/CFS amplimers (Lo/Alter) CHR11 8820301 CHR7 29324348 CHR11 6658083 CHR1 184094018 21Balb c CHR1 193634451 and PC1.67 CHR16 76026481 CHR10 41125197 CHR7 30397623 XMRV mouse DNA amplimers 0.01 Possible sources of contamination: mouse DNA (HMRV): microtome? monoclonals (XMRV - 22rv1) cell cultures (XMRV - 22rv1) reverse transcriptases (MLV) Conclusions Different kinds of contamination are known or just implicated The possibility of a ”stealth” infection should be considered There may remain a ”core” of true XMRV/HMRV infections Consequences for blood donation: There are no tests for mass testing of blood donations for XMRV/HMRV, neither nucleic acid nor antibody based. Simplest is to exclude people with ME/CFS diagnosis from blood donation Collaborators Amal Elfaitouri, UU Xingwu Shao, UU Sanna Hessel, UU Ali Sheikholvaezin, UU Anna Sjösten, UU Christina Öhrmalm, UU Fredrik Elgh, UmU Jan Olsson, UmU Carl-Gerhard Gottfries, GU Rüdiger Pipkorn, DKFZ Klas Källander, Cavidi Tech and many others Thanks to our contributors! Replico Medical AB MERUK