Dr J. Blomberg

Jonas
XMRV
Blomberg
1
Section
of
Clinical Virology
Department
of
Medical
Sciences
Xenotropic
Murine
Email
Farid. Benachenhou@medsci.uu.se
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