XMRV and MLV-related viruses are highly infectious

Frequent detection of infectious
xenotropic murine leukemia
virus (XMLV) in human cultures
established from mouse
xenografts

Yu-An Zhang, Anirban Maitra,
Jer-Tsong Hsieh, Charles M. Rudin, Craig Peacock, Collins Karikari, Rolf A. Brekken, Victor Stastny, Boning Gao, Luc Girard, Ignacio Wistuba, Eugene
Frenkel, John D. Minna and Adi F. Gazdar


Cancer Biology & Therapy 12:7, 1-12;
October 1, 2011; © 2011 Landes
Bioscience


Abstract

Purpose:

To investigate the frequency of
xenotropic murine leukemia virus (MLV)
presence in human cell lines established
from mouse xenografts and to search for
the evidence of horizontal viral spread to
other cell lines.

Methodology:

We examined xenograft tumor cell lines
from 7 independent laboratories and 128
non-xenografted tumor cell lines.

Cell line DNA was examined for mouse DNA
contamination, and by three Taqman qPCR
assays targeting the gag, env or pol
regions of MLV.

Sequencing was used for viral strain
identification. Supernatant fluids were
tested for reverse transcriptase (RT)
activity.

Results:

Six of 23 (26%) mouse DNA free xenograft
cultures were strongly positive for MLV
and their sequences had greater than
99% homology to known MLV strains.

Four of five available supernatant fluids
from these viral positive cultures were
strongly positive for RT activity.

Three of these supernatant fluids were
studied to confirm the infectivity of the
released virions for other human culture
cells.

Of the 78 non-xenograft derived cell lines
maintained in the xenograft
culture-containing facilities, 13 (17%)
were positive for MLV, including XMRV, a
virus strain first identified in human
tissues.

By contrast, all 50 cultures maintained in
a xenograft culture-free facility were
negative for viral sequences.


Conclusions:

Human cultures derived after mouse
xenografting frequently contain and
release highly infectious xenotropic MLV
viruses.

Laboratories working with xenograft-
derived human cultures should be aware
of the risk of contamination with
potentially biohazardous human-tropic
mouse viruses and their horizontal spread
to other cultures.



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Discussion


Reports of XMLV strains being present in
human xenograft cultures appeared in the
1970s. 10,12,33

While several reports have documented
this phenomenon, most reports are
sporadic case reports without systematic
analysis of true frequency.

Two reports examined multiple xenograft
cultures 12,33 and found frequencies of 7
and 67% respectively.

However, because of the relatively crude
detection methods available at that time
and failure to check for mouse cell
contamination, the true frequencies
cannot be determined from these reports.

Because of our initial failure to culture
SCLC tumors directly from tumor samples,
we developed long-term cultures from
xenografted tumors. 34

In the mid 1980s we detected (by
electron microscopy) large numbers of
retroviral particles released by SCLC cell
line NCI-N417.

We destroyed stocks of this cell line and
persuaded the American Type Culture
Collection to stop distribution.


However, prior to this finding, the cell line
had been distributed extensively to the
scientific community and we obtained a
culture from Gerold Bepler, Moffitt Cancer
Center, Tampa, FL.

While we did not report on the viral
findings, others have done so 14,15 and
we refer to this XMLV strain as N417
although it has also been referred to as
VB3.2. 21

Recent reports of squirrel monkey
retroviruses (SMRV) infecting human
cultures (by unknown routes or sources)
and their possible large scale horizontal
spread have also been published, 35,36
and several human cell lines for human
immunodeficiency virus research were
found to release infectious XMLV. 22,37

However, there are virtually no reports of
contamination of xenograft cultures
published during the past 15 y and most
scientists appear unaware of the
contamination problem (authors’
observations).

The purpose of our study was to
determine the frequency of xenograft
culture contamination by xenotropic
viruses and their horizontal spread in
multiple laboratories.

By sequencing the viruses so isolated we
could determine whether a single or
multiple strains of XMLV were responsible
for contamination and horizontal spread.


For our initial study we obtained 26
xenograft cultures from seven
independent laboratories. Only two of the
laboratory chiefs that participated in this
study were previously aware of the XMLV
contamination issue.

We utilized three primer sets that
detected virtually all XMLV stains as well
as most MLV strains, covering the three
major structural and functional regions of
the mouse retroviral genome.

Our multiple qPCR approaches had
advantage to detect all possible MLV
sequences (increased sensitivity) with
one or two additional probe confirmation
(increased specificity) as compared with a
single qPCR assay.

Nine of the samples were positive by
either two or all three of the probes used.

However, three of the nine positive
samples contained varying amounts of
mouse DNA, presumably as a result of
survival of mouse stromal cells from the
mouse xenograft.

Apparently the mouse stromal cells may
persist for lengthy periods in culture,
occasionally in excess of one year.

These three cultures were removed from
further study as the mouse genome
contains multiple endogenous MLV
provirus sequences which make
interpretation difficult.

Six of the remaining 23 xenograft cultures
(26%) were positive for one (or in one
case, two) strains of XMLV or related
viruses.

These six cultures came from six
independent labs indicating the
widespread nature of the contamination.

Of interest, our viral sequencing homology
analysis indicated that multiple strains of
XMLV were present in the six positive
lines, demonstrating that there are
multiple strains of XMLV or MLV-related
viruses capable of infecting human cells
individually or simultaneously after
xenografting.

The LAPC-4 cell line, widely used as a
model for androgen independent prostate
cancer, with over 120 citations in
PubMed, contained two strains.

Early and late passage cultures from the
originator (Dr. Charles Sawyers) as well as
a culture distributed to the Hsieh lab
contained the same strain mixture.


Supernatant fluids were available from five
of the six viral positive xenograft cell
lines. Four of these released large
numbers of potentially infectious viral
particles, indicating possibilities of
horizontal spread to other human cultures
as well as posing a biohazard of unknown
potential to laboratory personnel.

We presume that in one of these five cell
lines the XMLV virus existed in a latent
form. Because of the possibility of spread
to non-xenografted human cultures, we
examined cultures maintained in the same
culture facilities.

A total of 78 cultures were obtained from
five of the laboratories.

Thirteen of these cultures (17%) from
four of the laboratories were positive for
XMLV.

The viruses identified as being responsible
for horizontal spread were identical to
those identified in the positive xenograft
cultures in the respective laboratories.

By contrast, 50 cell lines from the Gazdar
lab maintained in a facility free of
xenograft cultures tested negative for
virus.

These differences were significant,
indicating the potential for horizontal viral
spread to other cultures maintained in the
same laboratory facility as xenograft
cultures.

In one case XMRV virus infection to a
non-xenograft colorectal carcinoma cell
line RKO38 was demonstrated from an
XMRV containing prostate xenograft
derived cell line 22Rv1 even though the
two cell lines had been maintained in the
same culture facility for only a few days.

Our results indicated the frequent
presence of XMLV viruses in cultures
initiated from human xenografted tumors,
with integration of the viral genomes and
production of abundant virions.

We also found evidence of widespread
contamination of non-xenografted
cultures maintained in the same tissue
culture facilities with xenografted
cultures.

However, to rigorously prove that the
virions released by the xenografted
cultures were infectious for human cells,
we collected supernatant fluids from three
XMLV-positive xenografted cultures
including LAPC-4 and used them to infect
five other human cell lines.

All three viruses readily infected two SCLC
lines, one NSCLC line and one virus
infected a non-tumorigenic immortalized
bronchial epithelial cell culture.

The fifth cell line, NSCLC line NCI-H460,
appeared to undergo an abortive
infection, as monitored by reverse
transcriptase activity.

However genomic analyses demonstrated
the presence of relatively low amounts of
integrated forms of two of three infected
viruses, indicating latent infection.

Thus human cells demonstrate
heterogeneity for XMLV viral sensitivity
and the XMLV viruses demonstrate
variable infective potential.

Several reports, have described the
finding, usually of partial XMRV or XMLV
related sequences at low abundance, in
human tumors and tissues. 19,39,40

These methods often utilize nested PCR
techniques for detection, and multiple
other labs have failed to confirm the
findings. Considerable recent evidence
indicates contamination as the probable
cause of these sequences in human cells.
41,42

Because of sequence similarities, the
presence of XMRV virus in human tumors
and cells has been attributed to
contamination, especially from the
frequently used 22Rv1 prostate cancer
cell line 41 but it has not been confirmed
in any cell lines, to our knowledge. 23


Hue et al. screened human tumor 411 cell
lines from the COSMIC collection and
found XMLV sequences positive in nine
cell lines (2.2%), in which five are closely
related to DG-75 strains but none of
these cell lines are confirmed to be
infected with XMRV and the verification of
mouse DNA contamination was not
addressed. 23

Our findings demonstrated that the XMLV
virus in colorectal cell line RKO from the
Maitra Lab is an XMRV isolate
contaminated from the virus containing
xenograft derived 22Rv1 prostate cancer
culture.

The window during which both cell lines
were maintained together in the same
culture facility was only a few days,
indicating that horizontal spread may
occur rapidly. This XMRV-positive RKO
culture probably represents the first
report of horizontal spread of the XMRV
virus.


Our results indicate that human tumor
cells frequently become infected with MLV
virus after xenografting and subsequent
culture.

We have observed that mouse stromal
cells may persist in culture for lengthy
periods. Mouse stromal cells, while they
contain abundant provirus forms of MLV,
including ecotropic, polytropic and
xenotropic strains, seldom spontaneously
release large amounts of infectious virus
(authors’ unpublished findings).

Virus infection of xenografted cells may
require activation of XMLV virus by
chemical or immunological induction in
mouse and by prolonged mouse and
human cell contact.

Viral transfer may occur in the mouse host
or during subsequent xenograft culture.

Our findings of infectivity of
XMLV-positive supernatant fluids
demonstrated that XMLV can readily
infect other human cultures without
presence of mouse cells or other aiding
factors, indicating that these viruses are
highly infectious.


In conclusion, our studies demonstrated
that several MLV strains were present in
over one fourth of xenograft cell lines.

Infected cell lines were identified in most
laboratories working with or establishing
xenograft cultures, indicating that such
contamination was widespread.

Infected cultures usually release large
numbers of infectious virions, and
intra-laboratory spread of MLV virus to
other cell lines maintained in the same
facilities may occur, confirming the highly
infectious nature of MLV virus.

Retroviruses have been associated with
multiple diseases including solid and
hematologic malignancies, AIDS as well as
with non-malignant diseases.

The high susceptibility of human cells to
infection with XMLV, the high levels of
reverse transcriptase activity present in
culture supernatant fluids and the
demonstrated infectivity of the shed
virions suggest that such viruses may
present potential biohazards to laboratory
personnel involved in cell culture facilities
or to those handling human xenografts.

In addition, the effects of the integrated
provirus or the released virions on the
biology of infected tumor cells are
unknown.

Provirus integration into the genome is not
random, and occurs preferentially at
transcription start sites, CpG islands,
DNase-hypersensitive sites and
gene-dense regions, suggesting that
provirus integration may influence
transcription in the host cell. 43

Thus laboratories handling or culturing
human xenografts should monitor for the
presence of MLV, and should consider
monitoring personnel for viral antigens or
antibodies to them.

Laboratories working with xenograft
cultures should have full knowledge and
understanding of the potential biological
and biohazardous risks and should not
distribute or publish their findings without
full disclosure of the virus status of their
xenograft-derived materials.

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