Xenotropic Murine Leukemia Virus¨Crelated Gammaretrovirus in Respiratory Tract

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DOI: 10.3201/eid1606.100066

Suggested citation for this article: Fischer N, Schulz C, Stieler K, Hohn O,

Lange C, Drosten C, et al. Xenotropic murine leukemia virus¨Crelated

gammaretrovirus in respiratory tract. Emerg Infect Dis. 2010 Jun; [Epub ahead of

print]

Xenotropic Murine Leukemia Virus¨Crelated Gammaretrovirus in Respiratory Tract

Fischer, Schulz, Stieler, Oliver Hohn, Christoph Lange,

Christian Drosten, and Aepfelbacher

Author affiliations: University Medical Center Hamburg-Eppendorf, Hamburg,

Germany (N. Fischer, C. Schulz, K. Stieler, M. Aepfelbacher);

Koch-Institute, Berlin, Germany (O. Hohn); Leibniz-Center for Medicine and

Biosciences, Borstel, Germany (C. Lange); and University of Bonn Medical Centre,

Bonn, Germany (C. Drosten)

Xenotropic murine leukemia virus¨Crelated gammaretrovirus (XMRV) has been

recently associated with prostate cancer and chronic fatigue syndrome. To

identify nucleic acid sequences, we examined respiratory secretions by using

PCR. XMRV-specific sequences were detected in 2%¨C3% of samples from 168

immunocompetent carriers and ¡Ö10% of samples from 161 immunocompromised

patients.

Xenotropic murine leukemia virus¨Crelated gammaretrovirus (XMRV) was originally

discovered in tissue from patients with familial prostate cancer homozygous for

a missense mutation in the RNase L gene, R462Q (1). Detection of viral nucleic

acid in tissue sections of cancerous prostate glands and cloning of the viral

integration sites confirmed XMRV as a bona fide human infection with a murine

leukemia virus¨Crelated retrovirus (1). Whether XMRV is actively involved in

prostate cancer tumorigenesis or whether it is just a bystander virus (2,3)

remains unclear.

On the basis of its close homology (up to 94% nt identity) to endogenous and

exogenous full-length sequences from Mus musculus mice (1), XMRV most likely

originated in mice, although they are probably not the current reservoir of

infection (4). Recent findings of XMRV sequences in up to 67% of peripheral

blood mononuclear cells (PBMCs) of patients with chronic fatigue syndrome and in

3.4% of PBMCs of healthy controls raise the question whether XMRV

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could be a blood-borne pathogen (5). However, the finding of XMRV in PBMCs from

patients with chronic fatigue syndrome is controversial because multiple studies

in Europe have failed to detect XMRV (6¨C8). Similarly, frequency of XMRV in

prostate cancer samples ranges from 0 to 23%, depending on geographic

restriction of the virus or, more likely, diagnostic techniques used (PCR,

quantitative PCR, immunohistochemistry) (1¨C3,9,10). Indirect evidence has

suggested sexual transmission (9). Questions remain about worldwide

distribution, host range, transmission routes, and organ tropism of the virus.

To begin to answer some of them, we looked for XMRV in respiratory samples from

267 patients with respiratory tract infection (RTI) and 62 healthy persons.

The Study

During 2006¨C2009, the 267 samples were collected from 3 groups of patients

(Table). Group 1 comprised patients who had traveled from Asia to Germany;

location of their permanent residency was unknown. Groups 2 and 3 and the

control group comprised only persons from northern Germany. From group 1, a

total of 75 sputum and nasal swab specimens were collected from patients who had

unconnected cases of RTI and who had recently traveled by air (11). From group

2, a total of 31 bronchoalveolar lavage (BAL) samples were collected from

patients with chronic obstructive pulmonary disease (defined by a forced

expiratory volume in 1 second/forced vital capacity <70% and forced expiratory

volume in 1 second <80% of the predicted value) who had signs of RTI. From group

3, a total of 161 BAL and tracheal secretion samples were collected from

patients with severe RTI and immunosuppression as a result of solid organ or

bone marrow transplantation. From the control group, throat swabs were collected

from 52 healthy persons and BAL samples were collected from 10 healthy

volunteers who had no signs of RTI and no known underlying disease.

All samples were analyzed by culture for pathogenic bacteria and fungi and by

PCR for rhinoviruses, adenoviruses, enteroviruses, influenza viruses A and B,

parainfluenza viruses 1¨C3, respiratory syncytial virus, cytomegalovirus,

Epstein-Barr virus, and human metapneumovirus. All samples were tested in

duplicates obtained by individual RNA extractions. XMRV RNA was reverse

transcribed from total RNA, after which nested PCR or real-time PCR were

conducted as

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recently described (1,12). No serum samples were available from these patients

to confirm the results by serologic testing.

For group 1, XMRV-specific sequences were detected with relatively low frequency

(2.3%). For group 2, XMRV-specific sequences were amplified in 1 BAL sample,

which was also positive for Staphylococcus aureus by routine culture methods.

For group 3, XMRV-specific sequences were detected at a frequency of 9.9%, which

was significantly higher than that for the healthy control group (3.2%) at the

90% confidence level but not at the 95% level (p = 0.078, 1 sample t-test). Of

16 group 3 samples, 10 showed no signs of co-infection. The remaining 6 samples

showed co-infection with rhinovirus or adenovirus (1 sample each); S. aureus (3

samples); or mixed infection with pathogenic fungi, Candida albicans and

Asperigillus fumigatus (1 sample).

All samples that were positive for XMRV by gag-nested PCR, together with a set

of those that were negative for XMRV, were retested by real-time PCR. Results

showed low XMRV RNA concentrations, 103 ¨C104/mL of specimen.

To confirm the validity of XMRV detection, a subset of 6 specimens (3 XMRV

positive and 3 XMRV negative) were tested by using an alternative PCR assay for

viral RNA (3) and a C-Type RT Activity Kit (Cavidi, Uppsala, Sweden) for type C

reverse-transcription activity. XMRV sequences from alternative targets in the

gag and env regions were confirmed in 2 of the 3 XMRV-positive samples but in

none of the controls. One XMRV-positive BAL specimen showed an 8-fold increase

above background of specific type C retroviral reverse-transcriptase activity,

suggesting presence of active type C retrovirus within this sample. This assay

is substantially less sensitive than reverse transcription¨CPCR.

All XMRV gag sequences (390-bp fragment) were 98%¨C99% identical to previously

published XMRV sequences from persons with prostate cancer (1,2). Phylogenetic

analysis showed close clustering (Figure).

Conclusions

XMRV, originally identified in RNase L¨Cdeficient patients with familial

prostate cancer, has gained interest since recent work showed its protein

expression in as many as 23% of prostate cancer cases (10) and XMRV-specific

sequences were detected in PBMCs of 67%

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patients with chronic fatigue syndrome (5). These results, however, could not be

confirmed by others (6¨C8). Both studies also detected XMRV protein or sequences

in their control cohorts with frequencies of 6% and 4%, respectively.

Among the most pressing information gaps with regard to XMRV is its preferred

route of transmission. Detection of XMRV in PBMCs and plasma of patients with

chronic fatigue syndrome raises the possibility of blood-borne transmission;

sexual transmission has also been hypothesized on the basis of indirect evidence

(5,9). We detected XMRV in respiratory secretions of immunocompetent patients

with and without RTI at a frequency of ¡Ö3.2%, which is in good concordance with

the recently reported prevalence in the general population of up to 4% (5).

Frequency of XMRV detection in group 1 patients (2.25%) was comparable to that

of human metapneumovirus and rhinovirus within this group and considerably less

frequent than that of parainfluenzavirus (15.5%) or influenza A virus (7.6%)

detection (11).

Our findings indicate that XMRV or virus-infected cells might be carried in and

transmitted by the respiratory tract. Attempts to isolate infectious virus from

XMRV sequence¨Cpositive respiratory samples failed, possibly because of

inadequate storage of samples before virus culturing attempts or relatively low

copy numbers of the virus within the samples. Thus, whether the respiratory

tract serves as a putative transmission route for XMRV cannot be determined at

this time. The observed increase in prevalence among immunosuppressed patients

with RTI suggests that XMRV might be reactivated in absence of an efficient

antiviral defense. Together with earlier observations on increased XMRV

replication in RNase L¨Cdeficient cells (1,12), this finding implies that the

immune system plays a role in controlling XMRV replication. It remains unknown

whether immunosuppression predisposes a patient to secrete infectious XMRV from

the respiratory tract or whether presence of virus might be meaningless for

epidemiology in a way similar to HIV-1 (15). Future studies should address

whether the respiratory tract might serve as a source of XMRV infection or

whether immunosuppression might cause an increased risk for primary infection.

This study was supported by the Werner Otto Stiftung grant no. 4/69 to N.F. The

study was approved by the ethics committee at the board of physicians of the

Free and Hanseatic City of Hamburg (No.WF-005/09).

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Dr Fischer works as a group leader at the Institute for Medical Microbiology and

Virology at the University Medical Center Hamburg-Eppendorf. Her main research

interests are emerging viruses, in particular the gammaretrovirus XMRV.

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Address for correspondence: Fischer, Institute for Medical Microbiology

and Virology, University Medical Center Hamburg-Eppendorf, istrasse 52,

20246 Hamburg, Germany; email: nfischer@...

Table. Detection of XMRV in respiratory tract secretions from 329 persons*

Group

Patient median age, y

Underlying disease

Sample

XMRV+

1 (75 patients with RTI)

42

None

Sputum, nasal swab

3/75 (2.3%)

2 (31 patients with RTI)

60

COPD

BAL

1/31 (3.2%)

3 (161 patients with RTI)

32

Immunosuppression after SOT or BMT

BAL, TS

16/161 (9.9%)

Control (62 persons with no RTI)

35

None

BAL, throat swab

2/62 (3.2%)

*XMRV, xenotropic murine leukemia virus; +, positive for XMRV¨Cspecific

sequences by PCR; RTI, respiratory tract infection; COPD, chronic obstructive

pulmonary disease; BAL, bronchoalveolar lavage; SOT, solid organ

transplantation; BMT, bone marrow transplantation; TS, tracheal secretion. Page

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Figure. Xenotropic murine leukemia virus¨Crelated gammaretrovirus (XMRV) gag

sequences derived from respiratory tract secretions. Phylogenetic tree comparing

the 390-nt gag fragment of all respiratory samples of this study with recently

published XMRV sequences from patients with familial prostate cancer (1). The

edited sequences were aligned with ClustalX version 1.82 (13,14) by using

default settings. The tree was generated on the basis of positions without gaps

only. Sequences are labeled as X, xenotropic; P, polytropic; mP, modified

polytropic; S, sputum, IS, immunosuppression; TS, tracheal secretion; and C,

control. Scale bar indicates nucleotide substitutions per position. Page 7 of 7