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Commentary
Nature of the Virus Associated
with Endemic Balkan Nephropathy
Cristina Riquelme,* David Escors,* Javier Ortego,* Carlos M. Sanchez,*
Branislava Uzelac-Keserovic,† Konstantin Apostolov,‡ and Luis Enjuanes*
*Centro Nacional de Biotecnologia, CSIC, Campus Universidad Autónoma,
Madrid, Spain; †Institute of Virology “Torlak,” Belgrade, Yugoslavia;
and ‡Royal Postgraduate Medical School, London, United Kingdom
Suggested
citation for this article: Riquelme C, Escors
D, Ortego J, Sanchez CM, Uzelac-Keserovic B, Apostolov K. Nature of
the virus associated with endemic Balkan nephropathy. Emerg Infect Dis
[serial online] 2002 Aug [date cited];8. Available from: URL:
http://www.cdc.gov/ncidod/EID/vol8no8/02-0042.htm
Endemic Balkan nephropathy (EBN), a disease restricted to three Balkan
countries (Bulgaria, Rumania, and Yugoslavia), is characterized by a progressive
shrinking of the kidneys and, in some cases, tumors in the proximal regions
of the urinary tract (1,2). A coronavirus was reported
to be involved in the etiology of the disease, mostly on the basis of
the isolation of a virus in cultures of kidney cells from a patient with
EBN (1,3). In addition, EBN-associated virus is reported
to share serologic homology with human coronaviruses OC43 and 229E, as
well as the porcine transmissible gastroenteritis coronavirus (formal
name: Transmissible gastroenteritis virus [TGEV]), a virus that
our laboratory has been studying for 16 years (4,5).
The objective of this commentary is to clarify whether the EBN-associated
virus is in fact related to members of the Coronaviridae family
(6).
Characterization
of the Virus in EBN Primary Kidney Cell Cultures
The EBN-associated virus was isolated from primary kidney cells cultures,
grown from fresh renal biopsy specimens of clinically confirmed cases
of EBN (3). The virus grown in the primary kidney cultures
was used to infect Vero cells (ATCC CRL 1586) and sent to our laboratory
for further identification.
A titration method was set up for the EBN-associated virus in Vero cells,
as described for coronaviruses (7). The virus had a small
plaque phenotype and titers of 106 to 107 PFU/mL.
No specific neutralization was observed when polyvalent or monoclonal
antibodies that neutralized TGEV or the human coronaviruses OC43 or 229E
were used in a standard neutralization assay. Furthermore, we observed
no reactivity by immunofluorescence microscopy with the same antisera
and specific monoclonal antibodies (7) on cells infected
with the EBN-associated virus. In contrast, cell cultures infected with
human coronaviruses or TGEV were positive with the corresponding antibodies.
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Click to view
enlarged image
Figure.
Electron microscopy images of thin sections and partially purified
virions from cells infected with coronavirus or a virus tentatively
associated with the endemic Balkan nephropathy (EBN)....
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Since coronavirus morphology is easily recognized by electron microscopy,
Vero cells infected with the EBN-associated virus were embedded in resin
for electron microscopy, and ultrathin sections were examined. Coronaviruses
interacting with the cell membrane or inside the cell cytoplasm were easily
seen when grown in swine testicle cells (Figure, A and
C). Coronaviruses bud at the intermediate compartment and Golgi membranes
(Figure, C) and the cytopathic effect on infected
cells differed from that observed after infection by the EBN-associated
virus. The most characteristic features of EBN-associated virus infection
were the accumulation of stacked intracellular membranes and a general
disorganization of the cytoplasmic membranous system (Figure,
D). No apparent effect on the nucleus structure was observed. Electron-dense
spherical virus particles approximately 30 nm in diameter were observed
in the cytoplasm of infected cells but not in the nucleus. The size of
these particles corresponds to that of the virions partially purified
from the same cells. The particles appear to have an internal dense nucleocapsid
(Figure, B). No viral factories were identified
in association with the membranous structures. No viruslike particles
were observed in uninfected Vero cells that resembled the virions described
in the infected ones.
Supernatants of Vero cells infected with the EBN-associated virus were
concentrated 100-fold by ultracentrifugation and visualized by negative
staining with 2% uranyl acetate. Using purified TGEV as a standard, we
observed only one type of spherical virion with a homogeneous mean virion
size 28.4 nm ± 2 nm in diameter (coefficient of variation 7.1%; n=30)
(Figure, F). In contrast, electron microscopy preparations
of TGEV observed in parallel showed virions approximately 120 nm in diameter,
with a corona of typical projecting peplomers (8,9) (Figure,
E ). The morphology of TGEV clearly differed from that of the EBN-associated
virus that had no peplomers. The EBN-associated virion morphology and
size were similar to that of small nonenveloped viruses such as picornavirus
and parvovirus.
Conclusion
The virus source used in these experiments is the same as that previously
analyzed (3) and lead to the tentative conclusion that
a coronavirus was present in the primary cell cultures from patients with
the endemic nephropathy. The dominant and only virus detected in the cell
cultures infected with the EBN-associated virus was unrelated to coronaviruses.
Accordingly, we think that the involvement of a coronavirus should no
longer be considered in EBN induction. Further studies are needed to clarify
the nature of the 28.4-nm, non-enveloped virus particles found in the
kidney cells of patients with EBN and to determine whether this virus
is the causal agent of the disease.
Acknowledgments
We thank Dusko Vasic and the staff of the Department of Urology, District
Hospital, Doboj, Bosnia, for providing the kidney biopsy specimens.
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