AFSSA Alfort, 22 rue Pierre Curie, 94703 Maisons-Alfort cedex, France1
Author for correspondence: Stéphan Zientara. Fax +33 1 49 77 13 13. e-mail s.zientara{at}afssa.fr
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
Main text |
---|
![]() ![]() ![]() ![]() |
---|
The origin of the blood and brain samples is described in Table 1. Some of the blood samples were collected from French horses which had previously been diagnosed as BDV seropositive (Galabru et al., 2000
). The other blood samples were obtained from horses showing nervous disorders or in good health. Horse brains were sampled from autopsies done at the AFSSA Dozulé laboratory (France). Bovine brains were sampled from cattle with nervous disorders and were received from the AFSSA Lyon laboratory (France). All the BDV positive samples were confirmed as BSE negative. Red fox brain tissues were received from the Caen Diagnostic Veterinary Laboratory (France) after negative PCR results for Neospora caninum detection. Other red fox brain tissues were received from the AFSSA Nancy laboratory (France) after negative results for rabies virus detection.
|
The RTnested-PCR was estimated to detect around 140 copies of BDV RNA with the p40 primers and 14 copies of BDV RNA with the p24 primers (Legay et al., 2000 ). To prevent possible contamination of the samples with BDV amplicons, tissue preparation, RNA extraction and cDNA amplification were carried out in separate rooms following strict rules of separation of pre- and post-PCR environments (Belak & Ballagi-Pordany, 1993
).
Seven p40 and five p24 PCR products were directly sequenced in both directions by direct automated sequencing (Genome Express). In order to avoid laboratory contamination, PCR products were not cloned. The sequences were analysed using the Megalign program of the Lasergene software (DNAstar). These sequences were aligned and compared with those of the reference strains described by Cubitt & de la Torre (1994 ) (strain He/80, accession number L27077), Briese et al. (1994)
(strain V, accession number U04608) and Nowotny et al. (2000)
(accession number AF136236). The only laboratory strain handled in our laboratory is the BDV He/80 strain.
Because of the high asymptomatic carriage of BDV in various animal species (Dürrwald & Ludwig, 1997 ), we did not focus our study on animals with neurological disorders. In order to confirm the first set of RTnested-PCR results, total RNA was extracted from new aliquots of the same samples and was subjected to RTnested-PCR with the p40 and p24 primers (the results are summarized in Table 2
).
|
Some positive brain samples were coded and sent to the Vienna and the Giessen laboratories (numbers 63, 66, 99 and 175). The PCR and the antigen detection (by immunohistochemistry) were negative in both laboratories. These discrepancies between the French laboratory and the Austrian and German laboratory results may be due to differences in the sensitivity of the detection methods and the fact that no RNA mimic was used in the second set of tests. Furthermore, the p24 and p40 antigen and genome distribution are very heterogeneous in brain tissue (Gonzalez-Dunia et al., 1997 ; Nakamura et al., 2000
). As an illustration, in our laboratory, only five brain samples among 13 which were positive on one occasion with the p40 primers were positive in two or three aliquots. We cannot rule out the fact that storage and transport conditions may have resulted in partial or total degradation of viral RNA (Sailleau et al., 1997
).
To confirm the specificity of the PCR assays, BDV p40 and p24 cDNA amplified from horse, fox and bovine brain samples and from horse blood samples was sequenced. The PCR products were directly sequenced. The determined sequences were aligned and compared to the He/80 strain, to the No/98 strain and also to strain V. Considering that only short sequences were successfully aligned (200 and 294 nucleotides for the p40 and the p24 sequences, respectively), significant variations at the nucleotide level between the detected sequences and the reference strain sequences were observed (Table 3). No amino acid substitutions between any of the compared p40 sequences were observed (data not shown). The sequence divergencies determined from comparing our BDV sequences and the reference strains ranged between 0 and 6·5% (fox brain 189). This degree of genetic variability (based on short nucleotide sequences of 294 and 200 nucleotide fragments of the BDV p40 and p24 genes, respectively) is in accordance with the general finding of sequence divergency among BDV isolates (Binz et al., 1994
; Iwata et al., 1998
; Schneider et al., 1994
; Vahlenkamp et al., 2000
; Staeheli et al., 2000
).
|
In order to determine the degree of variation of the He/80 strain cultivated in our laboratory, we sequenced directly the PCR products of three RTnested-PCR assays carried out on this He/80 strain. Sequences obtained from the He/80 PCR products were 100% identical to the corresponding sequence published by Cubitt & de la Torre (1994 ) (data not shown). This identity confirmed the high degree of sequence conservation between different sources of the same BDV strain and the low number of mutations induced by our RTnested-PCR protocol.
Three aliquots of horse blood 4 were both p40 and p24 positive by PCR. This horse was also BDV seropositive. This seropositivity (established 6 months before PCR detection) was previously determined by three different laboratories and by three methods: Western blot, indirect immunofluorescence assay and ELISA (Galabru et al., 2000 ). Positive blood samples 6, 7 and 26 to 30 originated from the same stud in southwest France. Ponies 6 and 7 presented with nervous disorders while the other ones in contact with them were in good health. Vahlenkamp et al. (2000)
also detected BDV p40 RNA in healthy horses living with BDV-infected horses. BDV transmission (through salival, nasal or conjunctival secretions) is possible through close contact between animals (Richt et al., 1997
). The fact that, among a majority of 196 randomly collected samples, BDV RNA was detected in animals living on the same farm could confirm a focused transmission of the virus. Moreover, the fact that a mare (no. 16) and her foal (nos 8 and 15) are both PCR positive may indicate vertical transmission. This route of transmission has recently been described (Hagiwara et al., 2000
).
This study also reported BDV p40 RNA detection from one cow (no. 175) which showed neurological disorders (Table 1). The sequence of the p40 PCR product obtained from cattle brain was found to be identical to the He/80 sequence. This sequence identity between the cattle BDV sequence and the horse BDV isolates has already been described by Hagiwara et al. (1996)
.
In conclusion, this study reports for the first time the presence of the BDV genome in France. It also presents the first detection of the BDV genome in the brain of red foxes (Vulpes vulpes) (Table 2). Unfortunately, we had no information about the clinical status of these animals before death. This wild animal species has never been described as naturally infected with BDV. These data add to the epidemiological knowledge about BDV, confirming that this virus has a wide distribution and host range, including domestic and wild animals (Dürrwald & Ludwig, 1997
; Richt et al., 1992
; Sauder et al., 1996
; Nakamura et al., 2000
). Further studies are necessary in order to improve knowledge about this possible zoonotic pathogen, and in particular about the role of wild animals in BDV epidemiology (Herzog et al., 1997
; Dürrwald & Ludwig, 1997
).
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() |
---|
Ballagi-Pordany, A. & Belak, S. (1996). The use of mimics as internal standards to avoid false negatives in diagnostic PCR. Molecular and Cellular Probes 10, 159-167.[Medline]
Belak, S. & Ballagi-Pordany, A. (1993). Experiences on the application of the polymerase chain reaction in a diagnostic laboratory. Molecular and Cellular Probes 7, 241-248.[Medline]
Berg, A. L., Dorries, R. & Berg, M. (1999). Borna disease virus infection in racing horses with behavioral and movement disorders. Archives of Virology 144, 547-559.[Medline]
Binz, T., Lebelt, J., Niemann, H. & Hagenau, K. (1994). Sequence analysis of the p24 gene of Borna disease virus in naturally infected horse, donkey and sheep. Virus Research 34, 281-289.[Medline]
Bode, L., Dürrwald, R. & Ludwig, H. (1994). Borna virus infections in cattle associated with fatal neurological disease. Veterinary Record 135, 283-284.[Medline]
Boucher, J. M., Barbillon, E. & Cliquet, F. (1999). Borna disease: a possible emerging zoonosis. Veterinary Research 30, 549-557.[Medline]
Briese, T., Schneemann, A., Lewis, A. J., Park, Y. S., Kim, S., Ludwig, H. & Lipkin, W. I. (1994). Genomic organization of Borna disease virus. Proceedings of the National Academy of Sciences, USA 91, 4362-4366.[Abstract]
Caplazi, P., Waldvogel, A., Stitz, L., Braun, U. & Ehrensperger, F. (1994). Borna disease in naturally infected cattle. Journal of Comparative Pathology 111, 65-72.[Medline]
Cubitt, B. & de la Torre, J. C. (1994). Borna disease virus (BDV), a nonsegmented RNA virus, replicates in the nuclei of infected cells where infectious BDV ribonucleoproteins are present. Journal of Virology 68, 1371-1381.[Abstract]
Degiorgis, M.-P., Berg, A.-L., Hard, C., Segerstad, A. F., Morner, T., Johansson, M. & Berg, M. (2000). Borna disease in a free ranging lynx (Lynx lynx). Journal of Clinical Microbiology 38, 3087-3091.
de la Torre, J. C. (1994). Molecular biology of Borna disease virus: prototype of a new group of animal viruses. Journal of Virology 68, 7669-7675.[Medline]
de la Torre, J. C., Gonzalez-Dunia, D., Cubitt, B., Mallory, M., Mueller-Lantzsch, N., Grasser, F. A., Hansen, L. A. & Masliah, E. (1996). Detection of Borna disease virus antigen and RNA in human autopsy brain samples from neuropsychiatric patients. Virology 223, 272-282.[Medline]
Dürrwald, R. & Ludwig, H. (1997). Borna disease virus (BDV), a (zoonotic?) worldwide pathogen. A review of the history of the disease and the virus infection with comprehensive bibliography. Journal of Veterinary Medicine 44, 147-184.
Galabru, J., Saron, M. F., Berg, M., Berg, A. L., Herzog, S., Labie, J. & Zientara, S. (2000). Borna disease virus antibodies in French horses. Veterinary Record 147, 721-722.[Medline]
Gonzalez-Dunia, D. (1997). Borna disease virus and the brain. Brain Research Bulletin 44, 647-664.[Medline]
Hagiwara, K., Nakaya, T., Nakamura, Y., Asahi, S., Takahashi, H., Ishihara, C. & Ikuta, K. (1996). Borna disease virus RNA in peripheral blood mononuclear cells obtained from healthy dairy cattle. Medical Microbiology and Immunology 185, 145-151.[Medline]
Hagiwara, K., Momiyama, N., Taniyama, H., Nakaya, T., Tsunoda, N., Ishihara, C. & Ikuta, K. (1997). Demonstration of Borna disease virus (BDV) in specific regions of the brain from horses positive for serum antibodies to BDV but negative for BDV RNA in the blood and internal organs. Medical Microbiology and Immunology 186, 19-24.[Medline]
Hagiwara, K., Kamitani, W., Takamura, S., Taniyama, H., Nakaya, T., Tanaka, H., Kirisawa, R., Iwai, H. & Ikuta, K. (2000). Detection of Borna disease virus in a pregnant mare and her fetus. Veterinary Microbiology 72, 207-216.[Medline]
Herzog, S., Pfeuffer, I., Haberzettl, K., Feldmann, H., Frese, K., Bechter, K. & Richt, J. A. (1997). Molecular characterization of Borna disease virus from naturally infected animals and possible links to human disorders. Archives of Virology 13, 183-190.
Iwata, Y., Takahashi, K., Peng, X., Fukuda, K., Ohno, K., Ogawa, T., Gonda, K., Mori, N., Niwa, S.-I. & Shigeta, S. (1998). Detection and sequence analysis of Borna disease virus p24 RNA from peripheral blood mononuclear cells of patients with mood disorders or schizophrenia and of blood donors. Journal of Virology 132, 241-244.
Kao, M., Hamir, A. N., Rupprecht, C. E., Fu, Z. F., Shankar, V., Koprowski, H. & Dietzschold, B. (1993). Detection of antibodies against Borna disease virus in sera and cerebrospinal fluid of horses in the USA. Veterinary Record 132, 241-244.[Medline]
Legay, V., Sailleau, C., Dauphin, G. & Zientara, S. (2000). Construction of an internal standard used in RT nested PCR for Borna disease virus RNA detection in biological samples. Veterinary Research 31, 565-572.[Medline]
Lipkin, W. I., Travis, G. H., Carbone, K. M. & Wilson, M. C. (1990). Isolation and characterization of Borna disease agent cDNA clones. Proceedings of the National Academy of Sciences, USA 87, 4184-4188.[Abstract]
Ludwig, H. & Bode, L. (2000). Borna disease virus: new aspects on infection, disease, diagnosis and epidemiology. Revue Scientifique et Technique Office International des Epizooties 19, 259-288.
Ludwig, H., Bode, L. & Gosztonyi, G. (1988). Borna disease: a persistent virus infection of the central nervous system. Progress in Medical Virology 35, 107-151.[Medline]
Lundgren, A.-L., Zimmermann, W., Bode, L., Czech, G., Gosztonyi, G., Lindberg, R. & Ludwig, H. (1995). Staggering disease in cats: isolation and characterization of the feline Borna disease virus. Journal of General Virology 76, 2215-2222.[Abstract]
Malkinson, M., Weisman, Y., Perl, S. & Ashash, E. (1995). A Borna-like disease of ostriches in Israel. Current Topics in Microbiology and Immunology 190, 31-38.[Medline]
Matthias, D. (1954). Der Nachweis von latent infizierten Pferden, Schafen und Rindern und deren Bedeutung als Virusreservoir bei der Bornaschen Krankheit. Archiv fuer experimentelle Veterinaermedizin 8, 506-511.
Metzler, A., Ehrensperger, F. & Wyler, R. (1978). Natürliche Bornavirusinfektion beim Kaninchen. Zentralblatt fuer Veterinaermedizin Reihe B 25, 161-164.
Metzler, A., Ehrensperger, F. & Danner, K. (1979). Bornavirus-Infektion bei Schafen: Verlaufsuntersuchungen nach spontaner Infektion, unter besonderer Berücksichtigung der Antikörperkinetik im Serum und Liquor cerebrospinalis. Schweizer Archiv fuer Tierheilkunde 121, 37-48.
Nakamura, Y., Kishi, M., Nakaya, T., Asahi, S., Tanaka, H., Sentsui, H., Ikeda, K. & Ikuta, K. (1995). Demonstration of Borna disease virus RNA in peripheral blood mononuclear cells from healthy horses in Japan. Vaccine 13, 1076-1079.[Medline]
Nakamura, Y., Takahashi, H., Shoya, Y., Nakaya, T., Watanabe, M., Tomonaga, K., Iwahashi, K., Ameno, K., Nomiyama, N., Taniyama, H., Sata, T., Kurata, T., de la Torre, J. C. & Ikuta, K. (2000). Isolation of Borna disease virus from human brain tissue. Journal of Virology 74, 4601-4611.
Nicolau, S. & Galloway, I. A. (1928). Borna disease and enzootic encephalomyelitis of sheep and cattle. In Medical Research Council Special Reports Series, vol. 121. London: His Majestys Stationery Office.
Nowotny, N. & Kolodziejek, J. (2000). Human bornaviruses and laboratory strains. Lancet 355, 1462-1463.
Richt, A. J., Van de Woude, S., Zink, M. C., Clements, J. E., Herzog, S. & Stitz, L. (1992). Infection with Borna disease virus: molecular and immunobiological characterization of the agent. Clinical Infectious Diseases 14, 1240-1250.[Medline]
Richt, A. J., Pfeuffer, I., Christ, M., Frese, K., Bechter, K. & Herzog, S. (1997). Borna disease virus infection in animals and humans. Emerging Infectious Diseases 3, 343-352.[Medline]
Rott, R. & Becht, H. (1995). Natural and experimental Borna disease in animals. Current Topics in Microbiology and Immunology 190, 17-30.[Medline]
Sailleau, C., Moulay, S., Crucière, C. & Zientara, S. (1997). Detection of African horse sickness virus in the blood of experimentally infected horses: comparison of virus isolation and a PCR assay. Research in Veterinary Science 62, 229-232.[Medline]
Sailleau, C., Hamblin, C., Paweska, J. T. & Zientara, S. (2000). Identification and differentiation of the nine African horse sickness virus serotypes by RTPCR amplification of the serotype-specific genome segment 2. Journal of General Virology 81, 831-837.
Sauder, C. & de la Torre, J. C. (1998). Sensitivity and reproducibility of RTPCR to detect Borna disease virus (BDV) RNA in blood: implications for BDV epidemiology. Journal of Virological Methods 71, 229-245.[Medline]
Sauder, C., Muller, A., Cubitt, B., Mayer, J., Steinmetz, J., Trabert, W., Ziegler, B., Wanke, K., Mueller-Lantzsch, N., de la Torre, J. C. & Grasser, F. A. (1996). Detection of Borna disease virus (BDV) antibodies and BDV RNA in psychiatric patients: evidence for high sequence conservation of human blood-derived BDV RNA. Journal of Virology 70, 7713-7724.[Abstract]
Schneider, P. A., Briese, T., Zimmermann, W., Ludwig, H. & Lipkin, W. I. (1994). Sequence conservation in field and experimental isolates of Borna disease virus. Journal of Virology 68, 63-68.[Abstract]
Sorg, I. & Metzler, A. (1995). Detection of Borna disease virus RNA in formalin-fixed, paraffin-embedded brain tissues by nested PCR. Journal of Clinical Microbiology 33, 821-823.[Abstract]
Staeheli, P., Sauder, C., Hausmann, J., Ehrensperger, F. & Schwemmle, M. (2000). Epidemiology of Borna disease virus. Journal of General Virology 81, 2123-2135.
Stitz, L., Krey, H. & Ludwig, H. (1980). Borna disease in rhesus monkeys as a model for uveo-cerebral symptoms. Journal of Veterinary Medicine 6, 333-340.
Vahlenkamp, T., Enbergs, H. & Müller, H. (2000). Experimental and natural Borna disease virus infections: presence of viral RNA in cells of the peripheral blood. Veterinary Microbiology 76, 229-244.[Medline]
Van de Woude, S., Richt, J. A., Zink, M. C., Rott, R., Narayan, O. & Clements, J. E. (1990). A Borna virus cDNA encoding a protein recognized by antibodies in humans with behavioral diseases. Science 250, 1278-1281.[Medline]
Weissenböck, H., Nowotny, N., Caplazi, P., Kolodziejek, J. & Ehrensperger, F. (1998). Borna disease in a dog with lethal meningoencephalitis. Journal of Clinical Microbiology 36, 2127-2130.
Received 21 February 2001;
accepted 11 May 2001.