1 Federal Research Center for Virus Diseases of Animals, PO Box 1149, 72001 Tübingen, Germany
2 Boehringer Ingelheim Pharma KG, Biberach an der Riss, Germany
Correspondence
Eberhard Pfaff
eberhard.pfaff{at}tue.bfav.de
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ABSTRACT |
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Present address: Molecular Neurogenetics Unit, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA.
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INTRODUCTION |
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In humans, caliciviruses represent the main cause of non-bacterial gastroenteritis (Clarke & Lambden, 1997b), whereas animal caliciviruses can cause vesicular lesions in swine and sea lions, respiratory illness and conjunctivitis in cats, and severe haemorrhagic liver diseases in rabbits and hares (Neill et al., 1998
; Smith et al., 1973
). While VESV, SMSV or FCV can easily be propagated in cell culture and cause a cytopathogenic effect within a few hours (Studdert, 1978
), there is no cell culture system available for lagoviruses or human caliciviruses (Konig et al., 1998
; White et al., 1996
). Caliciviruses consist of non-enveloped virions 3240 nm in diameter. Electron microscopy has revealed that several, but not all species, display typical cup-shaped surface depressions, the characteristic calyx morphology, from which the family derives its name (calix, Latin for cup) (Granzow et al., 1996
; Prasad et al., 1994
, 1999
; Schaffer et al., 1980
).
Caliciviruses have a polyadenylated plus-stranded RNA genome of 7·38·3 kb with a viral VPg protein of 1015 kDa covalently attached to its 5' end and a coterminal subgenomic RNA of 2·22·4 kb. The non-structural proteins are encoded in the 5' end, the major capsid protein VP1 and the minor, basic structural protein VP2 in the 3' end of the genome (Clarke & Lambden, 1997a, 2000
).
Based on sequence homologies to picornavirus proteins, the putative function of several non-structural proteins of caliciviruses has been proposed and, by analogy, they have been designated 2C-like helicase, 3C-like protease and 3D-like polymerase (Neill, 1990; Sosnovtseva et al., 1999
; Wei et al., 2001
; Wirblich et al., 1996
). These proteins are expressed as a polyprotein and subsequently processed by the viral protease into mature, non-structural proteins (Konig et al., 1998
; Liu et al., 1996
, 1999a
; Meyers et al., 2000
; Sosnovtsev et al., 1998
, 1999
; Wirblich et al., 1996
).
The aim of the present study was to characterize a virus, designated isolate 2117, which caused cytopathogenic effects in Chinese hamster ovary (CHO) cells. Electron microscopic analyses of infected cell suspensions revealed viral particles with typical calicivirus morphology. The viral genome sequence and potential ORFs within it were identified and the presence of the 2117 protease and VP2 was shown. Furthermore, a diagnostic RT-PCR assay for rapid and sensitive viral RNA detection was developed.
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METHODS |
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The virus strain FCV-253 was obtained from Centeon Pharma, Germany. The 2117-virus was derived from a plaque purification, performed in the laboratory of Dr W. Werz. For virus propagation, monolayers of CRFK/CHO cells were infected with FCV/2117 virus at an m.o.i. of 5/0·05, respectively. After 90 min, cultures with the inoculum were supplemented with complete medium containing 1 % FBS (DMEM/1 % FBS), and maintained for another 2472 h at 37 °C and 5 % CO2.
To determine the titre of the 2117-virus stock, CHO cells were seeded into 24-well plates (1·25x105 cells per well) and cultivated for 8 h. Dilutions of the virus stock (four replicates per dilution) were added and the cells were propagated further for 4 days in DMEM/1 % FBS. Infected cells were then incubated for 20 min with 25 % Formalin/5 % crystal violet (in 100 % ethanol) and washed with H2O. Cytopathogenic effects could be identified as clear plaques in a violet background, and the TCID50 was determined according to Spearman and Kärber (Kärber, 1931
; Mayr et al., 1974
).
Preparation of 2117-virions and electron microscopy.
2117-virions were isolated from the medium of infected CHO cells 2472 h post-infection (p.i.). The medium was centrifuged (10 min, 800 g) and viral particles in the supernatant concentrated by either crude ultracentrifugation (1·5 h, 104 000 g, 4 °C) or centrifugation through a cushion of 17 % sucrose in TEN-buffer (100 mM NaCl; 50 mM Tris-HCl, pH 7·5; 1 mM EDTA) (3 h, 104 000 g, 4 °C). The pellet was resuspended in 0·051·0 ml medium or TEN-buffer, respectively, for 1224 h at 4 °C. The virus was then purified from the pellet by layering on a sucrose-gradient (60403020 % sucrose in TEN-buffer) and centrifugation for 5 h at 126 000 g at 4 °C. The virus-containing band was visualized and collected under scattered light.
For electron microscopy, an aliquot of the virus sample was adsorbed to grids and negatively stained with 1 % uranyl acetate.
For immune electron microscopy the virus on the grid was incubated with anti-2117-virion polyclonal rabbit serum, diluted 1 : 1000 in PBS with 0·5 % BSA for 45 min. Grids were washed with PBS/0·5 % BSA, then incubated with the secondary antibody (goat-anti rabbit IgG) (Biocell) and conjugated to colloidal gold particles (10 nm). After 45 min grids were washed in PBS/0·5 % BSA and H2O and negatively stained with 1 % uranyl acetate. Rabbit preimmune serum served as a control.
RNA preparation.
QIAamp RNA isolation kits (Qiagen) were used to extract RNA from medium as well as 2117-infected cells, according to the manufacturer's instructions. Otherwise, prior to the isolation of RNA, viral VPg was digested with 0·1 mg proteinase K (Roche) in a sample of either 107 cells or culture supernatant collected from 107 cells in 15 mM Tris, pH 6·8, 0·2 % SDS at 56 °C. After 1 h, 1 ml TRIzol (Life Technologies, originally described by Chomczynski & Sacchi, 1987) was added and the protocol followed according to the manufacturer's instructions. For the isolation of polyadenylated RNA, PolyATract mRNA isolation systems III/IV from Promega were used.
cDNA cloning.
For analysis of the 2117 nucleotide sequence, polyadenylated RNA was isolated from 5x107 virus-infected CHO cells by using the PK/TRIzol RNA preparation method and the PolyATract mRNA isolation system. The polyadenylated RNA was used for the synthesis of a cDNA library by means of the ZAP Express cDNA Synthesis-/ZAP Express cDNA Gigapack II/III Gold Cloning kit (Stratagene). The phage library was screened by plaque hybridization with a 2117-specific PCR-fragment, labelled with [-32P]dCTP, random primers and Klenow enzyme (Rediprime DNA labelling system, Amersham Life Science) (Feinberg & Vogelstein, 1983
). Subcloning of pBK-CMV plasmids was carried out by in vivo excision using a helper-phage, as recommended by the supplier.
RT-PCR.
For the RT reaction, RNA and 1·25 µM reverse primer (rev) were mixed in a volume of 10 µl, heated for 10 min at 65 °C and cooled on ice. 4 µl 5x RT-buffer (Roche), 2 µl dNTP-mix (2·5 mM each dNTP; Invitrogen) and 12·5 U avian myeloblastosis virus (AMV) reverse transcriptase (Roche) were then added and incubated for 1 h at 42 °C in a total volume of 20 µl, followed by 10 min at 85 °C. For the PCR reaction, the RT sample was mixed with 10 µl 10x PCR-buffer (Roche), 0·25 µM of each primer (reverse and forward), 2 µl dNTP-mix (2·5 mM each dNTP; Invitrogen) and 0·5 U Taq DNA polymerase (Roche) or 1·75 U Expand enzyme mix (Roche) to a total volume of 100 µl. Standard PCR amplification protocol: 3 min 95 °C, 2535 cycles [(30 s 95 °C), (30 s Tm-3 °C), (60 s/1000 bp 72 °C)] and 8 min 72 °C. All PCR amplifications were carried out in a Trio-Thermocycler (Biometra).
PCR samples were analysed by agarose gel electrophoresis and either extracted or purified using the QIAquick gel extraction or PCR purification kit (Qiagen), respectively. PCR products were either inserted into the pCR2.1-TOPO-TA cloning vector (Invitrogen), according to the instructions of the supplier, or cloned into the EcoRV site of pBS SK (Stratagene), according to standard procedures.
To demonstrate the presence of caliciviral RNA in 2117-infected cells, total RNA from 2117-infected CHO cells was prepared and RT-PCR carried out by using the PCR Optimizer kit (Invitrogen), according to the manufacturer's instructions. The primers, derived from the polymerase-coding region (1arev: 5'-TAMACRCCATCATCRCCATAMGT; 1afor: 5'-TGGGGCTGTGAYGTYGGYGTYGCC), amplified a fragment that was then gel-purified and inserted into pBS SK. As a control, RNA from FCV-infected CRFK-cells was analysed in parallel.
To confirm the nucleotide sequence of the middle part of the genome and to obtain the 5' end of the coding sequence of the 2117-virus, the following primers were selected: for the midgenome-PCR (midrev: 5'-TCACAAGAATGTTCTCGG; 2117-position 41064123; midfor: 5' TGGCAAGACTACTCTTGC; 2117-position 15421559) and for the 5' end of the genome as well as for a nested PCR (endrevpcr: 5'-GATTCCGGATTAGAGTCG; 2117-position 16681685; endrevnest; 5'-TGATGGCAGAGCATTTGG; 2117-position 11481165 and endfor: 5'-GTGTTTGAGATGGCT). The resulting PCR-fragments were cloned using the pCR2.1-TOPO-TA cloning-kit (middle fragment) or the pBS SK- vector (5' end fragment), respectively.
Evaluation of PCR sensitivity for detection of 2117 RNA in FBS.
To test the sensitivity of a 2117-specific nested PCR (PCR primer: 2117polrev, 5'-GCATCACATTCCAGAGTTGTG, 2117-position 52195239 and 2117polfor, 5'-CCATCCGGTATGCCACTAA, 2117-position 47774795; and nested-primer: 2117polrevnest, 5'-AATTCCGTTTGGGGTCTTCAC, 2117-position 50865106 and 2117polfornest, 5'-ACCTATGGTGATGATGGCG; 2117-position 49124930), aliquots of the 2117-virus stock, ranging from 1 to 500 TCID50 were mixed into 0·5 or 60 ml FBS, respectively. The 0·5 ml samples were used directly for the isolation of RNA (QIAamp viral RNA Mini kit), while the 60 ml samples were first concentrated (100 min at 104 000 g and 4 °C), the pellet resuspended in 0·5 ml medium and then RNA isolation performed. One-third of the RNA was taken for RT-PCR, and 1 µl of the PCR product for the subsequent nested PCR.
Nucleotide sequencing and sequence analyses.
All 2117-specific DNA sequences were analysed on both strands in opposite directions, with an ABI PRISM 377 DNA sequencer (Perkin Elmer) using Big Dye Terminator Cycle Sequencing (Perkin Elmer). Computer analysis of the sequence data was performed using Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison. The 2117 virus sequence has been deposited in GenBank with accession no. AY343325.
CLUSTALX was used for the phylogram, excluding positions with gaps, corrected for multiple substitutions and run with 1000 bootstrap trials. The compared region comprises the amino acid sequence of ORF 2 of 2117 as well as the canine calicivirus CaCV (NP_777374), SMSV-4 (P36285), VESV (NP_066256), the primate calicivirus PCV Pan-1 (AAC61759), FCV (AAL93148), EBHSV (NP_068828), RHDV (AAF69514), the porcine enteric calicivirus PECV (NP_051035) and a human calicivirus, isolate Manchester (CAA60262).
Bacterial protein expression, protein purification and immunization of rabbits.
Parts of the putative 2117-protease (pro) coding region, as well as the gene encoding the minor structural protein (VP2), were amplified in RT-PCRs by primers with a restriction site (rev: HindIII; for: BamHI) at the 5' end (small letters) and 2117-specific sequences at the 3' region (capital letters) (prorev 5'-cagctaattaagcttGTCCAACGATTCTCCCCC, 2117-position 37493766 and profor 5'-ctaggatccgtcgacGTCCCTAAGACGGGCTCC, 2117-position 34753492; VP2rev 5'-cagctaattaagcttTTTGTAAATTTGTGGGTT, 2117-position 79087925 and VP2for 5'-ctaggatccgtcgacAGTGTAGCCGCATTGATC, 2117-position 75277544). The PCR products and the bacterial expression vector pQE9 (Qiagen) were double-digested with HindIII and BamHI, gel purified, ligated and transformed into E. coli XL-1 Blue (Stratagene) according to standard protocols.
For expression of the protease and VP2, overnight cultures of the transformed E. coli cells were diluted (1 : 4) and incubated at 37 °C with shaking at 200 r.p.m. until the optical density at 600 nm was approximately 0·6. At this time IPTG (Sigma) was added to a final concentration of 1 mM. After 4 h, cells were harvested by centrifugation, the bacterial pellet resuspended in 4 : 1 PBS : sample buffer (0·25 M Tris/HCl, pH 6·8, 20 % -mercaptoethanol, 20 % glycerol, 8 % SDS, 0·04 % bromophenol blue), shaken for 20 min at 200 r.p.m. and 65 °C, and sonicated three times for 20 s.
For analysis and purification of proteins, the samples were subjected to SDS-PAGE (Laemmli, 1970). After electrophoresis on 12·517·5 % gels, proteins were visualized by staining for 20 min in 0·1 % Coomassie brilliant blue (in fixer) and destaining for 13 h in fixer (30 % ethanol, 10 % acetic acid).
For protein purification, the band of the expressed viral protein was excised and electroeluted overnight at 100 V in Tris/glycine buffer (25 mM Tris, 192 mM glycine) containing 0·025 % SDS, using an elution device (Bio-Trap, Schleicher & Schüll). The SDS-PAGE and electroelution were repeated twice; the eluted protein was dialysed (dialysis tubes, Roth) for 48 h at 4 °C in PBS and analysed by SDS-PAGE.
To obtain 2117-specific antibodies, 200 µg of the dialysed protein was mixed with PBS to a total volume of 250 µl. For the first immunization of rabbits, the sample was mixed with the same volume of complete Freund's adjuvant (Difco laboratories). For the first, second and third boost the sample was mixed with incomplete Freund's adjuvant (Difco laboratories) and emulsified. The injections were delivered subcutaneously in the back every 2 weeks. Blood was taken from the ear vein before the first immunization and 2 weeks after the second and third boost. The same immunization procedure was performed with rabbits immunized with 2117-virions purified from 2117-infected CHO cells.
Western blotting.
To identify structural proteins, the medium of infected cultures only was centrifuged for 10 min at 800 g and concentrated by centrifugation through a 17 % sucrose cushion in TEN-buffer for 3 h at 104 000 g and 4 °C. The pellet was resuspended in medium for 1224 h at 4 °C and sample buffer added. For the non-structural proteins the cells were harvested and homogenized in PBS : sample buffer 4 : 1. The samples were shaken at 200 r.p.m. for 20 min at 65 °C and sonicated three times for 20 s. The proteins were subjected to PAGE on 12·517·5 % SDS gels (Laemmli, 1970) and transferred onto Protran nitrocellulose transfer membrane (Schleicher & Schüll) in Tris/glycine buffer with 20 % methanol for 4060 min at 25 V (Trans-blot semi-dry transfer cell; Bio-Rad). The nitrocellulose was incubated overnight at 4 °C in TBS (150 mM NaCl; 10 mM Tris/HCl, pH 7·5), supplemented with 1 % BSA. The membrane was then shaken at room temperature in TBS-T (500 mM NaCl; 20 mM Tris/HCl, pH 7·5; 0·05 % Tween 20 for 2 h with virus-specific antibodies (1 : 1000) and 1 h with peroxidase-conjugated IgG anti-rabbit antibody (Nordic Immunology). After incubation with the various antibodies the membrane was washed three times for 10 min in TBS-T. The blot was developed for 1 min in a 1 : 1 ECL1/ECL2 mixture (Amersham) and exposed to X-ray film (BioMax; Kodak) for 0·5 to 7 min.
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RESULTS |
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Electron microscopy of 2117-virions
Supernatant from 2117-infected CHO cells was concentrated through a sucrose cushion, purified by density-gradient centrifugation and analysed by electron microscopy. Viral particles were about 40 nm in diameter with icosahedric symmetry. These virions displayed a structured surface consisting of regularly arranged cup-shaped depressions (Fig. 1a). Size and morphology were consistent with classical caliciviruses. No virus particles were observed in a matching fraction of samples from uninfected CHO cells.
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Analysis of the coding sequence of the 2117-virus
In order to demonstrate the presence of calicivirus RNA in 2117-infected CHO cell cultures, RT-PCR was performed using a vesivirus-specific primer pair 1arev and 1afor, specific for the RNA polymerase. The sequence of the 508 bp 2117-fragment revealed nearly 80 % identity with the nucleotide sequence of the mink calicivirus MCV/13/1980/US. In a second step, a 2117-specific cDNA library was established and screened using the 508 bp 2117-specific PCR fragment. From two independent cDNA libraries several 2117 positive clones were isolated and sequenced. Since most of the viral sequences had a poly(A) tail and mapped to the 3' part of published calicivirus genomes, the cDNA library was screened with a second probe specific for the 5' region of the 2117 genome. Two clones covering the 5' region of the 2117 genome were isolated.
To verify the results of this additional screening, RT-PCR spanning the region of the middle part of the genome was performed. Each of the sequences of four PCR-fragments, spanning 2581 nucleotides, from four independent RT-PCRs were determined.
The nucleotide sequence of the putative 5' end of the 2117 genome was also obtained by RT-PCR and a following nested reaction. The comparison of 5' ends of genomic and subgenomic RNAs of several caliciviruses, as well as the putative 5' end of the 2117 subgenomic RNA, led to the selection of the endfor primer, used in the PCR and nested PCR. The primer contains an ATG sequence as a potential start codon of the first ORF of the 2117 genome. In the nested PCR, a DNA fragment of 1174 bp was amplified and 13 different fragments were analysed.
The entire coding nucleotide sequence of the 2117 genome has been assembled from the totality of all clones from the different reactions. From the adenine of the postulated start codon of ORF 1 up to the polyadenylated 3' end, the 2117 genome is composed of 8091 nucleotides, excluding the poly(A) tail. Three ORFs were predicted from computer analysis of the 2117 nucleotide sequence and by comparing the results with the genomic organizations and ORFs of other caliciviruses (Clarke & Lambden, 1997a; Thiel & Konig, 1999
). ORF 1 extends to nucleotide (nt) 5451, and encodes a putative polyprotein of 1816 amino acids (aa). ORF 2 ranges from nt 5455 to 7527 and ORF 3 from nt 7524 to 7928, and they encode proteins of 690 and 134 aa, respectively. The 3'-untranslated region consists of 163 nt.
With regard to genomic organization, ORFs 1 and 2 are in the first frame, with the ORF 1 stop codon (54495451) followed by 3 nucleotides and then the start codon of ORF 2 (54555457). ORF 3 is in the third frame, overlapping by 1 nt with ORF 2 (Fig. 2).
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DISCUSSION |
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The calicivirus, designated 2117, was initially isolated from CHO cells showing apparent cytopathogenic effects. Electron microscopic analysis of the cell lysate revealed virions with typical calicivirus morphology. These kind of virions have been described for vesiviruses as well as lagoviruses and classical human caliciviruses (Prasad et al., 1994; Schaffer et al., 1980
).
The analysis of the 2117 genome revealed considerable similarities to sequences of the canine and the mink calicivirus, as well as to SMSV and FCV strains. The relationship to the calicivirus family was also evident in the conservation of several amino acid motifs distributed throughout the genome, with the same linear arrangement and relative distances. For example, the non-structural proteins, for example NTPase, protease and polymerase motifs, are in the 5' end (ORF 1) and the motifs for the structural protein VP1 (ORF 2) in the 3' end. Additionally, through the amino acid motif FRAES in ORF 2, the predicted processing of the primary translation product to the mature 2117 VP1 appears consistent, since in other vesiviruses this motif has already been reported as the potential (Matsuura et al., 2000; Neill, 1992
; Neill et al., 1998
) or actual (Sosnovtsev et al., 1998
) processing site of the protein through the viral protease.
Regarding genomic organization, a rare feature was observed in the 2117 genome. The separation of the first and second ORF by a stop-codon resembles the organization of vesiviruses and noroviruses. However, the location of the coding sequences for the non-structural and the major capsid protein within a single frame is more typical for the lagoviruses and sappoviruses. This kind of arrangement has so far been described only for the canine calicivirus (Roerink et al., 1999).
The viral protease of 2117 has been identified as part of the non-structural polyprotein. As known from other caliciviruses, the non-structural proteins are expressed as a polyprotein and cotranslationally processed to the individual mature proteins by the viral protease. For the Southampton virus, for example, initially a 113 kDa C-terminal protein was observed in the rabbit reticulocyte lysate system. Whereas in expression studies in E. coli the protein was further processed to 22 and 16 kDa proteins, in addition to the 19 kDa protease and 57 kDa polymerase (Liu et al., 1996, 1999b
). In RHDV, the translation of viral RNA in rabbit reticulocyte lysates resulted only in a proteasepolymerase protein of 69 kDa (Wirblich et al., 1996
); however, the 15 kDa protease and 58 kDa polymerase could additionally be detected in cultures of RHDV-infected primary rabbit liver cells (Konig et al., 1998
) and also after transient expression of RHDV cDNA in cell culture (Meyers et al., 2000
). In FCV, only a stable proteasepolymerase precursor protein of 78 kDa has been found so far (Sosnovtsev et al., 2002
; Sosnovtseva et al., 1999
). Only one immunoreactive protein of 68 kDa could be detected in 2117-infected CHO cells, with antibodies against the protease generated in this study. Since the C terminus of the polymerase is defined by the stop codon of ORF 1 and a predicted polyprotein of 68 kDa includes all known amino acid motifs of both proteins, this protein is concluded to be a stable protein of protease and polymerase. Consequently, there is efficient processing of the precursor protein at the N terminus of the protease in infected CHO cells, but no detectable processing at the 3C3D boundary.
Concerning structural proteins, the major capsid protein VP1 and the minor, basic structural protein VP2 have both been described as components of calicivirus particles (Glass et al., 2000; Sosnovtsev & Green, 2000
; Wirblich et al., 1996
). For VP2, proteins of 9 and 8·5 kDa have been identified in RHDV and FCV, respectively, while a 35 kDa protein, as well as multiple higher molecular mass proteins, was characterized from stool samples of Norwalk virus-infected volunteers (Glass et al., 2000
; Sosnovtsev & Green, 2000
; Wirblich et al., 1996
). In the present study, a protein with a molecular mass of about 9 kDa was present in 2117-virions. The function of this protein remains unclear. In experiments expressing the RHDV capsid protein in insect cells, this basic protein was not necessary for the formation of particles (Sibilia et al., 1995
). Possibly the basic protein plays a role in packaging of the viral RNA or in infectivity. The composition of this protein of mainly basic amino acids would support the speculation that it functions as nucleic acid-binding protein (Neill et al., 1991
; Wirblich et al., 1996
).
The origin of the virus, as well as the route of entry of the virus or viral RNA into cell culture, is still unknown. Aside from the cells, FBS as a culture supplement is the most likely source of viral contamination. In order to detect the presence of viral RNA in biological material, a 2117 specific RT-PCR was developed. The PCR enabled the detection of at least one infectious unit mixed into FBS, indicating the sensitivity of the PCR and also the capability of isolating RNA from FBS. This test will enable the screening of cell culture material, especially FBS, for 2117 RNA. Contamination of FBS with other viruses, especially bovine viral diarrhoea virus, but also parainfluenza 3 virus, or infectious bovine rhinotracheitis virus has already been published (Erickson et al., 1991). In addition, transplacental passage of caliciviruses has already been described for cats and sea lions, where FCV and SMSV, respectively, have been detected in aborted foetuses (Smith et al., 1973
; van Vuuren et al., 1999
). Recently, Smith et al. (2002)
published the detection of a vesicular exanthema of swine-like calicivirus in tissues from a naturally infected, spontaneously aborted bovine foetus.
However, the spread of the 2117-virus in nature or its association with any naturally occurring symptoms has not yet been reported. Future studies will involve tests of several FBS samples for the presence of 2117 RNA by RT-PCR and infections of CHO cells with FBS under different conditions. Further research on the origin of the 2117-virus will entail screening of dogs and cattle for 2117-antibodies as well as examinations for a potential link with diseases in different species.
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ACKNOWLEDGEMENTS |
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Received 13 December 2002;
accepted 4 April 2003.