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Virus Research 155 (2011) 514–519
Contents lists available at ScienceDirect
Virus Research
journa l homepage: www.e lsev ier .com/ locate /v i rusres
etection of chimpanzee polyomavirus-specific antibodies in captive andild-caught chimpanzees using yeast-expressed virus-like particles
nja Zielonkaa, Ernst J. Verschoorb, Alma Gedvilaitec, Uwe Roeslerd, Hermann Müllera, Reimar Johnee,∗
Institute for Virology, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 29, 04103 Leipzig, GermanyBiomedical Primate Research Centre, Rijswijk, The NetherlandsVilnius University, Institute of Biotechnology, Graiciuno 8, Vilnius, LithuaniaInstitute of Animal and Environmental Hygiene, Philippstr. 13, 10115 Berlin, GermanyFederal Institute for Risk Assessment, Diedersdorfer Weg 1, 12277 Berlin, Germany
r t i c l e i n f o
rticle history:eceived 29 October 2010eceived in revised form7 December 2010ccepted 17 December 2010
a b s t r a c t
Chimpanzee polyomavirus (ChPyV) was originally detected in the faeces of a captive chimpanzee byrandom screening using broad-spectrum PCR. Its pathogenicity and the distribution among chimpanzeesare unknown so far. Here, the major capsid protein VP1 of ChPyV was expressed in yeast cells. Virus-likeparticles (VLPs) with a diameter of approximately 45 nm were demonstrated although the efficiency ofVLP formation was low as compared to monkey polyomavirus SV40-VLPs. The ChPyV-VLP preparation did
vailable online 25 December 2010eywords:olyomavirusirus-like particleshimpanzee
not agglutinate human erythrocytes. Low cross-reactions between ChPyV- and SV40-VLP-specific serawere detected by immunoblotting, but not by ELISA. Testing of 163 sera derived from captive and wild-caught healthy chimpanzees using an ELISA based on the ChPyV-VLPs resulted in 11.7% positive results,ranging from 0% to 56% in different groups. The VLPs may be used in future to assess the distribution ofChPyV infections among other animal species and humans.
LISAaemagglutination
. Introduction
Polyomaviruses are widely distributed among humans, mam-alian animals and birds. Most of the mammalian polyomaviruses
ause subclinical infections in non-immunocompromized hosts,ut may induce disease after severe immunosuppression. BKolyomavirus is the etiological agent of polyomavirus-associatedephropathy in transplant patients (Azzi et al., 1994; Hirsch, 2002)nd JC polyomavirus can cause progressive multifocal leukoen-ephalopathy in AIDS patients (Imperiale and Major, 2007). Merkelell polyomavirus is involved in induction of Merkel cell carci-oma, whereas the human polyomaviruses WU and KI have noteen linked to disease (Feng et al., 2008; Gaynor et al., 2007;llander et al., 2007). Recently, three novel polyomaviruses haveeen detected in skin samples of humans (Schowalter et al.,010; van der Meijden et al., 2010). Many of the mammalianolyomaviruses induce tumours after inoculation into laboratory
odents (Imperiale and Major, 2007). This is particularly seen forhe monkey polyomavirus SV40, and involvement of this virus inuman tumour diseases has been repeatedly suspected. Severalolyomaviruses have also been detected in birds, with avian poly-∗ Corresponding author. Tel.: +49 30 18412 1006; fax: +49 30 18412 2064.E-mail address: [email protected] (R. Johne).
168-1702/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.virusres.2010.12.009
© 2010 Elsevier B.V. All rights reserved.
omavirus (APV) being the best-characterized member (Johne andMüller, 2007).
Members of the family Polyomaviridae are non-enveloped icosa-hedral viruses with a diameter of approximately 45 nm and agenome of circular double-stranded DNA, about 5 kbp in length.The viral capsid is composed of the major capsid protein VP1 andthe minor capsid proteins VP2 and VP3. VP1 assembles into pen-tamers, which bind one molecule of VP2 or VP3, and 72 of thesepentameric units form the viral capsid (Liddington et al., 1991;Stehle et al., 1996). It has been shown for several mammalian poly-omaviruses that recombinant expression of VP1 in prokaryotic andeukaryotic systems can lead to the formation of virus-like particles(VLPs) (Salunke et al., 1986; Pawlita et al., 1996; Ulrich et al., 2009).VLPs are widely used for viral antigen production and are knownas excellent antigens for serological tests and for safe and efficientvaccines (Ulrich et al., 2009).
In the past few years, several novel members of the fam-ily Polyomaviridae have been discovered due to the applicationof new molecular detection methods. Among them, four novelpolyomaviruses of monkeys have been described: Squirrel mon-
key polyomavirus, Bornean orang-utan polyomavirus, Sumatranorang-utan polyomavirus and a polyomavirus of chimpanzees(Verschoor et al., 2008; Groenewoud et al., 2009; Johne et al., 2005).The chimpanzee polyomavirus (ChPyV) was first detected by a ran-dom screening using a polyomavirus-specific broad-spectrum PCR
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A. Zielonka et al. / Virus
n the faeces of a captive juvenile chimpanzee showing diarrhoeaJohne et al., 2005). As also Salmonella have been detected in thisecal sample, the clinical significance of ChPyV infection remainednclear (Johne et al., 2005). Initially, only the nucleotide sequencef the ChPyV VP1 had been determined, but very recently, wholeenome sequences of two closely related ChPyV variants have beenublished (Deuzing et al., 2010). Phylogenetic analyses show thathPyV is related to the B-lymphotropic polyomavirus originally iso-
ated from monkeys, and to the human Merkel cell polyomavirusJohne et al., 2005; Halami et al., 2010). However, larger insertionsn the N-terminus and in the outer loops of the ChPyV-VP1 suggestdistinct antigenicity of this virus.
In order to get more insights into the ChPyV capsid structure,ntigenicity and the distribution of ChPyV infections among chim-anzees, the VP1 was expressed in yeast, and its ability to formLPs was assessed. The ability of the VLPs to agglutinate erythro-ytes and the cross-reactivity of polyomavirus-specific antibodiesith the VLPs was evaluated. Sera of chimpanzees were screened
or the presence of ChPyV-specific antibodies using a VLP-basedLISA, in order to determine the frequency of ChPyV infections inhimpanzees.
. Materials and methods
.1. Serum samples
One hundred and sixty-three serum samples were collectedrom clinically healthy chimpanzees. Out of these, 89 sera werebtained from chimpanzees living in the Biomedical Primateesearch Centre (BPRC) in Rijswijk (NL), 25 from chimpanzees fromnother European research centre, but previously used in biomed-cal research, and 19 chimpanzee serum samples from differentoos in Europe. In addition, 30 serum samples collected from wild-aught individuals that were housed in a rehabilitiation centre infrica were included in this study.
.2. Cloning of VP1 and generation of recombinant yeast
A plasmid containing the VP1-encoding region of ChPyV (Johnet al., 2005) was used as template for amplification by PCRsing primers containing NheI cleavage sites upstream the initia-ion codon and downstream the stop codon of the VP1-encodingegion. The amplification product was cloned into the XbaI sitef the yeast expression vector pFX7 (Sasnauskas et al., 1999),lacing it downstream of a galactose-inducible promoter. Theector allows the selection of yeast transformants by formalde-yde resistance. The generation of the positive control plasmidFX7-SV40 containing the VP1-encoding region of SV40 has beenescribed previously (Sasnauskas et al., 2002). The plasmids wereransformed into Saccharomyces cerevisiae AH22 derivate 214 (a,eu2-3,112 his4-519). Yeast transformation, cultivation of yeast
ith glucose medium and the induction of recombinant proteinxpression using galactose-containing medium were performed asescribed previously (Sasnauskas et al., 2002; Zielonka et al., 2006).east transformed with vector pFX7 without any insert was useds a negative control.
.3. Purification of VLPs and APV particles
After mechanical disruption of yeast cells using glass beadsnd buffer DB 450 (450 mM NaCl, 1 mM CaCl2, 0.001% Trition
-100, 0.25 M L-Arginine in 10 mM Tris/HCl-buffer pH 7.2), theupernatant was collected and loaded onto a caesium chlorideCsCl) gradient with densities of 1.31 and 1.42 g/ml. Subsequently,centrifugation at 25.000 rpm (Rotor SW28, Beckman, USA)or 3 h at 4 ◦C was followed. Thereafter, fractions of 1 ml each
ch 155 (2011) 514–519 515
were collected and aliquots of them were subjected to sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fol-lowed by Coomassie brilliant blue staining. Fractions showing aband with an apparent molecular mass between 40 and 50 kDacorresponding to polyomavirus VP1 were pooled and diluted inbuffer DB 150 (150 mM NaCl, 1 mM CaCl2, 0.001% Trition X-100,0.25 M L-Arginine in 10 mM Tris/HCl-buffer pH 7.2). The mixturewas subjected to ultracentrifugation on another multiple steppedCsCl gradient with densities of 1.24, 1.29, 1.31, 1.34, 1.38 and1.42 g/ml overnight at 25,000 rpm (Rotor SW28, Beckman, USA)and fractions were analyzed as described above. After pooling ofthe VP1-containing fractions, a last CsCl density gradient centrifu-gation with 1.31 und 1.42 g/ml CsCl was performed at 37,000 rpm(Rotor SW41, Beckman, USA) overnight at 4 ◦C. The buoyant densityof the collected VP1-containing fractions was determined by usinga refractometer. The preparation of purified APV particles frominfected chicken embryo cells, which were used as comparativecontrol, has been described previously (Johne et al., 2007).
2.4. Electron microscopy
CsCl-gradient fractions containing recombinant VP1 were dial-ysed overnight against PBS and negatively stained using 2% uranylacetate. The specimens were examined in a JEM-1010 electronmicroscope (JEOL, Tokyo, Japan).
2.5. Generation of VLP-specific antisera in rabbits
Rabbits were immunized intradermally with 100 �g antigendissolved in 500 �l PBS and emulsified with 500 �l of Freund’sincomplete adjuvant (Difco, Germany). One rabbit was immunizedwith ChPyV-VLPs, one with SV40-VLPs, and one with a prepara-tion of pFX7-transformed yeast lysate serving as negative control.A first and second booster immunization was performed at 3-weekintervals each with 100 �g antigen in PBS intramuscularly with-out adjuvant. Sera were collected 3 weeks after the second boosterimmunization.
2.6. SDS-PAGE and immunoblotting
Proteins were analysed by electrophoresis on 12.5% sodiumdodecylsulfate-polyacrylamide gels followed by Coomassie bril-liant blue staining or immunoblotting. The polyclonal serum �APVelicited in rabbits with purified APV particles and cross-reactingwith VP1 of other polyomaviruses (Stoll et al., 1993; Sasnauskaset al., 2002) was used for VP1 detection. Immunoblotting was per-formed as described by Stoll et al. (1993). The apparent molecularweight of VP1 was calculated by comparison of the band posi-tion with SDS-PAGE Standards, Low Range (Bio-Rad, Germany) andBiotinylated SDS-PAGE Standards, Low Range (BioRad, Germany).
2.7. Haemagglutination assay
25 �l volumes of the antigen solution (25 �g/ml) were dilutedin 2-fold steps with PBS in a U-shaped microtiter plate (Greiner,Germany). Thereafter, 25 �l of a 1% human erythrocyte suspensionin PBS were added to each well. After 1 h incubation at 4 ◦C, haemag-glutination was recorded and the titre was determined defined asthe reciprocal of the highest dilution of the antigen suspensionclearly showing haemagglutination.
2.8. Enzyme-linked immunosorbent assay (ELISA)
For determination of cross-reactions of the rabbit immune sera,wells of a Nunc MaxiSorb ELISA plate (Nunc, Germany) were coated
516 A. Zielonka et al. / Virus Research 155 (2011) 514–519
F omass2 gativel AGE Sm
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ig. 1. Analysis of purified yeast-expressed VP1 by SDS-PAGE and subsequent Co�g of purified proteins were used: ChPyV-VP1 (lane 1) and SV40-VP1 (lane 2). Ne
ysate (lane 3) and positive control contained purified APV (lane 4). Lane M: SDS-Polecular mass indicated left.
vernight at 4 ◦C with 50 �l coating buffer (0.1 M sodium carbon-te, pH 9.6) containing purified antigen (0.3 �g/ml); other wellsf the plate were coated with coating buffer without antigen.hereafter, the wells were washed with PBS-Tween (0.05%) and00 �l/well blocking solution (10% skim milk powder in PBS-0.05%-ween) were added and incubated for 1 h at 37 ◦C. After removinghe blocking solution, the wells were washed three times withBS-Tween. In order to eliminate yeast protein-specific antibod-es, the sera were incubated with pFX7-transformed yeast lysateor 1 h at 37 ◦C and thereafter centrifuged at 14,000 rpm for 10 min.00 �l dilutions of the supernatant were added per well. After
ncubation for 1 h at 37 ◦C, wells were washed three times withBS-Tween, and 100 �l of biotinylated “anti-Rabbit IgG” (Sigma,ermany), diluted 1:1000 in PBS-Tween, were added to each wellnd incubated for 1 h at 37 ◦C. After three times washing with PBS-ween, the plate was incubated for 1 h at 37 ◦C with 100 �l/welltreptavidin-horse radish peroxidase conjugate (Roche, Germany),iluted 1:2000 in PBS-Tween. A final washing was performed twoimes with PBS-Tween and one time with PBS. 100 �l substrateolution containing o-phenylenediamine hypochlorid was addedo each well and colour development was stopped using 50 �l 4 M2SO4. Optical densities were measured at 450 nm with a referenceavelength of 650 nm. The OD450 values from the wells without
ntigen were subtracted from each value with antigen. The valuesetermined in 3 independent tests were averaged and standardeviations were calculated. The resulting OD450 value generatedy a serum using the homologue antigen was set to 100% and theercentage of colour development with a heterologous antigen wasalculated using its respective OD450 value.
For testing of chimpanzee sera, the protocol was modifieds previously described (Verschoor et al., 2008). Briefly, serumamples were diluted 1:100 in blocking buffer consisting of 1%ewborn calf serum/1% normal rabbit serum in PBS and 0.01%Tri-on X-100/0.01% Tween 20, and 100 �l were added to each well.ound antibodies were detected by adding rabbit anti-monkey
gG–alkaline phosphatase conjugate (R&D Systems Europe Ltd.,bingdon, UK), diluted 1:1500 in PBS-Tween. Colour developmentas initiated by adding BluePhos substrate (KPL, Gaithersburg,
SA). The colorimetric reaction was allowed to develop for 15 mint room temperature, and the absorbance was read at 595 nm. Seraith an OD value greater than four times of the negative controlerum (an SV40-negative rhesus macaque serum) were consideredositive.
ie brilliant-blue staining (A) or immunoblotting with APV-specific antiserum (B).control was prepared after correspondent purification of pFX7-transformed yeast
tandards, Low Range (A) or biotinylated SDS-PAGE Standards, Low Range (B), with
3. Results
3.1. Generation and purification of VLPs using recombinant yeast
The pFX7-derived expression plasmid carrying the VP1-encoding region of ChPyV cloned downstream the galactoseinducible promoter was transformed into yeast S. cerevisiae strainAH22-214. The sequence of the inserted VP1-encoding region wasverified by DNA sequencing. Yeast transformed with pFX7 withoutthe VP1-sequence and yeast transformed with plasmid pFX7-SV40-VP1 containing the VP1-encoding region of SV40 (Sasnauskas et al.,2002) were used as negative and positive controls, respectively.After induction of protein expression for 24 h, yeast cells were har-vested and lysates were subjected to CsCl density centrifugation.
Analysis of VP1-containing gradient fractions by SDS-PAGE andimmunoblotting is shown in Fig. 1. In Coomassie brilliant bluestained gels, a prominent band with an apparent molecular weightof approximately 49 kDa was found in the fractions of ChPyV-and SV40-VP1-expressing yeast cells (Fig. 1(A), lanes 1 and 2). Inthe ChPyV-VP1 fractions, additional faint bands presumably repre-senting remaining yeast proteins or degradation products of VP1became evident; these could not be removed by further densitygradient centrifugations. The yeast negative control also showedfaint bands, but not the prominent 49 kDa band (Fig. 1(A), lane3). APV particles purified from infected tissue culture cells wereused as an additional control. In the case of purified APV particles,a prominent band of 45 kDa corresponding to VP1 was visible alongwith additional bands representing the minor structural proteins(Fig. 1(A), lane 4). In the immunoblot, the polyclonal serum directedagainst APV particles detected the 49 kDa band in case of SV40-VP1 (Fig. 1(B), lane 2) and the structural proteins of APV (Fig. 1(B),lane 4). No bands were visible in the case of ChPyV-VP1 and theyeast negative control (Fig. 1(B), lanes 1 and 3). The yield in puri-fied VP1 preparations differed remarkably and was high for SV40with 1.1 mg per g wet weight of yeast and 10-fold lower in the caseof ChPyV-VP1 (0.1 mg/g).
3.2. Characterization of VLPs
The VP1-containing fractions were negatively stained usinguranyl acetate and subjected to electron microscopy. Particles witha diameter of 42–46 nm and a typical shape of polyomaviruseswere found in preparations of ChPyV-VP1, SV40-VP1 and APV
A. Zielonka et al. / Virus Research 155 (2011) 514–519 517
Fig. 2. Negative stain electron microscopy of density gradient-purified fractions contanegative control prepared from pFX7-transformed yeast lysate (C) or a positive controlwith a diameter of approximately 45 nm, wide white arrows indicate smaller particlescapsomere-like structures with a diameter of approximately 6 nm.
Fig. 3. Haemagglutinating activity of VLPs composed of ChPyV-VP1 or SV40-VP1,aps
(CclbAVsawsnrcdc
tp1taH
the SV40-VLPs as antigen revealed only negative results (data not
Fcl
negative control prepared from yeast lysate and a positive control composed ofurified APV. A 1% human erythrocyte dilution was used for the test. A 2-fold dilutioneries of the antigens was performed and the concentrations are indicated above.
Fig. 2(A), (B) and (D)). The fraction with the highest yield ofhPyV-VP1-VLPs had buoyant densities of 1.3–1.31 g/ml. In theorresponding fractions of the yeast negative control, only iso-ated spherical structures with a diameter of approximately 40 nm,ut without the typical structured surface, were found (Fig. 2(C)).s previously observed for other yeast-expressed polyomavirus-LPs (Sasnauskas et al., 2002), the VLPs were heterogenous inize, with a predominant diameter ranging from 42 to 46 nm. Inddition, a smaller fraction of VLPs with diameters of 20–25 nmas observed in ChPyV- and SV40-VP1 preparations. Although the
ame amount of total protein was analysed, a markedly higherumber of both particle types were found in the SV40-VP1 prepa-ation as compared to the ChPyV-VP1 preparation. However, inase of ChPyV-VP1 a high number of additional structures with aiameter of approximately 6 nm were found, which may representapsomeres resulting from disassembly of VLPs.
In order to characterize the VLPs further, their ability to agglu-inate human erythrocytes was tested (Fig. 3). The purified APVreparation with known haemagglutinating (HA) activity (Godde,
994) was used as control showing an HA activity down to a concen-ration of 200 ng/ml. As described previously (Zielonka et al., 2006)nd expected, the preparation of SV40-derived VLPs did not showA activity. Also, the ChPyV-VP1-VLP preparation, and the prepa-ig. 4. Immunoblot analysis of cross-reacting activities of sera raised in rabbits using puriomposed of a similarly purified pFX7-transformed yeast lysate (C) as antigens. 2 �g of VLysate of pFX7-transformed yeast (lane 3) as well as purified APV (lane 4) were analyzed.
ining recombinant ChPyV-VP1 (A) and SV40-VP1 (B) expressed in S. cerevisiae, acontaining purified APV (D). The bar indicates 100 nm. Narrow arrows show VLPswith a diameter between 20 and 25 nm, and wide black arrows indicate smaller
ration derived from the pFX7-transformed yeast lysate serving asa control, did not show any HA activity.
3.3. Serological cross-reactions of VLP-specific sera
In order to investigate serological cross-reactions between themonkey polyomaviruses SV40 and ChPyV, specific sera were pro-duced by inoculation of the purified VLP preparations in rabbits.For control purposes, an additional rabbit was inoculated with theyeast-protein preparation. By immunoblot analysis, the ChPyV-VP1and SV40-VP1 showed strong reactions with their homologoussera (Fig. 4(A) and (B)). In addition, the SV40-VP1-specific serumdetected the ChPyV-VP1 band, however, with a weaker intensity(Fig. 4(B)). None of the antigens were detected by the control serumraised against the yeast protein preparation (Fig. 4(C)). By testingin an ELISA, the sera again showed a strong reaction with theirhomologous antigens; however, with different intensity. In orderto enable comparison of the cross-reactivity of the sera, the averageOD value detected by a serum with its homologous antigen was setto 100% and the percentages of staining intensity with heterologoussera were calculated accordingly. In contrast to the results of theimmunoblot analyses, no cross-reactions were detected betweenthe sera and heterologous antigens in the ELISA (Fig. 5).
3.4. Testing of chimpanzee sera for ChPyV-specific antibodies
Sera derived from 163 chimpanzees were tested for antibodiesto ChPyV by an ELISA using the purified VLPs as antigen. In total,19 sera (11.7%) reacted positive, which could all be confirmed by arepetition of the test (Table 1). A parallel testing of the sera using
shown), thus confirming the specificity of the reaction. By analysisof the results according to the origin of the sera, a large variation ofthe seroprevalence rates between 0% for a small group of chim-panzees housed in various European zoos, and 56% for a group
fied yeast-expressed ChPyV-VP1-VLPs (A), SV40-VP1-VLPs (B) or a negative controlPs consisting of VP1 from SV40 (lane 1) and ChPyV (lane 2), and a similarly purifiedThe arrows indicate a MWG of 45 kDa.
518 A. Zielonka et al. / Virus Resear
Table 1ELISA results of chimpanzee sera for ChPyV-specific antibodies using ChPyV-VP1-VLPs as antigen.
Origin No. of sera tested Positive
No. of sera %
BPRC 89 2 2.2Research centre 25 14 56
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experimental background of the 25 animals originating from the
F“w“i
Zoos 19 0 0Rehabilitation centre 30 3 10
Total 163 19 11.7
f animals that had previously been used in biomedical researchecame evident. The majority of tested animals originated from aormer breeding colony previously housed at the BPRC in RijswijkThe Netherlands) and showed a ChPyV-specific seroprevalence of.2%. Three out of 30 wild-caught animals (10%) that were housed
n a rehabilitation centre in Africa also reacted positive in the ELISA.
. Discussion
ChPyV had been originally detected in the faeces of a captivehimpanzee using PCR techniques and its VP1-encoding sequenceas determined (Johne et al., 2005). Very recently, ChPyV DNAas been detected in a number of captive and wild-caught chim-anzees and two complete ChPyV genomes have been sequencedDeuzing et al., 2010). As the virus has been found in different tis-ues and faeces of healthy animals as well as animals with variouslinical symptoms, the clinical significance of ChPyV infection inhimpanzees is unclear so far. In addition, the functionality of theetermined sequence of the VP1 gene was not tested until now.ere, we show that this protein is capable to assemble into VLPs.sing these VLPs as antigen in a serological test, we could demon-
trate that ChPyV-specific antibodies are present in chimpanzeesrom different countries in Europe and Africa indicating that ChPyVnfections are distributed worldwide.
Although the formation of ChPyV-VP1-VLPs with a typical diam-ter of 45 nm was demonstrated, the efficiency of VLP formationas low. The reasons for the low efficiency are not known so far,
ut differences according to the polyomavirus species have beenescribed previously (Sasnauskas et al., 2002; Zielonka et al. 2006).sing a similar yeast expression system, the efficiency of VLP for-ation was 10-fold lower for APV-VP1 as compared to SV40-VP1
Sasnauskas et al., 2002). By electron microscopy, a high amount ofapsomere-like structures were detected in the ChPyV-VP1 prepa-ation, which may originate from disassembled VLPs. Therefore,nstability of the ChPyV-VP1-VLPs may be postulated. According tohe sequence analysis, the ChPyV-VP1 has a unique insertion in the
80
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anti-ChPyV-Vanti-pFanti-A P
anti-SV40-VP
anti-ChPyV-VP
anti-pFX
ant
ig. 5. Analysis of serological cross-reactions of the specific antisera by indirect ELISA. Tanti-ChPyV-VP1” was elicited with purified ChPyV-VLPs, the serum “anti-pFX7” was elicas elicited with purified APV, the serum “K0” was collected from the respective rabbits b
PBS”. Purified ChPyV-VP1-VLPs (A), SV40-VP1-VLPs (B) or APV (C) were used as antigents homologous antigen was set to 100% and the percentages shown for the other antigen
ch 155 (2011) 514–519
EF-loop of this protein (Johne et al., 2005). As this loop is situatedat the interface of capsomeres in other polyomaviruses (Liddingtonet al., 1991; Stehle et al., 1996; Stehle and Harrison, 1997), it mayalso influence the stability of ChPyV-VP1-VLPs in the absence of theminor structural proteins VP2 and VP3. An effect of these minor pro-teins on VLP assembly has been described previously for the goosepolyomavirus, where VLPs composed of VP1 had a size of 25 nm,but VLPs composed of VP1 and VP2 proteins had diameter of 45 nm(Zielonka et al., 2006).
The HA experiments with human erythrocytes indicate thatChPyV has no HA activity. Some of the mammalian polyomaviruses,e.g. murine polyomavirus, show HA activity, which is conferred bybinding of VP1 to sialic acid residues (Imperiale and Major, 2007).The avian polyomaviruses APV and GHPV also exhibit HA activity,which is retained by VLPs composed of VP1 only (Zielonka et al.,2006). Other polyomaviruses like SV40 do not show HA activity,which may be caused by a different binding mechanism to the cel-lular receptor (Neu et al., 2008). The cellular receptor of ChPyV isnot known so far, but the negative HA result may indicate that thereceptor molecules are not properly exposed on human erythro-cytes. However, it should also be taken into consideration that thehigh amount of disassembled VLPs present in the preparation mayhave an inhibitory effect on HA activity.
The ChPyV-VP1- and SV40-VP1-specific sera showed only low orno cross reactions in immunoblot and ELISA, respectively. The anti-genic divergence of both viruses is supported by analysis of theirVP1 amino acid sequences, which show a high degree of sequencevariation, especially in the BC-, DE-, and EF-loops, which form theouter surface of virus particles and therefore may carry the anti-genic epitopes (Johne et al., 2005; Babe et al., 1989; Jin et al., 1993;Siray et al., 2000). Some cross-reactivity observed in immunoblotmay be involving conserved amino acid sequences situated in theinternal parts of the VP1 and exposed for recognition only afterdenaturation of this protein before SDS-PAGE, but not during theELISA procedure. The high specificity of ChPyV-specific antibod-ies in the ELISA may be useful for application of this method forfield-origin sera testing.
The overall seroprevalence rate among the tested chimpanzeeswas generally low, between 0% and 10%, with the exception ofthe group of chimpanzees previously used in animal experimentsthat showed a high seropositivity rate of 56%. Differences in hous-ing conditions and/or husbandry can be the cause of variations invirus transmission and seropositivity. It may be speculated that the
research centre, which may include unusual transmissions of thevirus (e.g. by parenteral injections) may have caused the very highincidence in this group. However, detailed information on the his-tory of these animals could not be retrieved. The incidence of ChPyV
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anti-ChPyV-Vanti-pFanti-A Pi-A P
he rabbit serum “anti-SV40-VP1” was elicited with purified SV40-VLPs, the serumited with a similarly purified pFX7-transformed yeast lysate, the serum “anti-APV”efore immunization. ELISA result using PBS without adding serum is shown in lanes with a concentration of 0.3 �g/ml. The OD450 value determined for a serum withs have been calculated accordingly using their OD450 values.
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nfection in free-ranging animals should be estimated by analysisf the sera from chimpanzees housed in the rehabilitation centren Africa. However, these data have to be interpreted carefully, as
ost individuals are wild-caught at younger age and housed forears until our analysis. Therefore, the incidence rate measured inhis group may be an underestimation of the actual situation in theild.
In conclusion, ChPyV infections seem to be common in chim-anzees worldwide, but generally with a relatively low incidence.s only clinically healthy animals have been tested in this study,
he pathogenic potential of ChPyV remains unclear so far. Theeveloped ELISA could be applied in future in connection withhPyV-specific PCRs to test chimpanzees with distinct diseases inrder to assess the involvement of ChPyV in disease development.pplication of the ELISA system may further analyse the distribu-
ion of ChPyV infections among other monkey species and humanshus assessing the host spectrum of ChPyV.
cknowledgements
We thank the laboratory staff for excellent technical assistance.he authors wish to thank Dr. Jochen Reetz and Maria M. VargasFederal Institute for Risk Assessment, Berlin) for digital picturesrom electron microscopic preparations. This work was funded bygrant from the Deutsche Forschungsgemeinschaft (JO 369/3-1/2).
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