MAJOR ARTICLE
Chikungunya Virus Infection of Corneal Grafts Thérèse Couderc,1,2 Nicolas Gangneux,1,2 Fabrice Chrétien,3,4 Valérie Caro,5 Tan Le Luong,6 Bernadette Ducloux,6 Hugues Tolou,7 Marc Lecuit,1,2,8,a and Marc Grandadam7,9,a 1
Microbes and Host Barriers Group, 3Unité Histopathologie humaine et modèles animaux, 5Plateforme de Génotypage des pathogènes et Santé Publique, and 9Centre National de Référence des Arbovirus, Institut Pasteur, 2Inserm avenir U604, and 8Université Paris Descartes, Assistance Publique-Hôpitaux de Paris, Service des Maladies Infectieuses et Tropicales, Hôpital Necker-Enfants Malades, Paris, 4AP-HP, Groupe Hospitalier Albert Chenevier-Henri Mondor, Département de Pathologie, Faculté de Médecine, Université Paris XII, Créteil, 6Agence de la Biomédecine, coordination interrégionale, Lyon, and 7IRBA-IMTSSA, Unité de Virologie tropicale, Laboratoire associé au CNR des arbovirus, Marseille, France
(See the editorial commentary by Long and Heise, on pages 806–7.)
Background. Chikungunya virus (CHIKV) is an arbovirus with a high potential to spread globally. We investigated whether CHIKV is transmittable via corneal grafts. Methods. Serum specimens from 69 potential corneal donors living in La Réunion during the 2005–2006 outbreak of CHIKV infection were screened for anti-CHIKV antibodies. Serum specimens and corneoscleral rims were subjected to quantitative reverse-transcription real-time polymerase chain reaction (qRT-PCR) for detection of CHIKV. CHIKV isolation and immunolabeling were performed on eye tissue specimens. Viral transmission via the ocular route was assessed in an animal model of human CHIKV infection. Results. Twelve apparently uninfected donors were viremic and/or positive for immunoglobulin M (IgM) and/or immunoglobulin G. Eye tissue specimens from 12 donors who were or were not viremic and were or were not seropositive were investigated. qRT-PCR detected CHIKV RNA in corneoscleral rims from 4 patients: 1 patient was viremic, 2 were viremic and IgM positive, and 1 was IgM positive. Infectious CHIKV was isolated from all qRT-PCR–positive samples, and antigens were detected in corneal and scleral specimens, the iris, the ciliary body, and oculomotor muscles. Conclusions. One-third of eligible corneas (4 of 12) from donors apparently uninfected with CHIKV were infected with CHIKV during the study period. CHIKV infects the human cornea and can be transmitted via the ocular route. In the absence of systematic CHIKV screening in donors, cornea donation should be banned in areas where CHIKV circulates.
Chikungunya virus (CHIKV) is an arbovirus transmitted to humans by Aedes mosquitoes. This Alphavirus has recently reemerged and triggered massive outbreaks of infection. It is at risk to spread globally, given the expanding dissemination of its mosquito vectors, Aedes aegypti and Aedes albopictus [1]. CHIKV causes a systemic infection characterized by polyarthralgia and myalgia, sometimes associated with rash and ocular pain [2, 3]. CHIKV infection is most
Received 29 July 2011; accepted 7 November 2011; electronically published 15 June 2012. a M. L. and M. G. share the senior authorship. Correspondence: Marc Lecuit, MD, PhD, Microbes and Host Barriers Group, Institut Pasteur, Inserm 28 rue du Dr Roux, 75724 Paris, France (marc.lecuit@ pasteur.fr). The Journal of Infectious Diseases 2012;206:851–859 © The Author 2012. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail:
[email protected]. DOI: 10.1093/infdis/jis296
often symptomatic, but 3%–15% of infected individuals remain asymptomatic [4]. In 2005, CHIKV emerged in islands of the Indian Ocean and caused a large outbreak, most notably in La Réunion, a French overseas district, where more than one-third of the population was infected [1, 5, 6]. Although CHIKV transmission by tissue or organ grafting had not been documented when the outbreak started in La Réunion, public health authorities raised the question of possible transmission of CHIKV by grafts and blood transfusion, given the ability of other arboviruses to be transmitted via these routes [7–10]. The French national agency overseeing organ donations (Agence de la Biomédecine) decided to implement screening of organ and tissue donors for CHIKV in La Réunion, beginning in May 2005. All potential organ or tissue donors, including corneal donors, even when presenting no sign of CHIKV infection, underwent systematic screening for CHIKV, based on the detection in serum specimens of CHIKV Infection of Corneal Grafts
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anti-CHIKV immunoglobulin M (IgM) and immunoglobulin G (IgG) and of viral genome by quantitative reverse-transcription real-time polymerase chain reaction (qRT-PCR). In November 2005, a serum specimen from a corneal donor was positive for both anti-CHIKV IgM and viral genome. We investigated the corneoscleral rim from this donor by means of qRT-PCR and found that it contained CHIKV genome. On the basis of these preliminary data, systematic qRT-PCR screening for CHIKV in eye tissue samples was, in December 2005, added to the systematic serum screening established earlier that year. The discovery of additional CHIKV-positive donors from December 2005 through February 2006 led to a sine die suspension of corneal collection for therapeutic purposes, but sampling of potential donors was maintained under the control of the Agence de la Biomédecine for further scientific investigations regarding CHIKV. We report here the findings of these investigations and provide evidence that CHIKV infects corneal tissue and is transmissible through the ocular route in vivo. Together, these data highlight the risk for viral transmission through corneal transplants in areas where CHIKV circulates. METHODS
Viral RNA in the E1 structural protein region was amplified by qRT-PCR, using the reverse primer CCAAATTGTCCYGG TCTTCCT and the forward primer AAGCTYCGCGTCCTTT ACCAAG, and was quantified with the probe CCAATGTCYT CMGCCTGGACACCTTT, as described elsewhere [12, 13]. Sera from controls were negative for anti-CHIKV IgM, antiCHIKV IgG, and viral RNA. Virus Isolation and Titration
Infectious virus isolation was performed by inoculation of mammal and mosquito cell lines (Vero and C6/36, respectively), as described elsewhere [13], with 10% tissue suspensions from all qRT-PCR–positive donor corneas (ie, corneoscleral rims from donors A, C, and D and a corneoscleral sample from donor B) and from a nonviremic and seronegative donor. Sixteen hours after inoculation, cell cultures were fixed in 4% paraformaldehyde and submitted to direct immunofluorescence with a monoclonal antibody against CHIKV E2 protein coupled to cyanin-3 [14]. Viral titer in preservative medium containing samples from donors C and D was determined by the Vero cell plaque assay and expressed as plaqueforming units (PFU) per milliliter. Viral Genome Sequencing
From May 2005 through April 2006 in La Réunion, a total of 69 corneal donors from whom written consent for organ donation was obtained and who were eligible for organ/tissue donation were screened. Before our investigations, CHIKV infection had been neither diagnosed nor identified as the cause of death in these donors. Donors died of cardiovascular arrest or of injuries sustained during a motor vehicle crash. Their age ranged from 4 to 90 years (mean age, 56.36 years), and the ratio of males to females was 3.7. Eye tissue was available from 12 donors.
Virus isolated on C6/36 cells after inoculation of homogenates of serum and eye tissue specimens from donor C was sequenced. Genome-length RT-PCR was performed with the Titan One Tube RT-PCR kit (Roche Applied Science), using primers previously described [15], and amplicons were purified by ultrafiltration (Millipore). Sequencing reactions were performed using the BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems). Sequence chromatograms from both strands were obtained on automated sequence analyzer ABI3730XL (Applied Biosystems).
Serological Analysis and Genetic Detection of Virus
Mouse Experiments
Serum specimens from the 69 corneal donors were analyzed by IgM antibody-capture enzyme-linked immunosorbant assay (ELISA) and IgG sandwich ELISA for the presence of IgM and IgG antibodies reactive to CHIKV, as described elsewhere [11]. RNA was extracted from serum specimens from the 69 corneal donors, from corneoscleral rims from 11, from corneoscleral and aqueous humor specimens from 1, and from preservative medium containing a corneoscleral rim from 2. Viral RNA was purified from 140 µL of human fluid samples, using the QIAamp viral RNA mini kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s recommendations. For eye tissue, RNA was extracted from a section of the corneal rim (50–100 mg) by incubation with 560 µL of QIAamp viral RNA lysis buffer and mechanical crushing. After centrifugation of eye tissue homogenates, supernatants were submitted to the same procedure as described above for fluid samples.
Mice in which the type I interferon (IFN) receptor gene was knocked out (IFN-α/βR−/− mice) were obtained as described elsewhere [16] and bred according to Institut Pasteur guidelines for animal husbandry. Outbread OF1 mice and 9-day-old C57BL/6 mice were obtained from Charles River Laboratories (France). Mice were handled in accordance with Institut Pasteur guidelines for animal husbandry and were kept in BSL-3 isolators upon infection. The principles for good laboratory animal care were followed during the experimental process. Mice were inoculated intradermally with the CHIKV-21 isolate [15]. Blood was collected by cardiac puncture, after which mice were perfused via the intracardiac route with phosphate-buffered saline, and their organs were harvested. Viral titers in tissue and serum specimens were determined as described previously [17] and were expressed as median tissue-culture infectious dose (TCID50) per gram and TCID50
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per milliliter, respectively. To determine the effect of incubation of eye tissue under the same conditions as those used for eye tissue banking, 1 eye from an infected mouse was frozen and the second eye was incubated in preservative medium for 1 week at 33°C, before titration. For intraocular inoculation, mice were anesthetized, and eyes were or were not scarified before deposition of 10 µL of a viral suspension diluted with phosphate-buffered saline. Histologic Analysis and Immunostaining of Eye Tissue Specimens
A quarter of the eye from donor B and from a CHIKVnonviremic and -seronegative donor were fixed in paraformaldehyde and embedded in paraffin. Sections were counterstained with hematoxylin-eosin or periodic acid-Schiff. Chromogenic immunohistochemistry was performed as described previously [17] after antigen unmasking (97°C for 20 minutes in a citric acid buffer) and was revealed with a biotinylated secondary
goat anti-human antibody, which in turn was revealed by streptavidin-peroxidase and diaminobenzidine (brown color) (Vector Laboratories; available at: http://www.vectorlabs.com). A quarter of the eye from donor B and from a CHIKVnonviremic and -seronegative donor, corneoscleral rims from donor C and from a donor negative for CHIKV infection, eyes from mice infected with CHIKV intradermally, and eyes from mock-infected mice were frozen. Cryosections were labeled with monoclonal antibodies against CHIKV E2 protein [14] or against CHIKV capsid coupled to cyanin-3. Laminin was labeled with polyclonal rabbit anti-laminin (Sigma). Slides were counterstaining with Hoechst (Vector Lab) and observed with an AxioObserver microscope (Zeiss). Pictures and Z-stacks were obtained using the AxioVision 4.5 software. Role of the Funding Source
Sponsors provided financial support for the study but had no role in study design, data collection, data analysis, data
Figure 1. Chikungunya virus (CHIKV) infection in potential corneal donors from La Reunion. A, Laboratory screening for CHIKV infection in potential corneal donors from La Reunion. Eye tissue specimens were corneoscleral rims from donors A, C, and D and a quarter of an eye from donor B. NA, not available; qRT-PCR, quantitative reverse-transcription real-time polymerase chain reaction. B, Cytopathic effect observed in Vero cells 24 hours after incubation with homogenate of eye tissue from a noninfected donor (left) and from donor C (right). C, CHIKV antigens detected by direct fluorescence in Vero cells 16 hours after incubation with homogenate of eye tissue from a noninfected donor (left) and from donor A (right). CHIKV Infection of Corneal Grafts
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Table 1. Summary of the Detection of Chikungunya Virus (CHIKV) Infection in Serum and Eye Tissue Samples From the 4 Potential Cornea Donors With a Corneoscleral Rim Specimen Positive for CHIKV RNA Viremia (copies/mL) Eye Tissuea (copies/mg) Preservation Medium (copies/mL) Aqueous Humor (copies/mL) Immunostaining
Donor Donor A
3.7 × 106
Donor B Donor C
7
1.0 × 103
NA
NA
NA
7.7 × 10 9.7 × 107
4.5 × 106 5.6 × 106 (right)
NA 6.6 × 108,b
1.2 × 108 NA
Positive Positive
Donor D
ND
1.9 × 105
1.4 × 108,b
NA
NA
Abbreviations: NA, not available; ND, not detected. a
Corneoscleral rims from donors A, C, and D and a quarter of an eye from donor B.
b
Infectious virus particles in the preservation medium were titered by means of the Vero cell plaque assay and found to be 1.3 × 105 plaque-forming units (PFU) per milliliter for sample C and 1.0 × 105 PFU/mL for sample D.
interpretation, or report writing. M. L. had final responsibility for the decision to submit for publication. RESULTS CHIKV Infection in Corneal Donors Originating From a CHIKV Infection Outbreak Area
Results of CHIKV infection testing for potential corneal donors are summarized in Figure 1 and Table 1. Fifty-eight serum samples were seronegative for anti-CHIKV IgM and IgG, 5 were seropositive for anti-CHIKV IgM but negative for IgG, and 6 were seropositive for both anti-CHIKV IgM and IgG. None were seropositive for IgG only. Viral genome was detected in serum specimens from 1 seronegative donor (donor A) and from 2 IgM-only seropositive donors (donors B and C), with viremia in the range of levels previously described (Figure 1 and Table 1) [18]. Viral genome was present in eye tissue specimens from donor A, who was viremic and seronegative; from donors B and C, who were viremic and IgM positive; and from donor D, who was nonviremic and IgM positive. The available vitreous humor from donor B and the medium used for preservation of eye tissue specimens from donors C and D all displayed high viral loads (Figure 1 and Table 1). Isolation of CHIKV from Corneal Donors and Genome Sequencing of CHIKV Isolates
Cultures of Vero cells inoculated with preservative medium containing eye tissue specimens from donors C and D and with eye tissue homogenates from donors A, B, C, and D that were positive for CHIKV by qRT-PCR showed a cytopathic effect typical of CHIKV as early as 24 hours after inoculation (Figure 1B) and demonstrated CHIKV antigens on direct immunofluorescence staining 16 hours after inoculation (Figure 1C). Similar findings were obtained on C6/36 cells (data not shown). Direct determination of viral titers in preservation medium showed a large amount of infectious virus (1.3 × 105 and 1.0 × 105 PFU/mL for donors C and D, respectively). 854
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Complete nucleotide sequence of infectious viruses isolated from serum and eye tissue specimens from donor C after inoculation of C6/36 cells was determined (GenBank accession numbers FR717336 and FR717337, respectively; available at: http://www.ncbi.nlm.nih.gov/genbank). Both isolates displayed the same nucleotide sequence, and phylogenetic analysis showed that they clustered with the CHIKV isolates from the 2006 La Réunion outbreak [15]. Histologic and Immunostaining Analysis of Eye Tissue Specimens From Corneal Donors
The anatomy of eye is presented in Figure 2A. Histological analysis performed on the available eye (from donor B) showed that extrinsic and intrinsic eye muscles, connective tissue, and annexes were morphologically normal (data not shown). Direct fluorescence antibody labeling of the cornea revealed CHIKV antigens in keratocytes of the anterior and posterior stroma (Figure 2B and C). Cells from the corneal endothelium (Figure 2D) and stromal fibroblasts from sclera (Figure 2E and F) were also positive for CHIKV antigens. Further analysis of the eye revealed CHIKV antigens in fibroblasts of the surrounding connective tissue of the sclera (data not shown) and in smooth muscle of the ciliary body (Figure 2F). Higher magnification observation of smooth muscle sections showed that only stromal cells were labeled (Figure 2G). CHIKV antigens were present in fibroblasts all along the iris stroma (Figure 2H and H′) and between muscle fibers in the endomysium of extrinsic muscles (Figure 2I). The retina and choroid did not stain positive for virus. The Cornea Is a Portal of Entry for CHIKV
IFN-α/βR−/− and 9-day-old C57BL/6 mice intradermally inoculated with CHIKV displayed high viral loads in serum, muscles, joints, and skin, as previously shown [17], as well as in eyes (Figure 3A). Outbread adult mice (OF1 mice) showed infection of the same tissues, including eye tissues, although to a lesser extent (Figure 3A). Moreover, the viral load in frozen eyes from IFN-α/βR−/− mice was identical to that in eyes incubated in the same conditions as used for eye tissue banking
Figure 2. Immunostaining for Chikungunya virus (CHIKV) in eye tissue samples from corneal donors. A, Anatomy of the human eye (available at: http:// www.wpclipart.com/medical/anatomy/eye/Anatomy_Eye.png). B–H, Red staining and green staining indicates the presence of CHIKV antigens and laminin, respectively, and blue staining indicates nuclei. B and C, CHIKV antigens in keratocytes of the posterior and anterior corneal stroma (CS) from donor C and B, respectively. Note that the corneal epithelium (CEp) is removed in panel C. D, CHIKV antigens were seen in the corneal endothelium (CEn) from donor C, and keratocytes display diffuse labeling for viral antigens. E, CHIKV antigens were present in fibroblastic cells of the scleral stroma (SS) from donor C. F, CHIKV staining in SS and in smooth muscle (SM) of ciliary body from donor B. Note the presence of viral antigens in SS and in SM. G, SM of ciliary body from donor B. Note the presence of CHIKV staining in fibroblastic cells and its absence in smooth muscle fibers. H, Part of the iris from donor B. CHIKV antigens are present along the iris stroma (IS). Panel H′ is the same as panel H, but with bright field to visualize pigmented cells. I, CHIKV antigens in small fibroblastic cells localized in the endomysium of extraocular muscles from donor B. Abbreviations: BL, Bowman’s layer; CF, circular fibers; DM, Descemet’s membrane; PPE, posterior pigmented epithelium; RF, radial fibers; SEn, scleral endothelium. Scale bars are in micrometers.
(1.3 × 106 TCID50/g), indicating that the infectivity of the virus remained stable in the preservative medium. Exactly as observed for human ocular samples (Figure 2), CHIKV antigens were detected in keratocytes of the corneal stroma and sclera, in fibroblasts of the iris stroma, and of muscle fiber endomysium (Figure 3B), and in fibroblasts of ciliary bodies (data not shown). Deposition of CHIKV onto the surface of intact mouse cornea resulted in lethal infection in 20% of inoculated IFNα/βR−/− mice, and it led to 100% lethality when deposited on a scarified cornea (Figure 3C). Mortality data and kinetics of viral replication in tissue specimens for mice subjected to ocular inoculation after cornea scarification were similar to
mortality and kinetics data observed upon intradermal injection [17] (data not shown). Importantly, deposition of CHIKV onto the surface of scarified cornea of OF1 mice resulted in systemic infection, as assessed by the viral load in serum and tissues (Figure 3C). DISCUSSION From May 2005 through April 2006, serum samples from 69 potential corneal donors living in La Réunion during the 2005–2006 outbreak of CHIKV infection were screened for CHIKV. Twelve donors (17.4%) displayed at least 1 marker of CHIKV infection, as assessed by detection of anti-CHIKV CHIKV Infection of Corneal Grafts
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Figure 3. Ocular Chikungunya virus (CHIKV) infection in an experimental mouse model for human CHIKV disease. A, Viral load in serum and tissue specimens from mice infected intradermally with CHIKV. Mice in which the type I interferon (IFN) receptor gene was knocked out (IFN-α/βR−/−) were infected with 102 plaque-forming units (PFU) of CHIKV, and 9-day-old C57BL/6 mice and OF1 mice with 106 PFU and viral loads in serum and tissues were determined by day 2 after infection. Each data point represents the arithmetic mean ± SD for at least 4 mice. A broken line indicates the detection threshold. B, Immunostaining of CHIKV antigens on eye tissues from infected mice, with viral antigens in red and nuclei in blue. Panels a and a’ correspond to the same transversal cut of the eye. In panels b, c, d and d′, laminin is in green. Panel b shows CHIKV antigens in stroma of cornea, and panel c chows CHIKV antigens in stroma of sclera. In panel d, CHIKV antigens are present along the iris stroma. Panel d′ is the same as panel d but with bright field to visualize pigmented cells. C, Mortality among IFN-α/βR−/− mice following CHIKV inoculation through the ocular route, with or without scarification of the cornea, and viral load in serum and tissue specimens from OF1 mice following CHIKV inoculation through the ocular route after corneal scarification.
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IgM, anti-CHIKV IgG, and viral RNA in a serum specimen and/or viral RNA in a corneoscleral rim, although none were known to be infected when they were identified as potential organ donor. Infectious CHIKV was detected in corneoscleral rims from 4 of 12 donors who were tested for eye infection, indicating a prevalence of 33.3% of silent ocular CHIKV infection in corneal donors in the context of an outbreak.
A summary of the data obtained from 12 donors who were tested for eye infection is presented in Figure 4. Eight donors were negative for ocular viral infection, whereas 4 showed evidence of ocular CHIKV infection. Donors negative for ocular infection had either no marker of CHIKV infection (ie, no viremia and no anti-CHIKV immunoglobulin) or were no longer viremic but were positive for anti-CHIKV IgM and IgG, or for anti-CHIKV IgM only. Donors positive for ocular
Figure 4. Summary of the data obtained on the corneal donors tested for ocular Chikungunya virus (CHIKV) infection during the outbreak of infection in La Réunion. A, Chronology of detection of CHIKV in eye tissues from corneal donors before the decision to suspend corneal collection, and the distribution of symptomatic cases of CHIKV infection per week of onset of symptoms during La Réunion outbreak [30]. B, Twelve donors who displayed at least 1 marker of CHIKV infection as assessed by detection of anti-CHIKV immunoglobulin M (IgM) or immunoglobulin G (IgG), viral RNA in serum, and/or viral RNA in corneoscleral rim are represented as follows: patients A, B, C, and D, who had positive for cornea grafts, are noted in boxes, and each patient who had negative cornea grafts is symbolized by a diamond. For each patient, the presence or absence of viremia, IgM, and IgG is indicated. Note that the incubation period is usually 3–7 days in clinically apparent infection. CHIKV Infection of Corneal Grafts
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infection were thus either viremic or in the immediate postviremic phase (ie, positive for anti-CHIKV IgM only). One of them was viremic and still seronegative and, thus, most likely still in the incubating phase of the clinical disease. It is noteworthy that no corneal specimen from an anti-CHIKV IgG–positive donor was found to be positive for CHIKV, indicating that (1) these corneas did not contain infectious CHIKV, (2) they were likely safe for transplantation, and (3) CHIKV does not persist in corneal tissue. Analysis of a higher number of cases will be needed to confirm the safety of corneas from donors positive for anti-CHIKV IgG. Moreover, comparison of complete nucleotide sequence of infectious viruses isolated from serum and eye tissue showed no difference, indicating that viral replication in the eye compartment did not result from the selection of viral variants. We identified keratocytes in corneal and scleral stroma, as well as cells of the corneal endothelium, as target cells of CHIKV. Studies of the eye showed that CHIKV also targets fibroblasts in scleral connective tissue, in stroma of smooth muscles of ciliary bodies, in stroma of the iris, and between muscle fibers of ocular muscle. Together, these data demonstrate that CHIKV infects fibroblasts of eye tissues and that active replication in eye tissues leads to production of infectious viral particles in corneoscleral rims of donors. In a mouse model for human CHIKV infection, CHIKV infected eye tissues following intradermal injection and targeted fibroblastic cells in the same areas as those of human eye tissue samples. Interestingly, fibroblasts are the main target cells of CHIKV in the eye, in agreement with our previous findings involving human and mouse tissues (ie, joint, muscle, and skin) that are classically targeted during CHIKV infection [17]. The main ocular manifestation associated with CHIKV infection is periorbital or ocular pain and anterior uveitis [3, 19, 20]. Heterochromatic iridocyclitis was recently reported in a patient with a past history of CHIKV-associated fever for whom CHIKV RNA was found in aqueous humor [21]. However, active viral replication has never been reported in eye tissues. Our histological data provide a virological explanation for uveitis, a frequent ocular symptom observed in humans. Our report reveals the risk of iatrogenic CHIKV transmission through corneal transplantation. Viral transmission through corneal transplants requires that the corneal button to be grafted contains infectious viral particles at the time of the graft and that the cornea is a portal of entry for the virus. We showed that donor corneas can indeed contain infectious CHIKV in patients not identified as clinically infected with CHIKV and that conventional conservation conditions in eye banks do not eradicate infectious viral particles present in eye tissues. Finally, we showed that ocular inoculation of CHIKV results in systemic infection. We have thus demonstrated that the ocular route constitutes a portal of entry for CHIKV. 858
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Donor-to-host viral transmission through corneal transplantation, although rare, is a well-documented complication. It has been reported for rabies virus [22], hepatitis B virus (HBV), and herpes simplex virus 1 [23–25], and transplantation is contraindicated if a donor is infected with one of these viruses. To avoid viral transmission by organ and tissue transplantation, viral security in tissue banks is achieved by screening for viral infection in donors. The legal reference for donor selection in the European Union is Directive 2006/17/EC, Annex 1 (available at: http://eur-lex.europa.eu/LexUriServ/Lex UriServ.do?uri=OJ:L:2006:038:0040:0052:EN:PDF), in which the criteria for selection of cell and tissue donors and for testing are specified. These tests include systematic screening for HBV, for hepatitis C virus, for human immunodeficiency virus types 1 and 2, and for human T-lymphotropic virus types 1 and 2, if the donor has a risk factor for HTLV infection. They are the same as those defined by the Eye Bank Association of America. In the European directive, criteria for selecting donors state that donors must be excluded from donation if there is “evidence of any risk factors for transmissible diseases on the basis of a risk assessment, taking into consideration donor travel and exposure history and local infection prevalence.” During an outbreak of CHIKV infection in La Réunion during 2005, the Agence de la Biomédecine implemented a systematic control for CHIKV in all potential organ or tissue donors. This control was first based on serum investigations by detection of anti-CHIKV IgM and IgG and viral genome. When the first viremic corneal donor was identified, his cornea was tested for CHIKV and found to be positive, leading the Agence de la Biomédecine to add a supplementary criterion for corneal graft, namely, a test for the presence of CHIKV genome in eye tissues themselves. Our data on the screening of serum and eye tissue specimens for CHIKV infection show that ocular infection does not correlate with clinical symptoms, viremia, or presence of anti-CHIKV IgM. Consequently, none of these criteria can be used for safe selection of corneal graft donors. Except in the case of donors who are positive for anti-CHIKV IgG (for whom we found no detectable virus in available eye tissue specimens), the systematic screening for CHIKV in eye tissue specimens by qRT-PCR is thus highly recommended in donated corneas from an area where CHIKV circulates, to avoid CHIKV transmission via corneal graft. Indeed, during the outbreak in La Réunion, the Agence de la Biomédecine suspended corneal collection for therapeutic purposes until the epidemic was over, in May 2007, and this has certainly avoided CHIKV transmission by graft corneas. Our findings are relevant to other organ transplantations, and a systematic viral screening by qRT-PCR of donated organs and tissues is warranted in areas where CHIKV circulates. CHIKV is now regarded as the arbovirus most likely to spread globally, including in European countries and the United
States, given the wide distribution of its mosquito vector [26]. In the wake of the outbreak of CHIKV infection in India during 2006, which caused more than 1 million cases of infection, CHIKV was imported and propagated into Europe, causing an outbreak in Italy during 2007 and autochthonous cases in the French Riviera in late summer 2010 [27]. This underlines the potential risk for establishment of CHIKV in Europe and the Americas [28, 29]. In this context, our data strongly suggest that graft of ocular tissues containing infectious CHIKV represents an actual risk of viral transmission that can be avoided by testing eye tissue obtained for grafting, as was done during the outbreak in La Réunion. In the absence of systematic screening of donors for CHIKV, cornea donation should be banned in areas where CHIKV circulates. Notes Acknowledgments. We thank Philippe Desprès, for his generous gift of anti-E2 antibody; and Cécile Delval, Olivia Chény, and Jean Jacques Colpart, for their help in clinical study management. Financial support. This work was supported by Institut Pasteur, Inserm, Fondation pour la Recherche Médicale, Fondation BNP-Paribas, Mairie de Paris, Agence de la Biomédecine, and Institut de Veille Sanitaire. Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
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