BK polyomavirus-seroreactivity increases with virus replication

Abstracts / Journal of Clinical Virology 82S (2016) S1–S142

of infections occurred after the engraftment (>30 days from TX) (p = 0.02). Conclusions: Among pediatric HSCT recipients, viral respiratory infections in the post-transplant period are frequent and sometimes prolonged. Preventive measures must be tightened in this population in order to reduce the derived morbidity and mortality. http://dx.doi.org/10.1016/j.jcv.2016.08.196 Abstract no: 259 Presentation at ESCV 2016: Poster 157 Normalizing ELISPOT to quantify human cytomegalovirus (HCMV) and Epstein Barr-virus (EBV) specific T-cell response in kidney transplant recipients Fornara 1,∗ ,

Cassaniti 1 ,

Calarota 1 ,

C. I. S.A. K.M.G. Adzasehoun 1 , L. Scaramuzzi 2 , G. Comolli 3 , F. Baldanti 1,4 1

Molecular Virology Unit, Microbiology and Virology Department, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy 2 Nephrology, Dialysis and Transplantation Unit, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy 3 Molecular Virology Unit, Microbiology and Virology Department – Experimental Research Laboratories, Biotechnology Area, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy 4 Department of Clinical, Surgical, Diagnostic and Pediatric Sciences, University of Pavia, Italy Background: Herpes virus infection or reactivation are major complications in solid organ transplant recipients. Virus-specific T-cell response is crucial to control infection. Methods: HCMV and EBV specific CD4+ and CD8+ T-cell response were investigated in 29 kidney transplant recipients by a novel approach of enzyme-linked immunospot assay (ELISPOT). Overlapping 15-mer peptide pools of HCMV proteins immediate early IE-1, IE-2 and phosphoprotein pp65, and of EBV lytic (BZLF1 and BMRF1) and latent (EBNA1, EBNA3a, EBNA3b, EBNA3c, LMP1 and LMP2) proteins were used for stimulation of both CD4+ and CD8+ HCMV-specific and EBV specific T-cells, respectively. Virological and immunological monitoring were performed for one year of follow-up. Results: As for HCMV infection, 13/19 (68.4%) HCMV seropositive recipients showed levels of HCMV replication <100,000 DNA copies/ml blood and did not required anti-viral treatment, while 6/19 (31.6%) HCMV-seropositive patients were treated since showing ≥100,000 HCMV DNA copies/ml blood. Patients with spontaneous control of infection showed, at 120 days after transplant, levels of HCMV specific CD4+ T-cells significantly higher with respect to patients who needed treatment. HCMV specific T-cell response to single HCMV proteins (pp-65, IE-1, IE-2) was examined: pretransplant number of both CD4+ and CD8+ specific T cells directed against IE-1 showed significantly higher level in patients controlling infection and their level remained significantly higher until 120 days. No difference was shown for pp-65 and IE-2 between the two groups of patients. In addition, 5 HCMVseronegative recipients receiving organ from HCMV seropositive donor (D+/R−), were examined: 4/5 developed a primary infection within one month from transplantation and required antiviral treatment. HCMV-specific CD4+ T-cells remained significant lower with respect to patients able to control infection until 120 days after transplantation.

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As for EBV infection, 3/29 (10.3%) EBV-seropositive patients reaching levels of EBV ≥10,000 DNA copies/ml blood did not showed EBV-specific T-cell response for the entire period considered. However, EBV-specific T-cell response was detected in only 8/20 (40%) patients examined at 1 year follow-up, regardless of the presence of EBV DNA in blood. Conclusions: Normalizing ELISPOT may be a simple and useful tool to perform immunological monitoring in solid organ transplant recipients and to detect herpes virus specific response. However, while the importance of HCMV-specific T-cell response to control HCMV infection is evident, further studies are required to better define the role of EBV-specific T-cell response. http://dx.doi.org/10.1016/j.jcv.2016.08.197 Abstract no: 272 Presentation at ESCV 2016: Poster 158 BK polyomavirus-seroreactivity increases with virus replication H.F. Wunderink 1,∗ , E. van der Meijden 1 , C.S. van der Blij-de Brouwer 1 , H.L. Zaaijer 2 , A.C.M. Kroes 1 , J.I. Rotmans 1 , J.N. Bouwes Bavinck 1 , M.C.W. Feltkamp 1 1 2

Leiden University Medical Center, The Netherlands Sanquin Blood Supply, The Netherlands

Background: BK polyomavirus (BKPyV) infection causes nephropathy in 1–10% of kidney transplant recipients. This condition results in graft-loss in up to 50% of cases unless immunosuppression is lowered. Specific antiviral treatment is not available. In immunocompetent individuals, BKPyV resides latently in kidney tubular epithelium after primary infection during childhood. In order to predict which recipients will develop BKPyV nephropathy, we recently analyzed a cohort of kidney donorrecipient pairs prior to transplantation for several immunological and virological parameters. That study showed a strong correlation between the strength of BKPyV-seroreactivity measured in the donor and BKPyV infection and nephropathy in the recipient [1]. We hypothesized that BKPyV-seroreactivity of the donors mirrors the load of infectious virus in the transplanted kidney. To further investigate the relation between BKPyV-seroreactivity and BKPyVreplication, we analyzed the dynamics of BKPyV-seroreactivity in individuals that did or did not experienced a detectable BKPyV infection. Methods: A group of 101 kidney transplant recipients was analyzed for BKPyV-seroreactivity (VP1-antigen; Luminex immunoassay) and for BKPyV viremia (viral load measured by q-PCR). Serum and blood plasma samples were obtained before transplantation and at 3-month intervals until 18 months after. As controls 87 healthy blood donors were analyzed with a 12-month interval. Descriptive statistics and mixed model analysis was used to analyze the association between measured peak BKPyV-loads and BKPyV-IgG seroreactivity. Results: At baseline the overall BKPyV-seropositivity was high in both transplant recipients (92%) and blood donors (99%). The mean baseline BKPyV-IgG level in both groups was comparable. In 85% of the kidney recipients, BKPyV viremia was detected at some point during follow-up, with peak viral loads ranging from 10 to 579700 copies/ml, while no viremia was detected in the blood donors. After a year, in the healthy blood donors, the mean level of seroreactivity remained the same (p = 0.929). This was also the case among kidney recipients without BKPyV viremia (p = 0.981). Among kidney recipients that did develop viremia, however, a statistically significant increase in BKPyV-seroreactivity was observed

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Abstracts / Journal of Clinical Virology 82S (2016) S1–S142

(p < 0.001). Stratified analysis showed that the increase in BKPyVseroreactivity was correlated with the height of the peak viral load measured after transplantation. Conclusion: In kidney recipients that experience active BKPyV infection, BKPyV-seroreactivity is correlated with BKPyV viremia and dependent of the peak viral load. Based on our previous findings showing that donor BKPyV-seroreactivity predicts BKPyV infection in recipients [1], we believe our current findings provide a template to understand BKPyV infection in general, where BKPyVseroreactivity in an individual reflects primary BKPyV replication in the past and the level of latent virus in the kidneys. In that way BKPyV-seroreactivity in kidney donors can predict the amount of latent, potentially infectious virus in the kidney allograft. Reference [1] Wunderink, et al., Am. J. Transplant. (2016).

http://dx.doi.org/10.1016/j.jcv.2016.08.198 Abstract no: 323 Presentation at ESCV 2016: Poster 159 Frequent HHV6 DNA positivity in children with severe burn injury K. Labská 1,∗ , E.Marinenko 2 , E. Matˇejková 2 , 3,4 , ˇ H. Pˇribylová 1 , M. Pumannová 1 , H. Suca R. Zajíˇcek 3,4 1

National Institute of Public Health, Prague, Czech Republic 2 Prague Burn Centre, University Hospital Kralovske Vinohrady, Prague, Czech Republic 3 Third Faculty of Medicine, Charles University, Prague, Czech Republic 4 Prague Burn Centre, University Hospital Kralovske Vinohrady, Czech Republic Background: Burn injury harms human immunity at multiple levels including decrease of cell mediated immunity and the cytokine storm. Such conditions are well known trigger of herpesvirus reactivation. Aim of our study is to elucidate the frequency and possible impact of herpesvirus reactivation in children with severe burn injury. Methods: Enrollment criteria: age under 10, severe burn injury (grade II, >5% BSA), fever (day 3 after onset) and low inflammatory parameters (CRP, PCT, normal WBC). Enrolled children were tested for HSV, VZV, EBV, CMV and HHV6 DNA in blood by multiplex PCR (Seeplex Meningitis – V1, Seegene) and real-time PCR (CMV HHV6,7,8 R-gene, Biomerieux). Tests for anti HHV6 antibodies and avidity were performed by IIF and EIA (IgM – Anti HHV6 IIFT IgM, Euroimmun, IF-VIDITEST anti-HHV-6 IgG, Vidia and IgG ELISA-VIDITEST anti-HHV-6 IgG, Vidia). Results: Up to date we have enrolled 17 children. The mean age of affected children was 2 (median 2, range from 1 to 7 years). The most frequently detected virus was HHV6 (10/17), among these twice in combination with CMV and EBV, once in combination with EBV. All of them were HHV6 type B with viral load ranging from 91 to 2410 copies per ml of whole blood (median 323 cp/ml). Only one HHV6 DNA positive child was negative for HHV6 IgG and IgM antibodies (ELISA and IIF). Two children with HHV6 DNA were positive for anti HHV6 IgM antibodies. The remaining 7 were IgM negative. All patients positive for HHV6 IgG by EIA had high-avid antibodies. History of exanthema subitum was known only in one child. No rashes or cytopenias were observed during the hospitalization period.

Conclusion: We have not observed any typical clinical signs previously described for active HHV6 infection in immunocompromised patients in our group. Our patients are of age close to time of HHV6 primary infection. The presence of HHV6 DNA in blood is probably a residue of HHV6 integration to the blood progenitor cells during the primary infection, although none of the children had recent primary infection due to the presence of high-avid antibodies. http://dx.doi.org/10.1016/j.jcv.2016.08.199 Abstract no: 329 Presentation at ESCV 2016: Poster 160 BK polyomavirus infection activates the type I interferon response in human fibroblasts, depending on the cGAS-STING pathway Celine Bressollette-Bodin 1,∗ , Cécile Peltier 1 , Lise Chauveau 2 , Olivier Schwartz 2 1 UMR1064 Centre de Recherche en Transplantation et Immunologie, Nantes University, Nantes, France 2 Unité Virus Immunité, Institut Pasteur, Paris, France

BK polyomavirus (BKPyV) associated nephropathy is one of the major causes of renal allograft dysfunction. Immunosuppressive therapy favors viral reactivation and nephropathy can develop due to local inflammation and insufficiency of the antiviral immune response. Recent studies have focused on the BKPyV-specific T cell responses, however how BKPyV is detected and whether it induces an innate immune response remain mostly unknown. Our aim was to investigate innate responses to BKPyV infection in permissive cells and identify intracellular sensors involved in the recognition of the virus. Human foreskin fibroblastes (HFF) and human renal proximal epithelial cells (hRPTECs) were infected with BKPyV (Gardner strain), and Chikungunya or IFNa as positive controls. IFNb, MxA, IL1b and IL18 gene expression were measured by RT-qPCR, normalized to GAPDH, and results expressed as fold induction over mock-infected cells. At the protein level, MxA expression was measured using flow cytometry and IFN type I levels were measured using a bioassay (HL116 cells) in the supernatant of infected and control cells. We show that BKPyV infection activates MxA expression and type I IFN in HFF, but not in hRPTECs. To identify intracellular receptors involved in the sensing of BKPyV during infection of HFF, we transfected cells with different siRNA targeting molecules known to be involved in the recognition of viral nucleic acids (IFI16, cGAS, DNA PK, STING, IRF3). Total RNA was harvested at 3 and 6 dpi. Silencing was assessed by RTqPCR, normalized to GAPDH and plotted as fold induction over the siSCR condition. We observed a significant inhibition of MxA and IFN type I induction after six days post infection when cGAS, STING and IRF3 were silenced, suggesting the involvement of this sensing pathway in the recognition of BKPyV during viral multiplication in permissive cells. Moreover, BKPyV DNA replication was measured using in house quantitative PCR with and without IFN␣. DNA viral loads after three and six days post infection were significantly lower in cells cultivated with IFN ␣, compared to untreated cells, showing that IFN␣ restricts BKPyV replication in HFF and HRPTECs. Altogether, our results show that BKPyV is restricted by IFN type I. BKPyV infection in hRPTECs does not activate strongly the type I interferon response, when this response is significant in HFF. In HFF, this activation depends on the cGAS-STING-IRF3 pathway. http://dx.doi.org/10.1016/j.jcv.2016.08.200