Letermovir in lung transplant recipients with cytomegalovirus infection: A retrospective observational study.

Letermovir is a new antiviral drug approved for the prophylaxis of CMV infection in allogeneic stem cell transplants. The aim of the study was to assess the therapeutic efficacy of letermovir in difficult to treat CMV infections in lung transplant recipients. All lung transplant recipients between March 2018 and August 2020, who have been treated with letermovir for ganciclovir-resistant or refractory CMV infection were included in the study and analysed retrospectively. In total, 28 patients were identified. CMV disease was present in 15 patients (53.6%). In 23 patients (82.1%), rapid response was noticed, and CMV-viral load could be significantly decreased (>1 log10 ) after a median of 17 [14-27] days and cleared subsequently in all of these patients. Five patients (17.9%) were classified as non-responder. Thereof, development of a mutation of the CMV UL56 terminase (UL-56-Gen: C325Y) conferring letermovir resistance could be observed in three patients (60%). Common side effects were mild and mostly of gastrointestinal nature. Mild adjustments of the immunosuppressive drugs were mandatory upon treatment initiation with letermovir. In addition to other interventions, letermovir was effective in difficult to treat CMV infections in lung transplant recipients. However, in patients with treatment failure mutation conferring letermovir, resistance should be taken into account.

BK virus infection activates the TNFα/TNF receptor system in Polyomavirus-associated nephropathy.

Polyomavirus-associated nephropathy due to BK virus infection (BKVAN) is recognized as an important cause of significant kidney transplant dysfunction often leading to renal graft loss. The activation of innate immune defense mechanisms during BKVAN is still poorly understood and an altered regulation of inflammatory mediators by resident kidney cells upon viral infection can be expected to contribute to the onset and progression of disease. TNFα interacting with its receptors, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2), is largely accepted to be involved in viral responses, exhibiting both proinflammatory and immunosuppressive effects. Our aim was to examine the expressions of TNFα and TNFR1 and 2 in human collecting duct epithelial cells (HCDC) after infection with BKV as well as to study the effect of TNFα and poly(I:C), a synthetic analog of viral RNA, on the expressions of TNF receptors and proinflammatory cytokines and chemokines in HCDC. Quantitative RT-PCR analyses showed a downregulation of TNFα and an upregulation of both TNFR1 and 2 upon exposure of HCDC to the BK virus. TNFα stimulation induced the expressions of IL-6, IL-8, RANTES, and TNFR2. Poly(I:C) upregulated the expressions of both TNFR1 and TNFR2, a response that could be effectively blocked by siRNA to TLR3 and RIG-I, two double-stranded (ds) RNA receptors of the innate immune system. Poly(I:C)-dependent expression of TNFR2 but not TNFR1 was enhanced by TNFα. Taken together, our results suggest an involvement of TNF/TNFR system in virus-associated nephropathy.

The polyomaviruses WUPyV and KIPyV: a retrospective quantitative analysis in patients undergoing hematopoietic stem cell transplantation.

BACKGROUND: The polyomaviruses WUPyV and KIPyV have been detected in various sample types including feces indicating pathogenicity in the gastrointestinal (GI) system. However, quantitative viral load data from other simultaneously collected sample types are missing. As a consequence, primary replication in the GI system cannot be differentiated from swallowed virus from the respiratory tract. Here we present a retrospective quantitative longitudinal analysis in simultaneously harvested specimens from different organ sites of patients undergoing hematopoietic stem cell transplantation (HSCT). This allows the definition of sample types where deoxyribonucleic acid (DNA) detection can be expected and, as a consequence, the identification of their primary replication site. FINDINGS: Viral DNA loads from 37 patients undergoing HSCT were quantified in respiratory tract secretions (RTS), stool and urine samples as well as in leukocytes (n = 449). Leukocyte-associated virus could not be found. WUPyV was found in feces, RTS and urine samples of an infant, while KIPyV was repeatedly detected in RTS and stool samples of 4 adult patients.RTS and stool samples were matched to determine the viral load difference showing a mean difference of 2.3 log copies/ml (p < 0.001). CONCLUSIONS: The data collected in this study suggest that virus detection in the GI tract results from swallowed virus from the respiratory tract (RT). We conclude that shedding from the RT should be ruled out before viral DNA detection in the feces can be correlated to GI symptoms.

Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences.

Viral vectors based on various naturally occurring adeno-associated virus (AAV) serotypes are among the most promising tools in human gene therapy. For the production of recombinant AAV (rAAV) vectors, researchers are focusing predominantly on cross-packaging an artificial AAV genome based on serotype 2 (AAV2) into capsids derived from other serotypes. Within the packaged genome the inverted terminal repeats (ITRs) are the only cis-acting viral elements required for rAAV vector generation and depict the lowest common denominator of all AAV2-derived vector genomes. Up to now, no quantitative PCR (qPCR) for the detection and quantification of AAV2 ITRs could be established because of their extensive secondary hairpin structure formation. Current qPCR-based methods are therefore targeting vector-encoded transgenes or regulatory elements. Herein we establish a molecular biological method that allows accurate and reproducible quantification of AAV2 genomes on the basis of an AAV2 ITR sequence-specific qPCR. Primers and labeled probe are located within the ITR sequence and have been designed to detect both wild-type AAV2 and AAV2-based vectors. This method is suitable for detecting single-stranded DNA derived from AAV2 vector particles and double-stranded DNA derived from vector plasmids. The limit of detection has been determined as 50 ITR sequence copies per reaction, by comparison with a plasmid standard. In conclusion, this method describes the first qPCR system facilitating the detection and quantification of AAV2 ITR sequences. Because this method can be used universally for all AAV2 genome-based vectors, it will significantly simplify rAAV2 vector titrations in the future.

Systematic screening for novel, serologically reactive Hepatitis E Virus epitopes.

BACKGROUND: The National Institutes of Health classified Hepatitis E as an emerging disease since Hepatitis E Virus (HEV) is the major cause of acute hepatitis in developing countries. Interestingly, an increasing number of sporadic cases of HEV infections are described in industrialized countries as zoonosis from domestic livestock. Despite the increasing relevance of this pathogen in clinical virology, commercial antibody assays are mainly based on fragments of HEV open reading frame (ORF) 2 and ORF3. The largest ORF1 (poly-)protein, however, is not part of current testing formats. METHODS: From a synthesized full length HEV genotype 1 cDNA-bank we constructed a complete HEV gene library consisting of 15 respective HEV ORF domains. After bacterial expression and purification of nine recombinant HEV proteins under denaturating conditions serum profiling experiments using 55 sera from patients with known infection status were performed in microarray format. SPSS software assessed the antigenic potential of these nine ORF domains in comparison to seven commercial HEV antigens (genotype 1 and 3) by performing receiver operator characteristics, logistic regression and correlation analysis. RESULTS: HEV antigens produced with our method for serum profiling experiments exhibit the same quality and characteristics as commercial antigens. Serum profiling experiments detected Y, V and X domains as ORF1-antigens with potentially comparable diagnostic significance as the well established epitopes of ORF2 and ORF3. However no obvious additional increase in sensitivity or specificity was achieved in diagnostic testing as revealed by bioinformatic analysis. Additionally we found that the C-terminal domain of the potential transmembrane protein ORF3 is responsible for IgG and IgM seroreactivity. Data suggest that there might be a genotype specific seroreactivity of homologous ORF2-antigens. CONCLUSIONS: The diagnostic value of identified ORF1 epitopes might not necessarily improve sensitivity and specificity, but broaden the overall quality of existing test systems. ORF2 and ORF3-antigens are still commonly used in diagnostic assays and possibly hold the potential to serologically differentiate between genotype 1 and 3 infections. Our systematic approach is a suitable method to investigate HEV domains for their serologic antigenicity. Epitope screening of native viral domains could be a preferable tool in developing new serologic test components.

Activation of innate immune defense mechanisms contributes to polyomavirus BK-associated nephropathy.

Polyomavirus-associated nephropathy (PVAN) is a significant complication after kidney transplantation, often leading to premature graft loss. In order to identify antiviral responses of the renal tubular epithelium, we studied activation of the viral DNA and the double-stranded RNA (dsRNA) sensors Toll-like receptor 3 (TLR3) and retinoic acid inducible gene-I (RIG-I) in allograft biopsy samples of patients with PVAN, and in human collecting duct cells in culture after stimulation by the dsRNA mimic polyriboinosinic:polyribocytidylic acid (poly(I:C)), cytokines, or infection with BK virus. Double staining using immunofluorescence for BK virus and TLR3 showed strong signals in epithelial cells of distal cortical tubules and the collecting duct. In biopsies microdissected to isolate tubulointerstitial lesions, TLR3 but not RIG-I mRNA expression was found to be increased in PVAN. Collecting duct cells in culture expressed TLR3 intracellularly, and activation of TLR3 and RIG-I by poly(I:C) enhanced expression of cytokine, chemokine, and IFN-ß mRNA. This inflammatory response could be specifically blocked by siRNA to TLR3. Finally, infection of the collecting duct cells with BK virus enhanced the expression of cytokines and chemokines. This led to an efficient antiviral immune response with TLR3 and RIG-I upregulation without activation of IL-1ß or components of the inflammasome pathway. Thus, PVAN activation of innate immune defense mechanisms through TLR3 is involved in the antiviral and anti-inflammatory response leading to the expression of proinflammatory cytokines and chemokines.

Polyomavirus DNA and RNA detection in renal allograft biopsies: results from a European multicenter study.

Polyomavirus mediated nephropathy is an increasingly recognized complication in renal transplant recipients. In all, 362 renal biopsies collected from 15 European transplant centers were analyzed for presence of Polyomavirus nucleic acid (BK virus [BKV] and JC virus [JCV]). We evaluated 302 biopsies of patients with renal allograft dysfunction, including three with known BKV allograft nephropathy (BKVAN), and 60 native kidney biopsies. BKV DNA was detected in 8 of the 302 (2.6 %) biopsies obtained for transplant dysfunction, but in none of the controls. BKV RNA, indicating active viral replication, was found in all BKV DNA positive biopsies available for mRNA expression studies. Retrospective immunohistochemical staining was positive for SV40 large T antigen in all seven evaluated biopsies. BKV DNA and RNA were detected in biopsy tissues from patients with inconspicuous light microscopy for BKVAN. Further studies will evaluate the potential of intrarenal viral BKV RNA as an early predictor for BKVAN.

Rapid enzymatic test for phenotypic HIV protease drug resistance.

A phenotypic resistance test based on recombinant expression of the active HIV protease in E. coli from patient blood samples was developed. The protease is purified in a rapid one-step procedure as active enzyme and tested for inhibition by five selected synthetic inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) used presently for chemotherapy of HIV-infected patients. The HPLC system used in a previous approach was replaced by a continuous fluorogenic assay suitable for high-throughput screening on microtiter plates. This reduces significantly the total assay time and allows the determination of inhibition constants (Ki). The Michaelis constant (Km) and the inhibition constant (Ki) of recombinant wild-type protease agree well with published data for cloned HIV protease. The enzymatic test was evaluated with recombinant HIV protease derived from eight HIV-positive patients scored from 'sensitive' to 'highly resistant' according to mutations detected by genotypic analysis. The measured Ki values correlate well with the genotypic resistance scores, but allow a higher degree of differentiation. The non-infectious assay enables a more rapid yet sensitive detection of HIV protease resistance than other phenotypic assays.

Serial detection of Epstein-Barr virus DNA in sera and peripheral blood leukocyte samples of pediatric renal allograft recipients with persistent mononucleosis-like symptoms defines patients at risk to develop post-transplant lymphoproliferative disease.

We tested blood samples of 25 pediatric renal transplant recipients for Epstein-Barr virus (EBV) DNA load by quantitative polymerase chain reaction (PCR). Eleven of these transplant recipients showed clinical persistent mononucleosis-like symptoms years after transplantation (Tx). A quantitation of EBV DNA by PCR in peripheral blood lymphocyte (PBL) and serum samples revealed variable EBV DNA titers. The majority of EBV PCR results in samples of the 14 asymptomatic transplant recipients was repeatedly below detection limit. In contrast, patients with mononucleosis-like symptoms showed persistent EBV genome titers over a period of 6 months, ranging from 75 to 18 750 copies/10 000 PBL and from 680 to 335 000 copies/mL serum, respectively. One child suffering from this mononucleosis-like condition developed an EBV-associated Burkitt-like lymphoma 29 months after Tx. Whereas clinical and histological investigations did not indicate a post-transplant lymphoproliferative disorder (PTLD) until tumor detection, EBV titers in PBL and serum had been high for at least 8 months. We propose that pediatric transplant recipients who show both, recurrent mononucleosis-like symptoms and a sustained high EBV genome load, are at increased risk for severe EBV-related post-transplant complications.