Structural implications of BK polyomavirus sequence variations in the major viral capsid protein Vp1 and large T-antigen: a computational study.

BK polyomavirus (BKPyV) is a double-stranded DNA virus causing nephropathy, hemorrhagic cystitis, and urothelial cancer in transplant patients. The BKPyV-encoded capsid protein Vp1 and large T-antigen (LTag) are key targets of neutralizing antibodies and cytotoxic T-cells, respectively. Our single-center data suggested that variability in Vp1 and LTag may contribute to failing BKPyV-specific immune control and impact vaccine design. We, therefore, analyzed all available entries in GenBank (1516 VP1; 742 LTAG) and explored potential structural effects using computational approaches. BKPyV-genotype (gt)1 was found in 71.18% of entries, followed by BKPyV-gt4 (19.26%), BKPyV-gt2 (8.11%), and BKPyV-gt3 (1.45%), but rates differed according to country and specimen type. Vp1-mutations matched a serotype different than the assigned one or were serotype-independent in 43%, 18% affected more than one amino acid. Notable Vp1-mutations altered antibody-binding domains, interactions with sialic acid receptors, or were predicted to change conformation. LTag-sequences were more conserved, with only 16 mutations detectable in more than one entry and without significant effects on LTag-structure or interaction domains. However, LTag changes were predicted to affect HLA-class I presentation of immunodominant 9mers to cytotoxic T-cells. These global data strengthen single center observations and specifically our earlier findings revealing mutant 9mer epitopes conferring immune escape from HLA-I cytotoxic T cells. We conclude that variability of BKPyV-Vp1 and LTag may have important implications for diagnostic assays assessing BKPyV-specific immune control and for vaccine design. IMPORTANCE: Type and rate of amino acid variations in BKPyV may provide important insights into BKPyV diversity in human populations and an important step toward defining determinants of BKPyV-specific immunity needed to protect vulnerable patients from BKPyV diseases. Our analysis of BKPyV sequences obtained from human specimens reveals an unexpectedly high genetic variability for this double-stranded DNA virus that strongly relies on host cell DNA replication machinery with its proof reading and error correction mechanisms. BKPyV variability and immune escape should be taken into account when designing further approaches to antivirals, monoclonal antibodies, and vaccines for patients at risk of BKPyV diseases.

Impact of nucleic acid extraction procedures on human papillomavirus (HPV) detection and genotyping.

Human papillomavirus (HPV) infections are often asymptomatic, but some of the >200 HPV genotypes confer a high risk for precancerous cervical lesions and cervical cancer. Current clinical management of HPV infections relies on reliable nucleic acid testing detection and genotyping. We prospectively compared nucleic acid extraction without and with prior centrifugation enrichment for detecting and genotyping HPV in cervical swabs with atypical squamous or glandular cells. Consecutive swabs were analyzed from 45 patients with atypical squamous or glandular cells. Nucleic acids were extracted in parallel using three procedures, Abbott-M2000, Roche-MagNA-Pure-96 Large-Volume Kit without (Roche-MP-large) and with prior centrifugation (Roche-MP-large/spin) and tested using Seegene-Anyplex-II HPV28. In total, 54 HPV-genotypes were detected in 45 samples, 51 by Roche-MP-large/spin, 48 by Abbott-M2000 and 42 by Roche-MP-large. The overall concordance was 80% for detecting any HPV and 74% for specific HPV-genotypes. Roche-MP-large/spin and Abbott-M2000 showed the highest concordance for HPV detection (88.9%; kappa 0.78), and genotyping (88.5%). Two and more HPV-genotypes were detected in 15 samples, often with one HPV being more abundant. Dilution series confirmed the specific detection of multiple HPV-genotypes and their relative abundance. In 285 consecutive follow-up samples extracted by Roche-MP-large/spin, the top three detected genotypes were the high-risk HPV16, HPV53, HPV56 and the low-risk HPV42, HPV54 and HPV61. Rate and breadth of HPV detection in cervical swabs depends on extraction protocols being highest after centrifugation/enrichment. As multivalent HPV-vaccine coverage increases, detecting the evolving HPV virome depends on improved extraction and broader genotype coverage.

Molecular Characterization of BK Polyomavirus Replication in Allogeneic Hematopoietic Cell Transplantation Patients.

BACKGROUND: High-level BK polyomavirus (BKPyV) replication in allogeneic hematopoietic cell transplantation (HCT) predicts failing immune control and BKPyV-associated hemorrhagic cystitis. METHODS: To identify molecular markers of BKPyV replication and disease, we scrutinized BKPyV DNA-loads in longitudinal urine and plasma pairs from 20 HCT patients using quantitative nucleic acid testing (QNAT), DNase-I treatment prior to QNAT, next-generation sequencing (NGS), and tested cell-mediated immunity. RESULTS: We found that larger QNAT amplicons led to under-quantification and false-negatives results (P < .001). DNase-I reduced urine and plasma BKPyV-loads by >90% (P < .001), indicating non-encapsidated BKPyV genomes. DNase-resistant urine BKPyV-loads remained infectious in cell culture. BKPyV genome fragmentation of ≤250 bp impaired NGS coverage of genetic variation using 1000-bp and 5000-bp amplicons. Conversely, 250-bp amplicons captured viral minority variants. We identified genotype-specific and genotype-independent changes in capsid Vp1 or T-antigen predicted to escape from antibody neutralization or cytotoxic CD8 T-cells, respectively. Genotype-specific changes in immunodominant 9mers were associated with reduced or absent CD8 T-cell responses. Thus, failure to control BKPyV replication in HCT Patients may involve insufficient genotype-specific cytotoxic CD8 T-cell responses, potentially predictable by low neutralizing antibodies as well as genotype-independent immune escape. CONCLUSIONS: Our results provide new insights for patient evaluation and for designing immune protection through neutralizing antibodies, adoptive T-cell therapy, or vaccines.

BK Polyomavirus Evades Innate Immune Sensing by Disrupting the Mitochondrial Network and Promotes Mitophagy.

Immune escape contributes to viral persistence, yet little is known about human polyomaviruses. BK-polyomavirus (BKPyV) asymptomatically infects 90% of humans but causes premature allograft failure in kidney transplant patients. Despite virus-specific T cells and neutralizing antibodies, BKPyV persists in kidneys and evades immune control as evidenced by urinary shedding in immunocompetent individuals. Here, we report that BKPyV disrupts the mitochondrial network and membrane potential when expressing the 66aa-long agnoprotein during late replication. Agnoprotein is necessary and sufficient, using its amino-terminal and central domain for mitochondrial targeting and network disruption, respectively. Agnoprotein impairs nuclear IRF3-translocation, interferon-beta expression, and promotes p62/SQSTM1-mitophagy. Agnoprotein-mutant viruses unable to disrupt mitochondria show reduced replication and increased interferon-beta expression but can be rescued by type-I interferon blockade, TBK1-inhibition, or CoCl2-treatment. Mitochondrial fragmentation and p62/SQSTM1-autophagy occur in allograft biopsies of kidney transplant patients with BKPyV nephropathy. JCPyV and SV40 infection similarly disrupt mitochondrial networks, indicating a conserved mechanism facilitating polyomavirus persistence and post-transplant disease.

JC polyomavirus-specific antibody responses in pediatric kidney transplant recipients.

BKPyV is widely recognized in KTRs, but little is known about rates of primary and secondary JCPyV exposure in pediatric KTRs. We evaluated JCPyV exposure in pediatric KTRs using antibody responses in the first 12 months post-transplant. Of 46 children transplanted between 2009 and 2014, 6 lacked any samples for serologic testing, leaving 40 KTRs for study. JCPyV-specific IgG and IgM antibodies were measured using a normalized VLP ELISA. Significant JCPyV exposure was defined as IgG seroconversion, increasing IgG levels of >0.5 nOD units, or IgM detection. Of 40 recipients (median age 3.2 years), 11 (27.5%) were seropositive, 20 (50%) seronegative for JCPyV-IgG, while 9 (22.5%) had no specimen at the time of transplantation, but were confirmed as seronegative in post-transplant samples. Of 29 (72.5%) at risk, JCPyV-IgG seroconversion occurred in 15/29 (51.7%) including JCPyV-IgM in 6 patients (20.7%). Two patients (6.9%) developed only JCPyV-IgM. Among JCPyV-IgG-positive KTRs, six (12.5%) had significant IgG increases. Altogether 23 of 40 patients (57.5%) had serological evidence of primary or secondary JCPyV exposure. In these patients, kidney function tended to be lower during the 2 years of follow-up, but only one patient lost the graft due to JCPyV nephropathy. Thus, JCPyV exposure is common in pediatric KTR and may present serologically as primary or secondary infection. Although only one case of JC-PyVAN occurred, a trend toward lower renal function was seen. Dedicated studies of larger cohorts are warranted to define impact of JCPyV in pediatric KTR.

Correction to: Seroprevalence of hepatitis E virus (HEV) in a general adult population in Northern Norway: the Tromsø study.

Unfortunately, the supplement tables are missing in the original article. The missing files have been included here.

Seroprevalence of hepatitis E virus (HEV) in a general adult population in Northern Norway: the Tromsø study.

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in many parts of the world but only a few cases have been diagnosed in Norway. To investigate the HEV exposure rate in a presumed low-risk area, we have conducted a population-based study of anti-HEV IgG seroprevalence in Northern Norway. A total of 1800 serum samples from 900 women and 900 men, age 40-79 years, were randomly selected from the 21,083 participants in the 7th Tromsø Study, representing the 32,591 inhabitants of the Tromsø municipality that were ≥ 40 years. All samples were analyzed by ELISA-1 (recomWell HEV IgG). Samples testing positive or borderline, as well as a 1.5-fold excess of negative samples, were retested by ELISA-2 (DiaPro HEV IgG). If still borderline or a result discordant from ELISA-1, the sample was retested by ELISA-3 (Wantai HEV IgG) and strip-immunoassay (recomLine HEV IgG). Anti-HEV IgG was detected in 205 individuals (11.4%), yielding an estimated seroprevalence of 10.4% in the age-matched population of Tromsø. Using logistic regression analysis followed by multivariable backward elimination analysis, increasing age (OR 1.036 per year; p < 0.001) and higher education (OR 2.167; p < 0.001) were found as potential risk factors, whereas travel abroad or eating of red meat were not. Our results indicate that HEV-infection is common in Northern Norway and suggest that HEV testing should be included in the evaluation of elevated liver enzymes.

BK Polyomavirus MicroRNA Levels in Exosomes Are Modulated by Non-Coding Control Region Activity and Down-Regulate Viral Replication When Delivered to Non-Infected Cells Prior to Infection.

In immunosuppressed patients, BKPyV-variants emerge carrying rearranged non-coding control-regions (rr-NCCRs) that increase early viral gene region (EVGR) expression and replication capacity. BKPyV also encodes microRNAs, which have been reported to downregulate EVGR-encoded large T-antigen transcripts, to decrease viral replication in infected cells and to be secreted in exosomes. To investigate the interplay of NCCR and microRNAs, we compared archetype- and rr-NCCR-BKPyV infection in cell culture. We found that laboratory and clinical rr-NCCR-BKPyV-strains show higher replication rates but significantly lower microRNA levels than archetype virus intracellularly and in exosomes. To investigate whether rr-NCCR or increased EVGR activity modulated microRNA levels, we examined the (sp1-4)NCCR-BKPyV, which has an archetype NCCR-architecture but shows increased EVGR expression due to point mutations inactivating one Sp1 binding site. We found that microRNA levels following (sp1-4)NCCR-BKPyV infection were as low as in rr-NCCR-variants. Thus, NCCR rearrangements are not required for lower miRNA levels. Accordingly, Sp1 siRNA knock-down decreased microRNA levels in archetype BKPyV infection but had no effect on (sp1-4)- or rr-NCCR-BKPyV. However, rr-NCCR-BKPyV replication was downregulated by exosome preparations carrying BKPyV-microRNA prior to infection. To explore the potential relevance in humans, urine samples from 12 natalizumab-treated multiple sclerosis patients were analysed. In 7 patients, rr-NCCR-BKPyV were detected showing high urine BKPyV loads but low microRNAs levels, whereas the opposite was seen in 5 patients with archetype BKPyV. We discuss the results in a dynamic model of BKPyV replication according to NCCR activity and exosome regulation, which integrates immune selection pressure, spread to new host cells and rr-NCCR emergence.

Novel Human Polyomavirus Noncoding Control Regions Differ in Bidirectional Gene Expression according to Host Cell, Large T-Antigen Expression, and Clinically Occurring Rearrangements.

Human polyomavirus (HPyV) DNA genomes contain three regions denoted the early viral gene region (EVGR), encoding the regulatory T-antigens and one microRNA, the late viral gene region (LVGR), encoding the structural Vp capsid proteins, and the noncoding control region (NCCR). The NCCR harbors the origin of viral genome replication and bidirectional promoter/enhancer functions governing EVGR and LVGR expression on opposite DNA strands. Despite principal similarities, HPyV NCCRs differ in length, sequence, and architecture. To functionally compare HPyV NCCRs, sequences from human isolates were inserted into a bidirectional reporter vector using dsRed2 for EVGR expression and green fluorescent protein (GFP) for LVGR expression. Transfecting HPyV NCCR reporter vectors into human embryonic kidney 293 (HEK293) cells and flow cytometry normalized to archetype BKPyV NCCR revealed a hierarchy of EVGR expression levels with MCPyV, HPyV12, and STLPyV NCCRs conferring stronger levels and HPyV6, HPyV9, and HPyV10 NCCRs weaker levels, while LVGR expression was less variable and showed comparable activity levels. Transfection of HEK293T cells expressing simian virus 40 (SV40) large T antigen (LTag) increased EVGR expression for most HPyV NCCRs, which correlated with the number of LTag-binding sites (Spearman's r, 0.625; P < 0.05) and decreased following SV40 LTag small interfering RNA (siRNA) knockdown. LTag-dependent activation was specifically confirmed for two different MCPyV NCCRs in 293MCT cells expressing the cognate MCPyV LTag. HPyV NCCR expression in different cell lines derived from skin (A375), cervix (HeLaNT), lung (A549), brain (Hs683), and colon (SW480) demonstrated that host cell properties significantly modulate the baseline HPyV NCCR activity, which partly synergized with SV40 LTag expression. Clinically occurring NCCR sequence rearrangements of HPyV7 PITT-1 and -2 and HPyV9 UF1 were found to increase EVGR expression compared to the respective HPyV archetype, but this was partly host cell type specific.IMPORTANCE HPyV NCCRs integrate essential viral functions with respect to host cell specificity, persistence, viral replication, and disease. Here, we show that HPyV NCCRs not only differ in sequence length, number, and position of LTag- and common transcription factor-binding sites but also confer differences in bidirectional viral gene expression. Importantly, EVGR reporter expression was significantly modulated by LTag expression and by host cell properties. Clinical sequence variants of HPyV7 and HPyV9 NCCRs containing deletions and insertions were associated with increased EVGR expression, similar to BKPyV and JCPyV rearrangements, emphasizing that HPyV NCCR sequences are major determinants not only of host cell tropism but also of pathogenicity. These results will help to define secondary HPyV cell tropism beyond HPyV surface receptors, to identify key viral and host factors shaping the viral life cycle, and to develop preclinical models of HPyV persistence and replication and suitable antiviral targets.

Comparison of Two Commercial Tick-Borne Encephalitis Virus IgG Enzyme-Linked Immunosorbent Assays.

Despite the availability of protective vaccines, tick-borne encephalitis virus (TBEV) infections have been increasingly reported to the European Centre for Disease Prevention and Control in the past 2 decades. Since the diagnosis of TBEV exposure relies on serological testing, we compared two commercial enzyme-linked immunosorbent assays (ELISAs), i.e., Immunozym FSME IgG assay (ELISA-1) and Euroimmun FSME Vienna IgG assay (ELISA-2). Both assays use whole TBEV antigens, but they differ in viral strains (Neudoerfl for ELISA-1 and K23 for ELISA-2) and cutoff values. In testing of samples from 398 healthy blood donors, ELISA-1 showed higher reactivity levels than ELISA-2 (P < 0.001), suggesting different assay properties. This finding was supported by Bland-Altman analysis of the optical density at 450 nm (OD450) (mean bias, +0.32 [95% limits of agreement, -0.31 to +0.95]) and persisted after transformation into Vienna units. Concordant results were observed for 276 sera (69%) (44 positive and 232 negative results). Discordant results were observed for 122 sera (31%); 15 were fully discordant, all being ELISA-1 positive and ELISA-2 negative, and 107 were partially discordant (101 being ELISA-1 indeterminate and ELISA-2 negative and 6 having positive or indeterminate reactivity in both ELISAs). Neutralization testing at a 1:10 dilution yielded positive results for 33 of 44 concordant positive sera, 1 of 15 fully discordant sera, and 1 of 33 partially discordant sera. Indirect immunofluorescence testing revealed high antibody titers of ≥100 for yellow fever virus in 18 cases and for dengue virus in one case, suggesting that cross-reactivity contributed to the ELISA-1 results. We conclude that (i) cross-reactivity among flaviviruses remains a limitation of TBEV serological testing, (ii) ELISA-2 revealed reasonable sensitivity and specificity for anti-TBEV IgG population screening of human sera, and (iii) neutralization testing is most specific and should be reserved for selective questions.

Inter- and intralaboratory comparison of JC polyomavirus antibody testing using two different virus-like particle-based assays.

JC polyomavirus (JCPyV) can cause progressive multifocal leukoencephalopathy (PML), a debilitating, often fatal brain disease in immunocompromised patients. JCPyV-seropositive multiple sclerosis (MS) patients treated with natalizumab have a 2- to 10-fold increased risk of developing PML. Therefore, JCPyV serology has been recommended for PML risk stratification. However, different antibody tests may not be equivalent. To study intra- and interlaboratory variability, sera from 398 healthy blood donors were compared in 4 independent enzyme-linked immunoassay (ELISA) measurements generating >1,592 data points. Three data sets (Basel1, Basel2, and Basel3) used the same basic protocol but different JCPyV virus-like particle (VLP) preparations and introduced normalization to a reference serum. The data sets were also compared with an independent method using biotinylated VLPs (Helsinki1). VLP preadsorption reducing ≥35% activity was used to identify seropositive sera. The results indicated that Basel1, Basel2, Basel3, and Helsinki1 were similar regarding overall data distribution (P = 0.79) and seroprevalence (58.0, 54.5, 54.8, and 53.5%, respectively; P = 0.95). However, intra-assay intralaboratory comparison yielded 3.7% to 12% discordant results, most of which were close to the cutoff (0.080 < optical density [OD] < 0.250) according to Bland-Altman analysis. Introduction of normalization improved overall performance and reduced discordance. The interlaboratory interassay comparison between Basel3 and Helsinki1 revealed only 15 discordant results, 14 (93%) of which were close to the cutoff. Preadsorption identified specificities of 99.44% and 97.78% and sensitivities of 99.54% and 95.87% for Basel3 and Helsinki1, respectively. Thus, normalization to a preferably WHO-approved reference serum, duplicate testing, and preadsorption for samples around the cutoff may be necessary for reliable JCPyV serology and PML risk stratification.